Note: Descriptions are shown in the official language in which they were submitted.
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VIGILANCE NUCLEIC ACIDS AND RELATED DIAGNOSTIC,
SCREENING AND THERAPEUTIC METHODS
BACKGROUND OF THE INVENTION
Sleep is a naturally occurring, periodic,
reversible state of unconsciousness that is ubiquitous in
mammals and birds, although its precise function is not
known. The importance of sleep is suggested by its
homeostatic regulation: the longer an animal is awake,
the more it needs to sleep.
In humans, obtaining less than the required
number of hours of sleep, particularly over several
nights, leads to a decreased ability to retain new
information, impaired productivity, altered mood, lowered
resistance'to infection and an increased susceptibility
to accidents. Sleep-related traffic accidents annually
claim thousands of lives, and operator fatigue has also
been shown to play a contributory role in airplane
crashes and other catastrophic accidents.
Besides lifestyle factors, a variety of
physiological and psychological disorders can affect
sleep patterns. The most common sleep disorder is
primary insomnia, or a difficulty in initiating or
maintaining sleep, which affects a large percentage of
the population at some point in their lives. Other
common sleep disorders include hypersomnia, or excessive
daytime sleepiness, and narcolepsy, which is
characterized by sudden and irresistible bouts of sleep.
Currently available drugs used to modulate
vigilance, such as drugs that induce sleep, prolong
wakefulness, or enhance alertness, suffer from a number
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of shortcomings. For example, available sleep-inducing
drugs often do not achieve the fully restorative effects
of normal sleep. Often such drugs cause undesirable
effects upon waking, such as anxiety or continued
sedation. Many available drugs that increase vigilance
do so with a characteristic "crash" when the effect of
the drugs wears off. Furthermore, many of the currently
available drugs that modulate sleep and wakefulness are
addictive or have adverse effects on learning and memory.
Clearly, there is a need to identify drugs that
induce restorative sleep or that increase vigilance,
without undesirable side effects. Unfortunately, current
methods for screening for such drugs, using mammals, are
slow, burdensome and expensive. Thus, there exists a
need for improved methods for screening for drugs that
modulate sleep and vigilance.
Sleep disorders are very common, yet often go
undiagnosed or misdiagnosed because the molecular
correlates of these disorders are poorly understood.
Additionally, drugs that alter vigilance in normal
individuals and individuals suffering from vigilance
disorders may not be effective, or may have undesirable
side effects, because the drug does not target the
relevant genes or gene products that regulate the
vigilance state or mediate the vigilance disorder.
Thus, there also exists a need to identify
genes whose expression or activity is associated with
vigilance level or with particular vigilance disorders.
Identification of such genes and their expression and
activity profiles would allow more accurate diagnosis of
vigilance disorders and more accurate and rapid
determination of vigilance levels. Identification of
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such genes also provides rapids methods of identifying
therapeutic agents that specifically modulate the
expression or activity of the relevant genes associated
with vigilance. Such therapeutic agents can be used to
effectively treat vigilance disorders or to appropriately
alter vigilance levels or states in normal individuals.
The present invention satisfies these needs and
provides related advantages as well.
SUMMARY OF THE INVENTION
The invention provides a method of identifying
a compound that alters vigilance. The method consists of
contacting an invertebrate with a candidate compound,
evaluating a vigilance property in the contacted
invertebrate, and determining if the candidate compound
alters the vigilance property in the contacted
invertebrate. A candidate compound that alters the
vigilance property in the contacted invertebrate is
identified as a compound that alters vigilance.
In one embodiment, the vigilance property
evaluated is a behavioral property, including activity,
latency to sleep or arousal threshold. In another
embodiment, the vigilance property evaluated is a
molecular property, including expression of one or more
vigilance-modulated genes.
The invention also provides a method of
identifying a vigilance enhancing compound that modulates
homeostatic regulation. The method consists of
contacting an invertebrate with a compound that increases
vigilance, and determining the effect of the compound on
a homeostatic regulatory property of vigilance. A
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compound that alters the homeostatic regulatory property
is characterized as being a vigilance enhancing compound
that modulates homeostatic regulation.
Also provided is a method of identifying a
vigilance diminishing compound that modulates homeostatic
regulation. The method consists of contacting an
invertebrate with a compound that decreases vigilance,
and determining the effect of the compound on a
homeostatic regulatory property of vigilance. A compound
that alters the homeostatic regulatory property is
characterized as being a vigilance diminishing compound
that modulates homeostatic regulation.
The invention further provides an isolated
vigilance nucleic acid molecule, containing a nucleotide
sequence selected from the group consisting of SEQ ID
NOS:1-6 and 8-27, or modification thereof. Further
provides is an isolated oligonucleotide, containing at
least 15 contiguous nucleotides of the nucleotide
sequence of SEQ ID NOS:1-6 and 8-27, or the antisense
strand thereof. Also provides are kit containing two or
more isolated vigilance nucleic acid molecules or
oligonucleotides. The vigilance nucleic acid molecules
and olignucleotides can be optionally attached to a solid
support.
Also provided is a method of diagnosing a
vigilance disorder in an individual. The method consists
of determining a vigilance gene profile of the
individual, and comparing the profile to a control
profile indicative of the vigilance disorder.
Correspondence between the profile of the individual and
the control profile indicates that said individual has
the vigilance disorder. Further provided is a method of
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determining vigilance level in an individual. The method
consists of determining a vigilance gene profile of the
individual, and comparing the profile to a control
profile indicative of a predetermined vigilance level.
5 Correspondence between the profile of the individual and
the control profile indicates that the individual
exhibits said vigilance level. In such methods, at least
one vigilance gene profiled is selected from the group
consisting of Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc,
Cytochrome P450, AA117313, aryl sulfotransferase IV,
human breast tumor autoantigen homolog, KIAA313 homolog,
E25, and a gene comprising a nucleotide sequence of any
of SEQ ID NOS:2-6, 8-14 and 16-27 or modification
thereof.
Further provided is a method of determining the
efficacy of a compound in ameliorating a vigilance
disorder. The method consists of administering the
compound to an individual having a vigilance disorder,
and determining an effect of the compound on the
vigilance gene profile of the individual. Modulation of
the vigilance gene profile of the individual to
correspond to a normal vigilance profile indicates that
the compound is effective in ameliorating the vigilance
disorder. The invention also provides a method of
determining the efficacy of a compound in modulating
vigilance. The method consists of administering the
compound to an individual, and determining an effect of
the compound on the vigilance gene profile of the
individual. Modulation of the vigilance gene profile
indicates that the compound modulates vigilance. In such
methods, at least one vigilance gene profiled is selected
from the group consisting of Fas, BiP, Cyp4e2, AANAT1
(Dat), Ddc, Cytochrome P450, AA117313, aryl
sulfotransferase IV, human breast tumor autoantigen
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homolog, KIAA313 homolog, E25, and a gene comprising a
nucleotide sequence of any of SEQ ID NOS:2-6, 8-14 and
16-27 or modification thereof.
The invention further provides a method of
ameliorating a vigilance disorder in an individual. The
method consists of administering to an individual having
a vigilance disorder an agent that modulates the
vigilance gene profile of the individual to correspond to
a normal vigilance gene profile. The invention also
provides a method of modulating vigilance level in an
individual. The method consists of administering to an
individual an agent that modulates the vigilance gene
profile of the individual to correspond to a control
vigilance gene profile. In such methods, at least one
vigilance gene profiled is selected from the group
consisting of Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc,
Cytochrome P450, AA117313, aryl sulfotransferase IV,
human breast tumor autoantigen homolog, KIAA313 homolog,
E25, and a gene comprising a nucleotide sequence of any
of SEQ ID NOS:2-6, 8-14 and 16-27 or modification
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A shows a schematic of the ultrasound
activity monitoring system. Figure 1B shows a trial
comparing Drosophila activity detected by the ultrasound
apparatus (gray columns) to three behavioral states
scored by a human observer (black lines). Figure 1C
shows Drosophila activity during the light period
(horizontal white bar) and the dark period (horizontal
black bar).
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Figure 2 shows the rest-activity system
monitored in 5-day old female flies using the infrared
system. Figure 2A shows amount of rest under base-line
conditions (open circles), following manual
rest-deprivation during the dark period (black squares),
and following automated rest-deprivation during the dark
period (gray triangles). Figure 2B shows amount of rest
under base-line conditions (open circles) and following
automated rest-deprivation during the light period (gray
triangles). Figure 2A Inset shows rest under constant
darkness in control per01 flies (open circles) and in
rest-deprived per01 flies (black squares). Figure 2B
Inset shows a plot of rest during recovery versus
activity during rest deprivation.
Figure 3A and 3B show rest as a function of
Drosophila age for a 24-hour period. Rest during the
light period (horizontal white bar) and the dark period
(horizontal black bar) for flies 1 day after eclosion
(black squares), 2 days after eclosion (gray triangles),
3 days after eclosion (open circles), 16 days after
eclosion (gray diamonds), and 33 days after eclosion
(black circles) is shown. Figure 3C shows rest during
dark period in Drosophila given the indicated doses of
caffeine beginning in the final hour of the light period.
Figure 3D shows rest in the first hour of the dark
period, and Figure 3E shows latency to first dark rest,
in Drosophila given the indicated doses of hydroxyzine
beginning in the final hour of the light period.
Figure 4A shows the three experimental
conditions used to evaluate changes in gene expression,
waking (W), rest (R) and rest deprivation (RD). White
bars indicate the light period, black bars indicate the
dark period. The graphs in Figures 4B-4D show
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densitometric analysis of mRNA levels of
vigilance-modulated genes evaluated using ribonuclease
protection assays. Figure 4B shows levels of Fas and
Cyp4e2 mRNA in flies. Figure 4C shows levels of
Cytochrome oxidase C subunit I mRNA in flies and rats.
Figure 4D shows levels of BiP in flies and rats.
Figure 5A shows the number of infrared beam
crossings per day in wild-type, Datl /Datl and Datl /Df
flies (p>.05, n=25). Figure 5B shows activity patterns
as measured by the ultrasound system in wild-type,
Datl /Datl and Datl /Df flies (representative activity
records for 1 h during the light period are shown).
Figure 5C rest rebound in wild-type, Datl /Datlo and
Datl /Df flies during the first 6h of recovery. Figure 5D
shows rest rebound in wild-type Datl /Datl and Datl /Df
flies during the second 6h of recovery.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods of
rapidly and efficiently identifying compounds that alter
vigilance, including compounds that promote sleep,
prevent sleep, or increase vigilance. The compounds
identified by the methods of the invention can thus be
used to treat individuals suffering from psychological,
physiological or genetic conditions that deprive them of
restorative sleep or that cause excessive sleepiness.
These compounds can also be used to prolong wakefulness,
such as when it is desired to extend an individual's
productivity, or to increase attentiveness, learning or
memory.
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Sleep in mammals has been defined by several
criteria, including electrophysiological and behavioral
criteria. Behavioral criteria for sleep include
sustained quiescence, increased arousal threshold, and
"sleep rebound," or increased sleep or increased sleep
intensity following prolonged waking. The criterion of
sleep rebound indicates that sleep is under homeostatic
control and is thus distinguishable from mere inactivity.
Recently, physiological correlates of sleep in
mammals have been extended to the level of gene
expression. Molecular screening has revealed that brain
levels of mitochondrial enzymes and of several genes
implicated in neural plasticity are high during waking
and low during sleep (see, for example, (see Cirelli et
al., Mol. Brain Res. 56:293 (1998); Cirelli et al., Ann.
Med. 31:117 (1999); and Cirelli et al., Sleep 22(5):113
(1999)). Therefore, sleep in mammals can also be
characterized by a distinct pattern of gene expression.
Although it is well-known that most organisms
exhibit circadian rest-activity cycles, prior to the
present invention it was not known that invertebrates
exhibit a sleep-like state that is comparable, by
behavioral, physiological, developmental, molecular and
genetic criteria, to mammalian sleep. This sleep-like
state in invertebrates is henceforth referred to as
"sleep."
As described herein, invertebrate sleep is very
similar, by behavioral criteria, to mammalian sleep.
More specifically, as shown in Example I, sleep in an
exemplary invertebrate, Drosophila melanogaster, is
associated with sustained behavioral quiescence and
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increased arousal threshold. Additionally, sleep
deprivation during the normal sleep period led to a
rebound effect comparable to sleep rebound in mammals,
indicating that sleep is under similar homeostatic
5 control in invertebrates.
Furthermore, as described herein, sleep in
invertebrates is dependent on age, and follows a similar
pattern of age dependency as mammalian sleep, indicating
that sleep in invertebrates is developmentally regulated.
10 Likewise, sleep remains homeostatically regulated in
older invertebrates, as it is in older mammals (see
Example II). Additionally, sleep and wake in
invertebrates are subject to pharmacological manipulation
using compounds that are known to act as stimulants or
hypnotics in mammals (see Example III).
Furthermore, of importance to the determination
that sleep and wake in invertebrates are truly similar to
mammalian sleep and wake, it is also described herein
that several classes of genes, and several individual
genes, whose regulation is dependent on vigilance state
in mammals are similarly regulated in invertebrates (see
Example IV). Additionally, as disclosed herein,
mutations in genes that regulate sleep in invertebrates
affect vigilance properties, including homeostatic
regulation of sleep (see Example IV). Likewise,
mutations have been identified in mammalian genes that
affect sleep, including orexin (see Chemelli et al., Cell
98:437-451 (1999)), indicating that in both invertebrates
and mammals, vigilance is under genetic control.
The discovery that invertebrates exhibit sleep
and wake states that are similar by behavioral,
developmental, pharmacological, genetic and molecular
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criteria to mammalian sleep and wake, provides a basis
for the methods disclosed herein of identifying novel
compounds that can be used to modulate vigilance in
mammals by screening compounds for their effect on
vigilance properties in invertebrates.
The invention provides a method of identifying
a compound that alters vigilance. The method consists of
contacting an invertebrate with a candidate compound,
evaluating a vigilance property in the contacted
invertebrate, and determining if the candidate compound
alters the vigilance property in the contacted
invertebrate. A candidate compound that alters the
vigilance property in the contacted invertebrate is
identified as a compound that alters vigilance.
As used herein, the term "vigilance" is
intended to mean the degree or extent to which an
organism exhibits sleep or wake behaviors. Thus, the
term "altering vigilance" is intended to encompass a
change in state from wake to sleep or vice-versa, as well
as any increase or decrease in intensity or duration of
behaviors associated with a sleep or wake state.
The methods of the invention can be used to
identify compounds that either increase or decrease
vigilance. A compound that increases vigilance can, for
example, cause the animal to wake from sleep, prolong
periods of wakefulness, prolong normal latency to sleep,
restore normal sleep patterns following sleep
deprivation, or enhance beneficial wake-like
characteristics, such as alertness, responsiveness to
stimuli, energy, and ability to learn and remember. In
contrast, a compound that decreases vigilance can, for
example, cause an animal to sleep, prolong periods of
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sleep, promote restful sleep, decrease latency to sleep,
or decrease unwanted wake-like characteristics, such as
anxiety and hyperactivity.
As used herein, the term "vigilance property"
is intended to mean a behavioral, physiological or
molecular property in invertebrates that is correlated
with mammalian sleep and wake states. As described
further below, invertebrates can exhibit a variety of
behavioral properties that are closely correlated with
mammalian sleep and wake states, including activity,
arousal threshold and latency to sleep. Additionally, as
described further below, invertebrates can exhibit a
variety of molecular properties that are closely
correlated with mammalian sleep and wake states,
including expression of vigilance-modulated genes.
Invertebrates can also exhibit physiological properties
that are closely correlated with mammalian sleep,
including the frequency, type and intensity of neuronal
signals, heart rate, and the like.
Generally, invertebrates exhibit circadian
patterns of rest and activity, with most rest occurring
during the night in diurnal animals and most activity
occurring during the day. In contrast, in nocturnal
animals most rest occurs during the day, whereas most
activity takes place during the night. Under laboratory
conditions, it is possible to regulate the circadian
rest-activity cycle by regulating the length of light and
dark, and thus establish what are referred to herein as
"normal wake periods" and "normal sleep periods." For
example, in Drosophila melanogaster subjected to a
12h:12h light:dark cycle, the "normal wake period" is the
12 hour light period, whereas the "normal sleep period"
is the 12 hour dark period. Those skilled in the art can
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readily determine or establish normal wake and sleep
periods for other invertebrates.
An example of a behavioral vigilance property
that can be evaluated in invertebrates is activity during
all or part of a normal wake or sleep period. As used
herein, the term "activity" is intended to encompass all
behavioral activities normally exhibited by that
invertebrate including, for example, locomoting,
movements of body parts, grooming, eating, and the like,
in contrast to "inactivity" or "rest." Activity can be
evaluated throughout a normal wake period or throughout a
normal sleep period, or both, or evaluated for only part
of a normal wake or sleep period, such as for at least 10
minutes, 30 minutes, 1, 2, 4, 6, 8 or 12 hours. Once
activity during a normal sleep period or normal wake
period is established, those skilled in the art can
readily evaluate whether a candidate compound increases
or decreases intensity of activity or alters the pattern
of activity during all or part of that period.
For certain applications of the method, it will
be preferable to evaluate activity following sleep
deprivation. As described previously, sleep rebound
following sleep deprivation is a characteristic of
homeostatically regulated sleep. Thus, by establishing
the normal sleep rebound behavior of the invertebrate,
those skilled in the art can readily evaluate whether a
candidate compound affects the normal homeostatic
regulation of sleep.
