Note: Descriptions are shown in the official language in which they were submitted.
V095/02605 2 1 ~ 7 2 9 5 PCT~S94/080~
SEX~ lC DNA PROBE FOR PARROTS, METHODS AND KITS
Field of the Invention
The present invention relates to DNA or RNA
probes for binding specifically to female DNA of
parrots to determine the sex of parrots within a
single day and methods and kits.
Bac~ r ~
Parrots is the collective name for
approximately 350 species of birds scientifically
known as the Psittacidae genus. Parrots include
well-known species such as cockatoos, macaws,
parakeets and amazones.
Parrots are among the most popular pet
birds because of the ability of many species to mimic
human speech and to develop strong bonds with their
caretakers. However, as a result of habitat loss and
the pet bird trade, many species of parrots have
become rare or endangered in the wild.
WO 95/02605 PCT/US94/08r
G;~ , 6 '`Z ~ 9
In order to protect populations of wild
parrots, many countries have restricted or completely
forbidden the exportation of parrots. Moreover,
captive breeding has become an important procedure to
prevent further depletion of wild populations and to
satisfy the demand for parrots in the pet bird market.
For such breeding projects, male and female
birds are needed. Therefore, the ability to
determine the sex of individual birds is of major
importance. Unfortunately, most species of parrots
are monomorphic, meaning that there are no
recognizable visible or audible differences between
male and female birds. More specifically, virtually
all juveniles and about 60% of the adults of all
avian species are monomorphic.
Presently, there are basically five methods
relied upon today to determine the sex of birds.
Unfortunately, the methods are not without
drawbacks. In one method, the external reproductive
organs are physically examined to the extent
-possible. This method, called "vent-sexing," is
applicable for a few bird families where the external
reproductive organs are large enough that they can be
observed with relative ease in the cloaca. This
method is, in practice, only applicable to waterfowl,
such as ducks, geese, and swans, and gallinaceous
birds, such as chickens, where the method can be
-~095l0~05 ~1 6 7 2 9 5 PCT~S94/08023
-3-
applied at an early age. However, the difficulty in
handling, as well as the difficulties in observing
the external reproductive organs, make '~vent-sexing~
unsuitable for use with parrots, as well as other
s birds in which the external reproductive organs
cannot be observed.
A second method, which is an invasive
method, involves the examination of internal
reproductive organs. According to this method, a
bird must be placed under general anaesthesia and an
incision made so that the internal reproductive
organs can be observed with an endoscope. Although
this is the most often used procedure, it causes
great trauma to the bird and often results in
infection. Moreover, the method cannot be used with
very young birds, because of their sensitivity to
surgery, and the insufficient development of the
internal reproductive organs in such young birds.
A third method involves biochemical
determinations of the concentrations of the steroid
sex hormones, estradiol and testosterone, from blood
and/or faecal matter. This procedure is based on the
observation that male animals are characterized by a
high concentration of testosterone in their blood,
while females have high levels of estradiol. A
disadvantage of this procedure is that it is
technica~ly very complicated and requires experienced
WO95l02605 PCT~S94/080
~,~G~I ~9~
--4--
personnel for its operation. Another problem is that
the procedure has no diagnostic value when used on
young animals having low steroid hormone
concentrations. Although the method can be applied
to faecal matter rather than blood samples, it is
often difficult to assign faecal samples to
individuals birds when more than a few individual
birds are under observation.
The fourth method, which is also an
invasive method, is based on the occurrence of a
specific W chromosome in female birds. Male birds
have two Z sex chromosomes, "ZZ," whereas female
birds have one W sex chromosome and one Z sex
chromosome ("ZW"). In many, but not all bird
species, the W chromosome has some unique staining
characteristics and can, therefore, be observed and
distinguished by microscopic examination of bird
chromosome preparations. A disadvantage is that an
expensive laboratory equipment is needed and highly
qualified personnel are required for the performance
of this procedure. As a further drawback associated
with this method, in a number of bird species,
including some parrots, an unambiguous identification
of the W sex chromosome is not possible.
The fifth method available today, is a
fingerprint based DNA analysis which involves the
identifieation of sex specific DNA fragments by DNA
`~095/02605 ~1 ~ 7~9 ~ PCT~S94/08023
--5--
fingerprinting. According to this method, the DNA of
individual birds is degraded with specific enzymes,
resulting in the generation of specific fragments,
which upon fractionation by electrophoresis forms a
pattern specific for each individual bird, including
its sex. The disadvantage of a fingerprint based DNA
analysis is that it is technically very complicated
and takes approximately one week to perform.
