Method of creating a cDNA library enriched in signal sequences

The present invention relates to a novel method of identifying cDNA's which encode secreted and membrane-bound proteins. The present invention also relates to a novel method for preparing cDNA libraries enriched for signal sequences. The methods of the invention provide for an improved signal sequence detection system which results, when compared to the prior art, in a greater number of correctly identified signal sequences and less total time required to complete the procedure.

BACKGROUND OF THE INVENTION 
The present invention relates to a novel method of identifying nucleic acid 
encoding secreted and membrane-bound proteins based upon the presence of 
signal sequences. The present invention also relates to a novel method for 
preparing cDNA libraries enriched for signal sequences. 
Extracellular proteins are essential in the formation, differentiation and 
maintenance of multicellular organisms. The determination by individual 
cells of whether to live, proliferate, migrate, differentiate, interact 
with other cells or secrete are governed by information received from the 
cells neighbors and the immediate environment. This information is often 
transmitted by secreted polypeptides (e.g., mitogenic factors, survival 
factors, cytotoxic factors, differentiation factors, neuropeptides, and 
hormones) which are in turn received and interpreted by diverse cell 
receptors. These secreted polypeptides or signaling molecules normally 
pass through the cellular secretory pathway to reach their site of action 
in the extracellular environment. 
The targeting of both secreted and transmembrane proteins to the secretory 
pathway is accomplished via the attachment of a short, amino-terminal 
sequence, known as the signal peptide or signal sequence. von Heijne, G. 
(1985) J. Mol. Biol. 184, 99-105; Kaiser, C. A. & Botstein, D. (1986), 
Mol. Cell. Biol. 6, 2382-2391. The signal peptide itself contains several 
elements necessary for optimal function, the most important of which is a 
hydrophobic component. Immediately preceding the hydrophobic sequence is 
often a basic amino acid or acids, whereas at the carboxyl-terminal end of 
the signal peptide are a pair of small, uncharged amino acids separated by 
a single intervening amino acid which defines the signal peptidase 
cleavage site. While the hydrophobic component, basic amino acid and 
peptidase cleavage site can usually be identified in the signal peptide of 
known secreted proteins, the high level of degeneracy within any one of 
these elements makes difficult the identification or isolation of secreted 
or transmembrane proteins solely by searching for signal peptides in DNA 
data bases (e.g. GeneBank, GenPept), or based upon hybridization with DNA 
probes designed to recognize cDNA's encoding signal peptides. 
Secreted and membrane-bound cellular proteins have wide applicability in 
various industrial applications, including pharmaceuticals, diagnostics, 
biosensors and bioreactors. For example, most protein drugs commercially 
available at present, such as thrombolytic agents, interferons, 
interleukins, erythropoietins, colony stimulating factors, and various 
other cytokines are secretory proteins. Their receptors, which are 
membrane proteins, also have potential as therapeutic or diagnostic 
agents. Significant resources are presently being expended by both 
industry and academia to identify new native secreted proteins. 
According to a screening method recently reported by Klein R. D. et al. 
(1996), Proc. Natl. Acad. Sci. 93, 7108-7113 and Jacobs (U.S. Pat. No. 
5,536,637 issued Jul. 16, 1996), cDNAs encoding novel secreted and 
membrane-bound mammalian proteins are identified by detecting their 
secretory leader sequences using the yeast invertase gene as a reporter 
system. The enzyme invertase catalyzes the breakdown of sucrose to glucose 
and fructose as well as the breakdown of raffinose to sucrose and 
melibiose. The secreted form of invertase is required for the utilization 
of sucrose by yeast (Saccharomyces cerevisiae) so that yeast cells that 
are unable to produce secreted invertase grow poorly on media containing 
sucrose as the sole carbon and energy source. Both Klein R. D., supra, and 
Jacobs, supra, take advantage of the known ability of mammalian signal 
sequences to functionally replace the native signal sequence of yeast 
invertase. A mammalian cDNA library is ligated to a DNA encoding a 
nonsecreted yeast invertase, the ligated DNA is isolated and transformed 
into yeast cells that do not contain an invertase gene. Recombinants 
containing the nonsecreted yeast invertase gene ligated to a mammalian 
signal sequence are identified based upon their ability to grow on a 
medium containing only sucrose or only raffinose as the carbon source. The 
mammalian signal sequences identified are then used to screen a second, 
full-length cDNA library to isolate the full-length clones encoding the 
corresponding secreted proteins. 
Given the great efforts presently being expended to discover novel secreted 
and transmembrane proteins as potential therapeutic agents, there is a 
great need for an improved system which can simply and efficiently 
identify the coding sequences of such proteins in mammalian recombinant 
DNA libraries. While effective, the invertase yeast selection process 
described above has several disadvantages. First, it requires the use of 
special yeast cells in which the SUC2 gene encoding the invertase protein 
has been deleted or the coding sequence of the native invertase signal has 
been mutated so that the invertase is not secreted. Second, even 
invertase-deficient yeast may grow on sucrose or raffinose, albeit at a 
low rate, therefore, the invertase selection may need to be repeated 
several times to improve the selection for transformants containing the 
signal-less yeast invertase gene ligated to a mammalian secretory leader 
sequence. See, Jacobs, supra. Third, the invertase selection process is 
further inadequate because a certain threshold level of enzyme activity 
needs to be secreted to allow growth. Although 0.6-1% of wild-type 
invertase secretion is sufficient for growth, certain mammalian signal 
sequences are not capable of functioning to yield even this relatively 
moderate level of secretion. Kaiser, C. A. et al. (1987), Science 235; 
312-317. As a result, there still exists the need for an improved and 
simplified technique for selecting genes encoding signal 
sequence-containing (secreted or membrane-bound) polypeptides. 
SUMMARY OF THE INVENTION 
The present invention concerns a novel method for identifying genes 
encoding secreted and membrane-bound proteins using a starch degrading 
enzyme as a reporter molecule. Preferably, this reporter molecule is 
amylolytic. More specifically, according to the present invention 
mammalian signal sequences are detected based upon their ability to effect 
the secretion of a starch degrading enzyme (e.g. amylase) lacking a 
functional native signal sequence. The secretion of the enzyme is 
monitored by the ability of the transformed yeast cells, which cannot 
degrade starch naturally or have been rendered unable to do so, to degrade 
and assimilate soluble starch. 
In one embodiment, the invention relates to a method of detecting DNA 
comprising the coding sequence of a mammalian signal peptide which 
comprises: 
a) screening non-amylolytic yeast cells, transformed with DNA containing 
the coding sequence of a mammalian signal peptide ligated to DNA encoding 
an amylase lacking a functional native signal peptide, for their ability 
to degrade starch; and 
b) isolating an d purifying the DNA from the cells of step a). 
The yeast cells are preferably cells of a Saccharomyces cerevisiae strain, 
lacking a native amylolytic enzyme and containing an auxotrophic marker 
suitable for the selection of plasmid maintenance following 
transformation. The mammalian coding sequence is inserted amino terminal 
to, and in-frame with the secretion defective amylase gene. In a 
particularly preferred embodiment, the ATG start codon is eliminated or 
mutated at the N-terminus of the signal sequence as well as at the 
N-terminus of the mature amylase gene, such that translation is initiated 
only from the start codon of the mammalian signal peptide to be 
identified. 
In another embodiment, the invention relates to a method of detecting, in a 
mammalian recombinant DNA library, DNA encoding a secreted or 
transmembrane protein or an N-terminal fragment thereof, which comprises: 
a) screening non-amylolytic yeast cells, transformed with DNA obtained by 
the ligation of said mammalian recombinant DNA library to DNA encoding an 
amylase lacking a functional native signal peptide, for their ability to 
degrade starch; and 
b) isolating and purifying DNA from the cells of step a). 
Just as in the previous embodiment, the yeast transformed is preferably a 
Saccharomyces cerevisiae strain, or another yeast strain lacking a native 
amylase gene, while the recombinant DNA library preferably is a mammalian 
cDNA library. The DNA identified preferably is a full-length cDNA encoding 
a novel secreted or transmembrane polypeptide. 
It should further be appreciated that the previously enumerated embodiments 
may also be modified such that yeast cells which can naturally degrade 
starch may be employed, provided that the native amylase signal sequence 
has been rendered inoperable or preferably deleted prior to the ligation 
of the mammalian recombinant DNA library. 
In yet another embodiment, the invention relates to a method of creating a 
cDNA library enriched in signal sequences which comprises: 
a) creating a full-length cDNA library containing a first unique 
restriction site and a DNA promotor region 5' to the inserted cDNA; 
b) transcribing an mRNA transcript from the cDNA of step a); 
c) preparing random DNA oligonucleotide primers and reverse transcribing to 
create cDNA fragments of the full-length cDNA clone; 
d) preparing the cDNA fragments of step c) for ligation, and ligating to an 
adapter oligonucleotide coding for a second unique restriction site; 
e) digesting the cDNA of step d) with a restriction enzyme which cuts at 
the first unique restriction site; 
f) isolating cDNA fragments which have been cut by the restriction enzyme 
of step e) and correspond to a size of about 500 to about 1000 base pairs 
in length; and 
g) ligating the isolating cDNA fragment of step f) into an amylase 
expression vector previously digested with enzymes compatible with the 
first and second restriction site of the cDNA of step f). 
In a still further embodiment, the invention relates to a method of 
detecting cDNA encoding a novel secreted or transmembrane protein or an 
N-terminal fragment thereof in a mammalian cDNA library which comprises: 
a) screening non-amylolytic yeast cells, transformed with cDNA obtained by 
the ligation of a mammalian cDNA library enriched for signal sequences, to 
DNA encoding an amylase lacking a functional native signal peptide, for 
their ability to degrade starch; 
b) isolating and purifying cDNA from the transformed yeast cells of step a) 
which are capable of degrading starch; and 
c) analyzing the cDNA from step b) to determine novelty. 