As used herein, the term "sleep deprivation"
refers to depriving the animal of rest. This deprivation
is generally for a sufficient period of time during a
normal sleep period to result in a detectable decrease in
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activity, increase in sleep, or increase in intensity of
sleep during the subsequent period, also known as a
"sleep rebound" effect. In general, sleep deprivation
results from depriving the animal of rest during at least
10%, such as at least 25%, including from 50% to 100% of
the normal sleep period.
Any method appropriate for the particular
invertebrate can be used to deprive an animal of sleep.
As described in Example I, Drosophila melanogaster can be
sleep-deprived for the entire normal sleep period, using
manual or automated physical stimulation, and the amount,
pattern and intensity of activity indicative of sleep
rebound evaluated (see Figure 2A). In other organisms,
it may be preferable to sleep-deprive the animals using
electrical stimulation, noise, or other stimuli, for
longer or shorter periods. The time period and method
for sleep-depriving an animal can be determined by those
skilled in the art for a particular application.
Various manual and automated assays can be used
to evaluate intensity and patterns of activity. For
example, activity can be detected visually, either by
direct observation or by time-lapse photography.
Alternatively, an ultrasound monitoring system can be
used, such as the system shown in Figure lA and described
in Example I, below. Such a system is advantageous in
detecting very small movements of the animals' body parts
and, as shown in Figure 1B, the output is closely
correlated with visual observations. An example of the
activity of Drosophila melanogaster during a normal wake
period (12 hour light period) and a normal sleep period
(12 hour dark period), as evaluated using an ultrasound
monitoring system, is shown in Figure 1C.
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As a further example, an infrared monitoring
system, such as the infrared Drosophila Activity
Monitoring System available from Trikinetics (described
in M. Hamblen et al., J. Neurogen. 3:249 (1986)), can be
5 used. As described in Example I, below, an infrared
monitoring system is advantageous when simultaneously
evaluating activity in large numbers of invertebrates.
An example of the activity of a population of Drosophila
melanogaster during a normal wake period (12 hour light
10 period) and a normal sleep period (12 hour dark period),
as evaluated using an ultrasound monitoring system, is
shown in Figure 1C.
Those skilled in the art can determine an
appropriate method to evaluate invertebrate activity in a
15 particular application of the method, depending on
considerations such as the size and number of
invertebrates, their normal activity level, the intended
number of data points, and whether a quantitative or
qualitative assessment of activity is desired.
A further example of a behavioral vigilance
property that can be evaluated in invertebrates is
latency to sleep. As used herein, the term "latency to
sleep" refers to the period of time to the first rest
bout following the change from the normal wake period to
the normal sleep period (ie. from light to dark in
diurnal animals, or from dark to light in nocturnal
animals). As shown in Figure 4E, latency to sleep in
control Drosophila melanogaster was about 40 minutes. If
desired, latency to sleep following sleep deprivation can
also be established. Once normal latency to sleep, or
latency to sleep following sleep deprivation are
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established for a particular invertebrate, one skilled in
the art can evaluate whether a candidate compound
increases or decreases this vigilance property.
Another example of a behavioral vigilance
property that can be evaluated in invertebrates is
arousal threshold. As used herein, the term "arousal
threshold" refers to the amount of stimulation required
to elicit a behavioral response, such as movement. Any
reproducible stimulus can be used to evaluate arousal
threshold including, for example, vibratory stimulus,
noise, electrical stimulation, heat, or light.
Invertebrates that are in a wake state will
exhibit a behavioral response at a lower level of
stimulation than invertebrates that are in a sleep state.
For example, as described in Example I, below, when
subjected to vibratory stimuli of varying intensities,
Drosophila melanogaster that were in a wake-like state,
as determined by activity criteria, responded to
low-level stimuli that did not elicit a response in flies
that were in a sleep state. Furthermore, an animal that
is deeply asleep will exhibit an increased arousal
threshold compared to an animal that less deeply asleep.
Accordingly, arousal threshold is a measure of sleep
versus wake, as well as intensity of sleep. Once normal
arousal threshold associated with sleep and wake are
established for a particular invertebrate, those skilled
in the art can readily evaluate whether a candidate
compound increases or decreases this vigilance property.
Other vigilance properties that can be measured
in invertebrates include molecular properties correlated
with sleep and wake states. As used herein, the term
"molecular property" refers to any property that can be
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evaluated in invertebrate tissues, cells or extracts,
including, for example, production or turnover of a
second messengers, GTP hydrolysis, influx or efflux of
ions or amino acids, membrane voltage, protein
phosphorylation or glycosylation, membrane voltage,
enzyme activity, protein-protein interactions, protein
secretion, and gene expression.
A specific example of a molecular vigilance
property that can be evaluated in invertebrates is
expression of one or more vigilance-modulated genes. As
used herein, the term "expression" is intended to
encompass expression at the mRNA or polypeptide level.
Accordingly, expression of a vigilance-modulated gene can
be evaluated by any qualitative or quantitative method
that detects mRNA, protein or activity, including methods
described further below. Once the abundance or pattern
of expression of vigilance-modulated genes are
established for a particular invertebrate, those skilled
in the art can readily evaluate whether a candidate
compound increases or decreases expression of one or more
vigilance-modulated genes.
As used herein, the term "vigilance-modulated
gene" refer-s to a gene whose expression level varies
according to vigilance state. For example, the
expression level of a vigilance-modulated gene can
normally vary by at least about 10%, such as at
least 25%, or at least about 50%, including at least
about 100%, 250%, 500%, 1000% more between sleep and
wake. As described herein, at least about 1% of the
transcripts in invertebrates are modulated by vigilance
state and, consequently, correspond to
vigilance-modulated genes. Therefore, in the methods of
the invention one can evaluate expression of at least one
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vigilance-modulated gene, such as at least 2, 5, 10, 20,
50, 100 or more vigilance-modulated genes. Although not
necessary for the practice of the invention, as described
below, these genes can be cloned and/or their sequences
determined using standard molecular biology procedures.
If desired for a particular application of the
method, genes whose expression is normally upregulated in
the wake-like state, or genes whose expression is
normally upregulated in sleep, or any combination, can be
evaluated.
Exemplary vigilance-modulated genes identified
in Drosophila melanogaster, with their sequence
identifiers or GenBank Accession Nos. in brackets, and
the GenBank Accession Nos. of their apparent rat or human
homologs, are as follows: an apparent homolog of
mammalian Fatty acid synthase (Fas) (contains SEQ ID
NO:1; human:NM_004104); Cytochrome oxidase C, subunit I
(mt:Col) (J01404, J01405, and J01407; rat:J01435);
Cytochrome p450 (Cyp4e2) (X86076; rat:U39206;
human:AF054821)); BiP (Hsc70-3) (L01498; contains SEQ ID
NO:7; human:AF188611); and arylalkyamine N-acetyl
transferase (Dat) (Y07964; human:NM 001088). Each of
these genes was expressed at higher levels during waking
than during sleep (see Example IV). In contrast, a gene
designated "Rest" was 45% higher during sleep than during
rest.
Other Drosophila genes that are upregulated
during wake contain the nucleotide sequences designated
SEQ ID NOS:4, 5 and 6. Other Drosophila genes that are
upregulated during sleep contain the nucleotide sequences
designated SEQ ID NOS:2 and 3.
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As disclosed herein, there is similarity
between vigilance-modulated gene expression in rats and
in Drosophila melanogaster, both in terms of number and
type of genes that are modulated. For example, as
described in Example IV, below, Cytochrome oxidase C,
subunit I shows a rapid increase in expression during the
first few hours of waking in both rats and Drosophila.
Likewise, expression of a Drosophila and a rat Cytochrome
P450 (U39206, U39207) were similarly upregulated in
waking and sleep deprivation. Therefore,
vigilance-modulated genes in invertebrates include
homologs of genes whose expression levels vary with the
vigilance state of mammals.
A variety of vigilance-modulated genes in rats
are described in Cirelli et al., Mol. Brain Res. 56, 293
(1998); Cirelli et al., Ann. Med. 31:117 (1999); Cirelli
et al., Sleep 22(S):113 (1999) and include the following
genes, with their GenBank Accession Numbers given in
brackets: immediate-early genes, transcription factors
and chaperones (e.g. NGFI-A (M18416), NGFI-B (U17254),
Zn-15 related zinc finger (rlf; U22377), Arc (U19866),
JunB (X54686) and IERS (AW142256)); mitochondrial genes
(e.g. Cytochrome oxidase C subunit 1 (J01435), Cytochrome
oxidase C subunit IV (X54802, M37831, AA982407), NADH
dehydrogenase subunit 2 (NC_001665), 12S rRNA (J01438)
and F1-ATPase subunit alpha (X56133); and other genes,
including neurogranin (Ng/RC3; U22062), bone
morphogenetic protein 2 (Z25868), glucose-regulated
protein 78 (GRP78; M19645), brain-derived neurotrophic
factor (BDNF; M61178), interleukin-1(3 (IL-10; D21835),
dendrin (Y09000), and Ca"/calmodulin-dependent protein
kinase II (a-subunit) (J02942).
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Other rat genes, previously undisclosed as
vigilance-modulated genes, identified by differential
display analysis performed according to the methods
described in Cirelli et al., Mol. Brain Res. 56, 293
5 (1998), include Cytochrome P450 (Cyp4F5) (U39206,
U39207), AA117313, aryl sulfotransferase IV (X68640;
S42994), human breast tumor autoantigen homolog (LM04;
U24576), an apparent KIAA313 homolog (contains SEQ ID
NO:15; similar to human gene AB002311), and membrane
10 protein E25 (AF038953). Additional rat genes that are
upregulated during wake contain the nucleotide sequences
designated SEQ ID NOS:14 and 16-27. Other rat genes that
are upregulated during sleep contain the nucleotide
sequences designated SEQ ID NOS:8-13. Therefore,
15 invertebrate homologs of each of these genes are
considered to be vigilance-modulated genes.
Those skilled in the art can determine the
extent of identity or similarity between two genes needed
20 to establish that an invertebrate sequence is the homolog
of a mammalian vigilance-modulated gene. Generally,
homologous genes will encode polypeptides having at least
about 25% identity, such as at least about 30%, 40%, 50%,
75% or greater identity across the entire sequence, or a
functional domain thereof. Methods for cloning homologs
from any invertebrate species, using PCR or library
screening, are well known in the art, and are described,
for example, in standard molecular biology manuals such
as Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1992)
and in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, MD (1998).
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21
Another example of a molecular vigilance
property that can be evaluated in invertebrates is
function of one or more vigilance-altering genes. As
used herein, the term "vigilance-altering gene" refers to
a gene whose expression level can, but does not need to,
vary with vigilance state, but whose function influences
or is required for inducing or maintaining a vigilance
level or a vigilance property. Exemplary functions of a
vigilance-altering gene that can be evaluated include
transcriptional or translational regulatory activity, and
phosphorylation, dephosphorylation, glycosylation or
other post-translational modification.
Vigilance-modulated genes and
vigilance-altering genes can be identified, or their
roles confirmed, by a variety of methods, including
genetic methods. For example, animals can be generated
or identified with mutations at selected or random loci,
and their vigilance properties evaluated in order to
determine whether vigilance-modulated or
vigilance-altering genes map to these loci. For example,
as described in Example IV, below, the gene for
arylalkylamine N-acetyl transferase (also known as
dopamine acetyltransferase, or Dat; GenBank Accession No.
Y07964)) is both a vigilance-modulated gene and a
vigilance-altering gene in invertebrates. Drosophila
homozygous for a naturally-occurring hypomorphic allele
of this gene, Dat1o, exhibit a sleep rebound following
sleep deprivation that is much greater than in wild-type
flies, indicating that the Dat gene functions in the
homeostatic regulation of sleep. Drosophila hemizygous
for the Dat1o mutation, generated by crossing homozygotes
with Drosophila deficient at the Dat locus (Df), exhibit
an even more severe sleep rebound effect. Other
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vigilance modulated genes and vigilance-altering genes
can be identified, or their roles confirmed, by similar
methods.
As described in Example IV, below, Dopa
decarboxylase (Ddc) (GenBank Accession Nos. X04661,
M24111, X16802; human:M88700) is a further example of a
vigilance-altering gene whose function affects
homeostatic regulation of sleep. More specifically, the
amount of Ddc enzymatic activity in the invertebrate is
directly correlated with the amount of sleep rebound
exhibited by the animal following sleep deprivation, with
animals severely mutant at the Ddc locus exhibiting less
rebound than more mildly affected flies, and mildly
affected flies exhibiting less rebound than wild-type
flies.
Genetic methods of identifying new
vigilance-modulated or vigilance-altering genes that are
applicable to a variety of invertebrates are known in the
art. For example, the invertebrate can be mutagenized
using chemicals, radiation or insertions (e.g.
transposons, such as P element mutagenesis), appropriate
crosses performed, and the progeny screened for
phenotypic differences in vigilance properties compared
with normal controls. The gene can then be identified by
a variety of methods including, for example, linkage
analysis or rescue of the gene targeted by the inserted
element. Genetic methods of identifying genes are
described for Drosophila, for example, in Greenspan, Fly
Pushing: The Theory and Practice of Drosophila Genetics,
Cold Spring Harbor Laboratory Press (1997).
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23
There is a distinction between genes that are
modulated by vigilance state and genes that are modulated
by circadian rhythms. Thus, a gene that is modulated by
vigilance state will have a particular expression level
during a normal wake period that is similar to the
expression level following sleep deprivation, and a
different expression level during a normal sleep period.
In contrast, a gene that is modulated by circadian
rhythms will have a particular expression level during
the light period, and a different expression level during
the dark period, independent of the vigilance state of
the animal. As shown in Example IV, below, D-fos is an
example of a gene whose expression is modulated by
circadian rhythm rather than by vigilance state.
Assays to evaluate expression of
vigilance-modulated genes can involve sacrificing the
animal at the appropriate time, such as during a normal
wake period, during a normal sleep period or following
sleep deprivation, homogenizing the entire animal, or a
portion containing the brain or sensory organs, and
extracting either mRNA or proteins therefrom.
Alternatively, such assays can be performed in biopsied
tissue from the invertebrate.
A variety of assays well known in the art can
be used to evaluate expression of particular
vigilance-modulated genes, including the genes described
above. Assays that detect mRNA expression generally
involve hybridization of a detectable agent, such as a
complementary primer or probe, to the nucleic acid
molecule. Such assays include, for example, Northern or
dot blot analysis, primer extension, RNase protection
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assays, reverse-transcription PCR, competitive PCR,
real-time quantitative PCR (TaqMan PCR), and nucleic acid
array analysis.
Additionally, constructs containing the
promoter of a vigilance-modulated gene and a reporter
gene (e.g. 3-galactosidase, green fluorescent protein,
luciferase) can be made by known methods, and used to
generate transgenic invertebrates. In such transgenic
invertebrates, expression of the reporter gene is a
marker for expression of the vigilance-modulated gene.
Assays that detect protein expression can also
be used to evaluate expression of particular
vigilance-modulated genes. Such assays generally involve
binding of a detectable agent, such as an antibody or
selective binding agent, to the polypeptide in a sample
of cells or tissue from the animal. Protein assays
include, for example, immunohistochemistry,
immunofluorescence, ELISA assays, immunoprecipitation,
and immunoblot analysis.
Those skilled in the art will appreciate that
the methods of the invention can be practiced in the
absence of knowledge of the sequence or function of the
vigilance-modulated genes whose expression is evaluated.
Expression of vigilance-modulated genes can thus be
evaluated using assays that examine overall patterns of
gene expression characteristic of vigilance state. It
will be understood that as these vigilance-modulated
genes are identified or sequenced, specific probes,
primers, antibodies and other binding agents can be used
to evaluate their expression more specifically using any
of the above detection methods.
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One assay to examine patterns of expression of
vigilance-modulated genes, that does not require prior
knowledge of their sequence, is mRNA differential
display, which is described, for example, in Cirelli et
5 al., Mol. Brain Res. 56:293 (1998) and exemplified in
invertebrates in Example IV, below. In such a method,
RNA from the animal is reverse-transcribed and amplified
by PCR using a particular combination of arbitrary
primers. A detectable label, such as an enzyme, biotin,
10 fluorescent dye or a radiolabel, is incorporated into the
amplification products. The labeled products are then
separated by size, such as on acrylamide gels, and
detected by any method appropriate for detecting the
label, including autoradiography, phosphoimaging or the
15 like.
Such a method allows concurrent examination of
expression of thousands of RNA species, the vast majority
of which are expected not to be modulated by vigilance
state. However, as described in Example IV, below, there
20 will be a characteristic, reproducible banding pattern
associated with vigilance state. It can be readily
determined whether a particular candidate compound alters
this pattern of gene expression, such as by increasing or
decreasing the intensity of vigilance-modulated bands.
25 A further assay to examine patterns of
expression of vigilance-modulated genes is array
analysis, in which nucleic acids representative of all or
a portion of the genome of the invertebrate, or
representative of all or a portion of expressed genes of
the invertebrate, are attached to a solid support, such
as a filter, glass slide, chip or culture plate.
Detectably labeled probes, such as cDNA probes, are then
prepared from mRNA of an animal, and hybridized to the
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array to generate a characteristic, reproducible pattern
of spots associated with vigilance state. It can be
readily determined whether a particular candidate
compound alters this pattern of gene expression, such as
by increasing or decreasing the intensity of
vigilance-modulated spots.
Following identification of patterns of
vigilance-modulated gene expression, those skilled in the
art can clone the genes, if desired, using standard
molecular biology approaches. For example, a
vigilance-modulated band identified by differential
display can be eluted from a gel and sequenced, or used
to probe a library to identify the corresponding cDNA or
genomic DNA. Likewise, a vigilance-modulated gene from
an array can be identified based on its known position on
the array, or cloned by PCR or by probing a library.