Moreover, it requires isolation of very pure DNA,
enzymatic digestion of the isolated DNA,
fractionation by electrophoresis of the DNA fragments
in the digest, and transfer of these fragments from
the gel to a nylon membrane, followed by probing with
a specific probe. Moreover, radioactive labelling of
the probe is essential to the procedure, thus
requiring the use of highly sophisticated equipment
and facilities.
In view of the foregoing, it is evident
that there is a demand for a simple, quick, and
accurate method to determine the sex of birds, such
as parrots.
SummarY of the Invention
In brief, the present invention overcomes
certain of the above-mentioned problems and
shortcomings of the present state of the art through
the discovery of novel nucleotide sequences derived
from the~W chromosome of a female parrot, the African
WO95/0~05 ~t6~ 2 PCT~S94/080
grey parrot, Psittacus erithacus. Uniquely, the
novel nucleotide sequences of the present invention
are highly female specific for this species of
parrot. Moreover, they are believed to be highly
female specific for almost all other species of
parrot.
According to the present invention, the
novel, universal nucleotide sequences are used as DNA
or RNA probes in a quick and reliable procedure for
sex determination of parrots. Unique to this
procedure and as an advantage over prior invasive
procedures utilized heretofore, the sex-typing
procedures of the present invention can be completed
within 24 hours. Moreover, the procedures of the
present invention are reliable because virtually all
species of parrots contain a sex specific component
or components on the W sex chromosome which is
closely similar to the sex specific component, i.e.,
the nucleotide sequences, isolated from the African
grey parrot in accordance with the present invention.
In carrying out the present invention, it
requires only a few microliters of blood, which can
be collected easily without anaesthesia, from a wing
vein or a toe of the parrot. As an alternative, the
blood sample may be acquired from a blood feather
such as a developing secondary or primary flight or
tail fea~her.
vos5lo~os ~ PCT~S94/080~
21 ~ 7
--7--
To summarize a procedure of the present
invention, a blood sample is first obtained from a
parrot of choice, and DNA is then obtained from this
blood sample (10 - 60 min). The DNA is denatured,
bound to a nylon membrane and prehybridized (1-2
hrs). The DNA bound to the nylon membrane is than
hybridized with a radioactive or nonradioactive DNA
probe solution (12 hrs) of the present invention, and
washed to remove the non-specifically bound DNA probe
(1 hr). The specifically bound DNA probe is then
visualized with the appropriate procedure (2 hrs) to
determine the sex of the parrot.
The DNA or RNA probes used in the present
invention are sex specific but not parrot-species
specific. Radioactively or nonradioactively ~biotin)
tagged DNA or RNA probes of the present invention are
successful in determining the sex of many parrot
species in avian collections. The advantages of
using the DNA or RNA probes, methods and kits of the
present invention for the determination of the sex of
parrots include, for example, 1) rapid determination
(24 hrs); 2) major surgical procedures are not
required: 3) the use of blood as a readily available
source of DNA; 4) the use of a safe, stable, highly
sensitive and highly sex specific but not
species-specific DNA probe; 5) the use of simple
procedures utilizing standard clinical and research
~1~729~ !S '' AUG 1996
--8--
laboratory equipment which require minimal technical
ex^pertise for their operation; and 6) technical
simplicity as compared to currently available
procedures. Thus, the ~,oce~res of the present
invention can be easily practiced by veterinarianS
and breeders in their offices and by other qualified
personnel.
Accordingly, it should now be appreciated
that the present invention is believed to provide a
solution to the s-x-typing parrot art that has long
sought rapid and reliable methods for determining the
sex of different species of parrots. This is
accomplished by the present invention, as indicated
above, through the identification of novel, universal
nucleotide seguences which are useful as DNA or RNA
probes that are complementary to DNA segments on the
w chromosome ~h~ re characteristic for female
parrots o~ P~ us genus. Applications of
the DNA or RNA p.-~obes contemplated by the present
invention therefore include, for example,
determination of the sex of parrots in captive
breeding programs. Moreover, the applications of the
probes include the determination of the sex of
parrots in wild populations as part of, for example,
research and ecological studies.
The above features and advantages will be
better understood with reference to the FIGS.,
A~dElYDE~
~5 ~1 ~ 7295 PCT~594/o8
Detailed Description and Examples set out
hereinbelow. It will also be understood that the
biological materials, methods and kits of this
invention are exemplary only and are not to be
regarded as limitations of this invention.
Brief DescriPtion of the FIGS.
Reference is now made to the accompanying
FIGS. in which are shown characteristics
corresponding to the novel nucleotide sequences of
the present invention from which certain of their
features and advantages will be apparent:
FIG. 1 describes the nucleotide sequences
of the two strands of the 461 bp sex specific DNA
repeat of the African grey parrot. A randomly chosen
second clone of the repeat shows 17 nts or 4%
difference with the first clone.