The invention further relates to a variation of the above embodiments 
wherein the screening method is selected from the following: 
a) growth on selective media followed by replica plating onto YEPD-starch 
media; 
b) growth on selective media wherein starch is directly incorporated; and 
c) growth on selective media wherein starch bonded to a visible dye is 
directly incorporated.

SEQ ID NO: 1 is the nucleotide sequence represented by the expression 
plasmid of FIG. 1. 
SEQ ID NO:2 is the forward oligonucleotide primer used in the PCR 
amplification of Example 3. 
SEQ ID NO:3 is the reverse oligonucleotide primer used in the PCR 
amplification of Example 3. 
DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
Terms employed throughout this application should be construed with their 
ordinary and common meanings as known to those of ordinary skill in the 
art. Additionally, Applicants desire that the following terms be given the 
following construction: 
The terms "signal sequence", "signal peptide" and "secretory leader" are 
used interchangeably and refer to a short (usually about 15-60 amino 
acids), continuous stretch of amino acids at the amino-terminus of 
secreted and membrane-bound polypeptides, which directs their delivery to 
various locations outside the cytosol. Thus, specific sorting or targeting 
signals, which include signal sequences, may direct the delivery of 
polypeptides into the nucleus, ER, mitochondria, peroxisomes, etc. Signal 
sequences usually contain a hydrophobic core of about 4-15 amino acids, 
which is often immediately preceded by a basic amino acid. At the 
carboxyl-terminal end of the signal peptide there are a pair of small, 
uncharged amino acids separated by a single intervening amino acid that 
defines the signal peptide cleavage site. von Heijne, G. (1990) J. 
Membrane Biol. 115: 195-201. Despite their overall structural and 
functional similarities, native signal peptides do not have a consensus 
sequence. 
The term "amylase" is used to refer to an amylolytic enzyme catalyzing the 
hydrolysis of .alpha.-D-glucosidic linkages of polysaccharides such as 
starch. The term specifically includes endoamylases (a.k.a. 
.alpha.-amylases), which are responsible for endohydrolysis of 
.alpha.-1,4-D-glucosidic bonds at random; exoamylases, which attacks the 
.alpha.-1,4-D-glucosidic linkages only from the non-reducing outer 
polysaccharide ends; .beta.-amylases, which break every alternate 
glucosidic bond to produce maltose; and glucoamylases (a.k.a. 
.gamma.-amylases), which hydrolyze terminal .alpha.-1,4-D-glucosidic 
linkages to produce .beta.-D-glucose, and sometimes also 
.alpha.-1,6-D-glucosidic bonds (Modena et al. (1986), Arch. Bioch. Biophys 
248: 138-150. The source of the amylase enzymes used in the present 
invention can be of any source, including enzymes of mammalian, e.g. 
human, bacterial, fungal, or plant origin, whether purified from natural 
sources, prepared by recombinant DNA technology, chemical synthesis or any 
combination of these and/or other techniques. The term "amylase", unless 
otherwise indicated, collectively refers to all amylases covered by this 
definition. Amylases are commercially available, or can be produced by 
conventional methods well known for those skilled in the art. The most 
commonly known .alpha.-amylases are those isolated from various mammalian 
sources, including, e.g., human saliva, human, mouse (murine), hog and rat 
pancreas, Bacillus subtilis, Bacillus coagulans, Bacillus 
amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, 
various Aspergillus strains (e.g., Aspergillus, awamori), Pseudomonas 
saccharophila, Streptomyces hygroscopicus, Streptomyces venezuelae, 
Streptomyces limosus, Schwanniomyces occidentalis, Saccharomycopsis 
fibuligera, Drosophila melanogaster, Saccharomyces diastaticus and barley 
malt. Saccharomyces cerevisiae also produces an intracellular 
glucoamylase, which can be used with the present invention, but this 
enzyme is only produced by diploid cells undergoing sporulation. The 
nucleotide sequences of the human salivary and pancreatic .alpha.-amylase 
genes have, for example, been disclosed by Nakamura et al. (1984), Gene 
28, 263-270, and Nishide et al. (1986) Gene 50, 371-372. The cloning of 
the .alpha.-amylase cDNA of Aspergillus shirousamii has been reported by 
Shibuya et al. (1992) Biosci. Biotech Biochem. 56, 174-179. The cloning of 
a glucoamylase from the same organism was first described in Shibuya et 
al. (1990) Agric. Biol. Chem., 54, 1905-1914. The cloning of a 
glucoamylase gene of the yeast Arxula adeninivorans from a genomic library 
is reported by Bui et al. (1996), Appl. Microbiol. Biotechnol. 44, 
610-619. Potato .alpha.-amylase genes are disclosed, for example, in U.S. 
Pat. No. 5,498,832, issued Mar. 12, 1996. The genes specifically mentioned 
are for illustration only. A large variety of additional amylase genes 
have been cloned and sequences from various sources, and further genes can 
be readily identified by similar techniques. All amylases contain a short, 
N-terminal signal sequence which directs their secretion. The amylase gene 
used in accordance with the present invention preferably is of mouse 
origin or is from an amylolytic yeast. (see, e.g. McCann and Barnett 
(1986) Yeast 2, 109-115). 
The phrase "non-amylolytic" is used to refer to yeast cells that do not 
produce native amylase or in which the signal sequence(s) of the 
amylase(s) naturally produced has/have been deleted or inactivated, such 
that the native amylase is not secreted. In contrast, "amylolytic" yeast 
are those that can degrade starch due to the presence of one or more 
native secreted amylase enzymes. A typical, and preferred, representative 
of yeast that cannot degrade starch naturally is Saccharomyces cerevisiae 
(Baker's yeast). Many genes from bacteria, filamentous fungi and yeasts 
that encode amylolytic enzymes have been cloned into, and expressed in, S. 
cerevisiae. Representatives of other yeast strains lacking native amylase 
genes and which can be used with the present invention are 
Schizosaccharomyces pombe, Hansenula polymorpha, Kluveromyces lactis and 
Pichia pastoris. Clementi, R. & Rossi, J. (1986) Antonie van Leeuwenhoek 
52; 343-352. 
The phrase "recombinant DNA library" is used to refer collectively to 
genomic and cDNA libraries. Preferably, a "recombinant DNA library" 
contains a substantially complete representation of all genomic or cDNA 
sequences from a particular cell or tissue source. 
The term "DNA" is used to refer collectively to genomic DNA and cDNA, 
prepared from any source, including bacteria, plant cells, and mammalian 
cells, preferably cells of high primates, most preferably humans. 
The term "plate" is used to refer to petri dishes or 96-well micro titer 
dishes filled with solid medium used to grow separated bacterial colonies 
or plaques. 
The terms "plating" or "plating out" refer to the placement of bacteria, 
phages or yeast on plates so that colonies or plaques are formed. 
"Transformation" means introducing DNA into an organism so that the DNA is 
replicable, either as an extrachromosomal element or chromosomal 
integrant. Transformation is usually performed by the CaCl.sub.2 
transfection (Mandel and Higa, J. Mol. Biol. 53, 159-162 (1970)), 
electroporation (Miller et al., Proc. Natl. Acad. Sci. USA 85, 856-860 
(1988), Shigekawa and Dower, BioTechnique 6, 742-751 (1988)), Ausubel et 
al., Current Protocals in Molecular Biology, Unit 9.3, John Wiley & Sons, 
Inc. (1995). DEAE-dextran technique (eukaryotic cells, Lopata et al., 
Nucleic Acids Res. 12(14):5707-5717 (1984)), and liposome-mediated 
transfection (Felgner et al., Proc. Natl. Acad. Sci. USA 84, 7413-7417 
(1987)). Unless otherwise provided, the method used herein for 
transformation of E. coli is electroporation. 
"Polymerase Chain Reaction" or "PCR" is a rapid procedure for in vitro 
enzymatic amplification of a specific DNA segment. The DNA to be amplified 
is denatured by heating the sample. In the presence of DNA polymerase and 
excess deoxynucleotide triphosphates, oligonucleotides that hybridize 
specifically to the target sequence prime new DNA synthesis. One round of 
synthesis results in new strands of indeterminate length which, like the 
parental strands, can hybridize to the primers upon denaturation and 
annealing. The second cycle of denaturation, annealing and synthesis 
produces two single-stranded products that together compose a discrete 
double-stranded product, exactly the length between the primer ends. This 
discrete product accumulates exponentially with each successive round of 
amplification. Over the course of about 20 to 30 cycles, many million-fold 
amplification of the discrete fragment can be achieved. PCR protocols are 
well known in the art, and are described in standard laboratory textbooks, 
e.g. Ausubel et al., supra, Unit 15. 
"Reporter molecule" is a gene which codes for a protein which possess 
unique enzymatic ability or is otherwise easily distinguishable from the 
mixture of intra- or extracellular proteins. Typically, they are operably 
linked to test DNA, the transcriptional capability of which can then be 
estimated from the in vitro activity of the reporter gene product in the 
culture medium. Reporter genes and their application to mammalian gene 
transcription are described by Alam and Cook, (1990), Anal. Biochem. 188: 
245-254. In the present invention, the reporter molecule is a starch 
degrading enzyme, which is preferably amylase. 
Detailed Description 
Starch is one of the most widely distributed, naturally occurring organic 
compound which is derived mainly from higher plants. Soloman, B. (1978) 
Advances in Biochemical Engineering, eds., Springer Berlin Heidelberg, New 
York, 10: 135-177. Starch comprises two major components: (1) amylose, 
which comprises mainly .alpha.-1,4-linked D-glucose residues; and (2) 
amylopectin, which comprises both .alpha.-1,4- and .alpha.-1,6-linked 
D-glucose residues. Jensen B. F. and Norman B. (1984), Process Biochem. 