If desired, any of the expression and activity
assays described above can be used in combination, either
sequentially or simultaneously. Such assays can also be
partially or completely automated, using methods known in
the art.
Given the teachings described herein that
behavioral vigilance properties are closely correlated
with molecular vigilance properties, and that behavioral
and molecular properties are highly conserved across
disparate species, for example, mammals and flies, it is
understood that the invention can be practiced using any
invertebrate that exhibits at least one behavioral or one
molecular vigilance property that is susceptible to
evaluation or measurement.
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As disclosed herein, Drosophila melanogaster is
an example of an invertebrate that exhibits a variety of
vigilance properties that can be evaluated, including
homeostatically regulated activity, arousal threshold,
latency to sleep, and expression of vigilance-modulated
genes. Those skilled in the art understand that other
Drosophila species are also likely to exhibit similar
vigilance properties, including D. simulans, D. virilis,
D. pseudoobscura D. funebris, D. immigrans, D. repleta,
D. affinis, D. saltans, D. sulphurigaster albostrigata
and D. nasuta albomicans. Likewise, other flies,
including, sand flies, mayflies, blowflies, flesh flies,
face flies, houseflies, screw worm-flies, stable flies,
mosquitos, northern cattle grub, and the like will also
exhibit vigilance properties.
Furthermore, insects other than flies can also
exhibit behavioral and molecular vigilance properties.
For example, species of cockroach exhibit rest rebound
following rest deprivation, as well as a higher arousal
threshold correlated with rest (Tobler et al., Sleep Res.
1:231-239 (1992)). Thus, the invention can also be
practiced with insects such as cockroaches, honeybees,
wasps, termites, grasshoppers, moths, butterflies, fleas,
lice, boll weevils and beetles.
Arthropods other than insects also can exhibit
behavioral and molecular vigilance properties. For
example, scorpions exhibit rest rebound following rest
deprivation, as well as a characteristic arousal
threshold and heart rate associated with rest (Tobler et
al., J. Comp. Physiol. 163:227-235 (1988)). Thus, the
invention can also be practiced using arthropods such as
scorpions, spiders, mites, crustaceans, centipedes and
millipedes.
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Due to the high degree of genetic similarity
across invertebrate species, invertebrates other than
arthropods, such as flatworms, nematodes (e.g. C.
elegans), mollusks (e.g. Aplysia or Hermissenda),
echinoderms and annelids will exhibit behavioral and
molecular properties correlated with vigilance state, and
can be used in the methods of the invention.
Those skilled in the art can determine, using
the assays described herein, whether a particular
invertebrate exhibits behavioral or molecular properties
correlated with vigilance state and, therefore, would be
applicable for use in the methods of the invention. The
choice of invertebrate will also depend on additional.
factors, for example, such as the availability of the
animals, the normal activity levels of the animals, the
availability of molecular probes for vigilance-modulated
genes, the number of animals and compounds one intends to
screen, the ease and cost of maintaining the animals in a
laboratory setting, the method of contacting and type of
compounds being tested, and the particular property being
evaluated. Those skilled in the art can evaluate these
factors in determining an appropriate invertebrate to use
in the screening methods.
For example, if it is desired to evaluate
molecular properties in the methods of the invention, an
invertebrate that is genetically well-characterized, such
that homologs of vigilance-modulate genes are known or
can be readily determined, may be preferred. Thus,
appropriate invertebrates in which to evaluate molecular
properties of vigilance can include, for example,
Drosophila, and C. elegans. If it desired to evaluate
behavioral properties in the methods of the invention, an
invertebrate that exhibits one or more behavioral
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properties now known to be consistent with sleep, such as
fruit flies, cockroaches, honeybees, wasps, moths,
mosquitos, scorpions, may be preferred.
As disclosed herein, invertebrate sleep
exhibits an age-dependence similar to mammalian sleep.
Therefore, it may be desirable to practice the methods of
the invention using invertebrates of different ages so as
to identify compounds that alter vigilance in the very
young or very old. Such compounds can be tailored for
use in pediatric or geriatric patients.
As also disclosed herein, invertebrate sleep
patterns differ between females and males. Therefore, it
may be desirable to practice the methods of the invention
using invertebrates of both genders separately to
identify compounds appropriate for use in females, males,
or both females and males.
If desired, invertebrates that contain
mutations of varying degrees of severity in
vigilance-altering genes can be used in the screening
methods described herein, and compounds identified that
correct these defects. In such screens, a vigilance
property is evaluated in mutant invertebrates and in
normal invertebrates. A compound that alters the
vigilance property in the mutant invertebrate to a level
or amount more similar to the property in the normal
animal can thus be identified. For example, a screen can
be conducted in a Drosophila that is mutant at the Dat
locus or the Ddc locus, both of which, as shown in
Example IV, alter, in different directions, the amount of
sleep rebound exhibited by the animal following sleep
deprivation. Accordingly, a compound that alters
homeostatic regulation of sleep can be identified as a
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compound that restores more normal sleep rebound in a Dat
or a Ddc mutant animal. Animals mutant in other
vigilance-modulated or vigilance-altering genes can
similarly be identified or generated, and used to
5 identify compounds that affect a particular function
implicated in vigilance (e.g. neurotransmitter synthesis
or degradation), or a particular property of vigilance,
including a homeostatically regulated property of
vigilance.
The methods of the invention are practiced by
contacting an invertebrate with a candidate compound, and
evaluating a vigilance property. Appropriate
invertebrates, candidate compounds and vigilance
properties to evaluate for various applications of the
method have been described above. As used herein, the
term "contacting" refers to any method of administering a
candidate compound to an invertebrate such that the
compound, or a metabolite thereof, is introduced into the
invertebrate in an effective amount so as to act on its
nervous system.
Exemplary methods of contacting an invertebrate
with a candidate compound include feeding the compound to
the animal, topical administration of the compound,
administration by aerosol spray, immersion of the animal
in a solution containing the compound, and injection of
the compound. An appropriate method of contacting an
invertebrate with a compound can be determined by those
skilled in the art and will depend, for example, on the
type and developmental stage of the invertebrate, whether
the invertebrate is sleeping or awake at the time of
contacting, the number of animals being assayed, and the
chemical and biological properties of the compound (e.g.
solubility, digestibility, bioavailability, stability and
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toxicity). For example, as shown in Example IV below,
Drosophila melanogaster can be contacted with stimulants
or hypnotics by dissolving the drugs in fly food and
providing the food to the flies.
A "candidate compound" used to contact the
invertebrate can be any molecule that potentially alters
vigilance. A candidate compound can be a naturally
occurring macromolecule, such as a peptide, nucleic acid,
carbohydrate, lipid, or any combination thereof, or a
partially or completely synthetic derivative, analog or
mimetic of such a macromolecule. A candidate compound
can also be a small organic or inorganic molecule, either
naturally occurring, or prepared partly or completely by
synthetic methods. If desired, a candidate compound can
be combined with, or dissolved in, an agent that
facilitates uptake of the compound by the invertebrate,
such as an organic solvent (e.g. DMSO, ethanol), aqueous
solvent (e.g. water or a buffer), or food.
A candidate compound can be tested at a single
dose, or at a range of doses. It is expected that the
effects on properties correlated with vigilance will be
dose dependent, as demonstrated with caffeine and
hydroxyzine in Example III, below. Appropriate
concentrations of candidate compound to test in the
methods of the invention can be determined by those
skilled in the art, and will depend on the chemical and
biological properties of the compound and the method of
contacting. Exemplary concentration ranges to test
include from about 10 g/ml to about 500 mg/ml, such as
from about 100 g/ml to 250 mg/ml, including from about
1 mg/ml to 200 mg/ml.
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The number of different compounds to screen in
the methods of the invention can be determined by those
skilled in the art depending on the application of the
method. For example, a smaller number of candidate
compounds would generally be used if the type of compound
that is likely to alter vigilance is known or can be
predicted, such as when derivatives of a lead compound
are being tested. However, when the type of compound
that is likely to alter vigilance is unknown, it is
generally understood that the larger the number of
candidate compounds screened, the greater the likelihood
of identifying a compound that alters vigilance.
Therefore, the methods of the invention can employ
screening individual compounds separately or populations
of compounds including small populations and large or
diverse populations, to identify a compound that alters
vigilance.
The appropriate time and duration to administer
the compound can be determined by those skilled in the
art depending on the application of the method. For
example, it may be desirable to administer a compound at
the beginning or end of the normal wake or sleep period,
continuously throughout a normal wake or sleep period, or
prior to, during, or after sleep deprivation, depending
on the vigilance property being evaluated and the desired
effect of the compound. As exemplified in Example III,
below, compounds that either increase or decrease
vigilance can be administered in the last hour of the
normal wake period, and their effect on activity during
the next sleep period or on latency to sleep can be
readily observed.
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Methods for producing libraries of candidate
compounds to use in the methods of the invention,
including chemical or biological molecules such as simple
or complex organic molecules, metal-containing compounds,
carbohydrates, peptides, proteins, peptidomimetics,
glycoproteins, lipoproteins, nucleic acids, antibodies,
and the like, are well known in the art. Libraries
containing large numbers of natural and synthetic
compounds also can be obtained from commercial sources.
Following contacting the invertebrate with the
candidate compound, any of the vigilance properties
described above can be evaluated, and a determination
made as to whether the compound alters, such as increases
or decreases, the vigilance property compared to a
baseline or established value for the property in an
untreated control. Such a compound will similarly alter
vigilance in mammals. However, it will be understood
that the efficacy and safety of the compound in
laboratory mammals can be further evaluated before
administering the compound to humans or veterinary
animals. For example, the compound can be tested for its
maximal efficacy and any potential side-effects using
several different invertebrates or laboratory mammals,
across a range of doses, in a range of formulations, and
at various times during the normal sleep and wake
periods.
Additionally, a compound that alters vigilance
can be tested for its effects on one or more additional
vigilance properties in order to determine its most
effective application in therapy. For example, it may be
desirable to determine whether a compound that increases
vigilance does so without significantly altering latency
to sleep when the effect of the compound wears off. Such
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a compound would be an improvement over many of the
currently known vigilance-enhancing drugs that cause a
characteristic "crash" afterwards. It may also be
desirable to determine whether the compound that alters
vigilance does so without a compensatory sleep rebound
effect.
Therefore, once a compound is identified that
alters a desirable vigilance property, the methods of the
invention can be used to determine other vigilance
characteristics of the compound. Such other
characteristics can be assessed either simultaneously
with the initial screen, or alternatively they can be
assessed in or more separate screens to identify or
characterize other vigilance properties of the compound.
For example, a vigilance altering compound identified
that promotes sleep can be further assessed to determine
whether that compound additionally reduces arousal
threshold to normal sleep levels, while preserving the
ability of the animal to be wakened normally, and with
subsequent normal wake-like behaviors. Such a compound
would be an improvement over many of the currently
available sleep-inducing drugs, which may not promote
truly restorative sleep or normal function on awakening.
Similarly, a vigilance altering compound identified that
promotes wakefulness can be further assessed, as
described above, to determine whether that compound
additionally reduces the rate or extent of the wake-sleep
transition, or "crash," following the vigilance enhancing
effects of the compound..
The methods of the invention are therefore
applicable for screening and identifying compounds that
exhibit preferred vigilance altering effects as well as
for identifying compounds that exhibit a combination of
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preferred vigilance altering effects to yield optimal
vigilance altering compounds. Such optimal vigilance
altering compounds can be identified which combine
preferred effects on vigilance levels together with
5 maintaining some or all homostatic regulatory properties
of vigilance.
As used herein, "homestatic regulatory
properties of vigilance" or "homeostatic regulatory
properties" is intended to mean those vigilance
10 properties that are compensatory changes in vigilance
resulting from, or correlating with, the quantity or
quality of vigilance from a previous time period.
Homeostatic regulatory properties are therefore vigilance
properties when viewed in light of the vigilance state of
15 a previous period. Such properties include, for example,
vigilance properties such as sleep rebound, wake period,
latency to sleep, the rate of the sleep-wake transition,
alertness or drowsiness when there has been a
corresponding and opposite change in vigilance in the
20 immediate, prior period, or when there has been a
correlative effect in the immediate, prior period.
For the specific homeostatic regulatory
property referred to as sleep rebound, prolonged or more
intense sleep periods occur as a compensatory change to
25 prior increases in vigilance periods. For the remaining
homeostatic regulatory properties specifically
exemplified above, such properties are, for example,
compensatory changes due to correlative effects in the
prior period. For example, the transition rate between
30 wake and sleep states will be correspondingly increased
or decreased depending on the amount and quality of the
previous wake or sleep vigilance state. Similarly, an
animal will be more alert following a more restful period
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and will be more drowsy following a less restful period.
Such compensatory vigilance states arise from the quality
and nature of vigilance state of the previous time
period. Homeostatic regulatory properties of vigilance
other than those described above also exist and are well
known to those skilled in the art.
Preferred or optimal vigilance altering
compounds can be identified using the methods of the
invention which exhibit, for example, predetermined
effects on the magnitude of vigilance levels or on the
period and duration of the effect. For example,
vigilance altering compounds can be identified that
either increase or decrease vigilance levels in small or
large increments or to a specified degree. Vigilance
altering compounds similarly can be identified that
increase or decrease vigilance levels to a maximum amount
allowable without affecting other vital or relevant
physiological processes. Preferred or optimal compounds
also can be selected that modulate the duration of the
vigilance altering effect for a predetermined period,
including maximal durations, without adversely affecting
other vital or relevant physiological processes.
Compounds exhibiting one or more combinations
of the above effects can similarly be identified using
the methods of the invention. A specific example of one
such preferred or optimal combination is a compound that
alters vigilance, either by increasing or decreasing
vigilance, to its maximal extent, but for a short and
specified time. Another example is a compound that
results in small alterations in vigilance levels but
exhibits a relatively prolonged, and predetermined
duration of the effect. Vigilance altering compounds
exhibiting other combinations of preferred or optimal
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vigilance effects can similarly be selected using the
methods of the invention, given the teachings and
descriptions herein.
Additionally, preferred or optimal vigilance
altering compounds can be identified using the methods of
the invention which modulate, for example, one or more
homeostatic regulatory properties of vigilance following
a prior perturbation in vigilance levels or periods. For
example, vigilance altering compounds can be identified
that modulate the sleep rebound, wake period, latency to
sleep, the rate of the sleep-wake transition, alertness
or drowsiness. Vigilance altering compounds can be
identified, for example, that increase or decrease the
period or amount of sleep rebound following prolonged
periods of increased vigilance. Similarly, vigilance
altering compounds can be identified, for example, that
increase or decrease the period or amount of wake period
as well as the level of vigilance following prolonged
periods of sleep. Such compounds can be preferred
because they increase the animal's alertness and
therefore decrease lethargic periods during the wake
state. Finally, vigilance altering compounds can be
identified that, for example, decrease the rate of the
wake-to-sleep transition so as to prevent a crash
following prolonged waking periods as well as increase
the rate of the sleep-to-wake transitions so as to
achieve normal levels of vigilance following prolonged or
induced periods of sleep.
Vigilance altering compounds exhibiting one or
more combinations of the above modulatory effects on
homeostatic regulatory properties can similarly be
identified using the methods of the invention. One
specific example is a compound that prevents or reduces
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sleep rebound to a specified extent and maintains normal
vigilance levels following prolonged wake periods.
Another specific example is a compound that increases the
rate of the sleep-to-wake transition while also
preventing lethargic periods during the wake state
following prolonged or induced sleep.
Likewise, the methods of the invention are also
applicable to identifying compounds that maintain or
mimic, for example, one or more homeostatic regulatory
properties following a prior perturbation. For example,
it can be desirable to maintain or induce normal
homeostatic regulatory properties following prior
preturbation of vigilance levels or periods. In such
instances, the methods of the invention can be used to
identify compounds that cause such effects following a
prior modulation of vigilance.
Finally, preferred or optimal vigilance
altering compounds can be identified using the methods of
the invention which exhibit combinations, including
optimal combinations, of one or more preferred vigilance
altering effects and modulation or maintenance of one or
more homeostatic regulatory properties of vigilance. For
example, vigilance altering compounds can be identified
that induce specific magnitudes or durations of vigilance
levels and which alter homeostatic regulatory properties
following the induced changes in vigilance levels. One
specific example, is a compound that maximally increases
vigilance levels over prolonged periods without a
subsequent sleep rebound effect. Alternatively, such a
vigilance increasing compound can also result in little
or no crash following the prolonged wake period. Another
example is a compound that decreases vigilance, such as
induces restful sleep states, for a predetermined period
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without a lethargic vigilance states following the sleep
period. Similarly, vigilance altering compounds can be
identified that induce specific magnitudes or durations
of vigilance levels and which alter homeostatic
regulatory properties simultaneously with the induced
changes in vigilance levels. Compounds exhibiting
various other combinations of vigilant altering effects
and modulation, or maintenance, of homeostatic regulatory
properties can similarly be identified using the method
and teachings described herein.
Therefore, the invention allows the
identification of compounds that alter vigilance levels
and modulate or maintain homeostatic regulatory
properties of vigilance. Such compounds can be
identified in the initial screen, or alternatively, such
compounds can be identified step-wise by first
identifying compounds that alter vigilance and
subsequently determining whether such identified
compounds affect homeostatic regulatory properties of
vigilance, such as sleep rebound and latency to sleep.
Similarly, compounds can be identified either in the
initial screen or in step-wise procedures that alter
vigilance properties and are devoid of deleterious
side-effects, such as a precipitous crash after the drug
wears off or lack of restfulness following drug induced
sleep. Therefore, the methods of the invention are
applicable for identifying compounds that alter vigilance
in mammals, as well as to identifying compounds that
alter vigilance levels with concomitant homeostatic
regulatory properties. Similarly, the methods of the
invention are also applicable to identifying compounds
that alter vigilance in mammals that are devoid of
deleterious and unwanted side-effects.
