Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR IDENTIFYING TRANSLATIONALLY
REGULATED GENES
BACKGROUND OF THE INVENTION
Technical Field
The present invention relates to a method for identifying genes that
are translationally regulated. More specifically, the present invention
relates to the
rapid isolation of differentially expressed or developmentally regulated gene
sequences through segregation of mRNAs into translated and untranslated pools
and comparing the relative abundance of the mRNAs found in these pools by
differential analysis.
Background Art
15 The identification and/or isolation of genes whose expression differs
between two cell or tissue types, or between cells or tissues exposed to
stress
conditions, chemical compounds or pathogens, is critical to the understanding
of
mechanisms which underlie various physiological conditions, disorders, or
diseases. Regulation of gene expression has been shown to play an important
part
2o in many biological processes including embryogenesis, aging, tissue repair,
and
neoplastic transformation. Gene regulation at the level of translation has
been
shown to be of critical importance. For example, it has been demonstrated that
a
group of mRNAs are stored in an egg as a pool of untranslated mRNAs which,
following fertilization, shift into the pool of translated mRNAs. Another
example
25 of a change in the translational state of mRNA is a subgroup of mRNAs which
code for heat shock proteins which are not translated under normal
physiological
conditions. These mRNAs begin to be translated following exposure of cells to
high temperatures.
A number of methods have been developed for the detection and
3o isolation of genes which are activated or repressed in response to
developmental,
physiological, pharmacological, or other cued events. One particular method is
described in United States Patent Number 5,525,47l to Zeng, is subtractive
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hybridization. Subtractive hybridization is a particularly useful method for
selectively cloning sequences present in one DNA or RNA population but absent
in
another. The selective cloning is accomplished by generating single stranded
complementary DNA libraries from both control cells/tissue (driver eDNA) and
cell/tissue during or after a specific change or response being studied
(tester
cDNA). The two cDNA libraries are denatured and hybridized to each other
resulting in duplex formation between the driver and tester cDNA strands. In
this
method, common sequences are removed and the remaining non-hybridized single-
stranded DNA is enriched for sequences present in the experimental cell/tissue
1 o which is related to the particular change or event being studied. (Davis
et al.,
1987).
Currently used methodologies to identify mRNAs encoding proteins
which are being induced/reduced following a cue or stimulus rely on changes in
the
mRNA levels following transcriptional induction/repression via screening of
differentially expressed mRNAs. One such method for the identification of
differentially expressed mRNAs is disclosed in United States Patent Number
5,459,037 to Sutcliffe et al. According to this method, an mRNA population is
isolated, double-stranded cDNAs are prepared from the mRNA population using a
mixture of twelve anchor primers, the cDNAs are cleaved with two restriction
2o endonucleases, and then inserted into a vector in such an orientation that
they are
anti-sense with respect to a T3 promotor within the vector. E. col i are
transformed
with the cDNA containing vectors, linearized fragments are generated from the
cloned inserts by digestion with at least one restriction endonuclease that is
different from the first and second restriction endonucleouseases and a cDNA
preparation of the anti-sense cDNA transcripts is generated by incubating the
Iinearized fragments with a T3 RNA polymerase. The cDNA population is divided
into subpools and the first strand cDNA from each subpool is transcribed using
a
thermostable reverse transcriptase and one of sixteen primers. The
transcription
product of each of the sixteen reaction pools is used as a template for a
polymerase
3o chain reaction (PCR) with a 3'-primer and a 5'-primer and the polymerase
chain
reaction amplified fragments are resolved by electrophoresis to display bands
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representing the 3'-ends of the mRNAs present in the sample. This method is
useful for the identification of differentially expressed mRNAs and the
measurement of their relative concentrations. This type of methodology,
however,
is unable to identify mRNAs whose levels remain constant but their
translatability
is variable or changes.
Schena et al. developed a high capacity system to monitor the
expression of many genes in parallel utilizing microarrays. The microarrays
are
prepared by high speed robotic printing of cDNAs on glass providing
quantitative
expression measurements of the corresponding genes (Schena et al., 1995).
to Differential expression measurements of genes are made by means of
simultaneous, two color fluorescence hybridization. However, this method alone
is
insufficient for the identification of translationally regulated genes.
The use of a known inhibitor of hypusine formation, mimosime,
was used to reversibly suppress the hypusine-forming deoxyhypusyl hydroxylase
15 in cells while differentially displaying their polysomal versus non-
polysomal
mRNA populations. (Hanauske-Abel et al., l995) Utilizing this method, several
species of mRNA were discovered which disappear and reappear, respectively, at
polysomes in connection with inhibition and disinhibition of hypusine
formation
and which are thought to code for translationally controlled enzymes. This
method
20 only teaches the use of a known stimulating element (i.e., inducer or
repressor) to
identify translationally regulated genes. This method does not provide a
mechanism for the detection and/or identification of translationally regulated
genes
where the stimulating element is unknown.
Generally, the translation of eukaryotic mRNAs is dependent upon
25 S' cap-mediated ribosome binding. Prior to translation, the ribosome small
sub
unit (40S) binds to the 5'-cap structure on a transcript and then proceeds to
scan
along the mRNA molecule to the translation initiation site where the large sub-
unit
(60S) forms the complete ribosome initiation site. In most instances, the
translation initiation site is the first AUG codon. This "scanning model" of
3o translation initiation accommodates most eukaryotic mRNAs. A few notable
exceptions to the "scanning model" are provided by the Picornavirus family.
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These viruses produce non-capped transcripts with long (600-1200 nucleotides)
S'-
untranslated regions (UTR) which contain multiple non-translation initiating
AUG
codons. Because of the absence of a cap structure, the translational
efficiency of
these RNAs is dependent upon the presence of specific sequences within the
untranslated regions (UTR) known as internal ribosome entry sites (IRES).
More recently, IRES containing mRNA transcripts have been
discovered in non-viral systems such as the mRNA encoding for immunoglobulin
heavy chain binding protein, the antenapedia gene in Drosophila, and the mouse
Fgl-2 gene. These discoveries have promoted speculation for the role of cap-
1 o independent translation in the developmental regulation of gene expression
during
both normal and abnormal processes.
The discovery of the above-mentioned non-viral IRES containing
mRNAs implies that eukaryotic IRES sequences could be more wide spread than
has been previously realized. The difficulty in identifying eukaryotic IRES
15 sequences resides in the fact that they typically cannot be identified by
sequence
homology. [Oh et al., 1993; Mountford et al., 199S; Macejak et al., 1991;
Pelletier
et al., l988; Vagner et al. 1995] It would, therefore, be advantageous to have
a
method for identifying IRES containing mRNA in order to identify
translationally
controlled genes operating via 5'-cap independent translation in order to
ascertain
2o and assess their association with both normal and abnormal processes.