19: 129-134. The relative content of amylose and amylopectin in starch 
varies with the source. Amylose generally accounts for 20%-30% of the 
starch weight and has an average chain length of 500-1000 glucose units. 
Manners, D. J. (1989), Carbohydr. Pol. 11: 87-112. Amylopectin represents 
the major fraction of starch (70%-80%) and usually has a bimodal pattern 
of distribution, with shorter chains having average lengths of 11-25 and 
the longer chains 40-60 D-glucosyl residues, Soloman, supra. The methods 
of the invention work as a means of screening because starch can be broken 
down into its various component parts by the operation of amylase, and the 
presence of starch (or lack thereof) can be made readily discernable to 
the naked eye. 
The methods of the present invention are preferably used for screening 
recombinant DNA libraries for the presence of novel secreted or 
membrane-bound proteins. 
The construction of a genomic DNA library typically includes the following 
steps: (1) isolation of genomic DNA, (2) partial or complete digestion of 
the DNA, and (3) size fractionation. The DNA is then ligated to a vector, 
and introduced into a host cell, e.g. E. coli (by transformation with a 
plasmid vector or by in vitro packaging into bacteriophage particles and 
subsequent infection of E. coli). The latter steps are substantially the 
same for genomic and cDNA libraries. The size of a library of random 
genomic DNA fragments that is required to ensure representation of all 
sequences present in the genome will depend on the size of the genome and 
the size of the cloned fragments (see, Clarke, Cell 9, 91-99 (1976)). 
There are a number of different procedures for the preparation of genomic 
DNA, all of which start with some form of cell lysis, followed by 
deproteinization and recovery of the DNA. Typical protocols for the 
preparation of genomic DNA from mammalian, plant tissues and bacteria are 
described, e.g. in Ausubel et al., supra, Units 2.2-2.4. Digestion of the 
genomic DNA is performed by restriction enzymes, following routine 
procedures of partial or complete digestion. In order to avoid 
distortions, it is important to select an enzyme that cuts the DNA with 
high frequency but without any bias in selection of one site over another. 
A partial digestion method for the maximization of the randomness of DNA 
sequence in genomic libraries is described, for example, in Seed et al., 
Gene 19, 201-209 (1982). Protocols for enzymatic manipulation of DNA are 
disclosed in Ausubel et al., supra, Unit 3. The completely or partially 
digested DNA must then be size fractionated to remove small and large 
fragments, which would interfere with subsequent cloning. Methods for size 
fractionation are well known in the art and are typically based on sucrose 
gradient fractionation or preparative gel electrophoresis. The DNA is then 
ligated into a vector, which is introduced into a host cell, typically E. 
coli. General techniques for the construction of genomic DNA libraries are 
disclosed, for example, in Ausubel et al., supra, especially in Units 
5.1.1-5.1.2; 5.3.2-5.3.6; 5.4.1-5.4.3; and 5.7.1-5.7.3. Introduction of 
the library into E. coli can be performed by any standard transformation 
techniques, including CaCl.sub.2 transfection, and electroporation. 
In a typical procedure of constructing recombinant cDNA libraries, 
poly(A).sup.+ mRNAs are isolated from cells, preferably a cell type in 
which the mRNA encoding the desired polypeptide is produced in large 
quantities. The mRNAs are then converted into double stranded cDNA 
(dscDNA) in vitro using the enzyme reverse transcriptase to synthesize 
complementary cDNA strands from the mRNA template. In order to obtain 
double-stranded DNA suitable for ligation into a vector, the dscDNA copy 
of the mRNA is methylated and equipped with suitable (usually EcoRI) 
linkers. Methods for methylation of DNA are well known in the art, and 
involve the use of commercially available methylases which covalently join 
methyl groups to adenine or cytosine residues within specific target 
sequences. For example, EcoRI methylates an adenine residue within the 
EcoRI recognition sequence. In the process of converting mRNA into double 
stranded cDNA in vitro, a first cDNA strand is synthesized by the reverse 
transcriptase and separated from the mRNA by treatment with alkali or 
using a nuclease such as the enzyme RNase H. Conveniently, this step can 
be achieved using a reverse transcriptase that also has RNase H activity. 
E. coli DNA polymerase then uses the first cDNA strand as a template for 
the synthesis of the second cDNA strand, thereby producing a population of 
dscDNA molecules from the original poly(A).sup.+ mRNA. After converting 
the 5' and 3' ends into blunt ends, the dscDNA can be ligated to 
linkers/adaptors and subsequently ligated into suitable vectors and 
transformed or packaged into a cell, thereby forming the library. For 
methods for preparing high-quality cDNA libraries see, for example, Gubler 
and Hoffman, Gene 25, 263-269 (1983); Okayama and Berg, Mol. Cell. Biol. 
2, 161-170 (1982); and Kato et al., Gene 150, 243-250 (1994). Typical 
protocols for making cDNA libraries are also described in Ausubel et al., 
supra, especially in Units 5.2.1; 5.5.2-5.5.7; 5.6.1-5.6.8; and 
5.8.1-5.8.11. A particularly advantageous method for converting mRNA into 
dscDNA is disclosed in copending patent application Ser. No. 08/872,861 
filed Oct. 15, 1996. According to this method, reverse 
transcriptase-producing cells are transformed with vectors in which the 5' 
end of a mRNA molecule having a 5' oligonucleotide cap is ligated to a 
single-stranded 5' overhang complementary to the oligonucleotide cap, and 
the 3' end of the mRNA molecule is ligated to a single-stranded 3' 
overhang complementary to the 3' end of the mRNA molecule, so that the 
reverse transcriptase produced by the cell converts the mRNAs into dscDNAs 
to form a cDNA library. 
In the preferred embodiment of carrying out the present invention, a 
library is created which is enriched in signal sequences. This library is 
enriched in amino terminal signal sequences which are within a cloning 
vector that possesses both a unique restriction site at the 5' end of the 
inserted cDNA clone and a DNA promotor 5' to the inserted cDNA. Next, the 
cDNA clone is transcribed using the corresponding RNA polymerase to create 
an RNA transcript which contains the sequence of the transcribed cDNA in 
addition to vector sequence containing the 5' unique restriction sequence. 
For example, an Sp6 promotor can be used in conjunction with Sp6 
polymerase or a T7 promotor with T7 polymerase. Suitable additional 
promoters and RNA polymerases will be apparent to one of ordinary skill in 
the art. The RNA sequence is then randomly primed and replicated to 
produce various single stranded DNA fragments. These fragments are in turn 
replicated into double stranded fragments and specific DNA adapters are 
ligated onto the ends of the DNA fragments. The adapters are used in order 
to convert the blunt end of the replicated terminus into an exposed 5' 
end, similarly to what would result if a restriction enzyme had been used. 
Exposed 5' ends are necessary to maximize the efficiency of T4 DNA ligase, 
an essential step for insertion of the cDNA fragment into the cloning 
vector. The ligated double stranded fragments are then digested with 
specificity for cutting at the unique restriction site. The DNA fragments 
within a defined size range may then be isolated by gel electrophoresis 
and cloned into restriction sites within the amylase vector that are 
compatible with the specific 5' unique restriction site and the DNA 
adapter. In this manner it is possible to identify only those DNA 
fragments which correspond to the 5' end of the initial cDNA within the 
full length library. These fragments may then be used in the next step, 
which is the creation of a cDNA fusion library. 
The use of the above-described enriched signal sequence library offers 
several advantages over the prior art. First, this library ensures that 
all cDNA fragments which are screened in the amylase assay as containing 
signal sequences will be derived from specific cDNA present in the actual 
full-length library. If the full-length library were created completely 
independently from the "amylase library" or the cDNA fragments actually 
tested in the screen, there would be some novel genes identified through 
chance which were not actually represented in the retained full-length 
library. As a result, time and effort would be spent not only searching 
for a clone which doesn't exist in the full-length library, but also in 
searching for a full-length library which actually does contain the gene 
of interest. 
Additionally, the enriched signal sequence library, which contains a 
population of mammalian cDNA which is enriched in fragments proximal to 
the 5' end of the cDNA insertion, provides for a greater number of 
functional signal sequences over that which would be obtained if the 
tested population were comprised of random cDNA derived from the full 
length cDNA. The number of selected functional signal sequences is 
increased because, any signal sequence, if present in the full-length 
library, will be proximal to the 5' end of the cDNA insertion. 
The next step is the creation of a cDNA-fusion library, in which the 
library is fused N-terminally to an amylase gene lacking a functional 
signal sequence. The fusion library is created in any suitable yeast 
cloning vector known in the art, which carries a non-secreted amylase gene 
having its signal sequence deleted or inactivated. Preferably, the vector 
is derived from a .lambda. phage or a filamentous phage. Vectors derived 
from a filamentous phage (phagemid vectors) contain an M13, f1 or fd 
origin of replication. Filamentous phages are very useful cloning vectors 
because they are suitable for packaging of any length of DNA, and permit 
the isolation of DNA in either single- or double-stranded form. Prototypes 
of filamentous phage vectors are M13 mp derivatives, that were originally 
developed by Messig and colleagues. M13-based cloning vectors, along with 
general techniques for working with them are disclosed, for example, in 
Messing, J. Methods Enzymol. 101, 20-78 (1983); and Messing et al., Proc. 
Natl. Acad. Sci. USA 74, 3642-3646 (1977). Particularly useful M13-based 
vectors are M13mp18 and pUC19 vectors (Xanisch Perron et al., Gene 33, 
103-119 (1985)), pRK5-based vectors (EP 307,247 published Mar. 15, 1989), 
pUC118 (Viera and Messing, Methods Enzymol. 153, 3-11 (1987)), pBluescribe 
(Stratagene), and pBluescript (Stratagene). Other phagemid vectors may 
contain the origins of replication of other phages, such as F1. Such 
vectors usually also contain a pBR322 origins of replication, a drug 
resistance coding gene, and a polylinker inserted in frame into the 
portion of the lacZ gene coding for the alpha peptide. (See, e.g. Ausubel 
et al., Current Protocols in Molecular Biology, John Wiley and Sons, publ. 