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Compounds identified by the methods of the
invention as compounds that alter vigilance can also have
an effect on neuronal plasticity, or the ability to learn
and form memories. Learning is not possible during sleep
5 in mammals, whereas learning and memory are positively
associated with the level of vigilance during waking.
Thus, by increasing vigilance, it is also possible to
increase learning and memory. Accordingly, in one
embodiment, the invertebrate is contacted with a
10 candidate compound, a vigilance property is evaluated,
and learning or memory is also evaluated.
A variety of assays are known in the art that
can be used to evaluate learning and either short-term or
15 long-term memory in invertebrates, including habituation
and sensitization assays, and conditioning assays.
Habituation refers to a decrease, and sensitization
refers to an increase, in a behavioral response on
repeated presentation of the same stimulus, and can be
20 considered rudimentary forms of learning. Exemplary
habituation assays that can be readily adapted for use in
a variety of invertebrates are described, for example,
for C. elegans in Rankin et al., Behav. Brain Res.
37:89-92 (1990); for Drosophila in Boynton et al.,
25 Genetics 131:655-672 (1992); and for Aplysia in Kandel et
al., Cold Spring Harb. Symp. Quant. Biol. 40:465-482
(1976).
Classical (Pavlovian) conditioning is an
30 accepted behavioral paradigm for learning and memory. In
an exemplary conditioning assay, invertebrates can be
exposed to two different stimuli, such as two odorants or
two colors of light, one of which is associated with
negative reinforcement, such as an electric shock. The
35 animals are then removed and tested in a new apparatus,
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similar to the training arrangement but without
reinforcement. Avoidance behavior is scored as learning,
and retention time of the learned behavior is scored as
memory. Exemplary conditioning assays that can be
readily adapted for use in a variety of invertebrates are
described, for example, for Drosophila in Quinn et al.,
Proc. Natl. Acad. Sci. USA 71:708-712 (1974); for
cockroach in Mizunami et al., J. Comp. Neruol.
402:520-537 (1998); and for crab in Hoyle, Behav. Biol.
18:147-163 (1976).
As described previously, invertebrate sleep,
exemplified by Drosophila sleep, is comparable to
mammalian sleep by behavioral, physiological,
developmental, molecular and genetic criteria. In
particular, individual genes, and classes of genes,
identified as vigilance-modulated genes in Drosophila are
also vigilance-modulated in mammals (see Example IV).
Other vigilance-modulated genes identified from
invertebrate molecular and genetic screens thus will also
likely be vigilance-modulated in mammals.
As exemplified in Example IV using mutants in
the Dat gene, deliberately altering the activity or
expression of vigilance-modulated genes in invertebrates
is an effective method of altering a desired vigilance
property. As further exemplified in Example IV using
mutants in the Ddc gene, deliberately altering the
activity or expression of genes that are
vigilance-altering, but not necessarily
vigilance-modulated, in invertebrates is also an
effective method of altering a desired vigilance
property. Deliberately altering the activity or
expression of vigilance-modulated or vigilance-altering
genes in mammals, collectively termed henceforth as
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"vigilance genes," are thus expected to be similarly
effective in altering desired vigilance properties.
There are numerous important diagnostic,
therapeutic, and screening applications that arise from
identification of novel vigilance genes, together with
knowledge that modulation of expression or activity of
such vigilance genes is an effective method of altering
vigilance. For example, an expression or activity
profile of one or many vigilance genes can be established
that is a molecular fingerprint of each mammalian
vigilance level, state or disorder of interest. Thus, in
diagnostic applications, it can readily be determined, by
comparing the vigilance gene profile of the individual to
control profiles, whether that individual suffers from,
or is susceptible to, a particular vigilance disorder.
Likewise, the vigilance level of an individual, and the
effect of medications or medical procedures on the
vigilance level, can be accurately determined at the
molecular level. Such determinations allow for more
appropriate determination and use of therapeutics for
treating vigilance disorders and for maintaining or
restoring normal sleep and wake patterns.
In screening applications, identification of
vigilance genes and their role in vigilance allows novel
vigilance-altering compounds to be identified, lead
compounds to be validated, and the molecular effects of
these compounds and other known vigilance-altering
compounds to be characterized, by determining the effect
of these compounds on a vigilance gene profile. For
example, the ability of a compound to alter a vigilance
gene profile of an individual to correspond more closely
to a desired vigilance level or state can be determined.
Likewise, the ability of a compound, administered to an
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individual with a particular vigilance disorder, to alter
the vigilance gene profile to correspond more closely to
the profile of a normal individual can be determined.
The compounds so identified, validated or characterized
from such assays can be administered to normal
individuals to enhance or reduce vigilance, as desired,
or to individuals having a vigilance disorder to
ameliorate the disorder and induce more normal sleep and
wake patterns.
The invention provides an isolated vigilance
nucleic acid molecule, containing a nucleic acid sequence
selected from the group consisting of SEQ ID NOS:1-6 and
8-27, or a modification thereof. An isolated nucleic
acid molecule containing a nucleotide sequence designated
SEQ ID NO:15, or modification thereof, will not consist
of the exact sequence of the human KIAA313 gene having
GenBank Accession No. AB002311. An isolated nucleic acid
molecule containing a nucleotide sequence designated SEQ
ID NO:1, or modification thereof, will not consist of the
exact sequence of the Drosophila Pi clone having GenBank
Accession No. A0005554.
In one embodiment, an isolated vigilance
nucleic acid molecule of the invention contains a nucleic
acid sequence selected from the group consisting of SEQ
ID NOS:l-6 and 8-27. An isolated nucleic acid molecule
containing a nucleotide sequence designated SEQ ID NO:1
will not consist of the exact sequence of the Drosophila
P1 clone having GenBank Accession No. AC005554. In
another embodiment, an isolated vigilance nucleic acid
molecule of the invention consists of a nucleic acid
sequence selected from the group consisting of SEQ ID
NOS:1-6 and 8-27.
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The isolated vigilance nucleic acid molecules
of the invention contain sequences from novel
vigilance-modulated genes identified from mRNA
differential display analysis performed in Drosophila
melanogaster (SEQ ID NOS:1-6), or in rat (SEQ ID
NOS:8-27). SEQ ID NOS:2, 3 and 8-13 correspond to genes
that are upregulated during sleep. SEQ ID NOS:4, 5, 6
and 14-27 correspond to genes that are upregulated during
wake.
The isolated vigilance nucleic acid molecules
of the invention hybridize to mammalian vigilance genes,
and thus can be used in the diagnostic and screening
methods described below. Additionally, the isolated
vigilance nucleic acid molecules of the invention can be
administered in gene therapy methods, including antisense
and ribozyme methods, to increase or decrease expression
of encoded vigilance polypeptides. The isolated
vigilance nucleic acid molecules of the invention can
also be used as probes or primers to identify larger
vigilance cDNAs or genomic DNA, or to identify homologs
of the vigilance nucleic acid molecules in other species.
The isolated vigilance nucleic acid molecules can further
be expressed to produce vigilance polypeptides for use in
producing antibodies or for rationally designing
inhibitory or stimulatory compounds. Other uses for the
isolated vigilance nucleic acid molecules of the
invention can be determined by those skilled in the art.
As used herein, the term "nucleic acid
molecule" refers to both deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) molecules, and can optionally
include one or more non-native nucleotides, having, for
example, modifications to the base, the sugar, or the
phosphate portion, or having a modified phosphodiester
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linkage. The term nucleic acid molecule includes both
single-stranded and double-stranded nucleic acids,
representing the sense strand, the anti-sense strand, or
both, and includes linear, circular or branched
5 molecules. Exemplary nucleic acid molecules include
genomic DNA, cDNA, mRNA and oligonucleotides,
corresponding to either the coding or non-coding portion
of the molecule, and optionally containing sequences
required for expression. A nucleic acid molecule of the
10 invention, if desired, can additionally contain a
detectable moiety, such as a radiolabel, a fluorochrome,
a ferromagnetic substance, a luminescent tag or a
detectable agent such as biotin.
The term "isolated" in reference to a vigilance
15 nucleic acid molecule is intended to mean that the
molecule is substantially removed or separated from
components with which it is naturally associated, or
otherwise modified by a human hand, thereby excluding
vigilance nucleic acid molecules as they exist in nature.
20 An isolated nucleic acid molecule of the invention can be
in solution or suspension, or immobilized on a filter,
glass slide, chip, culture plate or other solid support.
The degree of purification of the nucleic acid molecule,
and its physical form, can be determined by those skilled
25 in the art depending on the intended use of the molecule.
The term "comprising" or "containing" in
reference to a vigilance nucleic acid molecule of the
invention, is intended to mean that the nucleic acid
molecule can contain additional nucleotide sequences at
30 either the 5' or 3' end of the recited sequence, or
branching from an internal position within the recited
sequence. The additional nucleotide sequence can, if
desired, correspond to sequences that naturally occur
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within the vigilance gene, including intron or exon
sequences, promoter sequences, coding sequence, or
untranslated regions. Alternatively, the additional
nucleotide sequence can correspond to linkers or
restriction sites useful in cloning applications; to
other regulatory elements such as promoters and
polyadenylation sequences that can be useful in gene
expression; to epitope tags or fusion proteins useful in
protein purification; or the like. Those skilled in the
art can determine appropriate sequences flanking the
recited nucleotide sequences for a particular application
of the method.
The term "modification," in reference to a
vigilance nucleic acid molecule of the invention, is
intended to mean a nucleic acid molecule that contains
one or several nucleotide additions, deletions or
substitutions with respect to a reference sequence, yet
retains at least one function specific to the reference
sequence. The appropriate function to be retained will
depend on the desired use of the nucleic acid molecule.
For example, where it is desired to express a vigilance
polypeptide, a "modification" can encode substantially
the same polypeptide as the reference vigilance nucleic
acid molecule, such that the encoded polypeptide has
substantially the same immunogenicity, antigenicity,
enzymatic activity, binding activity, or other biological
property, including vigilance-altering therapeutic
activity, as the polypeptide encoded by the reference
vigilance nucleic acid molecule.
Where it is desired to use a vigilance nucleic
acid molecule in the diagnostic and screening assays
described herein, a "modification" of a vigilance nucleic
acid molecule can be a molecule that retains the ability
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to hybridize to the recited sequence under moderately
stringent conditions, or under highly stringent
conditions. The term "moderately stringent conditions,"
is intended to refer to hybridization conditions
equivalent to hybridization of filter-bound nucleic acid
in 50% formamide, 5 X Denhart's solution, 5 X SSPE, 0.2%
SDS at 42 C, followed by washing in 0.2 X SSPE, 0.2% SDS,
at 50 . In contrast, "highly stringent conditions" are
conditions equivalent to hybridization of filter-bound
nucleic acid in 50% formamide, 5 X Denhart's solution, 5
X SSPE, 0.2% SDS at 42 C, followed by washing in 0.2 X
SSPE, 0.2% SDS, at 65 . Other suitable moderately
stringent and highly stringent hybridization buffers and
conditions are well known to those of skill in the art
and are described, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York (1992) and in Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and
Sons, Baltimore, MD (1998).
Thus, a modification of a vigilance nucleic
acid molecule can be a sequence that corresponds to a
homolog of the vigilance gene in another animal species,
including other Drosophila species, other flies, other
arthropods, other invertebrates, as well as other
mammalian species, such as human, primates, rat, mouse,
rabbit, bovine, porcine, canine or feline. The sequences
of corresponding vigilance genes of desired species can
be determined by methods well known in the art, such as
by PCR or by screening genomic, cDNA or expression
libraries derived from that species.
A modification of a vigilance nucleic acid
molecule can also include substitutions that do not
change the encoded amino acid sequence due to the
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degeneracy of the genetic code. Such modifications can
correspond to variations that are made deliberately, or
which occur as mutations during nucleic acid replication.
Additionally, a modification of a vigilance nucleic acid
molecule can correspond to a splice variant form of the
recited sequence.
In general, a modification of a vigilance
nucleic acid molecule of the invention that retains at
least one function specific to the reference sequence
will have greater than about 60% identity, such as
greater than about 70% identity, including greater than
about 80%, 90%, 95%, 97% or 99% identity, to the
reference sequence over the length of the two sequences
being compared. Identity of any two nucleic acid
sequences can be determined by those skilled in the art
based, for example, on a BLAST 2.0 computer alignment,
using default parameters. BLAST 2.0 alignments can be
performed at http://www.ncbi.nlm.nih.gov/gorf/bl2.html,
as described by Tatiana et al., FEMS Microbiol Lett.
174:247-250 (1999).
The invention also provides isolated
oligonucleotides containing at least 15 contiguous
nucleotides of a nucleotide sequence referenced as SEQ ID
NOS:1-6 and 8-27, or the antisense strand thereof. The
isolated oligonucleotides of the invention are able to
hybridize to vigilance nucleic acid molecules under
moderately stringent hybridization conditions and thus
can be advantageously used, for example, as probes to
detect vigilance gene DNA or RNA in a sample; as
sequencing or PCR primers; as antisense reagents to
administer to an individual to block translation of
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vigilance RNA"in cells; or in other applications known to
those skilled in the art in which hybridization to a
vigilance nucleic acid molecule is desirable.
As used herein, the term "oligonucleotide"
refers to a nucleic acid molecule that includes at least
contiguous nucleotides from the reference nucleotide
sequence, can include at least 16, 17, 18, 19, 20 or at
least 25 contiguous nucleotides, and often includes at
least 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200
10 or more contiguous nucleotides from the reference
nucleotide sequence.
If desired, the oligonucleotide containing at
least 15 contiguous nucleotides of a nucleotide sequence
referenced as SEQ ID NOS:1-6 and 8-27 can further be
15 capable of specifically hybridizing with the reference
nucleic acid molecule. As used herein, the term
"specifically hybridize" refers to the ability of a
nucleic acid molecule to hybridize, under moderately
stringent conditions as described above, to the reference
nucleic acid molecule, without substantial hybridization
under the same conditions with nucleic acid molecules
that are not the reference nucleic acid molecules. Those
skilled in the art can readily determine whether an
oligonucleotide of the invention both hybridizes to the
recited nucleic acid sequence under moderately stringent
conditions, and also is able to specifically hybridize to
the sequence, by performing a hybridization assay in the
presence of other nucleic acid molecules, such as total
cellular nucleic acid molecules, and detecting the
presence or absence of hybridization to the other nucleic
acid molecules.
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Depending on the intended use of the
oligonucleotides of the invention, those skilled in the
art can determine whether it is necessary to use an
oligonucleotide that hybridizes to the recited vigilance
5 nucleic acid molecule and that also specifically
hybridizes to the recited vigilance nucleic acid
molecules. For example, when there are a large number of
potential contaminating nucleic acid molecules in the
sample, it may be desirable to use an oligonucleotide
10 that specifically hybridizes to the recited vigilance
nucleic acid molecule. However, when background
hybridization is not considered detrimental, when there
are few contaminating molecules, or when the
oligonucleotide is being used in conjunction with a
15 second molecule, such as a second primer, an
oligonucleotide of the invention can be used that does
not specifically hybridize to the recited nucleic acid
sequence.
In one embodiment, the invention provides a
20 primer pair for detecting vigilance nucleic acid
molecules. The primer pair contains two isolated
oligonucleotides, each containing at least 15 contiguous
nucleotides of one of the nucleotide sequences referenced
as SEQ ID NOS:1-6 and 8-27, with one sequence
25 representing the sense strand, and one sequence
representing the anti-sense strand. The primer pair can
be used, for example, to amplify vigilance nucleic acid
molecules by RT-PCR or PCR.
The isolated vigilance nucleic acid molecules
30 and oligonucleotides of the invention can be produced or
isolated by methods known in the art. The method chosen
will depend, for example, on the type of nucleic acid
molecule one intends to isolate. Those skilled in the
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art, based on knowledge of the nucleotide sequences
disclosed herein, can readily isolate the vigilance
nucleic acid molecules of the invention as genomic DNA,
or desired introns, exons or regulatory sequences
therefrom; as full-length cDNA or desired fragments
therefrom; or as full-length mRNA or desired fragments
therefrom, by methods known in the art.
One useful method for producing an isolated
vigilance nucleic acid molecule of the invention involves
amplification of the nucleic acid molecule using the
polymerase chain reaction (PCR) and vigilance nucleic
acid-specific oligonucleotide primers and, optionally,
purification of the resulting product by gel
electrophoresis. Either PCR or reverse-transcription PCR
(RT-PCR) can be used to produce a vigilance nucleic acid
molecule having any desired nucleotide boundaries.
Desired modifications to the nucleic acid sequence can
also be introduced by choosing an appropriate primer with
one or more additions, deletions or substitutions. Such
nucleic acid molecules can be amplified exponentially
starting from as little as a single gene or mRNA copy,
from any cell, tissue or species of interest.
A further method of producing an isolated
vigilance nucleic acid molecule of the invention is by
screening a library, such as a genomic library, cDNA
library or expression library, with a detectable agent.
Such libraries are commercially available or can be
produced from any desired tissue, cell, or species of
interest using methods known in the art. For example, a
cDNA or genomic library can be screened by hybridization
with a detectably labeled nucleic acid molecule having a
nucleotide sequence disclosed herein. Additionally, an
expression library can be screened with an antibody
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raised against a polypeptide encoded by a vigilance
nucleic acid disclosed herein. The library clones
containing vigilance molecules of the invention can be
isolated from other clones by methods known in the art
and, if desired, fragments therefrom can be isolated by
restriction enzyme digestion and gel electrophoresis.