FIG. 2 describes the structure of one of
the strands of the 461 nt sex specific repeat of the
African grey parrot in order to illustrate that it is
made of a linear array of subfragments in which oligo
dT fragments of 2-6 thymine (=T) residues are
repeated at an average distance of 10.5 nts, which
represents one complete turn of the DNA double helix.
FIG. 3 describes the signals obtained by
hybridizing the cloned p32-labelled African grey
parrot sex specific DNA repeat element with three
differen~ amounts of nylon immobilized male and
W095/02605 PCT~S94/08C
~6~ ~9~
--10--
female species: 1 = chattering lory (Lorius garulus);
2 = cherry-headed conure (Aratinga erythrogenys); 3 =
sulphur-crested cockatoo (Cacatua galerita); 4
blue-fronted amazon (Amazona aestiva); 5 = blue and
gold macaw (Ara ararauna); 6 = African grey parrot
(Pisttacus erithacus).
Detailed Descri~tion
By way of illustrating and providing a more
complete appreciation of the present invention and
many of the att~n~t advantages thereof, the
following detailed description is provided concerning
the novel nucleotide sequences, methods and kits.
The novel nucleotide sequences of this
invention, preferably used as DNA or RNA probes, have
been derived from the DNA of the female African grey
parrot Psittacus erithacus. More particularly, they
have been cloned as a fragment of 461 base pairs,
obtained with the restriction nuclease MspI, in the
vector pGEM3Z+ and the host Eschericia coli 31/17.
The 461 base pair repeat is derived and isolated from
-a base pair repeat of about 570 base pairs and is
made up of 43 subfragments in which oligo dT
fragments of 2-6 thymine residues are repeated at an
average distance of lO.Sbp. More specifically, the
2-6 long oligo T fragments are part of 43
subfragments of a minimum length of about 4 nts and a
maximum ~ength of about 20 nts, and an average length
~095/0~05 PCT~S94/080~
216729~
--11--
of about 10.5 nts which together form the 461 nt long
fragment which is cloned. This latter fragment is
again tandemly repeated 12,000 times. The DNA
component is tandemly repeated with a copy number of
approximately 12,000 copies per female African grey
parrot genome and forms a substantial part of the W
chromosomal DNA of this species. The nucleotide
sequence of the component is conserved among the DNA
of females of many other species of parrot. While
the 461 base pair fragment has been sequenced as
reported in FIG. 1, the remaining base pairs in the
570 base pair fragment from which the 461 base pair
fragment has been derived and isolated have not been
sequenced.
The novel procedure to demonstrate the
presence of female W chromosome specific components
in parrot DNA utilizes a probe comprising a trace
amount of radio labelled female parrot DNA and a
large excess (4000 fold) of unlabelled male DNA in an
analysis of enzymatically digested and
electrophoretically fractionated female parrot DNA.
FOCUS (BRL) 14: 106-108 (1992). The excess
unlabelled male DNA acts to dilute the radio-label in
the components which are common to male and female
DNA, so that common sequences do not produce any
signal in the analysis, and the only signal observed
is that_ produced by female specific components.
W095/0~05 ~6~ 295 PCT~S94/08
-12-
Utilizing this procedure with the restriction enzyme
MspI, in a digest of the female African grey parrot,
Psittacus erithacus, identification of a component of
approximately 450 base pairs, which does not occur in
the male DNA, is accomplished.
This fragment is isolated from the
electrophoretically fractionated MspI digest and
cloned in plasmid vector pGEMZ+ in the bacterium
Eschericia coli 81/17. This cloned component has
been used for the determination of the nucleotide
sequence of the female specific component, its copy
number and genomic organization, and the conservation
among other species of parrots.
FIG. 1 shows that the sequence of the
component has a molecular length of 461 base pairs.
FIG. 2 shows that the fragment has an internal repeat
structure in which groups of 2 - 6 thymine residues
are repeated an average of 10.5 nucleotides. This
sequence characteristic is typical for a rather
unusual so called "curved DNA element".
A curved DNA is a double stranded (native)
DNA which most often has short runs of 2-6 adenine or
thymine residues at an average distance of 10.5 nts,
which is just a complete winding of the DNA double
helix. The curved nature of such DNA has been
demonstrated by electronmicroscopy and
circular~zation experiments. Curved DNA exhibits an
vo 95~0260s 21 6 7295 PCT~S94/080~
-13-
anomalous, slow electrophoretic velocity in
polyacrylamide gels. This latter characteristic is
usually taken as diagnostic evidence for the curved
structure. The curved structure is an inherent
property of such DNA and should be distinguished from
so called bent DNA. The latter owes its curvature to
interaction with certain proteins.