(1991), Unit 1.15.7.) Derivatives of the phage .lambda. that are used as 
cloning vectors typically contain restriction sites that flank some or all 
of the dispensable genes in about the middle third of the .lambda. genome. 
DNA can be inserted and packaged into such phages in vitro. .lambda. phage 
cloning vectors are well known in the art, and are disclosed, for example, 
in Ausubel et al., supra, Units 1.10-1.11. Some representative .lambda. 
vectors include .lambda.RK18 (Klein et al., supra); .lambda. Max1 
(Clontech); .lambda. EMBL3, .lambda.2001, .lambda.gt10, .lambda.gt11, 
Charon 4a, Charon 40, .lambda.ZAP (e.g. .lambda.ZAP/R, .lambda.ZAP/L) the 
latter most 7 of which are disclosed in Ausubel et al., supra. 
.lambda.-based vectors allow for efficient cloning of large numbers of 
cDNAs, and subsequent conversion to a plasmid library (for example in 
suitable bacterial cells, e.g. E. coli) that can be introduced into yeast. 
Yeast cells may be transformed with the plasmid library obtained by any 
technique known in the art. For example, transformation may be performed 
using lithium acetate (LiAc) in TE buffer, essentially as described in 
Gietz et al., Nucl. Acid. Res. 20(6), 1425 (1992). Alternatively, 
efficient transformation may be achieved by using the spheroplast 
transformation procedure, described, for example, in Ausubel et al., 
supra, Unit 13.7.3. Preferably, transformation is done by electroporation, 
as described previously. While a Saccharomyces cerevisiae strain is 
preferably employed with the present invention, the practice and scope of 
this invention should not be construed as being limited exclusively to 
this species. Any yeast organism which natively lacks the ability to 
degrade starch (i.e is non-amylolytic) and which is capable of being 
transformed by the above listed techniques so as to be rendered capable of 
starch degradation is useable with this invention. For example, 
Schizosaccharomyces pombe, Hansenula polymorpha, Kluveromyces lactis and 
Pichia pastoris. Clementi, R. & Rossi, J. (1986) Antonie van Leeuwenhoek 
52; 343-352. 
Alternatively, yeast strains which naturally do produce starch degrading 
enzymes (i.e are amylolytic) may be employed with the present invention, 
provided that the natural signal sequence for such starch degrading enzyme 
has been inactivated. The inactivation or "knock-out" may be accomplished 
by any known technique commonly employed in the art, e.g. site-directed 
mutagenesis. Additional techniques of inactivation are described in 
Ausubel, Chapter 8, supra. A suitable technique typically employs 
inactivating the entire gene by removing a large portion of it and 
replacing the deletion with a selectable marker (e.g. URA3, LEU2, HIS3). 
This inactive gene-marker fused hybrid can then be used to replace the 
functional, chromosomal copy of the gene within the cell by homologous 
recombination following transformation. Rothstein, R. (1991) Methods 
Enzymol. 194; 281-301. Suitable amylolytic yeast strains which can be 
employed with this technique are described above under the definition of 
amylase. 
After transformation, the yeast colonies are grown on selective media in 
order to detect the desired transformants. Detection may be effected by 
any technique commonly employed in the art. For example, (1) replica 
plating from the growth medium onto a YEPD-starch medium, (2) growth on a 
selective medium wherein starch was also incorporated, and (3) growth on a 
selective medium wherein starch is covalently attached to a readily 
identifiable dye. 
Applicants have discovered that the replica plating step can be preferably 
deleted by incorporation of starch directly into the selective growth 
media. The elimination of the replica plating step saves considerable time 
and effort, since colonies typically take 7-10 days to grow in the 
invertase screen following replica plating. The starch concentration can 
also be varied in order to adjust the sensitivity of the detection screen. 
That is, lower starch concentrations would be expected to detect less 
"functional" or more weakly secreted signal sequences. Contrarily, 
increasing the starch concentrations lowers the sensitivity to eliminate 
false positives or "noise" within the detection system. Preferably, starch 
concentrations are varied from 0.5% to 2.0%. 
The yeast colonies may be stained by any technique known in the art. For 
example, by exposure to iodine vapor, which can be accomplished by 
inverting the agar plate over iodine crystals for a time sufficient to 
visibly stain the starch in the plate media, e.g. 2-5 minutes. The iodine 
will form a blue-black staining complex with the starch present in the 
media, but not with the amylase break down products. In the preferred 
embodiment, the starch is bonded to a visible dye. When amylase is 
secreted by the individual colonies, a clear halo is readily identifiable 
without further manipulation. Any dye suitable for attachment to starch 
can be used, as is known in the art. The dye-starch bonding technique and 
useable dyes preferably employed in the invention are as identified in 
Biely et al. Anal. Biochem. 172, 176-179 (1988). 
Once amylase secreting colonies have been identified, they can be 
restreaked across fresh selective media in order to obtain well defined 
single colonies. The restreaking process also ensures plasmid maintenance 
amongst the yeast transformants. In the preferred embodiment, which 
incorporates the starch directly into the selective media, we have 
discovered that the time for the restreaking process is diminished 
vis-a-vis the invertase process, e.g. 3 days v. 5-7 days. 
The restreaked, transformed colonies can then be analyzed by any technique 
known in the art to detect novel DNA sequences. For example, isolation and 
purification of the DNA, then comparison to known sequences or libraries 
via hydribization techniques as is known. Alternatively, and preferably, 
the DNA present in the yeast colonies can be directly amplified by 
Polymerase Chain Reaction or PCR. The PCR oligos are designed to start and 
stop amplification of the amylase vector on either side of the inserted 
cDNA. The exact sequences will vary depending upon the type of amylase 
yeast expression vector employed. Preferably, the plasmid employed is 
pSST-amy.1 and the PCR oligos are sequences which anneal to the ADH 
promotor region and the amylase-encoding gene of the vector as recited in 
Example 3. However, other suitable amylase expressing vectors and marker 
annealing PCR oligos will be readily determinable to those of ordinary 
skill in the art. Amplified DNA sequences are then further isolated and 
analyzed by known and available techniques, such as column purification, 
gel electroporation and/or DNA sequencing. The cDNA may then be compared 
to known sequences and novelty ascertained. 
The following examples are offered by way of illustration and not by way of 
limitation and should not be construed as limiting the invention in any 
way. The disclosures of all citations in the specification are expressly 
incorporated herein by reference. 
EXAMPLES 
Example I 
Construction of Full-Length cDNA Library 
Isolation of m-RNA 
Human fetal liver mRNA was obtained from Clontech Laboratories, Inc. Palo 
Alto, Calif. U.S.A., catalog no. 64018-1. 
The following protocol is described in "Instruction Manual: 
Superscript.RTM. Lamda System for cDNA Synthesis and .lambda. cloning," 
cat. No. 19643-014, Life Technologies, Gaithersburg, Md., U.S.A. which is 
herein incorporated by reference. Unless otherwise noted, all reagents 
were also obtained from Life Technologies. The overall procedure can be 
summarized into the following steps: (1) First strand synthesis; (2) 
Second strand synthesis; (3) Adaptor addition; (4) Enzymatic digestion; 
(5) Gel isolation of cDNA; (6) Ligation into vector; and (7) 
Transformation. 
First Strand Synthesis 
Not1 primer-adapter (Life Tech., 2 .mu.l, 0.5 .mu.g/.mu.l) was added to a 
sterile 1.5 ml microcentrifuge tube to which was added poly A+ mRNA (7 
.mu.l, 5 .mu.g). The reaction tube was heated to 70.degree. C. for 5 
minutes or time sufficient to denature the secondary structure of the 
mRNA. The reaction was then chilled on ice and 5.times. First strand 
buffer (Life Tech., 4 .mu.l), 0.1 M DTT (2 .mu.l) and 10 mM dNTP Mix (Life 
Tech., 1 .mu.l) were added and then heated to 37.degree. C. for 2 minutes 
to equilibrate the temperature. Superscript II.RTM. reverse transcriptase 
(Life Tech., 5 .mu.l) was then added, the reaction tube mixed well and 
incubated at 37.degree. C. for 1 hour, and terminated by placement on ice. 
The final concentration of the reactants was the following: 50 mM Tris-HCl 
(pH 8.3); 75 mM KCl; 3 mM MgCl.sub.2 ; 10 mM DTT; 500 .mu.M each dATP, 
dCTP, dGTP and dTTP; 50 .mu.g/ml Not 1 primer-adapter; 5 .mu.g (250 
.mu.g/ml) mRNA; 50,000 U/ml Superscript II.RTM. reverse transcriptase. 
Second Strand Synthesis 
While on ice, the following reagents were added to the reaction tube from 
the first strand synthesis, the reaction well mixed and allowed to react 
at 16.degree. C. for 2 hours, taking care not to allow the temperature to 
go above 16.degree. C.: distilled water (93 .mu.l); 5.times. Second strand 
buffer (30 .mu.l); dNTP mix (3 .mu.l); 10 U/.mu.l E. Coli DNA ligase (1 
.mu.l); 10 U/.mu.l E. Coli DNA polymerase I (4 .mu.l ); 2 U/.mu.l E. Coli 
RNase H (1 .mu.l). 10 U T4 DNA Polymerase (2 .mu.l) was added and the 
reaction continued to incubate at 16.degree. C. for another 5 minutes. The 
final concentration of the reaction was the following: 25 mM Tris-HCl (pH 
7.5); 100 mM KCl; 5 mM MgCl.sub.2 ; 10 mM (NH.sub.4).sub.2 SO.sub.4 ; 0.15 
mM .beta.-NAD+; 250 .mu.M each dATP, dCTP, dGTP, dTTP; 1.2 mM DTT; 65 U/ml 
DNA ligase; 250 U/ml DNA polymerase I; 13 U/ml Rnase H. The reaction has 
halted by placement on ice and by addition of 0.5 M EDTA (10 .mu.l), then 
extracted through phenol:chloroform:isoamyl alcohol (25:24:1, 150 .mu.l). 