Furthermore, isolated vigilance nucleic acid
molecules and oligonucleotides of the invention can be
produced by synthetic means. For example, a single
strand of a nucleic acid molecule can be chemically
synthesized in one piece, or in several pieces, by
automated synthesis methods known in the art. The
complementary strand can likewise be synthesized in one
or more pieces, and a double-stranded molecule made by
annealing the complementary strands. Direct synthesis is
particularly advantageous for producing relatively short
molecules, such as oligonucleotide probes and primers,
and nucleic acid molecules containing modified
nucleotides or linkages.
In one embodiment, the isolated vigilance
nucleic acid molecules or oligonucleotides of the
invention are attached to a solid support, such as a
chip, filter, glass slide or culture plate, by either
covalent or non-covalent methods. Methods of attaching
nucleic acid molecules to a solid support, and the uses
of nucleic acids in this format in a variety of assays,
including manual and automated hybridization assays, are
well known in the art. A solid support format is
particularly appropriate for automated diagnostic or
screening methods, where simultaneous hybridization to a
large number of vigilance genes is desired, or when a
large number of samples are being handled.
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In another embodiment, the invention provides
kits containing two or more isolated vigilance nucleic
acid molecules or oligonucleotides. At least one
vigilance nucleic acid molecule contains a nucleotide
sequence selected from the group consisting of SEQ ID
NOS:l-6 and 8-27 or modification thereof. An exemplary
kit is a solid support containing an array of isolated
vigilance nucleic acid molecules or oligonucleotides of
the invention, including, for example, at least 3, 5, 10,
20, 30, 40, 50, 75, 100 or more isolated vigilance
nucleic acid molecules or oligonucleotides.
A further exemplary kit contains one or more
PCR primer pairs, or two or more hybridization probes,
which optionally can be labeled with a detectable moiety
for detection of vigilance nucleic acid molecules. The
kits of the invention can additionally contain
instructions for use of the molecules for diagnostic
purposes in a clinical setting, or for drug screening
purposes in a laboratory setting.
If desired, the kits containing two or more
isolated vigilance nucleic acid molecules or
oligonucleotides can contain nucleic acid molecules
corresponding to genes that are upregulated during sleep,
during wake, or any combination of these genes.
Additionally, the kits containing two or more isolated
vigilance nucleic acid molecules or oligonucleotides can
contain nucleic acid molecules corresponding to sequences
identified from Drosophila screens, from rat screens,
from screens in other animals, or any combination
thereof.
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The invention also provides a vector containing
an isolated vigilance nucleic acid molecule. The vectors
of the invention are useful for subcloning and amplifying
an isolated vigilance nucleic acid molecule, for
recombinantly expressing a vigilance polypeptide, and in
gene therapy applications, described further below. A
vector of the invention can include a variety of elements
useful for cloning and/or expression of vigilance nucleic
acid molecules, such as enhancer sequences and promoter
sequences from a viral, bacterial or mammalian gene,
which provide for constitutive, inducible or
cell-specific RNA transcription; transcription
termination and RNA processing signals, including
polyadenylation signals, which provide for stability of a
transcribed mRNA sequence; an origin of replication,
which allows for proper episomal replication; selectable
marker genes, such as a neomycin or hygromycin resistance
gene, useful for selecting stable or transient
transfectants in mammalian cells, or an ampicillan
resistance gene, useful for selecting transformants in
prokaryotic cells; and versatile multiple cloning sites
for inserting nucleic acid molecules of interest.
A variety of cloning and expression vectors are
commercially available, and include, for example, viral
vectors such as a bacteriophage, baculovirus, adenovirus,
adeno-associated virus, herpes simplex virus and
retrovirus; cosmids or plasmids; bacterial artificial
chromosome vectors (BACs) and yeast artificial chromosome
vectors (YACs). Such vectors are commercially available,
and their uses are well known in the art.
The invention also provides host cells that
contain a vector containing a vigilance nucleic acid
molecule of the invention. Exemplary host cells include
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mammalian primary cells; established mammalian cell
lines, such as COS, CHO, HeLa, NIH3T3, HEK 293-T and PC12
cells; amphibian cells, such as Xenopus embryos and
oocytes; and other vertebrate cells. Exemplary host
5 cells also include insect cells (e.g. Drosophila), yeast
cells (e.g. S. cerevisiae, S. pombe, or Pichia pastoris)
and prokaryotic cells (e.g. E. coli). Methods of
introducing a vector of the invention into such host
cells are well known in the art.
10 The methods of isolating, cloning and
expressing nucleic acid molecules of the invention
referred to herein are routine in the art and are
described in detail, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring
15 Harbor Laboratory, New York (1992) and in Ansubel et al.,
Current Protocols in Molecular Biology, John Wiley and
Sons, Baltimore, MD (1998), which are incorporated herein
by reference.
The invention further provides transgenic
20 non-human animals that are capable of expressing
wild-type vigilance nucleic acids, dominant-negative
vigilance nucleic acids, antisense vigilance nucleic
acids, or ribozymes that target vigilance nucleic acids.
Such animals have correspondingly altered expression of
25 vigilance polypeptides, and can thus be used to elucidate
or confirm the function of vigilance molecules, or in
whole-animal assays to determine of validate the
physiological effect of compounds that potentially alter
vigilance. The transgene may additionally comprise an
30 inducible promoter and/or a tissue specific regulatory
element, so that expression can be induced or restricted
to specific cell types. Exemplary transgenic non-human
animals expressing vigilance nucleic acids and nucleic
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acids that alter vigilance gene expression include mouse
and Drosophila. Methods of producing transgenic animals
are well known in the art.
The invention also provides isolated vigilance
polypeptides encoded by the vigilance nucleic acid
molecules of the invention. Isolated vigilance
polypeptides of the invention can be used in a variety of
applications. For example, isolated vigilance
polypeptides can be used to generate specific antibodies,
or in screening or validation methods where it is desired
to identify or characterize compounds that alter the
activity of vigilance polypeptides.
The isolated vigilance polypeptides of the
invention can be prepared by methods known in the art,
including biochemical, recombinant and synthetic methods.
For example, vigilance polypeptides can be purified by
routine biochemical methods from neural cells or other
cells that express abundant amounts of the polypeptide.
A vigilance polypeptide having any desired boundaries can
also be produced by recombinant methods. Recombinant
methods involve expressing a vigilance nucleic acid
molecule encoding the desired polypeptide in a host cell
or cell extract, and isolating the recombinant
polypeptide, such as by routine biochemical purification
methods described above. To facilitate identification
and purification of the recombinant polypeptide, it is
often desirable to insert or add, in-frame with the
coding sequence, nucleic acid sequences that encode
epitope tags or other binding sequences, or sequences
that direct secretion of the polypeptide. Methods for
producing and expressing recombinant polypeptides in
vitro and in prokaryotic and eukaryotic host cells are
well known in the art. Furthermore, vigilance
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polypeptides can be produced by chemical synthesis. If
desired, such as to optimize their functional activity,
stability or bioavailability, such molecules can be
modified to include D-stereoisomers, non-naturally
occurring amino acids, and amino acid analogs and
mimetics.
Also provided are antibodies that specifically
bind vigilance polypeptides encoded by the vigilance
nucleic acid molecules of the invention. Such antibodies
can be used, for example, in diagnostic assays such as
ELISA assays to detect or quantitate the expression of
vigilance polypeptides; to purify vigilance polypeptides;
or as therapeutic agents to selectively target a
vigilance polypeptide. Such antibodies, if desired, can
be bound to a solid support, such as a chip, filter,
glass slide or culture plate.
As used herein, the term "antibody" is used in
its broadest sense to include polyclonal and monoclonal
antibodies, as well as antigen binding fragments of such
antibodies. An antibody of the invention is
characterized by having specific binding activity for a
vigilance polypeptide or fragment thereof of at least
about 1 x 105 M-1. Thus, Fab, F (ab') 2 , Fd and Fv fragments
of a vigilance polypeptide-specific antibody, which
retain specific binding activity for the polypeptide, are
included within the definition of an antibody. Methods
of preparing polyclonal or monoclonal antibodies against
polypeptides are well known in the art (see, for example,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1988)).
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In addition, the term "antibody" as used herein
includes naturally occurring antibodies as well as
non-naturally occurring antibodies, including, for
example, single chain antibodies, chimeric, bifunctional
and humanized antibodies, as well as antigen-binding
fragments thereof. Such non-naturally occurring
antibodies can be produced or obtained by methods known
in the art, including constructing the antibodies using
solid phase peptide synthesis, recombinant production, or
screening combinatorial libraries consisting of variable
heavy chains and variable light chains.
The invention provides diagnostic methods based
on the newly identified and characterized vigilance genes
described herein. In one embodiment, the invention
provides a method of diagnosing a vigilance disorder in
an individual. The method consists of determining a
vigilance gene profile of the individual, and comparing
that profile to a control profile indicative of the
vigilance disorder. Correspondence between the profile
of the individual and the control profile indicates that
the individual has the vigilance disorder. At least one
of the vigilance genes profiled is selected from the
group consisting of Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc,
Cytochrome P450, AA117313, aryl sulfotransferase IV,
human breast tumor autoantigen homolog, KIAA313 homolog,
E25, and a gene containing SEQ ID NOS:2-6, 8-14, or 16-27
or modification thereof.
The methods of diagnosing vigilance disorders
have numerous applications. For example, a variety of
different types of sleep disorders are known, many of
which are extremely common in a given population, some of
which are more rare. Often individuals suffering from
vigilance disorders are unaware of their disorder, or
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their illness has been misdiagnosed, so they are not
receiving appropriate treatment. Appropriate diagnosis
of the disorder will allow more effective treatments
using currently available vigilance-altering compounds or
methods, using compounds identified from the screens
described herein, using the therapeutic methods described
herein, or any combination of these treatments.
Likewise, the methods of diagnosing vigilance disorders
are applicable to monitoring the course of therapy for
the disorder, such that appropriate modifications can be
made if needed.
Furthermore, the methods of diagnosing
vigilance disorders are applicable to screening for
vigilance disorders among the general population, or
among populations in whom sleepiness presents significant
danger to the individual or to the general population
(e.g. transportation workers, individual operating heavy
machinery, and the like). Likewise, the methods of
diagnosing vigilance disorders can be used in conjunction
with diagnosis or prognosis of an associated medical or
psychiatric condition. Additional useful applications of
the diagnostic methods of the invention can be determined
by those skilled in the art.
As used herein, the term "vigilance disorder"
refers to any condition that disturbs the normal sleep
and wake patterns of an individual. A vigilance disorder
can have a genetic or familial basis; can have a
psychiatric or medical basis; can be induced by
substances including medications and drugs; or can have
any combination of these underlying causes. Exemplary
vigilance disorders include, but are not limited to,
various forms of insomnia, hypersomnia, narcolepsy,
parasomnias, sleepwalking disorder, sleep apnea, restless
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legs syndrome (RLS) and fatal familial insomnia. A
variety of vigilance disorders in humans are described in
Diagnostic and Statistical Manual of Mental Disorders,
4th Edition (1994), published by the American Psychiatric
5 Association.
Appropriate laboratory animal models of human
vigilance disorders of interest are known in the art or
can readily be developed by transgenic and knockout
methods that alter expression or activity of vigilance
10 genes, or by pharmacological, surgical or environmental
manipulation. For example, as described in Chemelli et
al., Cell 98:409-412 (1998), orexin (hypocretin) knockout
mice, as well as canarc-1 mutant dogs, are animal models
of human narcolepsy. Additionally, Michaud et al., Arch.
15 Int. Pharmacodyn. Ther. 259:93-105 (1982), describes a
rat model of insomnia that is applicable for
pharmacological research. Panckeri et al., Sleep
19:626-631(1996), describes that the English bulldog is a
natural model of sleep-disordered breathing (SDB), and
20 canine models of obstructive sleep apnea are described in
Kimoff et al., J. Appl. Physiol. 76:1810-1817 (1994).
The diagnostic methods of the invention can
also advantageously be used to characterize previously
unrecognized vigilance disorders, or newly categorize
25 vigilance disorders, based on characteristic patterns of
expression or activity of vigilance genes. Such newly
characterized or categorized disorders are also
encompassed by the term "vigilance disorder." The
diagnostic methods of the invention can also be
30 advantageously used to identify the specific vigilance
genes most closely associated with, and thus likely to
play a causative role, in particular vigilance disorders.
Such genes are targets for modulation by gene therapy
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methods or by selective targeting of the encoded product
with therapeutic compounds.
In a further embodiment of the diagnostic
methods of the invention, there is also provided a method
of determining vigilance level in an individual. The
method consists of determining a vigilance gene profile
of the individual, and comparing that profile to a
control profile indicative of a predetermined vigilance
level. Correspondence between the profile of the
individual and the control profile indicates that the
individual exhibits the predetermined vigilance level.
At least one of the vigilance genes profiled is selected
from the group consisting of Fas, BiP, Cyp4e2, AANAT1
(Dat), Ddc, Cytochrome P450, AA117313, aryl
sulfotransferase IV, human breast tumor autoantigen
homolog, KIAA313 homolog, E25, and a gene containing SEQ
ID NOS:2-6, 8-14, or 16-27 or modification thereof.
Physiological correlates of depth of sleep
(e.g. stages of REM and non-REM sleep) and degree of
alertness in laboratory animals and humans are well known
in the art. As described above, corresponding behavioral
correlates of sleep and wake states are now also known in
invertebrates. Thus, control vigilance gene profiles can
be established from invertebrates, other animals or
humans that are indicative of the range of potential
vigilance levels, from highly alert, to drowsy, to
lightly asleep, to deeply asleep, to unconscious.
Control vigilance gene profiles can also be established
indicative of the transition between normal sleep and
wake or between normal wake and sleep; indicative of
sleep deprivation or indicative of sleep rebound.
Control vigilance gene profiles can also be established
indicative of the quality or quantity of sleep or wake in
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the previous sleep or wake period. Thus, in a test
individual, a vigilance gene profile can be determined,
and compared to any of the established control profiles
to determine the vigilance level of that individual.
The methods of the invention for determining
vigilance level in an individual are advantageous over
previous methods of determining vigilance level (e.g.
cognitive tests, arousal assays, EEG) in that vigilance
gene profiles are precise molecular fingerprints
characteristic of every possible vigilance level and
state of interest. Accordingly, the precise effect of
anaesthesia, medications (including vigilance-altering
medications), medical procedures, stress, environmental
conditions, and the like, on vigilance level in an
individual can be readily determined by a simple assay
that can be performed on either sleeping or awake
individuals. Such information is valuable, for example,
in choosing an appropriate course of medical treatment
for a patient that will avoid undesirable effects on
vigilance, such as disrupting restorative sleep,
decreasing daytime alertness, or causing excessive sleep
rebound. Furthermore, should it be preferable to
continue treatment with a medication that causes such
undesirable side effects, by knowing which vigilance
genes are undesirably altered, a clinician can determine
which vigilance-altering therapeutic should concurrently,
previously or subsequently be administered to counteract
the medication to restore more normal activity or
expression of those vigilance genes, and thus reduce or
eliminate the undesirable side effects.
As used herein, the term "vigilance gene
profile" refers to any read-out that provides a
qualitative or quantitative indication of the expression
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or activity of a single vigilance gene, or of multiple
vigilance genes. A vigilance gene profile can, for
example, indicate the expression or activity of one, or
of least 2, 5, 10, 20, 50, 100 or more vigilance genes.
A vigilance gene profile can, for example, indicate the
expression or activity in mammals of mammalian homologs
of one or more vigilance genes identified as such from
the invertebrate screening assays described herein, such
as Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc, or a gene
containing any of SEQ ID NOS:2-6. A vigilance gene
profile can alternatively or additionally indicate the
expression or activity of one or more vigilance genes
identified as such from mammalian studies described
herein, such as the homolog in that mammal of Cytochrome
P450, AA117313, aryl sulfotransferase IV, human breast
tumor autoantigen, KIAA313, E25, or a gene containing any
of SEQ ID NOS:8-14 and 16-27. A vigilance gene profile
can additionally indicate the expression or activity of
one or more vigilance genes identified as such from
published mammalian studies described above, including
NGFI-A, NGFI-B, rlf, Arc, JunB, IERS, Cytochrome oxidase
C subunit 1, Cytochrome oxidase C subunit IV, NADH
dehydrogenase subunit 2, 12S rRNA Fl-ATPase subunit
alpha, Ng/RC3, bone morphogenetic protein 2, GRP78, BDNF,
IL-1(3, dendrin, and Ca++/calmodulin-dependent protein
kinase II a-subunit.
The appropriate number and type of vigilance
genes to profile will depend on the application of the
method and knowledge of the relevance of particular
vigilance genes to vigilance disorders or levels. For
example, where an association between expression or
activity of a particular vigilance gene, or of a network
of vigilance genes, and a particular vigilance disorder
or vigilance level has been established or becomes
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established, it may be desirable to profile only, or
mainly, those vigilance genes. For example, a causal
association between expression of Dat and Ddc and period
of sleep rebound has been established, as described
herein. Thus, it may be desirable in certain
applications of the method to selectively profile the
expression of Dat, Ddc, or both, such as in an individual
in whom altering sleep rebound is of interest.
It is estimated that at least about 1% of genes
in animals are vigilance-modulated. Thus, a vigilance
gene profile can indicate expression or activity of one,
a few, many, or all of these vigilance genes. A
vigilance gene profile can also indicate expression or
activity of other genes that not previously characterized
as vigilance genes, which may then be determined to be
vigilance genes.