Therefore, it would be desirable to have a rapid, reliable, and
reproducible method for the identification and cloning of clinically and
therapeutically relevant differentially expressed genes which will overcome
the
inherent problems associated with the prior art methods.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method for
identifying translationally regulated genes in an organism including the steps
of
selectively stimulating translation of an unknown target mRNA with a stress
3o inducing element, the target mRNA being part of a larger sample of mRNA,
dividing the sample of mRNA into pools of translated and untranslated mRNA and
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differentially analyzing the pools of mRNA to identify genes translationally
regulated by the stress inducing element. The stress inducing element can
include
pathologic, environmental including chemical and physical stressors or other
stimulus that induces mRNA translation. Also, in accordance with the present
invention, there is provided a method for identifying gene sequences coding
for
internal ribosome entry sites. The method includes inhibiting 5'cap-dependant
mRNA translation in a cell, collecting a pool of mRNA from the cells, and
differentially analyzing the pool of mRNA to identify genes with sequences
coding
for internal ribosome entry sites.
~o BRIEF DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be readily
appreciated as the same becomes better understood by reference to the
following
detailed description when considered in connection with the accompanying
drawings wherein:
Figure lA is an absorbance profile of a fractionation of cytoplasmic
RNA on a sucrose density gradient wherein the absorbance (at 254nm) is plotted
against the sedimentation rate of the cytoplasmic RNA;
Figure 1B is a photograph of purified RNA electrophoresed on an
agarous gel and stained with ethidium bromide illustrating the fractionation
of
2o RNA;
Figure 2 is a photograph of a 5% acrylamide gel illustrating a
differential translation analysis of mRNA from sucrose density gradients
according
to the present invention;
Figure 3A-C are schematic representations of plasmids that contain
the Polio virus 2A genes (A) in plasmid pTK-OP3-WT2A, (B) in the plasmid
rniniTK-WT2A, and (C) in a plasmid containing a hygromycin selectable marker;
Figure 4 is graph illustrating the induction of
Polio virus 2A protease leading to cell death after induction of the 2A
protease;
Figure 5 is a photograph of a gel illustrating the presence of Polio
3o virus 2A protease expression in transformed HEK-293 cells (293-2A)
following
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induction with IPTG and the absence of the Polio virus 2A protease in HEK-293
(293) parental cells following treatment with IPTG; and
Figure 6 is a photograph of a Western blot illustrating the activity of
the Polio virus 2A protease in cleaving the p220 protein component of the 40S
ribosomal subunit demonstrating that clones which were induced for Polio virus
2A protease generated cleavage products of the p220 protein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for identifying
translationally regulated genes in an organism by selectively stimulating
translation
of an unknown target mRNA with a stress inducing element, the target mRNA
being part of a larger sample. The organism may be any organism which provides
suitable mRNA. The mRNA sample is divided into pools of translated and
untranslated mRNA which are differentially analyzed to identify genes which
are
15 translationally regulated by the stress inducing element. This method is
designed
for identifying and cloning genes which are regulated at the translational
level.
That is, the present method is designed for identifying and cloning genes
which are
either up- or down- regulated including identifying genes responsive to a
specific
pathology or stress condition.
2o The method of the present invention provides a novel approach to
the identification and cloning of genes that are involved in fundamental
cellular
functions and which are regulated at the level of translation in an organism.
The
basic underlying theory for this method relies on the assumption that an mRNA
encoding a protein required for a quick response to an external cue is
generally
25 stored as an untranslated mRNA. Following the appropriate external cue, the
mRNA is translated and the encoded protein quickly appears. By comparing
mRNA populations that are "active" or "non-active" at a given time, genes that
are
regulated by a mechanism referred to as the "shift mechanism" can be
identified.
The method can also be applied to identify in addition to genes
3o regulated at the translational level; genes regulated at the transcription
level; genes
regulated by RNA stability; gene regulated by mRNA transport rate between the
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nucleus and the cytoplasm; and gene regulated by differential splicing. That
is,
genes whose expression in part, is controlled/regulated at the mRNA level can
be
identified.
The method will identify genes encoding secreted and membrane
proteins; genes encoding for nuclear proteins; genes encoding for
mitochondrial
proteins; and genes encoding for cytoskeletal proteins. In addition, any other
gene
whose expression can be controlled at the mRNA level can be identified by this
method.
As used herein, RNA refers to RNA isolated from cell cultures,
1 o cultured tissues or cells or tissues isolated from organisms which are
stimulated,
differentiated, exposed to a chemical compound, are infected with a pathogen
or
otherwise stimulated. As used herein, translation is defined as the synthesis
of
protein on an mRNA template.
As used herein, the term stimulating translation of unknown target
15 mRNA or stimulating element includes chemically, pathogenically,
physically, or
otherwise inducing or repressing an mRNA population from genes which can be
derived from native tissues and/or cells under pathological and/or stress
conditions
that are regulated by the "shift mechanism." In other words, stimulating the
translation of mRNA with a stress inducing element or "stressor" can include
the
2o application of an external cue, stimulus, or stimuli which stimulates or
initiates
translation of a mRNA stored as untranslated mRNA in the cells from the
sample.
In addition to stimulating translation of mRNA from genes in native
cells/tissues,
stimulation can include induction and/or repression of genes under
pathological
and/or stress conditions. The present method utilizes a stimulus or stressor
to
25 identify unknown target genes which are translationally regulated by the
stress
inducing element or stressor.
The method of the present invention integrates two previously
known methodologies which were otherwise used separately. The first method is
the division of an mRNA sample into separate translated and untranslated pools
of
3o mRNA. The second methodology involves the simultaneous comparison of the
relative abundance of the mRNA species found in the separate pools by a method
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of differential analysis such as differential display, representational
difference
analysis (RDA), gene expression microarray {GEM), suppressive subtraction
hybridization (SSH) (Diatchenko et al., 1996), and techniques such as chip
technology exemplified by United States Patent No. 5,545,53l to Rava et al.
assigned to Affymax Technologies N.V. and direct sequencing exemplified by WO
961l7957 patent application to Hyseq, Inc.
Briefly, subtractive hybridization is defined as subtraction of
mRNA by hybridization in solution. RNA that are common to the two pools form
a duplex that can be removed, enriching for RNAs that are unique or more
abundant in one pool. Differential Display is defined as reverse transcription
of
mRNA into cDNA and PCR amplification with degenerated primers. Comparison
of the amounts amplification products (by electrophoresis) from two pools
indicate
transcript abundance. RDA, GEM, SSH, SAGE are described herein above.
The specific cells/tissues which are to be analyzed in order to
identify translationally regulated genes, can include any suitable cells
and/or
tissues. Any cell type or tissue can be used, whether an established cell line
or
culture or whether directly isolated from an exposed organism.