The aqueous phase was removed, collected and diluted into 5M NaCl (15 
.mu.l) and absolute ethanol (-20.degree. C., 400 .mu.l) and centrifuged 
for 2 minutes at 14,000.times.g. The supernatant was carefully removed 
from the resulting DNA pellet, the pellet resuspended in 70% ethanol (0.5 
ml) and centrigued again for 2 minutes at 14,000.times.g. The supernatant 
was again removed and the pellet dried in a speedvac. 
Adapter Addition 
The following reagents were added to the cDNA pellet from the Second strand 
synthesis above, and the reaction was gently mixed and incubated at 
16.degree. C. for 16 hours: distilled water (25 .mu.l); 5.times. T4 DNA 
ligase buffer (10 .mu.l); Sal I adapters (10 .mu.l); T4 DNA ligase (5 
.mu.l). The final composition of the reaction was the following: 50 mM 
Tris-HCl (pH 7.6); 10 mM MgCl.sub.2 ; 1 mM ATP; 5% (w/v) PEG 8000; 1 mM 
DTT; 200 .mu.g/ml Sal 1 adapters; 100 U/ml T4 DNA ligase. The reaction was 
extracted through phenol:chloroform:isoamyl alcohol (25:24:1, 50 .mu.l), 
the aqueous phase removed, collected and diluted into 5M NaCl (8 .mu.l) 
and absolute ethanol (-20.degree. C., 250 .mu.l). This was then 
centrifuged for 20 minutes at 14,000.times.g, the supernatant removed and 
the pellet was resuspended in 0.5 ml 70% ethanol, and centrifuged again 
for 2 minutes at 14,000.times.g. Subsequently, the supernatant was removed 
and the resulting pellet dried in a speedvac and carried on into the next 
procedure. 
Enzymatic Digestion 
To the cDNA prepared with the Sal 1 adapter from the previous paragraph was 
added the following reagents and the mixture was incubated at 37.degree. 
C. for 2 hours: DEPC-treated water (41 .mu.l); Not 1 restriction buffer 
(REACT, Life Tech., 5 .mu.l), Not 1 (4 .mu.l). The final composition of 
this reaction was the following: 50 mM Tris-HCl (pH 8.0); 10 mM MgCl.sub.2 
; 100 mM NaCl; 1,200 U/ml Not 1. 
Gel Isolation of cDNA 
The cDNA is size fractionated by acrylamide gel electrophoresis on a 5% 
acrylamide gel, and any fragments which were larger than 1 Kb, as 
determined by comparison with a molecular weight marker, were excised from 
the gel. The cDNA was then electroeluted from the gel into 0.1.times.TBE 
buffer (200 .mu.l) and extracted with phenol:chloroform:isoamyl alcohol 
(25:24:1, 200 .mu.l). The aqueous phase was removed, collected and 
centrifuged for 20 minutes at 14,000.times.g. The supernatant was removed 
from the DNA pellet which was resuspended in 70% ethanol (0.5 ml) and 
centrifuged again for 2 minutes at 14,000.times.g. The supernatant was 
again discarded, the pellet dried in a speedvac and resuspended in 
distilled water (15 .mu.l). 
Ligation of cDNA Into pRK5 Vector 
The following reagents were added together and incubated at 16.degree. C 
for 16 hours: 5.times. T4 ligase buffer (3 .mu.l); pRK5, Xhol, Not1 
digested vector, 0.5 .mu.g, 1 .mu.l); cDNA prepared from previous 
paragraph (5 .mu.l) and distilled water (6 .mu.l). Subsequently, 
additional distilled water (70 .mu.l) and 10 mg/ml tRNA (0.1 .mu.l) were 
added and the entire reaction was extracted through 
phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous phase was 
removed, collected and diluted into 5M NaCl (10 .mu.l) and absolute 
ethanol (-20.degree. C., 250 .mu.l). This was then centrifuged for 20 
minutes at 14,000.times.g, decanted, and the pellet resuspended into 70% 
ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000.times.g. 
The DNA pellet was then dried in a speedvac and eluted into distilled 
water (3 .mu.l) for use in the subsequent procedure. 
Transformation of Library Ligation Into Bacteria 
The ligated cDNA/pRK5 vector DNA prepared previously was chilled on ice to 
which was added electrocompetent DH10B bacteria (Life Tech., 20 .mu.l). 
The bacteria vector mixture was then electroporated as per the 
manufacturers recommendation. Subsequently SOC media (1 ml) was added and 
the mixture was incubated at 37.degree. C. for 30 minutes. The 
transformants were then plated onto 20 standard 150 mm LB plates 
containing ampicillin and incubated for 16 hours (370.degree. C.) to allow 
the colonies to grow. Positive colonies were then scraped off and the DNA 
isolated from the bacterial pellet using standard CsCl-gradient protocols. 
For example, Ausuble et al., 2.3.1. 
Example 2 
Construction of Enriched 5'-cDNA Library 
The following process results in a bias of cDNA fragments which 
preferentially represents the 5' ends of those cDNA's contained within the 
previously prepared full length library of Example 1. 
Linearize the Full-Length Library 
10 .mu.g of the pooled isolated full-length library plasmid DNA (41 .mu.l) 
of Example 1 was combined with Not 1 restriction buffer (New England 
Biolabs, 5 .mu.l) and Not 1 (New England Biolabs, 4 .mu.l) and incubated 
at 37.degree. C. for one hour. The reaction was extracted through 
phenol:chloroform:isoamyl alcohol (25:24:1, 50 .mu.l), the aqueous phase 
removed, collected and resuspended into 5M NaCl (5 .mu.l) and absolute 
ethanol (-20.degree. C., 150 .mu.l). This was then centrifuged for 20 
minutes at 14,000.times.g, decanted, resuspended into 70% ethanol (0.5 ml) 
and centrifuged again for 2 minutes at 14,000.times.g. The supernatant was 
then removed, the pellet dried in a speedvac and resuspended in distilled 
water (10 .mu.l). 
Sp6 Transcripts Synthesis 
The following protocol was taken from InvitroScript Cap Kit (Invitrogen). 
The following reagents were brought together and incubated at 37.degree. 
C. for 2 hours: distilled water (3 .mu.l); linearized DNA library 
(prepared previously, 1 .mu.g, 1 .mu.l); Ribonucleotide mix (Invitrogen, 
10 .mu.l); transcription buffer (Invitrogen, 2 .mu.l) and Sp6 enzyme mix. 
The reaction was then extracted through phenol:chloroform:isoamyl alcohol 
(25:24:1, 50 .mu.l) and the aqueous phase was removed, collected and 
resuspended into 5M NaCl (5 .mu.l) and absolute ethanol (-20.degree. C., 
150 .mu.l) and centrifuged for 20 minutes at 14,000.times.g. The pellet 
was then decanted and resuspended in 70% ethanol (0.5 ml), centrifuged 
again for 2 minutes at 14,000.times.g, decanted, dried in a speedvac and 
resuspended into distilled water (10 .mu.l). 
cDNA Production From Sp6 RNA Overview 
The procedure employed was the same as that used to create the full-length 
cDNA library described in Example 1, except with the following noted 
differences: 
First strand synthesis: 6 Nucleotide oligomer random primers were used to 
prime the first strand cDNA synthesis from the Sp6 RNA transcript in order 
to create random fragments of cDNA instead of priming from the poly A tail 
to create a long full-length cDNA. 
Adapter ligation: A Sal I adapter (Life Tech.) was alternatively 
substituted in place of the Not 1 adapter. 
Restriction enzyme digestion: Cla 1 or alternatively Sfi 1 were used in 
place of Not 1. This cuts at a restriction site 5' to the inserted cDNA 
within the full length library. As a result, only those fragments which 
correspond to the 5' ends of cDNA present in the full length library were 
identified. 
Gel isolation: Fragments of cDNA corresponding in size to 500-1000 base 
pairs were isolated. This size range was selected because it is believed 
that significantly shorter fragments may generate greater numbers of 
fortuitous and spurious signal sequences during the yeast screen. 
First Strand Synthesis 
The following reagents were brought together and heated at 70.degree. C. 
for 5 minutes in order to denature the secondary structure of the mRNA: 
Sp6 transcript prepared previously (5 .mu.g, 7 .mu.l); N6 primer-adapter 
(Life Tech., 2 .mu.l). The reaction was halted by placement on ice and 
First strand buffer (Life Tech., 4 .mu.l), 0.1 M DTT (2 .mu.l) and dNTP 
mix (1 .mu.l, 10 mM each dNTP) were added and then heated to 37.degree. C. 
for 2 minutes to equilibrate the temperature. Superscript II.RTM. reverse 
transcriptase (Life Tech., 5 .mu.l) was then added and the reaction 
incubated at 37.degree. C. for 1 hour, then terminated by placement on 
ice. 
Second Strand Synthesis 
While on ice, the following reagents were added to the reaction tube from 
the first strand synthesis and then reacted at 16.degree. C. for 2 hours: 
distilled water (93 .mu.l); Second strand buffer (Life Tech., 30 .mu.l); 
dNTP mix (3 .mu.l); 10 U/.mu.l E. Coli DNA ligase(1 .mu.l); 10 U/.mu.l E. 