DNA components can occur either as unique
or as repeated sequence elements. A quantitative
analysis shows that the sex specific component
amounts to approximately 0.3% of the genome of the
female African grey parrot, whereas it amounts to at
most 0.005% of the male genome. See Table I. Hence,
the female African grey parrot genome contains about
12,000 copies of the sex specific DNA component of a
length of 463 base pairs. These copies are tandemly
organized in the genome. In situ hybridization to
chromosomes of the African grey parrot shows strong
hybridization of the DNA or RNA probes of the present
invention to the W chromosome in the female whereas
such hybridization is not observed in the male.
The DNA probes of the present invention can
be produced by chemical synthesis, recombinant or
cloning technology or any other methods available in
the art so long as the methodology selected does not
interfere with their utilities stated herein.
Moreover F the DNA probes of the present invention may
~167~?9S- PCT~JS 94/0~423
IP~ JS 3 :AUG 1995
-14-
be modified by adding, deleting and/or substitutingnucleotides to form DNA probes of varying lengths
which are functionally equivalent to the nucleotide
sequences set forth in FIG. 1. In addition, RNA
probes with nucleotide sequences contemplated by the
present invention may be substituted for the DNA
probes. Therefore, it is to be understood by those
versed in this art that any DNA or RNA nucleotide
sequence, including equivalents and active segments
of the nucleotide sequences depicted in FIG. 1, which
is complimentary to a DNA~segment on a W chromosome
which is characteristic for a female parrot of the
Psittacus genus is contemplated by the present
invention. Examples of active nucleotide fragments
in accordance with the present invention are those
fragments derived by, for example, digesting the 461
base pair fragment with t.~ el,zyme SAU3Al at a GATC
site using diges~ion t.~hn.i ~ known to those
skilled in the art. When` ~h~ 461 base pair fragment
is digested with the enzyme SAU3Al, the 461 base pair
fragment is cleaved at the GATC sites beginning with
the nucleotide designated as 245 in the 5'-3'
sequence and with the nucleotide designated as 213 in
the 3'-5' sequence in FIG. 1 to generate four active
fragments having, for example, bases co~e~onding to
bases 1-248 and 249-461 in the 5'-3' sequence and
_~9~J
2167,~95 - PC~IS 94/08 023
IPEAJUS ~}~AUG ~
-15-
corresponding to bases 1-216 and 217-461 in the 3'-5'
sequence as designated in FIG. 1.
The DNA or RNA probes of the present
invention may be formed into kits which can be easily
used by, for instance, veterinarians, breeders, as
well as other gualified personnel interested in
sex-typing parrots~ of the Psittacus genus. A
typical kit in accordance with this invention
includes blood sample stabilizing Solution A;
lo proteinase-K and sodium sarcosylate Solutions, or a
commercially available fast DNA isolation kit; D~A
denaturing and neutralizing Solutions; nylon
membranes; biotin-labelled sex specific DNA or RNA
probe; hybridization Solution ~: wash Solutions C and
D; and a detection system specific for the biotin tag
so that all that is required to carry out the
sex-typing methods of the pres~nt invention is
standard laboratory e~uipm~t.
According to the prese~lt invention, blood
is used as a source of DNA because avian erythrocytes
are nucleated and contain DNA. Avian blood contains
on average approximately 5 to 10 mgs of DNA per ml.
In carrying out the sex typing of parrots in
accordance with the present invention, blood samples,
approximately 25 to 100 ~1, are obtained by brachial
(wing) vein puncture or by clipping of a toe nail or
by removing a blood feather such as a secondary, or
,~D ~r
Yo 95,~.605 ~ 1 6 7 2 9S PCT~S94/08023
-16-
primary flight feather or a tail feather. Clotting
of the blood is prevented by immediate dilution,
after collection, with an equal volume of a solution
containing about 0.15M NaCl and about 0.05 M sodium
EDTA, pH 7.5 (Solution A). In this solution, blood
samples can be stored for at least a week in a
refrigerator at 4 c.
The major contaminant of the DNA in avian
blood is protein, which can be removed from the
sample by protease digestion, e.g., phenol, or
chloroform extraction in the presence of (cationic or
anionic) detergents. ~When chloroform extraction is
used, the chloroform should be removed by for example
centrifugation.~ Accord~ 1 of the 1:1
diluted blood sample ~(containing on average 50 mgs of
DNA) is diluted with 90 ~1 of Solution A and
incubated for one hour at about 65-C with proteinase
K (2 ~gs per 100 ~1) and sodium lauryl sarcosylate
(0.2%). A commercially available DNA isolation
system (e.g. from Invitrogen, 3985 Sorrento Valley
Blvd, San Diego, CA 92121 or Washington Biotechnology
Inc., 6917 Arlington Rd., Bethesda, MD 20814) can be
used for this purpose.