The cells/tissues to be analyzed under the present method are
selectively stimulated utilizing a physiological, chemical, environmental
and/or
2o pathological stress inducing element or stressor, in order to stimulate the
translation of mRNA within the sample tissue and identify genes whose
expression
is regulated at least in part at the mRNA level. Following the stimulation of
the
translation of RNA, the RNA from the cells/tissues is isolated or extracted
from the
cells/tissues. The isolation of the RNA can be performed utilizing techniques
which are well known to those skilled in the art and are described, for
example, in
"Molecular Cloning; A Laboratory Manual" (Cold Springs Harbor Laboratory
Press, Cold Spring Harbor, New York, 1989). Other methods for the isolation
and
extraction of RNA from cells/tissue can be used and will be known to those of
ordinary skill in the art. (Mach et al., 1986, Jefferies et al., 1994).
3o Following the isolation of the pool of translated and untranslated
mRNA, the mRNAs which are actively engaged in translation and those which
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remain untranslated can be separated utilizing a procedure such as
fractionation on
a sucrose density gradient, high performance gel filtration chromatography, or
polyacrylamide gel matrix separation (Ogishima et al., 1984, Menaker et al.,
1974,
Hirama et al., 1986, Mechler, 1987, and Bharucha and Murthy, 1992), since
mRNAs that are being translated are loaded with ribosomes and, therefore, will
migrate differently on a density gradient than ribosome-free untranslated
mRNAs.
By comparing mRNA populations that are active or non-active in translation at
a
given time, genes that are regulated by the "shift mechanism" can be
identified.
Polysomal fractionation and specific analysis can be facilitated by
1 o treatment of target cell/tissue with drugs that will specifically inhibit
or modulate
transcription or translation. Examples of such drugs are actinomycin D and
cyclohexamide, respectively.
The fractionation can be completed to create polysomal
subdivisions. The subdivisions can be made to discriminate between total
15 polyribosomes or membrane bound ribosomes by methods known in the art
(Mechler, 1987). Further, the mRNA sample can be in addition fractionated into
one or more of at least the following subsegments or fractions: cytoplasmatic,
nuclear, polyribosomal, sub polyribosomal, microsomal or rough endoplasmic
reticulum, mitochondria) and splicesome associated mRNA by methods known in
2o the art (see also Table 1 ).
Following isolation and division of the total mRNA population into
separate translated and untranslated pools of mRNA, the relative abundance of
the
many mRNA species found in these pools are simultaneously compared using a
differential analysis technique such as differential display, representational
25 difference analysis (RDA), GEM-Gene Expression Microarrays (Schena et al.,
1995, Aiello et al., 1994, Shen et al., 1995, Bauer et al., 1993, Liang and
Pardee,
1992, Liang and Pardee, 1995, Liang et al., 1993, Braun et al., 1995, Hubank
and
Schatz, 1994) and suppressive subtraction hybridization (SSH). The RNA
isolated
from the fractions can be further purified into mRNA without the ribosomal RNA
3o by poly A selection. It should be noted that multiple pools can be analyzed
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utilizing this method. That is, different cell aliquots subjected to different
stressors
can be compared with each other as well as with the reference sample.
Labeled mRNA {in a cDNA or PCR product form) from polysomal,
non-polysomal or mRNPs (pools or individual fractions) can be used as probes,
to
identify clones of cDNA, genomic clones, and mRNA species that are fixed onto
a
solid matrix-like microarrays such as (GEM), that shown in United States
Patent
Number S,545,531 to Rava et al. and W096/17957 to Hyseq, Inc., and membranes
of any kind where clones can be either blotted after electrophoresis or
directly
loaded (dot blot) onto the membrane. The label can be radioactive,
fluorescent, or
1 o incorporating a modified base such as digoxigenin and biotin.
Comparison between the fractions derived from the polysomal or
polyribosomal fraction or other fractions to the total unfractionated material
is
essential to discriminate between differentials in expression levels that are
the
result of transcription modulation from those that result from modulation of
translation per se. The polysomal fractions or groups can include membrane
bound
polysomes, loose or tight polysomes, or free unbound polysome groups.
The importance of utilizing the polysomal sub-population in order
to identify differentially (translationally) expressed genes is shown in
Example 2
where a number of genes were not detected as translationally expressed under
heat
2o shock inducement when total mRNA was used as the detection probe but,
however,
when polysomal mRNA was used as a probe, a number of genes were identified as
differentially expressed. These genes were previously thought to be non-
differentially expressed when total mRNA was used as a probe. That is, as
shown
in Example 2, a number of genes that were not detected as translationally
expressed under heat shock inducement with total mRNA were detected when
probed with polysomal mRNA fractions.
The present method for identifying translationally regulated genes is
not limited by the source of the mRNA pools. Therefore, the present method can
be utilized to clone genes from native cells/tissue under pathological and/or
stress
3o conditions that are regulated by the "shift mechanism," as well as genes
that are
induced/repressed under pathological and/or stress conditions. Pathologies can
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include disease states including those diseases caused by pathogens and
trauma.
Stress conditions can also include disease states, physical and psychological
trauma, and environmental stresses. Following analysis by the selected method
of
differential analysis, the genes which have been identified as being regulated
by
translation can be cloned by any suitable cloning methodologies known to those
skilled in the art. (Lisitsyn and Wigler, 1993).
Differential comparisons can be made of all possible permutations
of polysomal vs. non-polysomal RNA where the definition of the fraction type
is
done, for example, by absorbance profile at 254nm, density of the sucrose
gradient
1 o as shown in Figure 1 A (or another size standard if high pressure liquid
chromatography or gel systems are used) and types of RNA that are stained with
ethidium bromide after electrophoresis of the fractions on agarous gels are
completed, as shown in Figure 1 B. In Figure 1 A, the polysomal fractions are
those
that have mRNA with more than two ribosomes loaded. The materials and
15 methods for this comparison are set forth below in the experimental
section.
Differential comparisons can also include polysomal vs. non-
polysomal fractions in each condition. By "condition" it is meant that cells
from
the same source, such as a cell line, a primary cell, or a tissue that
undergoes
different treatment or has been modified to have different features or to
express
2o different sets of genes. For example, this can be accomplished by
differentiation,
transformation, application of the stress such as oxygen deprivation, chemical
treatment, or radiation. Permutations can include, for example:
1. polysomal fractions between conditions individually (migrating
in the same density) or in a pool;
25 2. non-polysomal fractions between conditions individually
(migrating in the same density) or in a pool;
3. non-polysomal to polysomal between conditions and within each
condition individually (migrating in the same density) or in a pool; and
4. each of the fractions being polysomal and non-polysomal
3o individually (migrating in the same density) or in a pool that can be
compared to
total RNA that is unfractionated.