Coli DNA polymerase I (4 .mu.l); 2 U/.mu.l E. coli RNase H (1 .mu.l). 10 U 
T4 DNA Polymerase (Life Tech. added and the reaction continued to incubate 
at 16.degree. C. for another 5 minutes. The reaction was halt addition of 
0.5 M EDTA (10 .mu.l) and extracted through phenol:chloroform:isoamyl 
alcohol (25:24:1). The aqueous phase was removed, collected then diluted 
into 5M NaCl (15 .mu.l) and absolute ethanol (-20.degree. C., 400 .mu.l) 
and centrifuged for 20 minutes at 14,000.times.g. The DNA pellet was then 
decanted and resuspended in 70% ethanol (0.5 ml) and centrifuged again for 
2 minutes at 14,000.times.g. The supernatant was then removed and the 
pellet dried in a speedvac. 
Adapter Addition 
The following reagents were added to the DNA pellet obtained from the 
Second strand synthesis and reaction was incubated for 16 hours at 
16.degree. C.: distilled water (25 .mu.l); 5.times. T4 DNA ligase buffer 
(Life Tech., 10 .mu.l); Sal 1 adapters (10 .mu.l); T4 DNA ligase (5 
.mu.l). The reaction was extracted though phenol:chloroform:isoamyl 
alcohol (25:24:1, 50 .mu.l) and the aqueous phase was removed, collected 
and diluted into 5M NaCl (8 .mu.l) and absolute ethanol (-20.degree. C., 
250 .mu.l), then centrifuged for 20 minutes at 14,000.times.g. The DNA 
pellet was decanted and suspended into 70% ethanol (0.5 ml) and 
centrifuged again for 2 minutes at 14,000.times.g. The supernatant was 
removed and the residue pellet dried in a speedvac. 
Enzymatic Digestion 
The following reagents were added to the DNA pellet obtained from the 
adapter addition reaction and the reaction was incubated at 37.degree. C. 
for one hour: distilled water (41 .mu.l); Cla 1 restriction buffer (New 
England Biolabs, 5 .mu.l) and Cla 1 (New England Biolabs, 4 .mu.l). 
Gel Isolation of DNA 
The digested DNA from the digestion procedure above was size fractionated 
by acrylamide gel electrophoresis on a 5% acrylamide gel. cDNA fragments 
greater than 500-1000 base pairs in size, as determined by comparison with 
known molecular weight markers, were excised from the gel. The cDNA was 
electroeluted from the acrylamide gel into 200 .mu.l of 0.1.times.TBE 
buffer and extracted with phenol:chloroform:isoamyl alcohol (25:24:1, 200 
.mu.l). The aqueous phase was removed, collected and diluted by 5M NaCl 
(20 .mu.l) and absolute ethanol (-20.degree. C., 600 .mu.l). This was 
centrifuged for 20 minutes at 14,000.times.g, decanted, resuspended in 70% 
ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000.times.g. 
The supernatant was removed, the pellet dried in a speedvac and 
resuspended into distilled water (15 .mu.l). 
Ligation Into pSST-amy.1 Vector 
The following reagents were added together and incubated at 16.degree. C. 
for 16 hours: 5.times. T4 ligase buffer (Life Tech., 3 .mu.l); pRK5 
Cla-Sal digested vector, 0.5 .mu.g, 1 .mu.l); cDNA prepared from the 
digestion (5 .mu.l); distilled water (6 .mu.l). Subsequently, additional 
distilled water (70 .mu.l) and 10 mg/ml tRNA (0.1 .mu.l) was added and the 
entire reaction was extracted through phenol:chloroform:isoamyl alcohol 
(25:24:1, 100 .mu.l). The aqueous phase was removed, collected and diluted 
by 5M NaCl (10 .mu.l) and absolute ethanol (-20.degree. C., 250 .mu.l) and 
centrifuged for 20 minutes at 14,000.times.g. The DNA pellet was decanted, 
resuspended into 70% ethanol (0.5 ml) and centrifuged again for 2 minutes 
at 14,000.times.g. The supernatant was removed and the residue pellet was 
dried in a speedvac and resuspended in distilled water (3 .mu.l). 
Transformation 
The ligated cDNA/pSST-amy.1 vector DNA prepared previously was chilled on 
ice to which was added electrocompetent DH10B bacteria (Life Tech., 20 
.mu.l). The bacteria vector mixture was then electroporated as recommended 
by the manufacturer. Subsequently, SOC media (Life Tech., 1 .mu.l) was 
added and the mixture was incubated at 37.degree. C. for 30 minutes. The 
transformants were then plated onto 20 standard 150 mm LB plates 
containing ampicillin and incubated for 16 hours (370.degree. C.). 
Positive colonies were scraped off the plates and the DNA was isolated 
from the bacterial pellet using standard protocols, e.g. CsCl-gradient, 
Ausubel, Unit 2.3. The purified DNA was then carried on to the yeast 
protocols of Example 3. 
Example 3 
Overview: The yeast methods employed in the present invention were divided 
into three categories: (1) Transformation of yeast with the plasmid/cDNA 
combined vector; (2) Detection and isolation of yeast clones secreting 
amylase; and (3) PCR amplification of the insert directly from the yeast 
colony and purification of the DNA for sequencing and further analysis. 
While any yeast strain containing a stable mutant ura3 is useable with the 
present invention, the preferable yeast strain used with the practice of 
the invention was HD56-5A (ATCC-90785). This strain had the following 
genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, 
MAL.sup.+, SUC.sup.+, GAL.sup.+. Because this strain was MAL.sup.+, that 
is, it could use maltose as a sole carbon energy source, amylase was 
tested for whether it could be used as a selectable marker. However, we 
found insufficient secretion of amylase in combination with maltose 
activity to support colony growth in the absence of glucose. Instead, the 
detection of amylase secretion alone became the selection criteria. 
Transformation: Transformation was performed based on the protocol outlined 
by Gietz, D. et al., Nucl. Acid. Res. 20, 1425 (1992). With this 
procedure, we obtained transformation efficiencies of approximately 
1.times.10.sup.5 transformants per microgram of DNA. Transformed cells 
were then inoculated from agar into YEPD complex media broth (100 ml) and 
grown overnight at 30.degree. C. The YEPD broth was prepared as described 
in Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, 
Cold Spring Harbor, N.Y., U.S.A., p. 207 (1994). The overnight culture was 
then diluted to about 2.times.10.sup.6 cells/ml (approx. OD.sub.600 =0.1) 
into fresh YEPD broth (500 ml) and regrown to 1.times.10.sup.7 cells/ml 
(approx. OD.sub.600 =0.4-0.5). This usually took about 3 hours to 
complete. 
The cells were then harvested and prepared for transformation by transfer 
into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes, 
the supernatant discarded, and then resuspended into sterile water, and 
centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR 
centrifuge. The supernatant was discarded and the cells were subsequently 
washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM 
Li.sub.2 OOCCH.sub.3), and resuspended into LiAc/TE (2.5 ml). 
Transformation took place by mixing the prepared cells (100 .mu.l) with 
freshly denatured single stranded salmon testes DNA (Lofstrand Labs, 
Gaithersburg, Md., U.S.A.) and transforming DNA (1 .mu.g, vol. &lt;10 .mu.l) 
in microfuge tubes. The mixture was mixed briefly by vortexing, then 40% 
PEG/TE (600 .mu.l, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM 
EDTA, 100 mM Li.sub.2 OOCCH.sub.3, pH 7.5) was added. This mixture was 
gently mixed and incubated at 30.degree. C. while agitating for 30 
minutes. The cells were then heat shocked at 42.degree. C. for 15 minutes, 
and the reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 
seconds, decanted and resuspended into TE (500 .mu.l, 10 mM Tris-HCl, 1 mM 
EDTA pH 7.5) followed by recentrifugation. The cells were then diluted 
into TE (1 ml) and aliquots (200 .mu.l) were spread onto the selective 
media previously prepared in 150 mm growth plates (VWR). 
Alternatively, instead of multiple small reactions, the transformation was 
performed using a single, large scale reaction, wherein reagent amounts 
were scaled up accordingly. 
The selective media used was a synthetic complete dextrose agar lacking 
uracil (SCD-Ura) prepared as described in Kaiser et al., Methods in Yeast 
Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., U.S.A., p. 
208-210) (1994). Transformants were grown at 30.degree. C. for 2-3 days. 
Detection of clones containing secreted proteins: The detection of colonies 
secreting amylase was performed by any of the several methods: (1) Replica 
plating onto YEPD-starch agar; (2) Selective media growth including 
starch; (3) Selective media growth including red starch. The invention is 
preferably practiced by the latter of the following three described 
techniques. 
Replica plating: After growth on the SCD-Ura agar was complete, the 
transformants were transferred by replica plating onto YEPD agar 
containing 2.0% (w/v) soluble potato starch (Sigma). Following the replica 
plating step, the colonies were allowed to regrow at 30.degree. C. for 
24-48 hours. Colonies in which amylase was secreted by signal sequences 
were detected by inverting the agar plate containing the colonies over 
exposed iodine crystals for 2-5 minutes. The iodine formed a blue-black 
staining complex with the starch present in the media, but not with the 
amylase break down products. The amylase secreting colonies, and ergo 
secreted proteins or signal sequence containing cDNA's were identified by 
a distinct halo of non-stained agar around the positive colony. 
Starch concentrations in the YEPD agar was varied between 2.0% and 0.5% 
(w/v) and it was discovered that these starch concentrations could 
differentiate cells secreting amylase from those not secreting amylase. 
Selective media growth including starch: Starch was also directly 
incorporated into the SCD-Ura agar at concentrations of 2.0%, 1.0% and 
0.5% (w/v) and it was discovered that amylase secreting positives were 
detected provided that the media was buffered to a final pH of 7.0 with a 
potassium phosphate buffering system (final concentration 50 or 100 mM). 
This effectively eliminated the replica plating step. Buffering was 
necessary because the optimum pH for the mouse pancreatic amylase used in 
this example was 7.0 and the yeast media often is or becomes acidic due to 
the fermentation of glucose by the yeast. 