A "vigilance gene profile" can be, for example,
a quantitative or qualitative measure of expression of
mRNA expressed by a vigilance gene. A variety of methods
of detecting or quantitating mRNA expression have been
described above in connection with invertebrate screening
assays and include, but are not limited to, Northern or
dot blot analysis, primer extension, RNase protection
assays, differential display, reverse-transcription PCR,
competitive PCR, real-time quantitative PCR (TaqMan PCR),
and nucleic acid array analysis.
A "vigilance gene profile" can also be a
quantitative or qualitative measure of expression of
polypeptides encoded by vigilance genes. Methods of
detecting or quantitating protein expression have been
described above in connection with invertebrate screening
assays, and include, but are not limited to,
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immunohistochemistry, immunofluorescence,
immunoprecipitation, immunoblot analysis, and various
types of ELISA analysis, including ELISA analysis using
arrays of vigilance-polypeptide specific antibodies bound
5 to solid supports. Additional methods include
two-dimensional gel electrophoresis, MALDI-TOF mass
spectrometry, and Prot einChipT"'/SELDI mass spectrometry
technology.
A "vigilance gene profile" can also be a
10 direct or indirect measure of the biological activity of
polypeptides encoded by vigilance genes. A direct
measure of the biological activity of a vigilance
polypeptide can be, for example, a measure of its
enzymatic activity, using an assay indicative of such
15 enzymatic activity. An indirect measure of the
biological activity of a polypeptide can be its state of
modification (e.g. phosphorylation or glycosylation) or
localization (e.g. nuclear or cytoplasmic), where the
particular modification or localization is indicative of
20 biological activity. A further indirect measure of the
biological activity of a polypeptide can be the abundance
of a substrate or metabolite of the polypeptide, such as
a neurotransmitter, where the abundance of the substrate
or metabolite is indicative of the biological activity of
25 the polypeptide. Appropriate assays for measuring enzyme
activity, polypeptide modifications, and substrates and
metabolites or vigilance polypeptides, will depend on the
biological activity of the particular vigilance
polypeptide.
30 The appropriate method to use in determining a
vigilance gene profile can be determined by those skilled
in the art, and will depend, for example, on the number
of vigilance genes being profiled; whether the method is
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performed in vivo or in a sample; the type of sample
obtained; whether the assay is performed manually or is
automated; the biological activity of the encoded
vigilance polypeptide; the abundance of the transcript,
protein, substrate or metabolite being detected; and the
desired sensitivity, reproducibility and speed of the
method.
A vigilance gene profile can be established in
vivo, such as by diagnostic imaging procedures using
detectably labeled antibodies or other binding molecules,
or from a sample obtained from an individual. As changes
in vigilance gene expression in the brain are likely to
be most relevant to regulation of the sleep-wake cycle,
appropriate samples can contain neural tissue, cells
derived from neural tissues, or extracellular medium
surrounding neural tissues, in which vigilance
polypeptides or their metabolites are present. Thus, an
appropriate sample for establishing a vigilance gene
profile in humans can be, for example, cerebrospinal
fluid, whereas in laboratory animals an appropriate
sample can be, for example, a biopsy of the brain.
However, expression of vigilance genes can also
be modulated during the sleep-wake cycle in other tissues
than neural tissue, and vigilance polypeptides or their
metabolites can be secreted into bodily fluids. In
particular, in the case of genetic vigilance disorders,
including monogenic vigilance disorders, any alteration
in vigilance gene expression or function will be
manifested in every cell in the body that normally
expresses the vigilance gene. Thus, a vigilance gene
profile can be established from any convenient cell or
fluid sample from the body, including blood, lymph,
urine, breast milk, skin, hair follicles, cervix or
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cheek. Additionally, cells can readily be obtained using
slightly more invasive procedures, such as punch biopsies
of the breast or muscle, from the bone marrow or, during
surgery, from essentially any organ or tissue of the
body.
The diagnostic methods of the invention are
practiced by determining a vigilance gene profile of an
individual, and comparing the profile of that individual
to a control profile. As used herein, the term
"individual" refers to any mammalian individual, such as
a human, a veterinary animal, or a laboratory animal.
The control profiles, which as described above include
profiles established from invertebrates or of
"individuals" can have be determined previously,
simultaneously or subsequently to determining the
vigilance gene profile of the test individual.
In the diagnostic methods described herein,
correspondence between the vigilance gene profile of the
individual and the control profile is evaluated. As used
herein, the term "correspondence" refers to a significant
degree of similarity, including identity, in pattern or
amount of expression or activity between the vigilance
gene profile in the individual and the control profile.
The degree of similarity or identity required to
establish correspondence can be determined by those
skilled in the art, and will depend on several factors
including the number of vigilance genes being examined;
the usual range of variation in expression or activity of
the vigilance genes between conditions or individuals;
the relevance of a particular vigilance gene to the
vigilance disorder or vigilance level being evaluated;
and the sensitivity of the assay being used. In general,
the term "correspondence" refers to a vigilance gene
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profile that is more similar to the control vigilance
profile than to a vigilance profile that is indicative of
a different vigilance disorder, level or state than the
control vigilance profile.
Those skilled in the art understand that the
methods described above for diagnosing vigilance
disorders and determining vigilance level can readily be
applied to methods of screening for novel
vigilance-altering compounds; to methods of validating
the efficacy of vigilance-altering compounds identified
by other methods, such as by the invertebrate screening
methods described above; to methods of determining
effective dose, time and route of administration of known
vigilance-altering compounds; to methods of determining
the effects of vigilance-altering compounds on
homeostatic regulation of vigilance; to methods of
determining the molecular mechanisms of action of known
vigilance-altering compounds; and the like. Such methods
can be performed in laboratory animals, such as mice,
rats, rabbits, dogs, cats, pigs or primates, in
veterinary animals, or in humans.
Thus, in one embodiment, the invention provides
a method of determining the efficacy of a compound in
ameliorating a vigilance disorder. The method consists
of administering the compound to an individual having a
vigilance disorder, and determining an effect of the
compound on the vigilance gene profile of the individual.
A compound that modulates the vigilance gene profile of
the individual to correspond to a normal vigilance
profile indicates that the compound is effective in
ameliorating the vigilance disorder. At least one of the
vigilance genes profiled is selected from the group
consisting of Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc,
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Cytochrome P450, AA117313, aryl sulfotransferase IV,
human breast tumor autoantigen homolog, KIAA313 homolog,
E25, and a gene containing SEQ ID NOS:2-6, 8-14, or 16-27
or modification thereof.
As used herein, the term "ameliorating" is
intended to include preventing, treating, curing, and
reducing the severity of the vigilance disorder. Those
skilled in the art understand that any degree of
reduction in severity of a vigilance disorder can improve
the health or quality of life of the individual. The
effect of the therapy can be determined by those skilled
in the art, by comparison to baseline values for
vigilance properties affected in the disorder.
In another embodiment, the invention provides a
method of determining the efficacy of a compound in
modulating vigilance. The method consists of
administering the compound to an individual, and
determining an effect of the compound on the vigilance
gene profile of the individual. A compound that
modulates the vigilance gene profile indicates that the
compound modulates vigilance. At least one of the
vigilance genes profiled is selected from the group
consisting of Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc,
Cytochrome P450, AA117313, aryl sulfotransferase IV,
human breast tumor autoantigen homolog, KIAA313 homolog,
E25, and a gene containing SEQ ID NOS:2-6, 8-14, or 16-27
or modification thereof.
The vigilance genes to profile can be
determined by those skilled in the art, depending on the
type of vigilance-altering compound it is desired to
identify or characterize. For example, it may be
advantageous to examine the effect of a compound
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primarily on single genes whose causitive role in
vigilance has been established, including Dat, Ddc, or
orexin; or only or primarily on those vigilance genes
whose expression or activity is upregulated during sleep;
5 or only or primarily on those vigilance genes whose
expression or activity is upregulated during wake; or
only or primarily on those genes whose expression is
modulated during sleep rebound, during sleep-wake
transition, or in the period following restorative or
10 disrupted sleep.
The compounds so identified that alter
vigilance gene profile can, for example, enhance
vigilance, decrease vigilance and/or alter or maintain a
15 homeostatically regulated property of vigilance such as
period of sleep rebound, latency to sleep, rate of sleep-
wake transition, or vigilance properties in the period
following changes in sleep or wake, as described above in
relation to invertebrate screening methods. The effect*
20 of these compounds on any of these vigilance properties
can be corroborated, or further evaluated, in either
invertebrates or mammals. The effect of the compounds on
learning or memory in invertebrates or mammals can also
be assessed. Compounds that beneficially alter one or a
25 combination of vigilance properties can be administered
as therapeutics to humans and veterinary animals.
Once genes associated with vigilance disorders
and vigilance levels are identified, the expression or
activity of such genes in humans or veterinary animals
30 can be selectively targeted in order to prevent or treat
the vigilance disorder, or to beneficially alter
vigilance level, state or a homeostatically regulated
property of vigilance. The diagnostic, screening and
validation methods of the invention are useful in
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determining appropriate genes to target and appropriate
therapeutic compounds to use for a particular indication.
Additional vigilance genes can be identified by the
methods described herein or by other methods, including
differential display, arrays, and other forms of
expression or activity analysis in invertebrates and
mammals; genetic methods, such as by randomly or
specifically targeting genes in model organisms such as
Drosophila or mouse, or by mapping genes associated with
vigilance disorders or altered vigilance properties; or
from screens for genes associated with other behaviors or
molecular pathways that are subsequently determined to be
associated with vigilance.
Thus, in one embodiment, the invention provides
a method of ameliorating a vigilance disorder in an
individual. The method consists of administering to an
individual having a vigilance disorder an agent that
modulates the vigilance gene profile of the individual to
correspond to a normal vigilance gene profile. At least
one of the vigilance genes profiled is selected from the
group consisting of Fas, BiP, Cyp4e2, AANAT1 (Dat), Ddc,
Cytochrome P450, AA117313, aryl sulfotransferase IV,
human breast tumor autoantigen homolog, KIAA313 homolog,
E25, and a gene containing SEQ ID NOS:2-6, 8-14, or 16-27
or modification thereof. In one embodiment, the
vigilance gene modified is one of the recited genes.
In a further embodiment, the invention provides
a method of modulating vigilance level in an individual.
The method consists of administering to an individual an
agent that modulates the vigilance gene profile of the
individual. At least one of the vigilance genes profiled
is selected from the group consisting of Fas, BiP,
Cyp4e2, AANAT1 (Dat), Ddc, Cytochrome P450, AA117313,
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aryl sulfotransferase IV, human breast tumor autoantigen
homolog, KIAA313 homolog, E25, and a gene containing SEQ
ID NOS:2-6, 8-14, or 16-27 or modification thereof. In
one embodiment, the vigilance gene modified is one of the
recited genes.
The therapeutic methods of the invention
involve determining the effect of the agent on vigilance
gene profile. Thus, the therapeutic methods of the
invention are not intended to encompass administration of
vigilance-altering drugs which inherently may modulate
vigilance gene expression or activity, in the absence of
a determination that such drugs do predictably modulate
vigilance gene profile. The effect of the therapeutic
agent on vigilance gene profile in the particular
individual in whom the agent is administered need not be
determined, however, if the effect of the therapeutic
agent on vigilance gene profile in other individuals has
previously been established, and such effect on vigilance
gene profile can be shown to be reproducible across
individuals. Of course, it is understood that the
vigilance gene profile of the individual can, if desired,
be determined prior to administration of the therapeutic
agent, and/or monitored during the course of therapy,
using modifications of the diagnostic methods described
herein.
A variety of therapeutic agents can be used to
modulate vigilance gene profile in individuals having a
vigilance disorder or in whom alteration of vigilance
level is desired. Agents can be determined or designed
to alter vigilance gene expression or activity by a
variety of mechanisms, such as by directly or indirectly
increasing or decreasing the expression of a vigilance
gene. For example, a therapeutic agent can directly
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interact with the vigilance gene promoter; can interact
with transcription factors that regulate vigilance gene
expression; can bind to or cleave the vigilance gene
transcript (e.g. antisense oligonucleotides or
ribozymes); can alter half-life of the transcript; or can
be an expressible vigilance gene itself. A therapeutic
agent can also act by increasing or decreasing activity
of one or more encoded vigilance polypeptides. For
example, the agent can specifically bind to a vigilance
polypeptide and alter its activity or half-life; can bind
to a substrate or modulator of a vigilance polypeptide;
or can be the vigilance polypeptide or active portion
thereof.
The type of agent to be used can be determined
by those skilled in the art, and will depend, for
example, on factors such as the severity of the disorder;
the time period over which correction of the disorder or
alteration of the vigilance level is desired; the
cellular location of the vigilance molecule to be
targeted; whether the agent is administered in a clinical
setting or by the individual; and when during the
sleep-wake cycle the agent is administered. In general,
therapeutic agents useful in the methods of the invention
include "compounds," as described above, including small
molecules, and gene therapy molecules.
Therapeutic agents can be formulated in
pharmaceutical compositions in such a manner to ensure
proper distribution in vivo. For example, the
blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic
agents of the invention cross the BBB, they can be
formulated, for example, in liposomes, or chemically
derivatized. Methods of introduction of a therapeutic
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agent of the invention include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, oral, intranasal, intraspinal and
intracerebral routes. An agent can also appropriately be
introduced by rechargable or biodegradable polymeric
devices, which provide for the slow release or controlled
delivery of drugs. Appropriate formulations, routes of
administration and dose of a therapeutic agent can be
determined by those skilled in the art.
For more long-lasting effect, such as in the
case of genetic vigilance disorders, the therapeutic
agents of the invention can include gene therapy
molecules that modulate vigilance gene expression or
activity, including genes encoding vigilance polypeptides
or active or inhibitory portions thereof; genes
expressing antisense molecules that block expression of
vigilance genes; and genes expressing ribozymes that
target vigilance genes. Methods of introducing and
expressing genes in animals, including humans, are well
known in the art.
Gene therapy methods can be performed ex vivo,
wherein cells (e.g. hematopoietic cells, including stem
cells) are removed from the body, engineered to express a
vigilance polypeptide, and returned to the body. Gene
therapy methods can also be performed in situ, in which
an expressible nucleic acid molecule is placed directly
into an appropriate tissue, such as the brain or CNS, by
a direct route such as injection or implantation during
surgery. Gene therapy methods can also be performed in
vivo, wherein the expressible nucleic acid molecule is
administered systemically, such as intravenously.
Appropriate vectors for gene therapy can be determined by
those skilled in the art for a particular application of
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the method, and include, but are not limited to,
retroviral vectors (e.g. replication-defective MuLV,
HTLV, and HIV vectors); adenoviral vectors;
adeno-associated viral vectors; herpes simplex viral
5 vectors; and non-viral vectors. Appropriate formulations
for delivery of nucleic acids can also be determined by
those skilled in the art, and include, for example,
liposomes; polycationic agents; naked DNA; and DNA
associated with or conjugated to targeting molecules
10 (e.g. antibodies, ligands, lectins, fusogenic peptides,
HIV tat peptide). Gene therapy methods, including
considerations for choice of appropriate vectors,
promoters, formulations and routes of delivery, are
reviewed, for example, in Anderson, Nature 392:25-30
15 (1998).
It is understood that modifications which do
not substantially affect the activity of the various
embodiments of this invention are also included within
the definition of the invention provided herein.
20 Accordingly, the following examples are intended to
illustrate but not limit the present invention.
EXAMPLE I
Behavioral Correlates of Sleep in Drosophila
This example shows that Drosophila exhibits
25 sleep that is similar to mammalian sleep, as evidenced by
the main behavioral criteria for sleep, namely sustained
behavioral quiescence (rest), increased arousal
threshold, and increased sleep following prolonged waking
(homeostatic regulation).
30 In order to monitor fly behavior, five-day old
virgin female Canton-S Drosophila melanogaster were
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cultured at 25 C, 50-60% humidity, 12hr:12hr light:dark
cycle, on brewer's yeast, dark corn syrup and agar food,
following procedures modified from J. Bennett and D.L.
van Dyke, Dros. Inform. Serv. 46:160 (1971). Continuous,
high-resolution measurement of fly behavior was achieved
using an ultrasound activity monitoring system shown in
Figure 1A. Briefly, a 44kHz standing wave was passed
across an independent enclosure containing a single fly.
An integrated circuit sampled a portion of each wave as a
function of the transmit signal and compared it to the
output from the receive signal for the same time-window.
When the fly moved its mass within the field, it
perturbed the standing wave and the resulting difference
was counted as a movement. The output was sampled by a
PC at 200 Hz, the data were summed in 2-sec bins and
stored for later processing. This system detects very
small movements in Drosophila's behavioral repertoire,
including fine movements of the head, wings, and limbs.
In order to validate the output of the
ultrasound activity monitoring system, five behaviors
were visually scored in 2-sec bins by an observer blind
to the output of the ultrasound system on 18 independent
trials for a total of 8h. The correspondence rates for
specific states were as follows: Locomoting = 99%,
Inactive=97%, Grooming anterior limbs = 94%, Grooming
posterior limbs = 98%, and Eating = 97%. This
correspondence rate is similar to that found between
measures of activity and polysomnography in humans. A
representative validation trial lasting 60 min is shown
in Figure 1B, and indicates that the ultrasound output
and visual observation are in good agreement.
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As shown in Figure 1C, using the ultrasound
activity monitoring system, female flies maintained on a
12:12 light dark cycle were active throughout the light
period (horizontal white bar) and exhibited few periods
of sustained inactivity. In contrast, during the dark
period (horizontal black bar) there were extended bouts
of quiescence. Based on pilot studies, rest was defined
as uninterrupted behavioral quiescence lasting for at
least 5 min. Greater than 90% of rest occurred during
the dark period, as shown in Figure 1C.