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The method described above for the identif cation of translationally
regulated genes has a number of applications. A particular application for
this
method is its use for the detection of changes in the pattern of mRNA
expression in
cells/tissue associated with any physiological or pathological change. By
comparing the translated versus untranslated mRNAs, the effect of the
physiological or pathological cue or stress on the change of the pattern of
mRNA
expression in the cell/tissue can be observed and/or detected. This method can
be
used to study the effects of a number of cues, stimuli, or stressors to
ascertain their
effect or contribution to various physiological and pathological activities of
the
1 o cell/tissue. In particular, the present method can be used to analyze the
results of
the administrations of pharmaceuticals (drugs) or other chemicals to an
individual
by comparing the mRNA pattern of a tissue before and after the administration
of
the drug or chemical. This analysis allows for the identification of drugs,
chemicals, or other stimuli which affect cells/tissue at the level of
translational
regulation. Utilizing this method, it is possible to ascertain if particular
mRNA
species are involved in particular physiological or disease states and, in
particular,
to ascertain the specific cells/tissue wherein the external stimulus, i.e., a
drug,
affects a gene which is regulated at the translational level.
A further embodiment of the present invention provides a method
2o for identifying gene sequences coding for internal ribosome entry sites
(IRES) and
includes the general steps of inhibiting 5'cap-dependant mRNA translation in a
cell, collecting a pool of mRNA from the cells, and differentially analyzing
the
pool of mRNA to identify genes with sequences coding for internal ribosome
entry
sites.
As described above, it is known that an exception to the standard 5'-
cap dependent translation initiation exists. Sequences exist within
untranslated
regions (UTRs) of RNAs which can include the presence of specific sequences
known as internal ribosome entry sites (IRES). (Ehrenfeld, 1996) These
internal
ribosome entry sites have been shown to support translation initiation for
several
3o prokaryotic and eukaryotic systems as set forth above. However, in order to
identify translationally controlled genes via 5'-cap independent translation
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mechanisms and their association with both normal and abnormal processes, it
is
necessary to inhibit 5'-cap initiated translation so that 5'-cap independent
mRNA
translation can be selected for . This inhibition is necessary since IRES
sequences
are difficult, if not impossible, to identify by sequence homology.
In order to inhibit 5'-cap dependent translation and thereby select
for the presence of 5'-cap independent translation, cells or tissues which are
to be
analyzed for the presence of internal ribosome entry sites must be treated in
some
manner to prevent or discourage the 5'-cap translation initiation mechanism.
The
mechanisms) of standard scanning-type translation initiation should be
1 o substantially, if not totally, turned off or shut down to, in essence,
shift the
translation equilibrium in favor of IRES initiated translation. That is,
recognition
of the 5'-cap structure is inhibited by disrupting the normal mechanism for 5'-
cap
mediated initiation. The mechanism for inhibiting the 5'-cap translation can
include any known means or mechanisms for preventing the initiation of 5'-cap
15 mediated translation. One such mechanism for inhibiting 5'-cap mediated
translation is the expression of Polio virus 2A protease into a cell, cell
system, or
tissue to be analyzed for the presence of IRES sequences. The use of the Polio
virus 2A protease inhibits 5'-cap-dependent mRNA translation by inactivating
the
cellular 5'-cap-dependent translation machinery. This enables the
identification of
2o cellular IRES containing genes which rnay be translationally controlled and
play a
critical role in the immediate response of the cell following the application
of a
stress inducing element/stressor such as heat shock, hypoxia, or other stress
inducing elements as set forth above, prior to gene activation. The Polio
virus 2A
protease prevents 5'-cap-mediated translation by cleaving the large sub-unit
of eIF-
25 4y (p220) of eukaryotic translation initiation factor 4 (eIF-4) which is
involved in
the recognition of the mRNA 5'-cap.
In order to inhibit the 5'-cap-mediated translation, the Polio virus
2A protease must be incorporated into the cell or cells being analyzed for the
presence of gene sequences coding for internal ribosome entry sites and/or for
3o identifying translationally regulated genes. One such method for
incorporating the
Polio virus 2A protease into a cell involves the transformation of a target
cell with
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an expression vector containing the gene which codes for the Polio virus 2A
protease. Because the Polio virus 2A protease is deleterious to living cells
when it
is constitutively expressed, the expression vector containing the Polio virus
2A
protease gene is coupled with a bacterial LacI inducible system wherein a LacI
repressor is constituitively expressed under a CMV promoter. The Polio virus
2A
protease may be expressed under a number of suitable promoters including the
RSV, the TK, or the mini-TK promoter coupled at their 3' end to the LacI
repressor
binding sites. By transforming the target cells with an expression vector
containing the LacI repressor and the Polio virus 2A expression vector, the
1 o expression of the Polio virus 2A protease can be induced upon treatment of
the
cells with isopropyl-(3-D-thiogalatopyranoside (IPTG). Treatment of the target
cells with IPTG relieves the binding of the LacI repressor molecules bound at
the
repressor binding sites thus enabling transcription of the Polio virus 2A
protease.
By coupling the expression of the Palio virus 2A protease to an inducible
system,
such as the LacI system, this mechanism allows for the establishment of
control of
the expression of the gene coding for the Polio virus 2A protease.
Examples of an embodiment of the present invention for identifying
gene sequences coating for internal ribosome entry sites are set forth below
in the
examples.
2o Following induction of the expression of the Polio virus 2A
protease in the target cells, RNA, presumably containing internal ribosome
entry
sites, can be collected and analyzed utilizing the methods described above to
identify genes whose translation is up-regulated by the effects of the Polio
virus 2A
protease.
EXPERIMENTAL
DIFFERENTIAL TRANSLATION
MATERIALS AND METHODS
General Scheme
;o a. Total mRNA organic extraction of a11 RNA from the source tissue or cell.
(additional selection for polyA+ mRNA can be included).
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b. Nuclear RNA-lysis of cells (from a tissue or a cell line) by homogenization
in
hypotonic buffer. Collection of nuclei by centrifugation and organic
extraction of
the RNA.
c. Organic extraction of the RNA from the supernatant from 2 above.
d. Polyribosomal/subpolyribosomal fractionation. Lysis of cells by
homogenization hypotonic buffer, removal of nuclei and fractionation of
polyribosome on linear sucrose gradients and organic extraction of the RNA
from
each fraction of the gradient.
1o e. Secreted and membrane encoding transcripts.
1. Isolation of RER on Percol gradients (after homogenization of cells).
2. Preparation of microsomes containing the RER
3. Isolation of membrane-bound polyribosomes by successive treatment of cells
with detergents.