Selective media growth including red starch: Starch was coupled to the red 
dye (Reactive Red-120, Sigma) as per the procedure described by Biely et 
al, Anal. Biochem. 172, 176-179 (1988). The coupled starch was 
incorporated into the SCD-Ura agar plates at a final concentration of 
0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0 
(50-100 mM final concentration). The use of the red starch eliminated both 
the replica plating step as well as the need to perform any staining--the 
positive colonies were readily distinguishable without any further 
manipulation. 
The positive colonies from any of the above three detection methods were 
picked and streaked across fresh selective media (onto 150 mm plates) in 
order to obtain well isolated and identifiable single colonies. This step 
also ensured maintenance of the plasmid amongst the transformants. Well 
isolated single colonies positive for amylase secretion were detected 
either by replica plating onto YEPD/Starch agar, or by direct 
incorporation of starch or red starch into buffered SCD-Ura agar. Positive 
colonies were determined by their ability to break down starch resulting 
in a clear halo around the positive colony visualized either directly (red 
starch) or following iodine staining of the starch. 
Isolation of DNA by PCR Amplification: When a positive colony was isolated, 
a portion of it was picked by a toothpick and diluted into sterile water 
(30 .mu.l) in a 96 well plate. At this time, the positive colonies were 
either frozen and stored for subsequent analysis or immediately amplified. 
An aliquot of cells (5 .mu.l) was used as a template for the PCR reaction 
in a 25 .mu.l volume containing: 0.5 .mu.l Klentaq (Clontech, Palo Alto, 
Calif.); 4.0 .mu.l 10 mM dNTPs (Perkin Elmer-Cetus); 2.5 .mu.l Kentaq 
buffer (Clontech); 0.25 .mu.l forward oligo 1; 0.25 .mu.l reverse oligo 2; 
12.5 .mu.l distilled water. The sequence of the forward oligonucleotide 1 
was: 
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' [SEQ ID NO: 2] 
The sequence of reverse oligonucleotide 2 was: 
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' [SEQ ID NO: 3] 
PCR was then performed as follows: 
______________________________________ 
a. Denature 92.degree. C., 
5 minutes 
b. 3 cycles of Denature 92.degree. C., 30 seconds 
Anneal 59.degree. C., 30 seconds 
Extend 72.degree. C., 60 seconds 
c. 3 cycles of Denature 92.degree. C., 30 seconds 
Anneal 57.degree. C., 30 seconds 
Extend 72.degree. C., 60 seconds 
d. 25 cycles of Denature 92.degree. C., 30 seconds 
Anneal 55.degree. C., 30 seconds 
Extend 72.degree. C., 60 seconds 
e. Hold 4.degree. C. 
______________________________________ 
The underlined regions of the oligonucleotides annealed to the ADH promoter 
region and the amylase region, respectively, and amplified a 307 bp region 
from vector pSST-AMY1 when no insert was present. Typically, the first 18 
nucleotides of the 5' end of these oligonucleotides contained annealing 
sites for the sequencing primers. Thus, the total product of the PCR 
reaction from an empty vector was 343 bp. However, signal sequence-fused 
cDNA resulted in considerably longer nucleotide sequences. 
Following the PCR, an aliquot of the reaction (5 .mu.l) was examined by 
agarose gel electrophoresis in a 1% agarose using a Tris-Borate-EDTA (TBE) 
buffering system as described by Sambrook, J. et al., "Molecular 
Cloning--A Laboratory Manual" second edition, Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y., U.S.A. Clones resulting in a 
single strong PCR product larger than 400 bp were further analyzed by DNA 
sequencing after purification with a 96 Qiaquick PCR clean-up column 
(Qiagen Inc., Chatsworth, Calif., U.S.A.). 
Using the procedures outlines in Examples 1-3 cDNA signal sequences were 
isolated and identified by known DNA sequencing. The identities and 
frequency of occurrence of each sequence is reported in Table 1. 
TABLE 1 
______________________________________ 
Identified isolated protein from amylase screen of Example 1-3 
PROTEIN Number of Isolates 
Frequency (%) 
______________________________________ 
albumin 75 33.1 
fibrinogen beta 62 27.4 
novel sequences 38 16.8 
.alpha.-fetoprotein 12 5.3 
traG (E. Coli) 6 2.6 
vit. D binding protein 4 1.7 
heparin perenchall growth f. 3 1.3 
archain 3 1.3 
fibrinogen alpha 2 0.8 
fibrinogen rel. protein 2 0.8 
heparin cofactor II 1 0.4 
kininogen 1 0.4 
fibrinogen gamma 1 0.4 
compl. factor h-like 1 0.4 
.alpha.-1 antitrypsin 1 0.4 
aminopeptidase A 1 0.4 
HGF activator-like 1 0.4 
.beta.-2-glycoprotein 1 0.4 
uracil DNA glycoprotein 1 0.4 
eryth. 50 kD glycoprotein 1 0.4 
fragile X homolog 1 0.4 
neurotensin N 1 0.4 
C-reactive protein 1 0.4 
adrenomedullin 1 0.4 
inter-.alpha.-trypsin inhibitor 1 0.4 
mitochondrial COX-1 1 0.4 
GST1 GTP binding protein 1 0.4 
ribosomal protein s23 1 0.4 
______________________________________ 
Example 4 
Using the procedure published in Klein et al. describing the invertase 
selection process, signal sequences were detected, subject to DNA 
sequencing, and their identities and frequency of occurrence are reported 
in Table 2. 
TABLE 2 
______________________________________ 
Identified protein from invertase selection of Example 4 
PROTEIN Number of Isolates 
Frequency (%) 
______________________________________ 
albumin 76 50.6 
.beta.-2-glycoprotein 27 18.0 
fibrinogen beta 14 9.3 
novel 8 5.3 
kininogen 8 5.3 
afamin 2 1.3 
M130 antigen 2 1.3 
heparin parench. growth f. 2 1.3 
heparin cofactor ii 2 1.3 
hemopexin 1 0.6 
complement factor I 1 0.6 
.alpha.-1 anti-trypsin 1 0.6 
apolipoprotein B1 1 0.6 
.alpha.-2-hs-glycoprotein 1 0.6 
fibrinogen gamma 1 0.6 
.alpha.-fetoprotein 1 0.6 
tumor protein p21 1 0.6 
ribosomal protein s15 1 0.6 
______________________________________ 
In comparing Tables 1 & 2, it will be noted that the amylase screen 
identified 315 positives from 1.6.times.10.sup.6 Ura.sup.+ colonies, or a 
frequency of 1 in 5000. Among these, it was possible to sequence and 
identify 226 of these clones. In the invertase selection process, 272 
Suc.sup.+ positives were identified from 8.times.10.sup.6 Ura.sup.+ 
colonies, or a frequency of 1 in 29,000. From the invertase selection 
assay, it was possible to sequence 150 clones. 
As a result, it is clear that the method of the invention as practiced 
above resulted in a system which is about 6-fold more sensitive than the 
prior art method. Moreover, this increased sensitivity occurred with about 
4 fold fewer organisms screened. Furthermore, as is indicated on FIG. 2, 
while the prior art screening method can take up to 21 days, the present 
invention may be completed in as few as 6 days. This translates to over a 
3 fold decrease in time of completion of the screening procedure. 
Moreover, a comparison of Tables 1 and 2 indicates a larger diversity of 
proteins identified with amylase than with invertase. This ultimately 
results in 16% of the sequences obtained from the amylase screen being 
novel rather than the 5% obtained with invertase. The amylase process has 
eliminated the need for a replica plating step. Once the preliminary 
transformations have been performed, the positives can be directly 
identified; there is no need to either replica plate the transformants or 
to scrape and pool them prior to retesting for the invertase phenotype. 