To monitor rest-activity patterns in large
numbers of flies, an infrared Drosophila Activity
Monitoring System was used (Trikinetics; described in M.
Hamblen et al., J. Neurogen. 3:249 (1986)). To validate
the system, flies were visually monitored for a total of
17.75h (n=7). The number of times the fly crossed the
infrared beam was counted in 5-minute bins. Flies were
awake but did not cross the beam in 5 out of 213 bins
(miss rate = 2.35%). The results obtained with the
infrared activity monitoring system demonstrated robust
circadian organization of activity and showed good
correspondence with the ultrasound monitoring system.
In order to determine whether periods of rest
are associated with increased arousal thresholds, flies
were subjected to vibratory stimuli of increasing
intensity (.05g, .1g, and 6g). In these experiments,
flies were placed in glass tubes (65mm in length, 5mm
I.D.) maintained on a hard plastic platform above a Grass
speaker. The output of the speaker was controlled via a
Beckman signal generator and the resulting vibration of
the platform was measured with an accelerometer. Each
fly received a stimulus each hour (total of 8 stimuli) of
constant intensity. The behavioral state at the time of
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stimulus delivery and the ensuing response were
videotaped and scored off-line.
Flies that had been behaviorally awake readily
responded to intensities of .05g and lg (90% of trials).
Flies that had been behaviorally quiescent for 5 minutes
or more rarely showed a behavioral response to these
stimuli (-20% of trials; p<.001, XI). However, when the
intensity of the stimulus was increased to 6g, all flies
quickly responded regardless of behavioral state (p>.l,
X2) .
These results indicate that, like sleep in
mammals, sustained periods of quiescence in Drosophila
are characterized by increased arousal thresholds.
It was next investigated whether the amount of
rest in Drosophila is homeostatically regulated. Under
baseline conditions the amount of rest during the light
period was quite low (Figure 2A, open circles). Flies
(n=24) were deprived of rest individually by gentle
tapping of their containers at rest onset (about 4
stimuli/min) for 12h during the dark period. Efforts
were made to avoid disturbing the flies if they were
eating or grooming. During the first 12h of the
following light period, rest-deprived flies (Figure 2A,
black squares; p<.001, Wilcoxon signed-ranks test for
matched pairs) exhibited a seven-fold increase in rest
compared to baseline.
Additionally, an automated system was used to
rest-deprive large numbers of flies. Only flies that
were active (indicated by the number of infrared
crossings) for at least 66% of the light period and
inactive (no infrared crossings) for at least 66% of the
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dark-period were studied. Rest deprivation was achieved
by placing glass tubes, containing individual flies, into
a cylinder that was rotated in a hybridization oven
(Hybaid) at 10 revolutions/minute. At the nadir of the
arc the tubes would be carried to the apex and dropped
2.5cm. Note that flies were not forced to walk
throughout each cycle.
Automated rest deprivation for 12h during the
dark period resulted in a three-fold increase in rest
over baseline values during the first 6h of the following
light period (mean of 10 independent experiments, n=286,
Figure 2A, gray triangles; all z>3.1, p<.001). In the
first 24h following manual rest deprivation, flies
recovered 50% of the rest that was lost, a value
comparable to the sleep rebound seen in mammals following
short-term sleep deprivation.
To investigate whether the homeostatic
regulation is separable from circadian factors, per"
mutant flies, which are arrhythmic under constant
darkness, were examined. Under constant darkness, per"
flies had the same amount of rest as under.light-dark
conditions (p>0.5), but the amount of rest was evenly
distributed across the 24 hours (open circles). Twelve
hours of automated rest deprivation in constant darkness
resulted in a significant increase in rest during the
first 6h of recovery (black squares) compared to baseline
(n=25, p<.001). Since rest is evenly distributed in per"
flies, rest deprivation eliminated only about 50% of
daily rest, compared to 90% in wild-type flies.
Recordings with the ultrasound system showed
that the rest rebound after deprivation was characterized
by actual immobility and not simply an increase of
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stationary waking activities, such as eating or grooming,
that may result in reduced infrared beam crossing.
Moreover, the amount of activity during the deprivation
was not correlated with the size of the rest rebound,
5 indicating that the increase in rest was not due to
levels of prior activity (Fig. 2B, inset). Consistent
with this, when flies were stimulated in the apparatus
for 12h during the light period, rest not only failed to
increase, but was actually reduced by 16 +/- 4% during
10 the first 6h of recovery (Figure 2B, compare gray
diamonds (rest deprived) with open circles (baseline)).
Thus, the increase in rest is not due to physical
exhaustion induced by forced activity.
Additional controls were used to validate the
15 infrared system. Flies deprived of food for 12h during
the dark period and given food during the following light
period showed no change in the number of infrared
crossings. This result indicated that eating was not
miscoded as rest. Food deprivation has been shown to
20 increase activity in Drosophila (Connolly, Nature 209:224
(1966)) and waking in mammals (Jacobs et al., Exp.
Neural. 30:212 (1971)). It was determined that food
deprivation for 1 day increased waking by 50% in
Drosophila. In addition, dusting flies with Reactive
25 Yellow, as described in Phillis et al., Genetics 133:581
(1993), increased grooming behavior by 72% but did not
reduce the number of infrared crossings. This result
indicated that grooming was not miscoded as rest.
In additional experiments it was determined
30 that male flies obtain 70% of their daily rest during the
dark period and exhibit an additional rest peak between
03.00 and 07.00 during the light period. Rest
deprivation using the automated system revealed that both
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nighttime rest and rest during the day are
homeostatically regulated.
These results indicate that rest in Drosophila,
like sleep in mammals, is under homeostatic control.
EXAMPLE II
Age-Dependence of Sleep in Drosophila
This example shows that Drosophila sleep, like
mammalian sleep, exhibits age dependence. This example
also shows that homeostatic regulation of sleep is
preserved in older flies.
In mammals, sleep is prominent in the very
young, stabilizes during adolescence and adulthood, and
declines during old age (see Stone, Clin. Ger. Med. 5:363
(1989); Bliwise, in Principles and Practice of Sleep
Medicine, Kryger et al. Eds. (Saunders, Philadelphia, 2nd
ed., 1994), chap. 3; Dijk et al., J. Physiol. 516:611
(1999)). To determine whether sleep in Drosophila
follows a similar pattern, Drosophila rest was assayed at
various days after eclosion using the infrared system.
As shown in Figure 3A, on the first full day
after eclosion (black squares) rest was pronounced,
decreased on day 2 (gray triangles), and reached stable
adult values by day 3 (open circles; p<.001; ANOVA,
Bonferroni correction). As shown in Figure 3B, as the
flies aged the amount of rest during the night began to
decline (gray diamonds, 16 days of age) and was
significantly below that found in young adults (open
circles, 3 days of age) by 33 days of age (black circles,
P<.001).
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These results indicate that rest in Drosophila
follows a similar age-dependent pattern as sleep in
mammals.
Several studies indicate that the homeostatic
regulation of sleep is preserved in older humans (see
Stone, Clin. Ger. Med. 5:363 (1989); Bliwise, in
Principles and Practice of Sleep Medicine, Kryger et al.
Eds. (Saunders, Philadelphia, 2nd ed., 1994), chap. 3;
Dijk et al., J. Physiol. 516:611 (1999)). When 33 day
old flies were deprived of rest they exhibited a rest
rebound which was similar to that seen in young flies.
These results indicate that homeostatic
regulation of rest is preserved in older flies, as it is
in older mammals.
EXAMPLE III
Pharmacological Modulation of Sleep in Drosophila
This example shows that pharmacological
compounds that modulate mammalian vigilance level also
modulate fly vigilance level.
Sleep in mammals is modulated by several
classes of drugs that act as stimulants or hypnotics.
For example, caffeine increases wakefulness and motor
activity, while antihistamines reduce sleep latency
(Yanik et al., Brain Res., 403:177 (1987)). While the
mutagenic effects of caffeine in the fly are well-studied
(e.g. Legator et al., J. Environ. Sci. Hlth. 13: 135
(1979); Dudai, Israel J. Med. Sci. 15:802 (1979);
Itoyamaet al., Cytobios. 83:245 (1995); Nassel, Microsc.
Res. Tech. 44:121 (1999) ), little is known about its
behavioral effects.
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Drugs (caffeine or hydroxyzine) dissolved in
food were continuously available to flies beginning in
the final hour of the light period. As shown in Figure
3C, when flies were given caffeine, the amount of rest
during the dark period decreased in a dose-dependent
fashion (n=36/dose, *, p<.0001) and motor activity
increased.
Histamine has been shown to be a
neurotransmitter in the central and peripheral nervous
system of the fly (Nassel, Microsc. Res. Tech. 44:121
(1999)). When flies were given hydroxyzine, an
antagonist of the H1 histamine receptor, rest during the
first hour of the dark period was increased in a
dose-dependent manner (Figure 3D), and latency to first
dark period rest was decreased (Figure 3E) (n=40/dose,
p=.056; **, p<0.001). The increase in rest was not
associated with a general impairment of fly behavior.
The activity per waking minute was unchanged during the
dark period (both during the first hour and the
subsequent hours). The total amount of activity during
the light period was also unchanged. Furthermore,
responsiveness to arousing stimuli was preserved.
Thus, two agents that modulate waking and sleep
in mammals also modulate vigilance states in Drosophila.
EXAMPLE IV
Molecular Correlates of Sleep in Drosophila
This example shows that Drosophila gene
expression is modulated by vigilance state, in a similar
manner as it is in mammals.
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Recently, several genes have been identified
whose expression in the rat brain changes in relation to
sleep and waking (see Cirelli et al., Mol. Brain Res.
56:293 (1998); Cirelli et al., Ann. Med. 31:117 (1999);
Cirelli et al., Sleep 22(5):113 (1999)). In order to
determine whether there are any molecular changes
associated with the rest-activity cycle in the fly, gene
expression in Drosophila was systematically screened
using mRNA differential display as well as a targeted
approach with RNase protection assays (RPA) to search for
specific genes.
mRNA differential display and RPA were
performed as in Cirelli et al., Mol. Brain Res. 56:293
(1998), with the following modifications. For
differential display, reverse transcription was performed
with 0.5pg of pooled total RNA from fly heads (n=20).
Two independent pools were reverse-transcribed per
condition. PCR reactions were performed in duplicate for
each pool. One hundred and four combinations of primers
were used. For RPA, 1-2 g of total RNA from pooled fly
heads (n=60) were used. The amount of sample RNA was
normalized using a riboprobe specific for ribosomal
protein rp49.
RNA was extracted from whole heads of flies
that (I) had been spontaneously resting for 3h during the
dark period; (ii) had been rest deprived for 3h and were
collected at the same circadian time, or (iii) had been
spontaneously awake for 3h during the light period (see
Figure 4A). This allowed distinguishing between changes
in gene expression associated with behavioral state and
those associated with circadian time or with stimulation.
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The behavioral state was determined
individually for each fly; only flies that satisfied
specific criteria were selected for analysis. In
particular, a fly was considered to be awake if it was
5 active for at least 90% of the 3-hour light period and
100% of the hour before sacrifice. A fly was resting if
it was inactive for at least 66% of the 3-hour dark
period and 100% of the hour before sacrifice. Only about
60-70% of the flies examined satisfied these criteria.
10 It should be noted that failure to specifically identify
rest and waking, as has been done in circadian screens,
results in samples containing a mixture of behavioral
states.
Similar to what has been shown in rat, it was
15 determined that about 1% of the transcripts examined in
Drosophila were modulated by behavioral state. Out of an
estimated 5,000 RNA species screened, 54 were expressed
at higher levels during waking than during rest and 28
were higher during rest.
20 Several transcripts (46) showed a prominent
circadian, but not state-dependent, modulation (Van
Gelder et al., Curr. Biol. 5: 1424 (1995)). For example,
a transcript designated "Circadian" was increased by 400%
in the dark conditions (both rest and rest deprivation)
25 with respect to the light condition (waking). This
transcript did not correspond to any known sequence. An
additional gene which showed a circadian, but not
state-dependent, modulation was Drosophila fos (Perkins
et al., Genes Dev. 4:822 (1990)). D-fos was expressed at
30 higher levels during the dark hours, irrespective of
behavioral state. By contrast, in rat (and cat) c-fos is
high during waking and low during sleep, irrespective of
circadian time (Pompeiano et al., J. Sleep Res. 3:80
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(1994)). In the rat suprachiasmatic nucleus, c-fos
expression is modulated in a circadian way by light
(Schwartz et al., Sem. Neurosci. 7:53 (1995)). It should
be noted that the transcriptional activity of CREB, which
is necessary for fos induction, is also higher during the
dark hours in Drosophila (Belvin et al., Neuron 22:777
(1999)).
An example of a transcript whose expression was
higher after periods of rest was designated "Rest". As
confirmed using RPA, this mRNA was 45% higher in rest
than in rest deprivation. None of the rest-related
transcripts matched any published sequence, similar to
the results in the rat.
By contrast, several known genes were
identified that were expressed at higher levels during
waking than during rest, irrespective of circadian time
(p<0.1, ANOVA). One, with high homology to Fatty acid
synthase (Fas), was increased after 3h of spontaneous
waking or rest deprivation compared to rest (by 50% and
88%, respectively, using RPA, as shown in Figure 4B,
top). This sequence matched a Drosophila P1 Clone
(A0005554). Subsequent analysis using Genescan indicated
that the sequence matched a proposed peptide that had 49%
homology with rat FAS.
Since Fas expression had not been studied in
the fly, in situ hybridization with digoxigenin-labeled
probes was performed as described in Aronstein et al.,
Neuroscience 2:115 (1996). In situ analyis indicated
that the Fas transcript is expressed throughout the fly
brain, including the optic lobes, but not in the eye.
Although the role of this enzyme in the fly brain not
clear, fatty acids are increasingly being recognized as
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modulators of neural activity (see Clark, Evolution
44:637 (1990); Yehuda et al., Peptides 19:407 (1998)).
Significantly, several genes were identified
that were upregulated during waking vs. rest in the fly
that corresponded to genes upregulated during waking vs.
sleep in the rat, irrespective of circadian time. In the
rat, mitochondrial genes, including Cytochrome oxidase C,
subunit I, show a rapid increase in expression during the
first few hours of waking (Cirelli et al., Mol. Brain
Res. 56, 293 (1998); Cirelli et al., Ann. Med. 31:117
(1999); Cirelli et al., Sleep 22(S):113 (1999) and
Figure 4C, bottom). In Drosophila, mRNA levels of
Cytochrome oxidase C, subunit I, also show a rapid
increase during the first few hours of waking with
respect to rest (Figure 4C, top). Such rapid changes in
the expression of the mitochondrial genome are thought to
represent a local response of nervous tissue to the
increased metabolic requirements of waking (Wong-Riley et
al., Neuroscience 76, 1035 (1997); Cirelli et al., Mol.
Brain Res. 56:293 (1998)).
Cytochrome P450 (Cyp4e2), a member of a large
family of detoxifying enzymes (Dunkov et al., Mol. Gen.
Genet. 251:290 (1996)), was also increased in waking and
rest deprivation with respect to rest by 77% and 99%,
respectively (Fig. 4B, bottom). A related cytochrome
P450 (Cyp4F5) was upregulated after periods of waking in
rat cerebral cortex, as demonstrated by using gene
discovery arrays and RPA (Rat Atlas cDNA 1.2 expression
array (Clontech)).
BiP is a chaperone protein localized in the
endoplasmic reticulum that assists in the folding and
assembly of newly synthesized secretory and transmembrane
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proteins. BiP may also serve as a calcium buffer (Pahl
et al., Physiol. Rev. 79:683 (1999)). In Aplysia, the
homologue of BiP is upregulated within 3h of behavioral
training and is thought to promote the structural changes
necessary for the establishment of long-term memory (Kuhl
et al., J. Cell Biol. 119:1069 (1992)). Figure 4D
(bottom) shows that, in the rat, BiP mRNA is expressed at
higher levels after periods of spontaneous waking and
sleep deprivation (8h) than after periods of sleep. A
similar pattern is found in Drosophila (Figure 4D, top).
After spontaneous waking and rest deprivation (3h), BiP
mRNA exhibits a 2-fold and 3-fold increase above resting
values, respectively.
It was also determined that mRNA levels of
arylalkyamine N-acetyl transferase (Dat) were increased
by 48% after 2-3h of waking compared to rest. This
enzyme, which is found in Drosophila brain, is involved
in the catabolism of monoamines such as tryptamine,
tyramine, serotonin, dopamine, and octopamine (Hintermann
et al., Proc. Natl. Acad. Sci. USA 93:12315 (1996);
Brodbeck et al., DNA Cell Biol.17:621(1998)). In rats,
waking is associated with a marked increase in brain mRNA
for arylsulfotransferase, another enzyme implicated in
the catabolism of monoamines (Cirelli et al., Mol. Brain
Res. 56, 293 (1998); Cirelli et al., Ann. Med. 31:117
(1999); Cirelli et al., Sleep 22(S):113 (1999)). These
findings are of importance because, in the species tested
so far, waking is associated with high central
monoaminergic activity, while a reduction of such
activity is a hallmark of sleep (McGinty et al., Brain
Res. 101: 569 (1976); Aston-Jones et al., 1:876 (1981)).
This has led to the suggestion that sleep may serve to
counteract the effects of continued monoaminergic
discharge. According to this hypothesis, an impaired
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catabolism of monoamines should result in an increased
need for sleep (Hartmann et al., Functions of Sleep,
(Yale University Press, New Haven (1973); Siegel et al.,
Brain Res. Rev. 13:213 (1988); Jouvet, Neuropsychopharm.