DNA samples obtained are then denatured
with NaOH (final concentration about 0.5 M) and
neutralized with about 1 M NaH2PO4. Samples
containing approximately 1 mg of denatured parrot DNA
VOg5/02605 PCT~S94/080~
~` 2~1 6729~
-17-
are then loaded and immobilized onto nylon filters
(0.2 ~M pore size) with the aid of a slot or dot blot
apparatus. The filters are then dried in vacuo at
about 80 C for about two hrs to bind the DNA
irreversibly to the filters.
Prior to hybridization with the probe,
filters containing the DNA samples are prehybridized
for about one hr at about 65 C in a solution
containing about 0.9 M NaCl, O.l M Tris-HCl buffer pH
7.8, 0.05 M Na2EDTA, 0.2~ sodium lauryl sulphate and
500 ~g per ml heparin as a blocking agent (Solution
B).
The sex specific DNA fragment, obtained by
cloning from the genome of the female African grey
parrot as described above and recited in FIG. 1, is
tagged by enzymatic or nonenzymatic procedures with
radioactive p32 or nonradioactive biotin or
fluorescent groups like fluoresceine or rho~mine
using technologies available to these in the labeling
art. It should be understood that the DNA probe may
also be tagged with fluorescent dyes, such as
fluoreseine or rhodamine using techni~ues known to
those versed in this art. In the present example the
DNA probe is tagged by, e.g., labelling with biodUTP
using nick translation or random primer extension.
As to an RNA probe, it is labelled with, e.g., bioUTP
using T~ RNA polymerase (pGEM3Z+ has a T7 RNA
WO 9S/02C05 ; Z l 6 ~ PCT~S94/080
--18--
polymerase promoter site). The tagged, denatured DNA
probe is then incubated for approximately 60 - 65-C
and at a concentration of about 25 ng per ml of
Solution B with the nylon filters containing the
parrot DNA. Subsequent to incubation, the filters
are washed first at room temperature with 1 x SSC (=
0.15 M NaCl, 0.015 M sodium citrate pH. 7.0, Solution
C) (five times for about five minutes with about lO0
ml) and subse~uently one time for about two minutes
at about 55-C with approximately 100 ml 1 x SSC
containing about 0.1% sodium lauryl sulphate
(Solution D). Binding of radioactively labelled
probe is visualized and quantified by autoradiography
and/or a betascope. In this Example, bound probes
labelled nonradioactively with biotin are visualized
through a commercially available detection process
which involves a specific color reaction or
chemiluminescence. The specific colour reaction
involves, e.g., the binding of a streptavidin or
avidin phosphatase conjugate to the biotin residue
-(streptavidine and avidine are proteins which have a
ver yhigh specific affinity for biotein; phosphatase
as an enzyme which hydrolyes phosphate esters.) and
the subsequent hydrolysis of a so-called chromogenic
substrate which consists of an uncoloured phosphate
ester which becomes strongly coloured after removal
of the ~hosphate by the phosphatase. Examples of
VOg5/0~05 t 6 7~9 ~ PCT~Sg4tO8023
--19--
such esters are bromo-indoxyl phosphate which is
uncoloured, but which generates dark blue indigo
- after removal of the phosphate. Chemiluminescent
detection systems make use of chemiluminescent
substrates in a similar phosphatase catalyzed
reaction, followed by photographic detection of the
emitted light.
The present invention will now be further
illustrated with reference to the following Examples.
ExamPle I
SEX ~r.~ lC DNA F'177'r~F~IT
Female african grey parrot DNA (10 ug) is
digested in 100 ~1 of a buffer (containing 6 mM
MgC12, 6 mM Tris-HCl buffer pH 7.5, 50 mM NaCl) with
20 units of the restriction endonuclease Mspl for 3
hrs at 37- C. For identification purposes of the sex
specific DNA fragment, 10 ~1 of the digest is
fractionnated by electrophoresis through a 1% agarose
gel in TPE buffer (TPE = 0.08 M Tris-phosphate pH
7.5, 0.008 M EDTA). A southern blot of the gel is
made and probed with a genomic DNA probe made from 25
ng radioactively (p32) labelled female DNA [labelled
by random primer extension, Feinberg, A.P. et al.:
Anal. Biochem., 132:6-13 (1983), preannealed to 100
2 5 ~g reiterated, denatured male African grey parrot
Cot2 DNA (Cot indicates a degree of repetitivity of
the DNA -sample, see: De Kloet pH and de Kloet SR
W095l02605 PCT~S94/080
2 g ~
-20-
(1992) Molecular determination of the sex of parrots
(FOCUS (BRL) 14:106-108 (1992)).