This represents a considerable reduction in the time and resources 
necessary to complete the signal sequence analysis. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 3 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 7633 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: Artificial Sequence 
&lt;222&gt; LOCATION: 1-7633 
&lt;223&gt; OTHER INFORMATION: Amylase yeast expression - #plasmid 
- - &lt;400&gt; SEQUENCE: 1 
- - gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg - # 
50 
- - atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg - # 
100 
- - gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg - # 
150 
- - ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac - # 
200 
- - actcaaccct atctcggtct attcttttga tttataaggg attttgccga - # 
250 
- - tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg - # 
300 
- - aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg - # 
350 
- - cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca - # 
400 
- - gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag - # 
450 
- - ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac - # 
500 
- - gactcactat agggcgaatt gggtaccggg ccccccctcg aggcttagcg - # 
550 
- - atgcttcgtt gcttgcatgc aacttctttt cttttttttt cttttctctc - # 
600 
- - tcccccgttg ttgtctcacc atatccgcaa tgacaaaaaa aatgatggaa - # 
650 
- - gacactaaag gaaaaaatta acgacaaaga cagcaccaac agatgtcgtt - # 
700 
- - gttccagagc tgatgagggg tatcttcgaa cacacgaaac tttttccttc - # 
750 
- - cttcattcac gcacactact ctctaatgag caacggtata cggccttcct - # 
800 
- - tccagttact tgaatttgaa ataaaaaaag tttgccgctt tgctatcaag - # 
850 
- - tataaataga cctgcaatta ttaatctttt gtttcctcgt cattgttctc - # 
900 
- - gttccctttc ttccttgttt ctttttctgc acaatatttc aagctatacc - # 
950 
- - aagcatacaa tcaactccaa gctatcgata ggccacactg gccgtcgacg - # 
1000 
- - cggccgctgg ggtatctctc gagaaaagag aggcccaata tgacccacat - # 
1050 
- - actcaatatg gacgaactgc tattatccac ctgtttgagt ggcgctgggt - # 
1100 
- - tgatattgct aaggaatgtg agagatactt agctcctaat ggatttgcag - # 
1150 
- - gtgtgcaggt ctctccaccc aatgaaaaca tcgtagtcca cagcccttca - # 
1200 
- - agaccatggt gggaaagata tcaaccaatt agctacaaaa tatgttccag - # 
1250 
- - gtctggaaat gaagatgaat tcagggacat ggtgaacagg tgcaacaatg - # 
1300 
- - ttggtgtccg tatttatgtg gatgctgtca ttaaccacat gtgtggagtg - # 
1350 
- - ggggctcaag ctggacaaag cagtacatgt ggaagttatt tcaacccaaa - # 
1400 
- - taacagggac tttcctggag ttccctattc tggttttgac tttaatgatg - # 
1450 
- - gaaaatgtag aactgcaagt ggaggtatcg agaactacca agatgctgct - # 
1500 
- - caggtcagag attgtcgtct gtctggcctt ctggatcttg cacttgagaa - # 
1550 
- - agattatgtt cgaaccaagg tggctgacta tatgaaccat ctcattgaca - # 
1600 
- - ttggcgtagc agggttcaga cttgatgctt ctaagcacat gtggcctgga - # 
1650 
- - gacataaagg caattttgga caaactgcat aatctcaata caaaatggtt - # 
1700 
- - ctcccaagga agcagacctt tcattttcca agaggtgatt gatctgggtg - # 
1750 
- - gtgaggcagt gtcaagtaat gagtattttg gaaatggccg tgtgacagaa - # 
1800 
- - ttcaaatatg gagcaaaatt gggcaaagtt atgcgcaagt gggatggaga - # 
1850 
- - aaagatgtcc tacttaaaga actggggaga aggttggggt ttgatgcctt - # 
1900 
- - ctgacagagc ccttgtgttt gtggacaacc atgacaatca gcgaggacat - # 
1950 
- - ggtgctgggg gagcatccat cttgacattc tgggatgcta gactctataa - # 
2000 
- - aatggctgtt ggctttatgt tggctcatcc ttatggtttc acacgggtga - # 
2050 
- - tgtcaagtta ctattggcca agaaatttcc agaatggaaa agatgtcaat - # 
2100 
- - gactgggttg gaccaccaaa taacaatgga aaaaccaaag aagtgagcat - # 
2150 
- - taacccagac agcacttgtg gcaatgactg gatctgtgaa caccgatggc - # 
2200 
- - gtcaaataag gaacatggtt gccttcagaa atgtcgtcaa tggtcagcct - # 
2250 
- - tttgcaaact ggtgggataa tgacagcaac caggtagctt ttggcagagg - # 
2300 
- - aaacaaagga ctcattgtct ttaacaatga tgactgggct ttgtcagaaa - # 
2350 
- - ctttacagac tggtcttcct gctggcacat actgtgatgt catttctgga - # 
2400 
- - gataaagtcg atggcaattg cactggaata aaagtctatg ttggcaatga - # 
2450 
- - tggcaaagct cacttttcta ttagtaactc tgccgaagac ccatttattg - # 
2500 
- - caatccatgc agagtcaaaa atataaggat ccgcggaagc tttggacttc - # 
2550 
- - ttcgccagag gtttggtcaa gtctccaatc aaggttgtcg gcttgtctac - # 
2600 
- - cttgccagaa atttacgaaa agatggaaaa gggtcaaatc gttggtagat - # 
2650 
- - acgttgttga cacttctaaa taagcgaatt tcttatgatt tatgattttt - # 
2700 
- - attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga - # 
2750 
- - ctcttaggtt ttaaaacgaa aattcttgtt cttgagtaac tctttcctgt - # 
2800 
- - aggtcaggtt gctttctcag gtatagcatg aggtcgctct tattgaccac - # 
2850 
- - acctctaccg gcatgcgaat tcgagctcgg tacccgggta ataactgata - # 
2900 
- - taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt - # 
2950 
- - ataatacagt tttttagttt tgctggccgc atcttctcaa atatgcttcc - # 
3000 
- - cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt - # 
3050 
- - tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca - # 
3100 
- - catcatccac ggttctatac tgttgaccca atgcgtctcc cttgtcatct - # 
3150 
- - aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc - # 
3200 
- - acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct - # 
3250 
- - tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga tagggagccc - # 
3300 
- - ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac - # 
3350 
- - ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac - # 
3400 
- - cgtgtgcatt cgtaatgtct gcccattctg ctattctgta tacacccgca - # 
3450 
- - gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc - # 
3500 
- - gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg - # 
3550 
- - tgccctccat ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa - # 
3600 
- - caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt - # 
3650 
- - acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat - # 
3700 
- - taaatagctt ggcagcaaca ggactaggat gagtagcagc acgttcctta - # 
3750 
- - tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct - # 
3800 
- - gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac - # 
3850 
- - atatgcgtat atataccaat ctaagtctgt gctccttcct tcgttcttcc - # 
3900 
- - ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca - # 
3950 
- - aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagctta cattttatgt - # 
4000 
- - tagctggtgg actgacgcca gaaaatgttg gtgatgcgct tagattaaat - # 
4050 
- - ggcgttattg gtgttgatgt aagcggaggt gtggagacaa atggtgtaaa - # 
4100 
- - agactctaac aaaatagcaa atttcgtcaa aaatgctaag aaataggtta - # 
4150 
- - ttactgagta gtatttattt aagtattgtt tgtgcacttg cctgcaggcc - # 
4200 
- - ttttgaaaag caagcataaa agatctaaac ataaaatctg taaaataaca - # 
4250 
- - agatgtaaag ataatgctaa atcatttggc tttttgattg attgtacagg - # 
4300 
- - aaaatataca tcgcaggggg ttgactttta ccatttcacc gcaatggaat - # 
4350 
- - caaacttgtt gaagagaatg ttcacaggcg catacgctac aatgacccga - # 
4400 
- - ttcttgctag ccttttctcg gtcttgcaaa caaccgccaa ctctaagagg - # 
4450 
- - tgatacttat ttactgtaaa actgtgacga taaaaccgga aggaagaata - # 
4500 
- - agaaaactcg aactgatcta taatgcctat tttctgtaaa gagtttaagc - # 
4550 
- - tatgaaagcc tcggcatttt ggccgctcct aggtagtgct ttttttccaa - # 
4600 
- - ggacaaaaca gtttcttttt cttgagcagg ttttatgttt cggtaatcat - # 
4650 
- - aaacaataaa taaattattt catttatgtt taaaaataaa aaataaaaaa - # 
4700 
- - gtattttaaa tttttaaaaa agttgattat aagcatgtga ccttttgcaa - # 
4750 
- - gcaattaaat tttgcaattt gtgattttag gcaaaagtta caatttctgg - # 
4800 
- - ctcgtgtaat atatgtatgc taaagtgaac ttttacaaag tcgatatgga - # 
4850 
- - cttagtcaaa agaaattttc ttaaaaatat atagcactag ccaatttagc - # 
4900 
- - acttctttat gagatatatt atagacttta ttaagccaga tttgtgtatt - # 
4950 
- - atatgtattt acccggcgaa tcatggacat acattctgaa ataggtaata - # 
5000 
- - ttctctatgg tgagacagca tagataacct aggatacaag ttaaaagcta - # 
5050 
- - gtactgtttt gcagtaattt ttttcttttt tataagaatg ttaccaccta - # 
5100 
- - aataagttat aaagtcaata gttaagtttg atatttgatt gtaaaatacc - # 
5150 
- - gtaatatatt tgcatgatca aaaggctcaa tgttgactag ccagcatgtc - # 
5200 
- - aaccactata ttgatcaccg atatatggac ttccacacca actagtaata - # 
5250 
- - tgacaataaa ttcaagatat tcttcatgag aatggcccag ctgcctcgcg - # 
5300 
- - cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac - # 
5350 
- - ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg - # 
5400 
- - gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca - # 
5450 
- - cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag - # 
5500 
- - attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt - # 
5550 
- - aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact - # 
5600 
- - cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag - # 
5650 
- - gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat - # 
5700 
- - gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc - # 
5750 
- - tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga - # 
5800 
- - cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc - # 
5850 
- - gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc - # 
5900 
- - ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct - # 
5950 
- - catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa - # 
6000 
- - gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat - # 
6050 
- - ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca - # 
6100 
- - ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg - # 
6150 
- - tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga - # 
6200 
- - cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga - # 
6250 
- - gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt - # 
6300 
- - ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag - # 
6350 
- - atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca - # 
6400 
- - cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat - # 
6450 
- - ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt - # 
6500 
- - aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca - # 
6550 
- - gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta - # 
6600 
- - gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga - # 
6650 
- - taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag - # 
6700 
- - ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc - # 
6750 
- - catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag - # 
6800 
- - ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca - # 
6850 
- - cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag - # 
6900 
- - gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg - # 
6950 
- - gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg - # 
7000 
- - gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg - # 
7050 
- - cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta - # 
7100 
- - tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg - # 
7150 
- - ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg - # 
7200 
- - gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac - # 
7250 
- - ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt - # 
7300 
- - tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag - # 
7350 
- - ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt - # 
7400 
- - gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt - # 
7450 
- - atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt - # 
7500 
- - gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg - # 
7550 
- - ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct - # 
7600 
- - ttcgctttct tcccttcctt tctcgccacg ttc - # - # 
7633 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 43 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: Artificial Sequence 
&lt;222&gt; LOCATION: 1-43 
&lt;223&gt; OTHER INFORMATION: Forward PCR oligonucleotide - #primer 
- - &lt;400&gt; SEQUENCE: 2 
- - tgtaaaacga cggccagtta aatagacctg caattattaa tct - # 
- # 43 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 41 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: Artificial Sequence 
&lt;222&gt; LOCATION: 1-41 
&lt;223&gt; OTHER INFORMATION: Reverse PCR oligonucleotide - #primer 
- - &lt;400&gt; SEQUENCE: 3 
- - caggaaacag ctatgaccac ctgcacacct gcaaatccat t - # 
- # 41 
__________________________________________________________________________