21, 24S (1999) ) .
To evaluate this possibility, a Drosophila
mutant was used in which the activity of the Dat enzyme
is deficient (Dateo) . Dat1O is a hypomorphic allele of
AANATlb. Insertion of blastopia into the first intron
results in 10% of wildtype dopamine acetyltransferase
activity. As indicated by both the infrared and
ultrasound monitoring systems, flies homozygous for the
Datl mutation did not differ from wild-types in the
percentage and circadian distribution of rest and waking
under baseline conditions (Figure 5A). They also showed
normal amounts and patterns of activity (Figure 5B).
Each strain obtained >90% of their daily rest during the
dark period. However, following 12h of rest deprivation
during the dark period, it was found that Datl flies
displayed a rest rebound that was much greater than in
rest deprived controls (189%) (Figure 5C).
To confirm that this phenotype maps to the Dat
locus and to assay for gene dosage effects, flies with
one dose of the Dat1O mutation (hemizygous) were generated
by crossing Dat1O homozygotes with flies carrying a
deficiency (Df) of the Dat locus, Df(2R)Pxl. Flies
hemizygous for the Datl mutation (Dateo/Df) did not differ
from wild-types or Datl homozygotes in the percentage and
circadian distribution of rest and waking under baseline
conditions (Figure 5A). Datl /Df flies showed not only an
increased rest rebound during the first 6h of recovery
compared to wild-type flies (Figure 5C), but also a
persistent rebound during the second 6h of recovery
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(Figure 5D). These results indicate that the more
severely mutant the fly is at the Dat locus, the greater
the rebound. Although the mechanism responsible for the
increased homeostatic response to rest deprivation is not
5 clear, these results suggest a linkage between the
catabolism of monoamines and the regulation of sleep and
waking in Drosophila.
In order to evaluate whether other genes
involved in monoamine catabolism are associated with
10 altered vigilance, mutants in Dopa decaryboxylase (Ddc)
were evaluated. Dopa decaryboxylase (Ddc) is involved in
the final step in the synthesis of the neurotransmitter
dopamine. Two genotypes, Ddc(ts2]/+ and Ddc[27]/+, both
heterozygous for Ddc mutations, were tested. Ddc(ts2]/+
15 has somewhat more enzyme activity than Ddc[27]/+.
Ddc(ts2]/+ and Ddc(27]/+ Drosophila were tested initially
for activity and sleep, both of which were normal.
Ddc(ts2]/+ and Ddc(27]/+ Drosophila were then tested for
rebound effect after sleep deprivation. Both Ddc[ts2]/+
20 and Ddc(27]/+ Drosophila exhibited approximately half as
much rebound as wild-type flies. Moreover, the rebound
in Ddc(27]/+ flies (2 hr long) was shorter than in
Ddc(ts2]/+ flies(4 hr long), as compared to
wild-type (6 hr long). These results are consistent with
25 a role for Ddc in homeostatic regulation of sleep. More
specifically, the less Ddc enzyme activity, the less
rebound.
The results observed with Ddc mutants are also
consistent with the Dat results. Dat mutants fail to
30 degrade several neurotransmitters, including dopamine.
The less Dat activity the flies have, the more and longer
rebound they show. The Ddc mutants exhibit opposite
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behavior -- the less neurotransmitter produced, the less
rebound. Thus, there is an apparent correlation between
the accumulation of neurotransmitters such as dopamine
and the amount of rebound.
Taken together, the results shown in
Examples I-IV indicate that rest in invertebrates is very
similar to mammalian sleep, as evidenced by increased
arousal threshold, homeostatic regulation, dependence on
age, sensitivity to pharmacological manipulation, and
expression of similar vigilance-modulated genes.
Although the invention has been described with
reference to the examples provided above, it should be
understood that various modifications can be made without
departing from the spirit of the invention. Accordingly,
the invention is limited only by the claims.
SEQUENCE LISTING IN ELECTRONIC FORM
This description contains a sequence listing in
electronic form in ASCII text format. A copy of the sequence
listing in electronic form is available from the Canadian
Intellectual Property Office. The sequences in the sequence
listing in electronic form are reproduced in the following
Table.
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SEQUENCE TABLE
<110> Neurosciences Research Foundation, Inc.
TONONI, Giulio
CIRELLI, Chiara
SHAW, Paul J.
GREENSPAN, Ralph J.
<120> Vigilance Nucleic Acids and Related
Diagnostic, Screening and Therapeutic Methods
<130> FP-NI 4498
<150> US 09/449,175
<151> 1999-11-24
<150> US 09/456,785
<151> 1999-12-08
<160> 27
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 317
<212> DNA
<213> Drosophila sp.
<400> 1
catcccagtt acgcacagct aggagagata agttaacaga ttggatttgc gtcgatcgga 60
ttgaatctgc ctaactgctg tcttgcaggg ctgtaggctt cggtccaaaa ttaagtctac 120
agtctctgcg attccgggca gttgaaatga tgcaagatga gaggctcagt ccataacact 180
taacgctttg tgcaccgtac aatttagtac ccgacattcc ttgagagcta gacggcacca 240
gacgtcccaa cttaccaaat atatctttta tgctctatct atatgtatgt cgcatatcta 300
gtttatgcta gttgtag 317
<210> 2
<211> 356
<212> DNA
<213> Drosophila sp.
<400> 2
accttctaac ctcatacaca cacacgggat cctttcttat agggggcctg ctttcaaaac 60
tatttctgcg gactcctgta attcatcctg tcgtctgctt tccattatta cacgttctca 120
acgctcacaa gattgcttca aaccgatctg tctctatccc gcatctgcct gccccggtat 180
cgttttgata tgtgaatgcc ttacacgatc cacgcttttg aaagabccsc ccagtccaac 240
ccgaatcccg ccacggccct tttttccacc aatctcgcaa gatgccttgc tcgttttgta 300
aatagctttt yygaagacgr aagatcaaga caaatcaaat caaatcgatc caattt 356
<210> 3
<211> 393
<212> DNA
<213> Drosophila sp.
<400> 3
gcccttcgtg aattcgcttt ctacccgcgt tgacattctc cgctggagct ggtattaaag 60
tgctaatggg catcgacttt gctggcgtcg tgatgggctg gaagttcttc gatcacgcag 120
cscatttggg cggcgcatgt ttggcatctt ttgggccacg tatggggcac agatatgggc 180
aaagcgcatt ggtctactga attactacca tgacctgcgc cggacgaagc agaaatagta 240
CA 02391305 2009-11-16
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caggaggctt ggggattcgc caagtgaacg gacggggagc tgaggcaata gagcggtcrr 300
ggtagcacta cgctgtgtca ataagagtga catcgttctg ccttagaagt tcattgaaat 360
cagttaagtt gaataaatcg ttgacatcct gat 393
<210> 4
<211> 505
<212> DNA
<213> Drosophila sp.
<400> 4
gcccttcgga attcggtttt ttttttttcc ctcatgctcc cgactaaaaa gttcttttaa 60
gttaagtaag gcacaacgac ttatattagc aacaactgtt gtttcttggt ttttctcaca 120
attagaaaga aattgaaatg gaaactcatt tagatcatag gcttcttcct tattgacacg 180
atactcttct ttcgttttat ctattatctc ctctaaatac tcttttacat caacactatc 240
atcaattaaa ctagcaatct tcccattttt tacaataaag ttagtatgag gaggatccaa 300
agcataatca tttaagttaa accaataaag caatgcttta aacaactttt cttcttcctt 360
ttccttagta acttgctgtt tttttaggtt cctcattaga aacctcagga tttaagaatg 420
ttgaatcatc caatgctata ggagaagaaa tcsatgatga tctactccaa gatagctgkg 480
attgatcmtc agtggattga ctgaa 505
<210> 5
<211> 169
<212> DNA
<213> Drosophila sp.
<400> 5
gcccttctga ctcactgagc ctccgcccct accaacccca ggtgttcctc agcctcgtaa 60
ctggcccagc ttccaggata cggacctctg agtccggttt cgataccccc acaaccacaa 120
tcacgatcac gaagaagact ccggcgaaga taagcccaag gacaagtct 169
<210> 6
<211> 291
<212> DNA
<213> Drosophila sp.
<400> 6
tgccggatgt taacataggr gttagtctaa aggtctgcga gagcatcgac aaggagctta 60
gatcatccta caatcagcag aggcagcagc aacgcaaggv agccaaamvg gagcagcagg 120
tgacgcgggc agaaacgtac gattccatta gacgttttgg acgcgcccat caaaaagcca 180
ggcaaagggg aataccaggm aagggaaaga gaacgtgaaa aagaggcgtc agcggctctt 240
caggaaacgg acaaggaaag ggraammccc cccccccsaa ttccggaggg c 291
<210> 7
<211> 267
<212> DNA
<213> Drosophila sp.
<400> 7
gttagcctac ggatatgtaa agcggtcaag agaaacagac aaatgttata tgtataagcg 60
aataatgtgg tcccaattcc tgcgatatat gcgaatatat cggggaatcg gactgtattg 120
tattgatgag atatagaaga aagagagaga gagaggagcc agggagctgc gggtcgtgcg 180
gaggaggaga ataggtcgat gaggaatggg aacggcagaa gagcaagaga taaaccacga 240
aatcatacgg aaaccactag agagcag 267
<210> 8
<211> 225
<212> DNA
<213> rattus sp.
CA 02391305 2009-11-16
94
<400> 8
gttctttttt ttccggagct ggggactgaa cccagggcct tgcgcttcct aggtaaggga 60
tctacctcgc agctaaatcc ccagccccca gctcactggt cttaaagggc tccaggagtc 120
attttatata caagggaact gaagcatgaa gggttagatc acctgctcag gctccctaca 180
gcttgtcagt gattagcaaa ttactctgtc aggtttcctc aagta 225
<210> 9
<211> 219
<212> DNA
<213> rattus sp.
<400> 9
gctttggctt tttggcagta cagggtttct ctttgtagcc ctggatgtct tggaactcac 60
gttgtagaac aggctggcct tggaactcag aaatccatca gcctttgcct cacaccactt 120
tgcaactgtc atctcttaaa tgcaaattat attatttgct gaaatttaaa atattgtttt 180
gtgactacat tatgtggtgc tttgtatatg cttgcccca 219
<210> 10
<211> 213
<212> DNA
<213> rattus sp.
<400> 10
ttttccagtg ggagttataa ctcagcaatc tctttgtata ggagtgataa aaacaatcaa 60
atgttgtcaa gctagaacaa tgtacacaag aatttaattt gatgtcccat gaggggtata 120
ttttctctat gctcaaccct tagaggcaat cagggtaaat taccaaatta ccaaattata 180
cgaaaagcca ggctagataa agattatatt ttc 213
<210> 11
<211> 205
<212> DNA
<213> rattus sp.
<400> 11
tagctactta aagttaaaaa tcactctaat atttgtatca tagaaactca aaaaaaaaaa 60
gaaaatcaca gaaacataat caaatgggag aaggcaggag agaatggtct caacagattt 120
aagttggctg ttgggactga ggaggagagg acctgatgaa aagaccatgc tctggggaca 180
gggatacctt agattctctg tctac 205
<210> 12
<211> 154
<212> DNA
<213> rattus sp.
<400> 12
aagttcagtt atacttttaa atggtttgtt cgtgaatctt ctcttatgtt cttcttaaga 60
aaattgcgaa gttcatacga gttagacatt aagaaaataa taagaatatt gaggacgtgt 120
gttataggaa tgtaattttc caagcaacca gtac 154
<210> 13
<211> 167
<212> DNA
<213> rattus sp.
<400> 13
ctgaaaggtt gagttgatta gagaaaaaaa ataattaaga ccaaagtgct gtgtttgggt 60
ttccatttgg aactgtgaat cttggcaaag accaccctaa ctttgacttg ctacccaggc 120
actcaccttc tgtccttcta tctcttgtct tcaccttcag ctcaaga 167
CA 02391305 2009-11-16
<210> 14
<211> 244
<212> DNA
<213> rattus sp.
<400> 14
cggagctggg gaccgaaccc agggccttgc gcttcctagg caggcactct accactgagc 60
taaatcccca acccctcaat gttttgaaaa gacggtaaat accttgtgct ttaagaataa 120
acaggctaga gcgatggctc aggggataag gcctgtatat aagccatgct cacacgtcac 180
agtgaataca tagctcaaat gactaaactg agtctaaatt actaaaggga agcagcattt 240
gtgc 244
<210> 15
<211> 263
<212> DNA
<213> rattus sp.
<400> 15
catgttcttt tctacttggt tcaaaatacg gcagtaatct tgctgggcaa tgcagacaaa 60
ctggcagtca tccacctttg tcctcatgac tcctttcatg tactctttgt ccatggtggg 120
cgaaacacca aagctgtttc ccatgcacag tatttctgct ttcccatctg gatacgtcac 180
ctccaccgaa ccattgagaa tcactgacca ggaatccagc tcttctccat catttaacac 240
aatggttcca gctctttcca cca 263
<210> 16
<211> 121
<212> DNA
<213> rattus sp.
<400> 16
tatatttatg gacatctaag taggttccat ttcctctctg ttgtaaataa tgcattcata 60
aatgcaaatg tagaaggtgt ccttgtagta ggtagttaag agccctctga atatacagcc 120
t 121
<210> 17
<211> 82
<212> DNA
<213> rattus sp.
<400> 17
tgtggtactt catacaaaga atattagaaa agggtatgca aaaggaagac agttaagtgg 60
tagatggctg cccaagaaat gc 82
<210> 18
<211> 114
<212> DNA
<213> rattus sp.
<400> 18
ttacgggctg tgagctctcg agttgcgacc gccttattca gtttacagct gggtagattt 60
ttaaggagtg agacccaaag taataaacct gtgattgtag catgcacaac tcag 114
<210> 19
<211> 138
<212> DNA
<213> rattus sp.
<400> 19
gaataataga actttttaca gccaaggaca ttgcatgtgt acgacgcatc cctgaagtgt 60
CA 02391305 2009-11-16
96
tgtgcttcat ggtgttaaag ctgacccaag tcactgaaca caatattgca gccattcaac 120
tcacatttgt aacggagg 138
<210> 20
<211> 221
<212> DNA
<213> rattus sp.
<400> 20
cttctgcctt ctgactactg ggattaatga catgtgccac aacccccatc ttctaataat 60
gttttaaata cttaagatta aataaatagt acaggtgatt tttttaaaaa aaatgtacaa 120
cagtcatcat gtttttaaac ctcctgaaaa ttactgtatt ctcatcatat attttgaaag 180
gagctttaat aacaaaaatt atcacataca tttctcagag a 221
<210> 21
<211> 219
<212> DNA
<213> rattus sp.
<400> 21
agatttattt attatgtaga cagcgttcca cctgcatgta cacgtgcagg ccagaagagg 60
gcaccagatc tcattacaga tggttgtgag cccaccatgt ggttgctggg aattgaactc 120
atgacctctg gaagagcaat tagtgctctt aaccactgag ccatctctcc agccccacga 180
tgaggttctt aagagctgca accaagtggg ggacgataa 219
<210> 22
<211> 215
<212> DNA
<213> rattus sp.
<400> 22
ctgtcagggc tcaggaaaca ctgtgaaaga ggaagtgaat aaatgtgaga gtcagaggtt 60
ggggtggaag gctatggaat gctgccttat agatacgaca tggctactgc agatgtgcat 120
tcacagtaac cacgattacc tacataatat caaggcagtc acattccagc atggataagg 180
gagaaggtca taagaagtat tgtcagtggg tagaa 215
<210> 23
<211> 106
<212> DNA
<213> rattus sp.
<400> 23
aggaatgcat ctacactcta agtaaaattg attcgttcta atttccgtgt cagttactgc 60
tgtagtctgc tcctgcttag cgctatgatc cgaattcacg aagggc 106
<210> 24
<211> 154
<212> DNA
<213> rattus sp.
<400> 24
cagcagttct ttccatcttc ttaattggcg ataattttct tcattaagta gaactattca 60
ttatgcagag taccattgtg gagatgcaaa tacagcccag gtattcggac agcaaagaca 120
aagtgttatt gtggtaaggc ctgagttatc aaaa 154
<210> 25
<211> 337
<212> DNA
<213> rattus sp.
CA 02391305 2009-11-16
97
<400> 25
agactcaggt cataaatcaa agaacattgt gtacattgct tctttggatc tgagactggt 60
agtgtccctg ggctcctatg agggcatcat cagaagatga acaaggtgac tkttggggat 120
gctttctgga tggggaatga cttggctatg cctggscgca tgttgtgtgt kgaactgttt 180
cctcgsgttc cctcggtttc tctctttgta graagtgcta agktttgtac ctcaaagcat 240
actaggtcat gtctctatac tatattccta aagggtccac agctacccta atctaccctg 300
ttacctaaga tccacagaga gtctggaacc ttgttgt 337
<210> 26
<211> 160
<212> DNA
<213> rattus sp.
<400> 26
tcattaaaat cacggrtttt gctattatgc cttattatgt caagagtttg ttagatgtta 60
catcagcatc tcagggtagt gacttgatta tattcatctc tgtattctct aagaacaata 120
agatgtctac ataaaaccag tattgaaagt acatactttt 160
<210> 27
<211> 186
<212> DNA
<213> rattus sp.
<400> 27
aagatcgatg ctaccttggc agcaaagtaa gaccctgtgt gacagaagaa ggaagagaac 60
agaagggaaa gagaaaagga tggtgtccga gagacaggaa aagctaaact gtggttatgc 120
catttggggg acaggaccag gtgaagaaaa gggcactcca agttacatat atacaagctg 180
agaaaa 186