After identification of the sex specific
DNA fragment by autoradiography or with a betascope,
a preparative (larger) gel is run and the gel slice
containing the 461 bp sex specific fragment cut out.
The sex specific fragment is isolated from the gel by
the powdered glass procedure, Vogelstein, B. et al.:
Proc. Natl. Acad. Sci. USA, 76:615-619 (1979). The
fragment is ligated into the powdered glass
procedure. See Vogelstein B. et al.: Proc. Natl.
Aca. Sci. USA, 76:615-619 (1979). The fragment is
ligated into the ACC1 site of the plasmid pGEM3Z+ and
the ligation product is used to transform E. coli
71/18 using the CaC12 procedure (Maniatis, T. et al.
(1982) "Molecular cloning; a laboratory manual."
Cold Spring Harbour Laboratory. Cold Spring Harbour,
NY, 2) probe labelling with biotin:). The
transformed bacteria are spread on 20 ml agar plates
containing Luria bertani (L8) medium (LB = 1%
bacto=tryptone, 1% NaCl, 0.5% bacto yeast extract pH
7.4) containing per ml 50 ~gs of penicillin, 150 ~gs
bromo-idnolyl-beta-galactoside and 75 ~g isopropyl
beta thiogalactoside. After growth vernight at 37-C,
the white colonies (containing plasmids with inserts)
are selected and transferred to fresh FL plates
covered ~ith a Nylon filter. Control plates without
~095t02605 1 6 729 ~ PCT~S94/08023
-21-
a filter are also inocculated with the colonies in
the same pattern. After growth until the colonies
are detectable, the nylon filter is removed from the
plate and dried in a vacuum oven at 80-C. Colonies
with a sex specific insert are identified by colony
hybridization (Maniatis, T. et al. (1982) "Molecular
cloning; a laboratory manual." Cold Spring Harbour
Laboratory. Cold Spring Harbour, NY, 2) probe
labelling with biotin:) using the same genomic sex
lo specific DNA probe as is used initially for
determination of the sex of parrots (FOCUS (BRL)
14:106-108 (1992)). Positive colonies are identified
by autoradiography or a betascope, and identified on
the control plates which are incubated without a
nylon filter because of the similarity in pattern of
application which is used. After identification the
colonies are transferred into LB medium without agar,
containing 50 ~g penicillin per ml, and incubated
overnight at 37 C. The bacteria are collected by
centrifugation and resuspended in LB + ampicillin
containing 15% glycerol and stored at -80-C.
ExamDle II
L~ NATIoN OF THE SEX OF PARROTS WITH
THE AFRICAN GREY PARROT SEX SPECIFIC DNA OOl.~tl~
In carrying out a procedure in accordance
with the present invention to determine the sex of a
parrot, twenty-five (25) ~1 blood is removed from a
wing vein or toe with a heparinized capillary and
WO9~l02605 PCT~S94/08~
~16~29 l
suspended in 0.5 ml of buffer A (a solution
containing 0.05 M Na2EDTA and 0.15 M NaCl (pH 7.4)).
Of this suspension, 100 ~l (corresponding to 5 ~l of
blood) is taken and diluted with 200 ~l of a solution
containing 8~ sodium dodecyl trimethylammonium
bromide, 1.5 M NaCl, 100 mM Tris-HCl buffer pH 8.6,
50 mM EDTA. After heating for two minutes at 68 C,
vortexing for two minutes with 300 ~l of chloroform
and centrifugation for two minutes in an Eppendorf
centrifuge, the aqueous phase (top layer) (250 ~l) is
collected. Since avian blood contains approximately
5 ~g DNA per ~l, this aqueous phase contains
approximately 25 (5 x 5) ~g of DNA per 250 ~l, or l
~g of DNA per 10 ~l. Fifty ~l of the aqueous phase
is then taken ( containing approximately 5 ~g of DNA),
diluted with 50 ~l to TE (0.01 M Tris-HCl buffer pH
8.0, 0.001 M Na2EDTA), and the DNA is denatured (make
single stranded) by the addition of 100 ~l of l M
NaOH. After five (5) minutes, the denatured DNA is
neutralized with 200 ~l l M NaH2PO4 and 100 ~l of 1.5
M NaCl is added to a final volume of 500 ~l. 100 ~l
of this solution (containing approximately l ~g of
DNA) is applied to a Nylon filter with the aid of a
slot blot or dot blot apparatus. The filter is dried
and baked in an oven at 80-C. For hybridization, the
filter with the bound denatured DNA is then
preincub~ted for thirty minutes at 60-C with 0.2 ml
~095/02605 PCT~S94108023
- ~167~9S
-23-
per cm2 of a solution containing 0.9 M NaCl, 0.002 M
Na EDTA, 0.02 M Tris-HCL pH 8.0, 0.1% sodium dodecyl
sulphate and 500 ~g per ml of heparin (as the
blocking agent to prevent direct binding of the probe
to the filter without binding to the DNA) and 1000 ~g
per ml of sodium pyrrophosphate. Heat denatured
radioactively or nonradioactively (biotin) labelled
parrot sex specific probe (40 - 100 ng per 10 ml
buffer) is then added and the incubation continued
overnight. Subsequently, the filters are washed with
1 x SSC to remove the unhybridized probe, and the
bound radioactive label visualized by autoradiography
or a betascope. The biotin labelled probe is
visualized with any of the appropriate techniques
like streptavidine - alkaline phosphatase etc.