~ Nuclear proteins. Isolation of cytoskeletal associated polyribosomes by
treating
cells lyzates with different detergents.
g. Mitochondria) genes. Isolation of mitochondria on Percoll gradients.
i. Alternative splicing. Separation of nuclei and isolation of splicsosome
(proteins
and RNA complex) on linear sucrose gradients.
Preparation of cell extracts
Cells were centrifuged. The pellet was washed with PBS and recentrifuged. The
cells were resuspended in 4x of one packed cell volume (PCV) with hypotonic
lysis buffer (HLB: 20mM TrisHCL pH=7.4; lOmM NaCI; 3mM MgClz). The
celis were incubated five minutes on ice. IxPCV of HLB containing 1.2% Triton
X-I00 and 0.2M sucrose was added. The cells were homogenized with a Dounce
homogenizes (five strokes with B pestle). The cell lysate was centrifuged at
2300g
for ten minutes at 4°C. The supernatant was transferred to a new tube.
HLB
containing 1 Omg/ml heparin was added to a final concentration of i mg/ml
heparin.
3o NaCI was added to a final concentration of 0.15M. The supernatant was
frozen at
-70°C after quick freezing in liquid N~ or used immediately.
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Sucrose ~~radient fractionation
A linear sucrose gradient from 0.5M to 1.5M sucrose in HLB was prepared.
Polyallomer tubes (14X89mm) were used. 0.5 to 1.0m1 of cell extract was loaded
on the gradient. The cells were centrifuged at 36,000 RPM for 1 I 0 minutes at
4°C.
An ISCO Density Fractionator was used to collect the fractions and record the
absorbance profile.
RNA purification
1 o SDS was added to 0.5% and Proteinase K to 0.1 mg/ml and incubated at
37°C for
30 minutes. Extract with an equal volume of phenol+chloroform ( 1:1 ). The
aqueous phase was extracted with one volume of chloroform and the RNA was
precipitated by adding Na-Acetate to 0.3M and 2.5 volumes of ethanol and
incubating at -20°C overnight. Centrifuged ten minutes, the supernatant
was
aspirated and the RNA pellet was dissolved in sterile, diethylpyrocarbonate
(hereinafter referred to as "DEPC") DEPC-treated water.
DIFFERENTIAL ANALYSIS
Differential disnlay:
2o Reverse transcription: 2p,g of RNA were annealed with 1 pmol of oligo dT
primer
(dT), g in a volume of 6.5 p, l by heating to 70°C for five minutes and
cooling on ice.
2p1 reaction buffer (x5), lp,l of lOmM dNTP mix, and 0.51 of Superscript II
reverse transcriptase (GibcoBRL) was added. The reaction was carried out for
one
hour at 42°C. The reaction was stopped by adding 70p.1 TE ( 1 OmM Tris
pH=8;
O.ImM EDTA). Oligonucleotides used for Differential display: The
oligonucleotides were essentially those described in the Delta RNA
Fingerprinting
kit (Clonetech Labs. Inc.). There were 9 "T" oligonucleotides of the
structure: 5'
CATTATGCTGAGTGATATCTTTTTTTTTXY 3' (SEQ ID No: 1). The 10 "P"
oligonucleotides were of the structure: 3' ATTAACCCTCACTAAA
"TGCTGGGGA" 3' (SEQ ID No: 11 ) where the 9 or 10 nucleotides between the
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parenthesis represent an arbitrary sequence and there are 10 different
sequences
(SEQ ID Nos. 12-21), one for each "P" oligo.
Amplification reactions: each reaction is done in 20p 1 and contains SOp.M
dNTP
mix, 1 pM from each primer, 1 x polymerase buffer, 1 unit expand Polymerase
(Beohringer Mannheim), 2pCi [a-3zP]dATP and 1 ~ 1 cDNA template. Cycling
conditions were: three minutes at 95°C, then
three cycles of two minutes at 94°C, five minutes at 40°C, five
minutes at 68°C.
This was followed by 27 cycles of one minute at 94°C, two minutes at
60°C, two
1o minutes at 68°C. Reactions were terminated by a
seven minute incubation at 68°C and addition of 20p 1 sequencing stop
solution
(95% formamide, l OmM NaOH, 0.025% bromophenoI blue, 0.025% xylene
cyanol).
Gel analysis: 3-4p 1 were loaded onto a 5% sequencing polyacrylamide gel and
samples were electrophoresed at 2000 volts/40 milliamperes until the slow dye
(xylene cyanol) was about 2 cm from the bottom. The gel was transferred to a
filter paper, dried under vacuum and exposed to x-ray film.
2o Recovery of differential bands: bands showing any a differential between
the
various pools were excised out of the dried gel and placed in a
microcentrifuge
tube. SOp l of sterile H20 were added and the tubes heated to 100°c for
five
minutes. 1 p l was added to a 49~ 1 PCR reaction using the same primers used
for
the differential display and the samples were amplified for 30 cycles of: one
minute at 94°C, one minute at 60°C and one minute at
68°C. l Op 1 was analyzed
on agarous gel to visualize and confirm successful amplification.
REPRESENTATIONAL DIFFERENCE ANALYSIS
Reverse transcription: as above but with 2p,g polyA+ selected mRNA.
Preparation of double stranded cDNA: cDNA from previous step was treated with
alkali to remove the mRNA, precipitated and dissolved in 20p.1 H20. 5p,1
buffer,
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2p 1 l OmM dATP, HZO to 48p 1 and 2~ 1 terminal deoxynucleotide transferase
(TdT) were added. The reaction was incubated 2-4 hours at 37°C. 5p.1
oligo dT
( 1 p,g/~ 1 ) was added and incubated at 60°C for 5 minutes. 5 p,l 200
mM DTT, 10 pl
1 Ox section buffer ( 1 OOmM Mg C 1 z, 900 mM Hepes, pH 6.6) I 6 ul dNTPs ( 1
mM), and 16 U of Kienow were added and the mixture was incubated overnight at
room temperature to generate ds cDNA. 100 1 TE was added and extracted with
phenol/chloroform. The DNA was precipitated and dissolved in 50p.1 H20.
Generation of representations: cDNA with DpnII was digested by adding 3p,1
DpnII reaction buffer 20 V and DpnII to 25p.1 cDNA and incubated five hours at
37°C. 50p,1 TE was added and extracted with phenol/chloroform. cDNA was
precipitated and dissolved to a concentration of l Ong/p l .