In the event the blood sample is taken from
a blood feather, the procedure is as follows. A
growing primary or secondary feather is collected and
the featherpulp is squeezed out of the feather into 1
or 2 ml (depending on the size of the feather) ~f
buffer A (see above) or any other stabilizing
solution (Dulbecco's modified Eagles etc.). To this
suspension is added 75-150 ~1 of a solution of 10 ~g
per ml collagenase (Closteridium haemolyticum) in
water and the mixture incubated for two hours at
37-C. After gentle homogenization DNA is prepared
from the ~esulting cells as described above for blood
W095102605 21~ 7 2 9 ~ PCT~S94/08C
-24-
DNA. After measuring the amount of prepared DNA with
a spectrophotometer, the DNA is again alkali
denatured, applied to a nylon filter and the filter
prehybridized and hybridized with a probe as
described above.
It should be understood that the procedure
described is used to generate the results in Example
III and as depicted in FIG. 3. It should also be
understood that the procedures described herein are
used to sexually type the parrots listed in Table I.
'~095/0~05 ~6~5. PCT~S94/08023
-25-
TABLE I
Species Female male
- Blackmasked lovebird 8.3 0.3
Ring parrot 40.2 2.2
Bluefronted amazone 7.6 0.7
Double yellow headed amazone 8.4 o.7
Aymara parakeet 15.8 1.5
Crimsonwinged parrot 16.4 1.3
Blue and gold macaw 12.4 1.2
Scarlet macaw 14.7 O.g
Severes macaw 13.8 1.4
Redfronted conure 15.8 1.3
Sun conure 17.2 1.7
Goffins cockatoo 30.3 O.9
Moluccan cockatoo 35.0 1.3
Sulphur-creasted cockatoo 33.7 1.1
Gang-gang cockatoo 30.2 0.8
White-tailed black cockatoo 38.3 0.7
Red-tailed black cockatoo 35.3 o.g
Rosebreasted cockatoo 31.3 1.1
Chattering lory 13.5 1.3
Budgerigar 23.5 0.7
Cockatiel 34.5 0.6
Blue-headed pionus 12.4 0.9
Eastern rosella 25.6 0.4
Princess of Wales parakeet 24.3 0.5
Palm cockatoo 12.7 0.7
Alexandrine parakeet 20.6 1.3
Ring-necked parakeet 30.3 2.1
African grey parrot lOO.O 2.5
Pes~uet's parrot 8.5 0.3
Table I. Use of PARsexl for molecular sex determina-
tion of selected psittacines. Values represent the
average amount of PARSEXl present in DNA of 2 - 5
males and females of the species indicated,
calculated as percentage of the amount present in the
female African grey parrot. PARsexl refers to the
DNA probe having the sequence shown in FIG. 1.
PC~ 9 4 1 0 8 ~ 2 3
-;~93~ IPEA/US ~2AUG 199
--26--
~ ple IT~
RF~JT.'rS
FIG. 3 shows that in a blotting experiment,
DNA of the African grey parrot as well as of other
species of parrots, belonging to the different major
subfamilies of the Psittacus , namely, the conure,
the cockatoo, the lory, the mazone and the macaw
also produce a sex specific signal with the
radioactively labelled female specific DNA component
of the African grey parrot, showing that the sex
specific characteristics of the component is
conserved among the parrot family. In FIG. 3,
different amounts of DNA of males and females of the
species indicated are immobilized on a nylon filter
and hydribized with the p32-labelled African grey
parrot sex specific DNA repeat element as the probe.
The sex specific hybridization of this probe-with DNA
of all six species shows that the sex speci~ic DNA
component is structurally conserved between the
different species and can be used for the
determination of all species.
~0~
VOg5/0~05 - 27 21 6 72 9 5 PCT~S94/080~
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: de Kloet, Siwo R.