The following oligonucleotides are used in this procedure:
R-Bgl-12 5' GATCTGCGGTGA 3' (SEQ ID No: 22)
R-Bgl-24 5' AGCACTCTCCAGCCTCTCACCGCA 3' (SEQ ID No:23}
~5 J-Bgl-12 5' GATCTGTTCATG 3' (SEQ ID No: 24}
J-Bgl-24 5' ACCGACGTCGACTATCCATGAACA 3' {SEQ ID No:25)
N-Bgl-12 5' GATCTTCCCTCG 3' (SEQ ID No:26)
N-Bgl-24 5' AGGCAACTGTGCTATCCGAGGGAA 3' {SEQ IDNo:27)
R-Bgl-12 and R-Bgl-24 oligos were ligated to Tester and Driver: 1.2p,g DpnII
digested cDNA. 4p.1 from each oligo and 5p1 ligation buffer X10 and annealed
at
60°C for ten minutes. 2p,1 ligase was added and incubated overnight at
16°C. The
ligation mixture was diluted by adding 140p 1 TE. Amplification was carried
out
in a volume of 200p,1 using R-Bg 1-24 primer and 2p.1 ligation product and
repeated in twenty tubes for each sample. Before adding Taq DNA polymerase,
the tubes were heated to 72°C for three minutes. PCR conditions were as
follows:
five minutes at 72°C, twenty cycles of one minute at 95°C and
three minutes at
72°C, followed by ten minutes at 72°C.
Every four reactions were combined, extracted with phenol/chloroform and
precipitated. Amplified DNA was dissolved to a concentration of 0.5p,g/p l and
all
samples were pooled.
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Subtraction: Tester DNA (20pg) was digested with DpnII as above and separated
on a 1.2% agarous gel. The DNA was extracted from the gel and 2pg was ligated
to J-Bgl-12 and J-Bg124 oligos as described above for the R-oligos. The
ligated
Tester DNA was diluted to 1 Ong/p l with TE. Driver DNA was digested with
DpnII and repurifled to a final concentration of 0.5pg/pl. Mix 40p.8 of Driver
DNA with 0.4pg of Tester DNA. Extraction was carried out with
phenollchloroform and precipitated using two washes with 70% ethanol,
resuspended DNA in 4p1 of 30mM EPPS pH=8.0, 3mM EDTA and overlayed
~ o with 3 5 p l mineral oil. Denatured at 98°C for five minutes, cool
to 67°C and 1 p l
of 5M NaC 1 was added to the DNA. Incubated at 67°C for twenty hours.
Diluted
DNA by adding 400p 1 TE.
Amplification: Amplification of subtracted DNA in a final volume of 200p 1 as
follows: Buffer, nucleotides and 20p 1 of the diluted DNA were added, heated
to
72°C, and Taq DNA polymerase was added. Incubated at 72°C for
five minutes
and added J-Bg 1-24 oligo. Ten cycles of one minute at 95°C, three
minutes at
70°C were performed. Incubated ten minutes at 72°C. The
amplification was
repeated in four separate tubes. The amplified DNA was extracted with
2o phenol/chloroform, precipitated and ali four tubes were combined in 40p.1
0.2XTE, Digested with Mung Bean Nuclease as follows: To 20p 1 DNA 4p 1
buffer, 14p1 Hz0 and 2p.1 Mung Bean Nuclease (10 units/pl) was added.
Incubated at 30°C for thirty-five minutes + First Differential Product
(DPI).
Repeat subtraction hybridization and PCR amplification at driver: differential
ratio
of 1:400 (DPII) and 1:40,000 (DPIII) using N-Bgl oligonucleotides and J-Bgl
oligonucleotides, respectively. Differential products were cloned into a
Bluescript
vector at the BAM HI site for analysis of the individual clones.
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EXAMPLE 1
Differential Translation Analysis of mRNA From Sucrose Density Gradients
C6 glioma cells were grown under normal conditions (Normoxia) or
under oxygen deprivation conditions (Hypoxia) for eight hours. The cells were
then harvested and cytoplasmic extracts were applied onto sucrose gradients.
RNA
was extracted from the fractions obtained from the sucrose gradient and pooled
into polysomal and non-polysomal samples. Following reverse transcription, the
differential display technique was applied using the primers T1 and P10 as set
forth
in Table 2. The PCR products were separated on a 5% acrylamide sequencing gel.
1 o The gel was then dried and exposed to X-ray film. The results are shown in
Figure
2 wherein "A" shows an mRNA species apparent only in a non-polysomal fraction
of cells after eight hours of hypoxia. This represents a potentially
transcriptionally
induced mRNA species which was still translationally repressed but which could
be actively transcribed after prolonged hypoxia. "B" represents an mRNA
species
found in the non-polysomal fraction of cells grown under normal oxygen levels
which was transferred into the polysomal fraction following hypoxia.
The materials and methods were performed as set forth above. This
example demonstrates the utility of the present invention for identifying
translationally regulating genes which are regulated by a stress inducing
element.
EXAMPLE 2
Representative Heat Shock GEM Differential Expression Analysis
Materials and Methods
The experimental cells were grown under both normal temperature
(37°C) and heat shock temperature (43°C) for four hours. The
cells were then
harvested and cytoplasmic extracts were obtained and RNA extracted therefrom.
Then, the extracted RNA was analyzed utilizing GEM technology as disclosed
above.
Tables 3 and 4 demonstrate the utility of utilizing polysomal probes
3o versus total mRNA probes in differential expression analysis to identify
genes
which are differentially expressed in response to a stimulus such as heat
shock.
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These Tables illustrate that fibronectin, pyruvate kinase, protein disulfide
isomerese, poly(ADPribose) polymerise, thymopoietin, 90Kd heat shock protein,
acylamino acid-releasing enzyme, (3-spectrin, and pyruvate kinase were all
identified as being differentially expressed utilizing a polysomal probe
whereas,
with the exception of fibronectin, the other proteins were not identified as
being
differentially expressed when a total mRNA probe was utilized. This example
demonstrates the utility of the present invention for identifying
translationally or
differentially regulated genes which are regulated by a stress inducing
element.
Additionally, in Table 3, the results of heat shock differential gene
expression
1 o analysis with both polysomal probes and total mRNA probes is provided.
Table 3
illustrates that a number of differentially expressed genes were identified
using a
polysomal probe whereas when a total mRNA probe was used, these genes were
not necessarily identified as being differentially expressed. Table 4
statistically
illustrates the number of differentially expressed genes identified utilizing
either
total mRNA or polysomal mRNA as a probe. Table 4 clearly illustrates that
polysomal mRNA probes yielded between two and greater than ten fold increases
in the number of differentially expressed genes versus total mRNA probes.
EXAMPLE 3
2o Identification of IRES Containing Genes
Establishment of mammalian cells expressi~~2A protease
HEK-293 human (ATCC CRL-1573) cells were used as a model
system for Polio virus 2A protease induced expression, since preliminary study
indicated that 2A protease enhances expression of IRES containing genes in
this
cell line. HEK-293 cells were co-transfected with CMV-LacI - (constructed by
applicant using techniques known to those skilled in the art) in combination
with
either one of the Polio virus 2A protease expression vectors PTK-OP3-WT2A,
miniTK-WT2A, on PCIbb-LacI-Hyg (constructed by applicant on basis of vectors
from Stratagene} as shown in Figures 3A-C, respectively. The LacI expression
3o vector contained a hygromycin selectable marker, and the Polio virus 2A
protease
expression vector contained a neomycin selectable marker which enabled the
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isolation of clones resistant to both markers, presumably expressing both LacI
repressor and Polio virus 2A proteins.
Analysis of Polio virus 2A protease expression
Death assay: - Resistant clones which grew after selection on hygromycin
(50pg/ml) and neomycin (SOO~g/ml), were treated with IPTG (5mM for 48h +
SmM for further 48h). Cells were then monitored for their viability and the
clones
that showed full mortality upon Polio virus 2A protease induction, presumably
expressing the deleterious effect of the Polio virus 2A protease, were
selected for
1 o further analysis. Two such clones were isolated, HEK-293 cells expressing
Polio
virus 2A protease under the control of a TK promotor (clone # 14) and HEK-293
cells expressing the Polio 'virus 2A protease under the control of a miniTK
promoter (clone #1) as shown in Figure 4.
Analysis of 2A protease expression: - Direct analysis of the Polio virus 2A
protease expression in HEK-293miniTK#1 clones and HEK-293TK#14 clones
after IPTG induction was not performed due to the lack of antibodies against
the
protein. Several currently available techniques can be used to measure changes
in
gene expression including Northern blot analysis, RNase protection assay, in
situ
2o hybridization, and reverse transcriptase polymerase chain reaction (RT-
PCR). RT-
PCR is a very sensitive method, and was used to monitor the induction of the
mRNA encoding for Polio virus 2A protease in HEK-293miniTK#1 clones
following IPTG treatment. mRNA was prepared from HEK-293 parental cells and
HEK-293miniTK-2A clones following treatment with IPTG at different time
points. The RNAs were subjected to the RT-PCR reaction using Polio virus 2A
protease specific oligonucleotides:
5'GCAACTACCATTTGGCCACTCAGGAAG3', (SEQ ID No:28) and
5'GCAACCAACCCTTCTCCACCAGCAG3' and (SEQ ID No: 29).
Polio virus 2A protease mRNA was not detected in HEK-293
3o parental cells, however it was induced following IPTG treatment and reached
its
highest level after 48 hours of IPTG treatment as shown in Figure 5.
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Analysis of 2A protease activity
p220 cleavage: - A well characterized function of Polio virus 2A protease is
the
cleavage of the p220 protein (4Fy translational factor), a component of the
40S
ribosomal subunit. Cleavage of p220 yields three N-terminal cleavage products
of
100-120KDa molecular weight due to post-translational modification. p220 and
its
cleavage products were identified by 7% SDS PAGE and Western blot analysis
using polyclonal anti-p220 antibodies specifically directed against the N-
terminal
region p220 as shown in Figure 6. Figure 6 demonstrates such an analysis in
which HEK-293 miniTK2A#1 clone and HEK-293TK2A#14 clone were induced
1 o for Polio virus 2A protease expression to generate cleavage products of
p220. As
control, HEK-293 cell lysate was treated with Polio virus 2A protease produced
by
in vitro translation, and was found to generate identical cleavage products
with the
same mobility on 7% SDS PAGE as in the HEK-293 2A clones.
This system was used as the source of mRNA for polysomal
fractionation. RDA analysis was performed using the protocol described above
to
identify genes whose translation was up-regulated by the effects of the Polio
virus
2A protease. Table 5 summarizes the results of analyses performed according to
the above-described method and genes isolated thereby.
Throughout this application various publications are referenced by
2o citation and patents by number. Full citations for the publication are
listed below.
The disclosure of these publications in their entireties are hereby
incorporated by
reference into this application in order to more fully describe the state of
the art to
which this invention pertains.
The invention has been described in an illustrative manner, and it is
to be understood the terminology used is intended to be in the nature of
description
rather than of limitation.
Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. Therefore, it is to be
understood that within the scope of the appended claims, the invention may be
3o practiced otherwise than as specifically described.
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TABLE 1
FRACTIONATION MEASURES AND IDENTIFIES
RNA associated with:
no fractionation changes of transcript abundance
Total RNA
Nuclear Measures denovo synthesis of mRNA
Cytoplasmatic Changes of transcript abundance
Cytoplasmatic/Nuclear transport of mRNA from the nucleus
to the
Nuclear/Cytoplasmatic cytoplasm, increased or decreased
stability
of mRNA
Polyribosomal/subpoly translationally controlled genes
ribosomal
Rough Endoplasmic Reticulum differences in the abundance of
Microsomes transcripts encoding membrane and
membrane bound polysomes secreted proteins
Cytoskeletal polyribosomes differences in abundance of transcript
encoding for nuclear proteins
mitochondrial differences in the abundance of mRNA
encoding mitchondrial proteins
Splicesome differences in alternative splicing
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TABLE 2
Primers used in differential Display analysis
T Primers:
5'
s T 1: CATTATGCTGAGTGATATCTTTTTTTTTAA (SEQ ID No: 2)
T2: CATTATGCTGAGTGATATCTTTTTTTTTAC (SEQ ID No: 3)
T3: CATTATGCTGAGTGATATCTTTTTTTTTAG (SEQ ID No: 4)
T4: CATTATGCTGAGTGATATCTTTTTTTTTCA (SEQ ID No: 5}
T5: CATTATGCTGAGTGATATCTTTTTTTTTCC (SEQ ID No: 6)
1o T6: CATTATGCTGAGTGATATCTTTTTTTTTCG (SEQ ID No: 7)
T7: CATTATGCTGAGTGATATCTTTTTTTTTGA (SEQ ID No: 8)
T8: CATTATGCTGAGTGATATCTTTTTTTTTGC (SEQ ID No: 9)
T9: CATTATGCTGAGTGATATCTTTTTTTTTGG (SEQ ID No: I O}
15 P Primers:
5'
PI : ATTAACCCTCACTAAATGCTGGGGA (SEQ ID No: 12)
P2: ATTAACCCTCACTAAATGCTGGAGG (SEQ ID No: 13)
P3: ATTAACCCTCACTAAATGCTGGTAG (SEQ ID No: 14)
2o P4: ATTAACCCTCACTAAATGCTGGTAG (SEQ ID No: 1
S)
PS: ATTAACCCTCACTAAAGATCTGACTG (SEQ ID No: 16}
P6: ATTAACCCTCACTAAATGCTGGGTG (SEQ ID No: 17)
P7: ATTAACCCTCACTAAATGCTGTATG (SEQ ID No: 18)
P8: ATTAACCCTCACTAAATGGAGCTGG (SEQ ID No: 19)
25 P9: ATTAACCCTCACTAAATGTGGCAGG (SEQ ID No: 20)
P I 0 ATTAACCCTCACTAAATGCACCGTCC (SEQ ID No: 21
)
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TABLE 3
Heat Shock Differential Gene Expression
Analysis with Polysomal Probes
clone Gene Total Polysomal
13h04 Pyruvate kinase No Change Induced
IO
5b08 Saposin No Change Induced
>10
9f12 Na,K-ATPase a-1 subunit No Change Induced
x4
1a04 Thymopoietin a No Change Induced
x4
13h10 Poly(ADP-ribose) polymeraseNo Change Induced
x5
7c09 pM5 Reduced Induced
x2 >6
l4ell Ubiquitin Induced Induced
x2 x4
10c06 Initiation Factor 4B No Change Induced
x4
1b09 90-kDa heat-shock proteinNo Change Induced
10
1c06 Acylamino acid-releasing No Change Induced
enzyme 10
1e09 ~-spectrin Reduced Induced
x2 x5
3b04 Elongation, factor-1-gammaNo Charge Induced
x4
13a12 Fibronectin Induced Induced
x2 x10
7h12 Cytochrome C reductase No Change Induced
core I >10
9d12 Cytoskeletal y-actin No Change Induced
>6
13f09 Protein disulfide isomeraseReduced Induced
x2 >10
9g12 DAP5 Induced
x5
TABLE 4
Sfafis Tics
Probe Number of differentials Fold induction
Total mRNA 4hrs HS 2 2
Polysomal RNA 1hr HS 14 2-4
g ~g
15 >10
37
Polysomal RNA 4hrs HS I3 2-4
6 ~10
18 >10
37
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TABLE 5
Transtafionatty con~rotted genes
are idenfiified by the 2a protease system
A. Ribosomal proteins or proteins directly involved in
translation encoded by mRNAs containing 5' TOP#
S 1 7 gbM13932
S 9 gb U14971
EF-2 gbM19997
L27a gb U14968
L37a gbL06499
(Meyuhas et al., 1996)
B. Proteins encoded by mRNAs containing 5'TOP in their 5'
UTR
Laminin binding receptor
ø 1-tubutln gb J00314
C. Gene with GC rich 5'UTR that regulates their
translation
spermidtne synthase gbM34338
retinol binding protein 5'UTR X00129
D. Unknown genes potenfaly regulated by translation
EST gb1059051 EST gb AA043162 EST gbW76915
EST gbT54424 EST gb AA025896 D 4 5 2 8 2
EST gbH15523 EST gb R07358
EST gbW95821 EST gb H83477
EST gbW99369 EST T34436
E: Known genes that are potentially regulated by
translation (and may conatin IRES ~n their 5' UTR) .
mitocnondrtat hinge protein gbS61826
gp2&L2 m(tochondrial protein gp26L2
mRNA encoding a protein related to tysyt t-RNA
synthetase emb z31711
SAP14 human spttcesosome gb U41371
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Vagner et al. "Alternative Translation of Human Fibroblast Growth Factor 2
mRNA Occurs by Internal Entry of Ribosomes" Molecular and Cellular Biology,
Vol. 15, No. 1, pp. 35-44 (1995).
Welsh et al., "Arbitrary primed PCR fingerprinting of RNA",
Nuc. Ac. Res. 20:496S-4970 ( 1992).
Zhao et al., "New primer strategy improves precision of differential display"
2o Biotechniques 18: 842-850 (l995).
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Luria) Sylvie
Einat) Paz
Harris, Nicholas
Skaliter, Rami
Grosman, Zehava
(ii) TITLE OF INVENTION: METHOD FOR IDENTIFYING TRANSLATIONALLY
REGULATED GENES
(iii) NUMBER OF SEQUENCES: 29
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kohn & Associates
(B) STREET: 30500 Northwestern Hwy., Suite 4l0
(C) CITY: Farmington Hills
(D) STATE: Michigan
(E) COUNTRY: US
(F) ZIP: 48334
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kohn, Kenneth I.
(B) REGISTRATION NUMBER: 30,955
(C) REFERENCE/DOCKET NUMBER: 0168.0002l
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (248) 539-5050
(B) TELEFAX: (248) 539-5055
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CATTATGCTG AGTGATATCT TTTTTTTTVV 30
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{2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
CATTATGCTG AGTGATATCT TTTTTTTTAA 30
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
CATTATGCTG AGTGATATCT TTTTTTTTAC 30
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 30 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
CATTATGCTG AGTGATATCT TTTTTTTTAG 30
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 30 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii} MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
CATTATGCTG AGTGATATCT TTTTTTTTCA 30
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(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CATTATGCTG AGTGATATCT TTTTTTTTCC 30
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CATTATGCTG AGTGATATCT TTTTTTTTCG 30
(2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
CATTATGCTG AGTGATATCT TTTTTTTTGA 30
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CATTATGCTG AGTGATATCT TTTTTTTTGC 30
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(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CATTATGCTG AGTGATATCT TTTTTTTTGG 30
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATTAACCCTC ACTAAANNNN NNNNNN 26
{2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
ATTAACCCTC ACTAAATGCT GGGGA 25
{2) INFORMATION FOR 5EQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
{B) TYPE: nucleic acid
{C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
ATTAACCCTC ACTAAATGCT GGAGG 25
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(2} INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
ATTAACCCTC ACTAAATGCT GGTAG 25
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:15:
ATTAACCCTC ACTAAATGCT GGTAG 25
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
{B) TYPE: nucleic acid
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
ATTAACCCTC ACTAAAGATC TGACTG 26
(2) INFORMATION FOR SEQ ID N0:17:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
{A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
ATTAACCCTC ACTAAATGCT GGGTG 25
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(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
ATTAACCCTC ACTAAATGCT GTATG 25
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
ATTAACCCTC ACTAAATGGA GCTGG 25
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
ATTAACCCTC ACTAAATGTG GCAGG 25
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
ATTAACCCTC ACTAAATGCA CCGTCC 26
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(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: 5EQ ID N0:22:
GATCTGCGGT GA 12
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
AGCACTCTCC AGCCTCTCAC CGCA 24
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
{B) TYPE: nucleic acid
{C) STRANDEDNESS: single
{D) TOPOLOGY: linear
{ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GATCTGTTCA TG 12
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
ACCGACGTCG ACTATCCATG AACA 24
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(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
GATCTTCCCT CG I2
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primers'
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
AGGCAACTGT GCTATCCGAG GGAA 24
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
GCAACTACCA TTTGGCCACT CAGGAAG 27
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GCAACCAACC CTTCTCCACC AGCAG 25
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