(ii) TITLE OF INv~NllON: Sex-Specific DNA Probe For Parrots,
Methods And Kits
(iii) NUMBER OF SEQUENCES: 44
(iv) CORRESPONDENCE ~nn~.c,~
(A) ADDRESSEE: Ruden, Barnett, McClosky, Smith, Schuster &
Russell, P.A.
(B) STREET: 200 East Broward Boulevard
(C) CITY: Fort Lauderdale
(D) STATE: FL
(E) COUNTRY: USA
(F) ZIP: 33301
(v) CGI.~ul~:K ~An~RT.~ 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 #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/093,198
(B) FILING DATE: 15-JUL-1993
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT lN~O~IATION:
(A) NAME: Manso, Peter J.
(B) REGISTRATION NUMBER: 32,264
(C) REFERENCE/DOCKET NUMBER: FL20979-34
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 305-527-2498
(B) TELEFAX: 305-764-4996
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTTTTCGTCC ATTCCTAACC ACATTAAAGC CTAlllllCA CCCATTTCCA ACCAATTTTA 60
AGCAATTTGT GGTCATTTCA AACACAGTTT TCACCACTTT GAACAAGCTT TAAGTCCTTT 120
wo gs/o~os ~ 16 ~ 2 9 ~ - 28 - PCT~S94/080'
rTGGTTGCTT GTAAATGATT TTTGGAGTTT TCTAACCCCT TTTGAGCCAT TTTTTCTGTT 180
TCTAACCCAT TTTTTCAACA GTTCTAGCTC GGTTTAAGTA G~ GCTT TTTTCTAACC 240
CATTGATCCC ATGACTAATC AGTTTTAAGC C~lllllGlC CATTTCTGAC CCATCTTTGC 300
CCAGCTCTAG ~lll~lllAA GCC~lllllC TCCATTTCTA ACCCGTTTCT AGCCCATTCC 360
TGACCTGTTT TAAGCCTATT CCTAACCCAT TTCCAACCCA TTTTTGGTCT TGTCAAATGC 420
AlllllCACC TCTTCTGACT CGCTTGAAGA C~lllllGCC 460
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GTTTTCGTCC 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATTCCTAACC AC 12
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ATTAAAGCCT 10
~095102605 1 ~ 7~9~ PCT~S94/08023
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(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
AlllllCACC C - 11
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ATTTCCAACC A 11
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATTTTAAGCA 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA tgenomic)
W095/0~05 21 6 7 2 9 S PCT~S94/08Q
-30-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
A~ ~lGGTC 10
(2) INFORMATION FOR SEQ ID NO:9:
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(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATTTCAAACA CA 12
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GTTTTCACCA lO
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CTTTGAACAA G 11
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
V095l02605 ~ 6 7~9S PCT~S94/08023
-31-
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CTTTAAGTC 9
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GGTT G 11
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CTTGTAAATG lO
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
AlllllGGA 9
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
W095/02605 ~ ~ PCT~S94/080
~167 2 9~ -32-
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GTTTTCTAAC CC 12
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
~llllGAGCC 10
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
AllllllCT g
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GTTTCTAACC C 11
~O95/02605 21 6 7~ 9 ~ PCT~S94/08023
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AlrllllCAA CA 12
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GTTCTAGCTC G 11
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GTTTAAGTA 9
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
WO95/02605 21 6 7 2 9 ~ PCT~S94/08~
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
~lllllG 7
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
A ACCC 14
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATTGATCCCA TGACTAATCA 20
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GTTTTAAGCC 1
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
V095/02605 ~1 6 72 9~ PCT~S94/08023
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
i l CC 1 0
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
AlLl~lGACC CAT 13
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CTTTGCCCAG CTCTAG 16
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
CTTT 4
(2) INFORNATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
WOg5/02605 PCT~S94/080
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GTTTAAGCC 9
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CTCC 10
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
ATTTCTAACC C 11
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GTTTCTAGCC C 11
~095/02605 21 672~ PCT~S94/08023
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
ATTCCTGACC T 11
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GTTTTAAGCC T 11
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
ATTCCTAACC C 11
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
ATTTCCAACC C 11
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
ATTTTTGGT 9
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
CTTGTCAAAT GC 12
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
ATTTTTCACC T 11
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
W095/02605 39 PCT~S94/080
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
CTTCTGACTC G 11
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
CTTGAAGAC 9
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lO base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
C~ GCCG 10
W095/0~05 40 PCT~S94/08C
2l6~ ~9~
The present invention may, of course, be
carried out in other specific ways than those herein set
forth without departing from the spirit and essential
characteristics of the invention. The present
embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive and all
changes coming within the spirit and scope of the
appended claims are intended to be embraced herein.
Having described my invention, I claim:
