AU5485600A

AU5485600A - Characterization of the yeast transcriptome

Info

Publication number: AU5485600A
Application number: AU54856/00A
Authority: AU
Inventors: Kenneth Kinzler; Victor Velculescu; Bert Vogelstein
Original assignee: Johns Hopkins University
Current assignee: Johns Hopkins University
Priority date: 1999-06-16
Filing date: 2000-06-14
Publication date: 2001-01-02
Also published as: WO2000077214A3; EP1190044A2; JP2003516716A; WO2000077214A2; CA2378512A1

Description

WO 00/77214 PCT/USOO/16223 CHARACTERIZATION OF THE YEAST TRANSCRIPTOME This application is a continuation-in-part of co-pending application Serial No. 09/012,031 filed January 22, 1998, the disclosure of which is incorporated by reference herein. This invention was made with government support under CA57345 awarded 5 by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD OF THE INVENTION This invention is-related to the characterization of the expressed genes of the yeast genome. More particularly, it is related to the identification and use of previously 10 unrecognized genes. BACKGROUND OF THE INVENTION It is by now axiomatic that the phenotype of an organism is largely determined by the genes expressed within it. These expressed genes can be represented by a "transcriptome," conveying the identity of each expressed gene and its level of 15 expression for a defined population of cells. Unlike the genome, which is essentially a static entity, the transcriptome can be modulated by both.external and internal factors. The transcriptome thereby serves as a dynamic link between an organism's genome and its physical characteristics. The transcriptome as defined above has not been characterized in any eukaryotic 20 or prokaryotic organism, largely because of technological limitations. However, some general features of gene expression patterns were elucidated two decades ago through

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WO 00/77214 PCT/USOO/16223 RNA-DNA hybridization measurements (Bishop et al., 1974; Hereford and Rosbash, 1977). In many organisms, it was thus found that at least three classes of transcripts could be identified, with either high, medium, or low levels of expression, and the number of transcripts per cell were estimated (Lewin, 1980). These data of course 5 provided little information about the specific genes that were members of each class. Data on the expression levels of individual genes have accumulated as new genes were discovered. However, in only a few instances have the absolute levels of expression of particular genes been measured and compared to other genes in the same cell type. Description of any cell's transcriptome would therefore provide new 10 information useful for understanding numerous aspects of cell biology and biochemistry. SUMMARY OF THE INVENTION It is an object of the present invention to provide isolated DNA molecules and methods of using such molecules to affect the cell cycle and identify candidate drugs. 15 These and other objects of the invention are achieved by providing the art with one or more of the embodiments described below. According to one embodiment of the invention an isolated DNA molecule is provided. It comprises a coding sequence of a yeast gene selected from the group consisting of NORF genes comprising a SAGE tag as shown in SEQ ID NOS:67-811. 20 According to another embodiment of the invention a method of using NORF genes is provided. The method is for affecting the cell cycle of a cell. The method comprises the step of administering to a cell an isolated DNA molecule comprising a coding sequence of a NORF gene whose expression varies by at least 10% between any two phases of the cell cycle selected from the group consisting of log phase, S 25 phase, and G2/M. In yet another embodiment of the invention a method for screening candidate antifungal drugs is provided. The method comprises the steps of contacting a test substance with a yeast cell and monitoring expression of a NORF gene whose expression varies by at least 10% between any two phases of the cell cycle selected 30 from the group consisting of log phase, S phase, and G2/M, wherein a test substance 2 WO 00/77214 PCT/USOO/16223 which modifies the expression of the yeast gene is a candidate antifungal drug. In still another embodiment of the invention a method for identifying human genes which are involved in cell cycle progression is provided. The method comprises the step of contacting human DNA with a probe which comprises at least 14 5 contiguous nucleotides of a NORF gene whose expression varies by at least 10% between any two phases of the cell cycle selected from.the group consisting of log phase, S phase, and G2/M. A human DNA sequence which hybridizes to the probe is identified as a sequence of a candidate human gene which is involved in cell cycle progression. 10 The present invention provides probes which comprise at least 14 contiguous nucleotides of a NORF gene comprising a SAGE tag as shown in SEQ ID NOS:67 811. The invention also provides an array of probes on a solid support. At least one probe in the array comprises at least 14 contiguous nucleotides of a NORF gene 15 comprising a SAGE tag as shown in SEQ ID NOS:67-81 1. Still another embodiment of the invention is a method of identifying a candidate drug as a member of a class of drugs having a characteristic effect on gene expression in a yeast cell. A yeast cell is contacted with a candidate drug. Expression of at least one NORF gene whose expression is affected by the class of drugs is monitored in the 20 yeast cell. Detection of a difference in expression of the at least one NORF gene relative to expression in the absence of the candidate drug identifies the candidate drug as a member of the class of drugs. These and other embodiments of the invention which will be apparent to those of skill in the art upon reading the detailed disclosure provided below, make available 25 to the art hitherto unrecognized genes, and information about the expression of genes globally at the organismal level. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic of SAGE Method and Genome Analysis. In applying SAGE to the analysis of yeast gene expression patterns, the 3' most NiaIII site was 30 used to define a unique position in each transcript and to provide a site for ligation of 3 WO 00/77214 PCT/USOO/16223 a linker with a BsmFI site. The type Ils enzyme BsmFI, which cleaves a defined distance from its non-palindromic recognition site, was then used to generate a 15bp SAGE tag (designated by the black arrows), which includes the NlalII site. Automated sequencing of concatenated SAGE tags allowed the routine identification 5 of about a thousand tags per 36-lane sequencing gel. Once sequenced, the abundance of each SAGE tag was calculated, and each tag was used to search the entire yeast genome to identify its corresponding gene. The lower panel shows a small region of Chromosome 15. Gray arrows indicate all potential SAGE tags (NlaIII sites) and black arrows indicate 3' most SAGE tags. The total number of tags observed for each 10 potential tag is indicated above (+ strand) or below (- strand) the tag. As expected, the observed SAGE tags were associated with the 3' end of expressed genes. Figure 2. Sampling of Yeast Gene Expression. Analysis of increasing amounts of ascertained tags reveals a plateau in the number of unique expressed genes. Triangles represent genes with known functions, squares represent genes predicted on 15 the basis of sequence information, and circles represent total genes. Figure 3. Virtual Rot. (A) Abundance Classes in the Yeast Transcriptome. The transcript abundance is plotted in reverse order on the abscissa, whereas the fraction of total transcripts with at least that abundance is plotted on the ordinate. The dotted lines identify the three components of the curve, 1, 2, and 3. This is analogous 20 to a Rot curve derived from reassociation kinetics where the product of initial RNA concentration and time is plotted on the abscissa, and the percent of labeled cDNA that hybridizes to excess mRNA is plotted on the ordinate. (B) Comparison of Virtual Rot and Rot Components. Transitions and data from virtual Rot components were calculated from the data in Figure 3A, while data for Rot components were 25 obtained from Hereford and Rosbash, 1977. Figure 4. Chromosomal Expression Map for S. cerevisiae. Individual yeast genes were positioned on each chromosome according to their open reading frame (ORF) start coordinates. Abundance levels of tags corresponding to each gene are displayed on the vertical axis, with transcription from the + strand indicated above the 30 abscissa and that from the - strand indicated below. Yellow bands at ends of the expanded chromosome represent telomeric regions that are undertranscribed (see text 4 WO 00/77214 PCT/USOO/16223 for details). Figure 5. Northern Blot Analysis of Representative Genes. TDH2/3, TEF 1/2 and NORF 1, are expressed relatively equally in all three states (lane 1, G2/M arrested; lane 2, S phase arrested; lane 3, log phase), while RNR4, RNR2 , and NORF5 are 5 highly expressed in S-phase arrested cells. The expression level observed by SAGE (number of tags) is noted below each lane and was highly correlated with quantitation of the Northern blot by PhosphorImager analysis (r 2 =0.97). TABLE LEGENDS Table 1. Highly Expressed Genes. Tag represents the 10 bp SAGE tag adjacent 10 to the NlaIII site; Gene represents the gene or genes corresponding to a particular tag (multiple genes that match unique tags are from related families, with an average identity of 93%); Locus and Description denote the locus name and functional description of each ORF, respectively; Copies/cell represents the abundance of each transcript in the SAGE library, assuming 15,000 total transcripts per cell and 60,633 15 ascertained transcripts. Table 2. Expression of Putative Coding Sequences. Table column headings are the same as for Table 1. Table 3. Expression of the most abundant NORF genes. SAGE Tag, Locus, and Copies/cell are the same as for Table 1; Chr and Tag Pos denote the chromosome 20 and position of each tag; ORF Size denotes the size of the ORF corresponding to the indicated tag. In each case, the tag was located within or less than 250 bp 3' of the NORF. Table 4. Expression of NORF genes. SAGE tag and Copies/cell are the same as for Table 1. Chr and Tag Pos denote the chromosome and position of each tag. 25 Table 5. Gene expression changes in different cell cycle phases. L denotes log phase; S denotes synthesis phase; G2/M denotes the mitotic phase. Tag Sequence represents the 10 bp SAGE tag adjacent to the NlaIII site; "ratio L to S" denotes the ratio of expression in log phase to expression in synthesis phase; "ratio S to G2/M" denotes the ratio of expression in synthesis phase to expression in G2/M phase; "ratio 30 G2/M to L" denotes the ratio of expression in G2/M to log phase. #DIV/0! indicates 5 WO 00/77214 PCT/USOO/16223 an increase in expression from 0; a value of 0 indicates a decrease in expression to 0; a value of I indicates no change; a value less than 1 indicates a decrease in expression; and a value greater than 1 indicates an increase in expression. Table 6. Intergenic open reading frames that contain or are adjacent to observed 5 SAGE tags. Copies/cell represents abundance of each mRNA transcript as in Table 1. Positive expression level indicates the tag is on the + strand of the chromosome; Negative expression level indicates the tag is on the - strand. DETAILED DESCRIPTION 10 It is a discovery of the present invention that certain hitherto unknown genes (the NORFs) exist and are expressed in yeast. These genes, as well as other previously identified and previously postulated genes, can be used to study, monitor, and affect phases of cell cycle. The present invention identifies which genes are differentially expressed during the cell cycle. Differentially expressed genes can be 15 used as markers of phases of the cell cycle. They can also be used to affect a change in the phase of the cell cycle. In addition, they can be used to screen for drugs which affect the cell cycle, by affecting expression of the genes. Human homologs of these eukaryotic genes are also presumed to exist, and can be identified using the yeast genes as probes or primers to identify the human homologs. 20 New genes termed NORFs (not previously assigned open reading frames) have been found. They are uniquely identified by their SAGE tags. In addition their entire nucleotide sequences are known and publicly available. In general, these were not previously identified as genes due to their small size. However, they have now been found to be expressed. 25 Differentially expressed yeast genes are those whose expression varies by a statistically significant difference (to greater than 95% confidence level) within different growth phases, particularly log phase, S phase, and G2/M. Preferably the difference is at least 10%, 25%, 50%, or 100%. In some cases, differentially expressed genes are not expressed at detectable levels in one or more cell cycle phases 30 as determined by SAGE analysis. Genes which have been found to have differential expression characteristics include: NORF N 2 1, 2, 4, 5, 6, 17, 25, 27, TEFI/TEF2, 6 WO 00/77214 PCT/USOO/16223 ENO2, ADHI, ADH2, PGKI, CUPlA/CUPIB, PYKI, YKL056C, YMRI16C, YELO33W, YOR182C, YCRO13C, ribonucleotide reductase 2 and 4, and YJR085C. Differential expression can be detected by any means known in the art, such as hybridization to specific probes or immunological assays. 5 Isolated DNA molecules according to the invention contain less than a whole chromosome and can be genomic or cDNA, i.e., lacking introns. Isolated DNA molecules can comprise a yeast gene or a coding sequence of a yeast gene involved in cell cycle progression, such as NORF genes which comprise SAGE tags as shown in SEQ ID NOS:67-81 1. Isolated DNA molecules which comprise yeast genes or 10 coding sequences of yeast genes comprising SAGE tags as shown in SEQ ID NOS:37 12,203 are also isolated DNA molecules of the invention. Isolated DNA molecules can also consist of a yeast gene or a coding sequence of a yeast gene which comprises a SAGE tag as shown in SEQ ID NOS:37-12,203 or 67-811. Any technique for obtaining a DNA of known sequence may be used to obtain 15 isolated DNA molecules of the invention. Preferably they are isolated free of other cellular components such as membrane components, proteins, and lipids. They can be made by a cell and isolated, or synthesized using PCR or an automatic synthesizer. Methods for purifying and isolating DNA are routine and are known in the art. To administer yeast genes to cells, any DNA delivery techniques known in the 20 art may be used, without limitation. These include liposomes, transfection, mating, transduction, transformation, viral infection, electroporation. Vectors for particular purposes and characteristics can be selected by the skilled artisan for their known properties. Cells which can be used as gene recipients are yeast and other fungi, mammalian cells, including humans, and bacterial cells. 25 Antifungal drugs can be identified using yeast cells as described herein. Expression of a differentially expressed NORF gene can be monitored by any means known in the art. When a test substance modifies the expression of such a differentially expressed gene, for example by increasing or decreasing its expression, it is a candidate drug for affecting the growth properties of fungi and may be useful 30 as an antifungal agent. Expression of more than one NORF gene can be monitored. For example, expression of 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 250, 7 WO 00/77214 PCT/USOO/16223 300, 350, 400, 450, or 500 or more NORF genes can be monitored in single or multiple assays. Because differentially expressed genes are likely to be involved in cell cycle progression, it is likely that these genes are conserved among species. The 5 differentially expressed NORF genes identified by the present invention can be used to identify homologs in humans and other mammals by contacting DNA from these mammals with a probe which comprises at least 10 contiguous nucleotides of a differentially expressed NORF gene. The DNA can be genomic or cDNA, as is known in the art. Means for identifying homologous genes among different species 10 are well known in the art. Briefly, stringency of hybridization can be reduced so that imperfectly matching sequences hybridize. This can be in the context of inter alia Southern blots, Northern blots, colony hybridization or PCR. Any hybridization technique which is known in the art can be used. A DNA sequence which hybridizes to the probe is identified as a sequence of a candidate gene which is involved in cell 15 cycle expression. Probes according to the present invention are isolated DNA molecules which have at least 10, and preferably at least 12, 14, 16, 18, 20, or 25 contiguous nucleotides of a particular NORF gene or other differentially expressed gene. The probes may or may not be labeled. They may be used, for example, as primers for 20 PCR assays, or for detection of gene expression for Southern or Northern blots or in situ hybridization. Preferably the probes are immobilized on a solid support. The solid support can be any surface to which a probe can be attached. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, or particles such as beads, including but not limited to latex, 25 polystyrene, or glass beads. Any method known in the art can be used to attach the a probe to the solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the probe and the solid support. More preferably, probes are present on an array so that multiple probes can 30 simultaneously hybridize to a single biological sample. The probes can be spotted onto the array or synthesized in situ on the array. See Lockhart et. al., Nature 8 WO 00/77214 PCT/USOO/16223 Biotechnology, Vol. 14, December 1996, "Expression monitoring by hybridization to high-density oligonucleotide arrays." A single array contains at least one NORF probe, but can contain more than 100, 500 or even 1,000 different probes in discrete locations. If desired, one or more NORF probe(s) present on the array can be 5 nucleotide sequences from a NORF gene which is differentially expressed during the cell cycle. Genes identified by the present invention which are differentially expressed during the cell cycle can also be used to obtain gene expression profiles characteristic of the response of yeast genes of a yeast cell to a particular drug or class of drugs. 10 Classes of drugs of particular interest for which gene expression profiles can be generated include those drugs which affect cell cycle or other cell processes, such as chemotherapeutic agents. If desired, gene expression profiles characteristic of more than one drug of a particular class can be generated and used to make a composite gene expression profile. For example, microtubule poison drugs such as vinblastin, 15 taxol, vincristine, and taxotere can be used to generate gene expression profiles characteristic of microtubule poisons. To generate a gene expression profile characteristic of a particular drug or class of drugs, a yeast cell is contacted with a particular drug or a member of a particular class of drugs. Expression of at least one yeast gene is monitored, either before and 20 after contacting or in the contacted cell and in another yeast cell which has not been contacted with the drug. Genes which are monitored can be any yeast gene, including NORFS. Preferably, these genes are differentially expressed during the cell cycle. For example, yeast genes can be selected from genes comprising the SAGE tags shown in Tables 3, 4, 5, and 6 (SEQ ID NOS:67-12,203). If desired, genes such as NORF N1 25 1, 2, 4, 5, 6, 17, 25, or 27, TEF1/TEF2, ENO2, ADHI, ADH2, PGK1, CUPIA/CUP IB, PYKI, YKL056C, YMR116C, YELO33W, YOR182C, YCRO13C, ribonucleotide reductase 2 and 4, and YJR085C, can be used for monitoring alterations in gene expression. The expression of any number of these genes, such as 1, 2, 3, 4, 5, 10, 15, 20, 30 25, 30, 40, 50, 60, 75, 100, 150, 250, 500, 1000, 2000, 3000, 4000, 5000, or 5,500 genes, can be measured. It is particularly convenient to monitor expression of the 9 WO 00/77214 PCT/USOO/16223 differentially expressed genes using nucleic acids which are immobilized on a solid support or in an array, such as the gene arrays described above. Many genes, particularly cell cycle genes, are likely to be conserved between yeast and mammals, including humans. Thus, gene expression profiles characteristic 5 of a drug or class of drugs can be used to predict the effects of candidate drugs on human cells, by identifying the candidate drug as a member of a class of drugs whose characteristic gene expression profile is known. The candidate drugs can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The candidate drugs can be naturally 10 occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly or synthesized by chemical methods known in the art. The effect of a candidate drug on expression of at least one gene whose expression is affected by the class of drugs is monitored. A gene expression profile 15 obtained using the candidate drug which is similar to a gene expression profile for a particular drug or class of drugs identifies the candidate drug as a member of that class of drugs. The effect of modifying particular substituents of a known drug or of a candidate drug can be similarly tested. Such methods are useful for determining whether 20 alterations intended, for example, to increase solubility or absorption of a particular drug will have an unintended and possibly deleterious effect on genes which are differentially expressed during the cell cycle. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific 25 examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. EXAMPLE Summary We have analyzed the set of genes expressed from the yeast genome, herein 30 called the transcriptome, using serial analysis of gene expression (SAGE). Analysis 10 WO 00/77214 PCT/USOO/16223 of 60,633 transcripts revealed 4,665 genes, with expression levels ranging from 0.3 to over 200 transcripts per cell. Of these genes, 1,981 had known functions, while 2,684 were previously uncharacterized. Integration of positional information with gene expression data allowed the generation of chromosomal expression maps, 5 identifying physical regions of transcriptional activity, and identified genes that had not been predicted by sequence information alone. These studies provide insight into global patterns of gene expression in yeast and demonstrate the feasibility of genome wide expression studies in eukaryotes. Results 10 Characteristics and Rationale of SAGE Approach Several methods have recently been described for the high throughput evaluation of gene expression (Nguyen et al., 1995; Schena et al., 1995; Velculescu et al., 1995). We used SAGE (Serial Analysis of Gene Expression) because it can provide quantitative gene expression data without the prerequisite of a hybridization 15 probe for each transcript. The SAGE technology is based on two basic principles (Figure 1). First, a short sequence tag (9-11 bp) contains sufficient information to uniquely identify a transcript, provided that it is derived from a defined location within that transcript. Second, many transcript tags can be concatenated into a single molecule and then sequenced, revealing the identity of multiple tags simultaneously. 20 The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags and identifying the gene corresponding to each tag. Genome-wide expression In order to maximize representation of genes involved in normal growth and cell-cycle 25 progression, SAGE libraries were generated from yeast cells in three states: log phase, S phase arrested and G2/M phase arrested. In total, SAGE tags corresponding to 60,633 total transcripts were identified (including 20,184 from log phase, 20,034 from S phase arrested, and 20,415 from G2/M phase arrested cells). Of these tags, 56,291 11 WO 00/77214 PCT/USOO/16223 tags (93%) precisely matched the yeast genome, 88 tags matched the mitochondrial genome, and 91 tags matched the 2 micron plasmid. The number of SAGE tags required to define a yeast transcriptome depends on the confidence level desired for detecting low abundance mRNA molecules. 5 Assuming the previously derived estimate of 15,000 mRNA molecules per cell (Hereford and Rosbash, 1977), 20,000 tags would represent a 1.3 fold coverage even for mRNA molecules present at a single copy per cell, and would provide a 72% probability of detecting such transcripts (as determined by Monte Carlo simulations). Analysis of 20,184 tags from log phase cells identified 3,298 unique genes. As an 10 independent confirmation of mRNA copy number per cell, we compared the expression level of SUP44/RPS4, one of the few genes whose absolute mRNA levels have been reliably determined by quantitative hybridization experiments (Iyer and Struhl, 1996), with expression levels determined by SAGE. SUP44/RPS4 was measured by hybridization at 75 +/- 10 copies/cell (Iyer and Struhl, 1996), in good 15 accord with the SAGE data of 63 copies/cell, suggesting that the estimate of 15,000 mRNA molecules per cell was reasonably accurate. Analysis of SAGE tags from S phase arrested and G2/M phase arrested cells revealed similar expression levels for this gene (range 52 to 55 copies/cell), as well as for the vast majority of expressed genes. As less than 1% of the genes were expressed at dramatically different levels 20 among these three states (see below), SAGE tags obtained from all libraries were combined and used to analyze global patterns of gene expression. Analysis of ascertained tags at increasing increments revealed that the number of unique transcripts plateaued at -60,000 tags (Figure 2). This suggested that generation of further SAGE tags would yield few additional genes, consistent with the 25 fact that sixty thousand transcripts represented a four-fold redundancy for genes expressed as low as 1 transcript per cell. Likewise, Monte Carlo simulations indicated that analysis of 60,000 tags would identify at least one tag for a given transcript 97% of the time if its expression level was one copy per cell. The 56,291 tags that precisely matched the yeast genome represented 4,665 30 different genes. This number is in agreement with the estimate of 3,000 to 4,000 expressed genes obtained by RNA-DNA reassociation kinetics (Hereford and 12 WO 00/77214 PCT/USOO/16223 Rosbash, 1977). These expressed genes included 85% of the genes with characterized functions (1,981 of 2,340), and 76% of the total genes predicted from analysis of the yeast genome (4,665 of 6,121). These numbers are consistent with a relatively complete sampling of the yeast transcriptome given the limited number of 5 physiological states examined and the large number of genes predicted solely on the basis of genomic sequence analysis. The transcript expression per gene was observed to vary from 0.3 to over 200 copies per cell. Analysis of the distribution of gene expression levels revealed several abundance classes that were similar to those observed in previous studies using 10 reassociation kinetics. A "virtual Rot" of the genes observed by SAGE (Figure 3A) identified three main components of the transcriptome with abundances ranging over three orders of magnitude. A Rot curve derived from RNA-cDNA reassociation kinetics also contained three main components distributed over a similar range of abundances (Hereford and Rosbash, 1977). Although the kinetics of reassociation of 15 a particular class of RNA and cDNA may be affected by numerous experimental variables, there were striking similarities between Rot and virtual Rot analyses (Figure 3B). Because Rot analysis may not detect all transcripts of low abundance (Lewin, 1980), it is not surprising that SAGE revealed both a larger total number of expressed genes and a higher fraction of the transcriptome belonging to the low abundance 20 transcript class. Integration of Expression Information with the Genomic Map The SAGE expression data could be integrated with existing positional information to generate chromosomal expression maps (Figure 4). These maps were generated using the sequence of the yeast genome and the position coordinates of ORFs obtained 25 from the Stanford Yeast Genome Database. Although there were a few genes that were noted to be physically proximal and have similarly high levels of expression, there did not appear to be any clusters of particularly high or low expression on any chromosome. Genes like histones H3 and H4, which are known to have coregulated divergent promoters and are immediately adjacent on chromosome 14 (Smith and 30 Murray, 1983), had very similar expression levels (5 and 6 copies per cell, 13 WO 00/77214 PCT/USOO/16223 respectively). The distribution of transcripts among the chromosomes suggested that overall transcription was evenly dispersed, with total transcript levels being roughly linearly related to chromosome size (r2 =0.85, data not shown). However, regions within 10 kb of telomeres appeared to be uniformly undertranscribed, containing on 5 average 3.2 tags per gene as compared with 12.4 tags per gene for non-telomeric regions (Figure 4). This is consistent with the previously described observations of "telomeric silencing" in yeast (Gottschling et al., 1990). Recent studies have reported telomeric position effects as far as 4 kb from telomere ends (Renauld et al., 1993). Gene Expression Patterns 10 Table I lists the 30 most highly expressed genes, all of which are expressed at greater than 60 mRNA copies per cell. As expected, these genes mostly correspond to well characterized enzymes involved in energy metabolism and protein synthesis and were expressed at similar levels in all three growth states (Examples in Figure 5). Some of these genes, including ENO2 (McAlister and Holland, 1982), PDC1 (Schmitt 15 et al., 1983), PGK1 (Chambers et al., 1989), PYK1 (Nishizawa et al., 1989), and ADH1 (Denis et al., 1983), are known to be dramatically induced in the glucose-rich growth conditions used in this study. In contrast, glucose repressible genes such as the GAL1/GAL7/GAL1O cluster (St John and Davis, 1979), and GAL3 (Bajwa et al., 1988) were observed to be expressed at very low levels (0.3 or fewer copies per cell). 20 As expected for the yeast strain used in this study, mating type a specific genes, such as the a factor genes (MFAI, MFA2) (Michaelis and Herskowitz, 1988), and alpha factor receptor (STE2) (Burkholder and Hartwell, 1985) were all observed to be expressed at significant levels (range 2 to 10 copies per cell), while mating type alpha specific genes (MFal, MFa2, STE3) (Hagen et al., 1986; Kurjan and Herskowitz, 25 1982; Singh et al., 1983) were observed to be expressed at very low levels (<0.3 copies/cell). Three of the highly expressed genes in Table 1 had not been previously characterized. One contained an ORF with predicted ribosomal function, previously identified only by genomic sequence analysis. Analyses of all SAGE data suggested 30 that there were 2,684 such genes corresponding to uncharacterized ORFs which were 14 WO 00/77214 PCT/USOO/16223 transcribed at detectable levels. The 30 most abundant of these transcripts were observed more than 30 times, corresponding to at least 8 transcripts per cell (Table 2). The other two highly expressed uncharacterized genes corresponded to ORFs not predicted by analysis of the yeast genome sequence (NORF = Nonannotated ORF). 5 Analyses of SAGE data suggested that there were at least 160 NORF genes transcribed at detectable levels. The 30 most abundant of these transcripts were observed at least 9 times (Table 3 and examples in Figure 5). Interestingly, one of the NORF genes (NORF5) was only expressed in S phase arrested cells and corresponded to the transcript whose abundance varied the most in 10 the three states analyzed (> 49 fold, Figure 5). Comparison of S phase arrested cells to the other states also identified greater than 9 fold elevation of the RNR2 and RNR4 transcripts (Figure 5). Induction of these ribonucleoside reductase genes is likely to be due to the hydroxyurea treatment used to arrest cells in S phase (Elledge and Davis, 1989). Likewise, comparison of G2/M arrested cells identified elevation of RBL2 15 and dynein light chain, both microtubule associated proteins (Archer et al., 1995; Dick et al., 1996). As with the RNR inductions, these elevated levels seem likely to be related to the nocodazole treatment used to arrest cells in the G2/M phase. While there were many relatively small differences between the states (for example, NORF1, Figure 5), overall comparison of the three states revealed surprisingly few dramatic 20 differences; there were only 29 transcripts whose abundance varied more than 10 fold among the three different states analyzed (Tables 4 and 5). A comprehensive analysis for NORF genes was performed using the SAGE data. Yeast genome intergenic regions were defined as regions outside annotated ORFs or the 500bp region downstream of annotated ORFs (yeast genome sequence 25 and tables of annotated ORFs were obtained from SGD at http://genome-www.stanford.edu/Saccharomyces/). Based on sequence analysis a total of 9524 putative ORFs of 25-99 amino acids were present in the intergenic regions; 510 of these ORFs contain or are adjacent to observed SAGE tags (Table 6). Of the 60,633 SAGE tags analyzed, there were 302 unique SAGE tags either within 30 or adjacent to intergenic ORFs (100bp upstream or 500bp downstream of the ORF) (Table 6). Note that in some cases, more than one NORF contains or is adjacent to the 15 WO 00/77214 PCT/USOO/16223 SAGE tag. These tags matched the genome uniquely, were in the correct orientation, and were expressed at levels greater than 0.3 transcript copies per cell. The expression level for each NORF shown in Table 6 corresponds to the number of mRNA transcript copies per cell. If the expression level is positive it 5 means that the tag is on the + strand of the chromosome; if negative, the tag is on the - strand of the chromosome. Discussion Analysis of a yeast transcriptome affords a unique view of the RNA components 10 defining cellular life. Comparison of gene expression patterns from altered physiologic states can provide insight into genes that are important in a variety of processes. Comparison of transcriptomes from a variety of physiologic states should provide a minimum set of genes whose expression is required for normal vegetative growth, and another set composed of genes that will be expressed only in response to 15 specific environmental stimuli, or during specialized processes. For example, recent work has defined a minimal set of 250 genes required for prokaryotic cellular life (Mushegian and Koonin,- 1996). Examination of the yeast genome readily identified homologous genes for 196 of these, over 90% of which were observed to be expressed in the SAGE analysis. Detailed analyses of yeast transcriptomes, as well as 20 transcriptomes from other organisms, should ultimately allow the generation of a minimal set of genes required for eukaryotic life. Like other genome-wide analyses, SAGE analysis of yeast transcriptomes has several potential limitations. First, a small number of transcripts would be expected to lack an NlaIII site and therefore would not be detected by our analysis. Second, our 25 analysis was limited to transcripts found at least as frequently as 0.3 copies per cell. Transcripts expressed in only a minute fraction of the cell cycle, or transcripts expressed in only a fraction of the cell population, would not be reliably detected by our analysis. Finally, mRNA sequence data are practically unavailable for yeast, and consequently, some SAGE tags cannot be unambiguously matched to corresponding 30 genes. Tags which were derived from overlapping genes, or genes which have unusually long 3' untranslated regions may be misassigned. Increased availability of 16 WO 00/77214 PCT/USOO/16223 3' UTR sequences in yeast mRNA molecules should help to resolve the ambiguities. Despite these potential limitations, it is clear that the analyses described here furnish both global and local pictures of gene expression, precisely defined at the nucleotide level. These data, like the sequence of the yeast genome itself, provide 5 simple, basic information integral to the interpretation of many experiments in the future. The availability of mRNA sequence information from EST sequencing as well as various genome projects, will soon allow definition of transcriptomes from a variety of organisms, including human. The data recorded here suggest that a reasonably complete picture of a human cell transcriptome will require only about 10 10 20 fold more tags than evaluated here, a number well within the practical realm achievable with a small number of automated sequencers. The analysis of global expression patterns in higher eukaryotes is expected, in general, to be similar to those reported here for S. cerevisiae. However, the analysis of the transcriptome in different cells and from different individuals should yield a wealth of information regarding 15 gene function in normal, developmental, and disease states. Experimental Procedures Yeast cell culture The source of transcripts for all experiments was S. cerevisiae strain YPH499 (MATa ura3-52 lys2-801 ade2-101 leu2-,iJ his3-A200 trpl-163) (Sikorski and 20 Hieter, 1989). Logarithmically growing cells were obtained by growing yeast cells to early log phase (3 x 10' cells/ml) in YPD (Rose et al., 1990) rich medium (YPD supplemented with 6 mM uracil, 4.8 mM adenine and 24 mM tryptophan) at 30'C. For arrest in the GI/S phase of the cell cycle, hydroxyurea (0.1 M) was added to early log phase cells, and the culture was incubated an additional 3.5 hours at 30*C. For 25 arrest in the G2/M phase of the cell cycle, nocodazole (15 pg/ml) was added to early log phase cells and the culture was incubated for an additional 100 minutes at 30*C. Harvested cells were washed once with water prior to freezing at -70*C. The growth states of the harvested cells were confirmed by microscopic and flow cytometric analyses (Basrai et al., 1996). 17 WO 00/77214 PCT/USOO/16223 SAGE protocol The SAGE method was performed as previously described (Velculescu et al., 1995; Kinzler et al., U.S. Patents 5,866,330 and 5,695,937), with exceptions noted below. PolyA RNA was converted to double-stranded cDNA with a BRL synthesis 5 kit using the manufacturer's protocol except for the inclusion of primer biotin-5'-T, 8 3'. The cDNA was cleaved with NlalII (Anchoring.Enzyme). As NlaIII sites were observed to occur once every 309 base pairs in three arbitrarily chosen yeast chromosomes (1, 5, 10), 95% of yeast transcripts were predicted to be detectable with a NlaIII-based SAGE approach. After capture of the 3' cDNA fragments on 10 streptavidin coated magnetic beads (Dynal), the bound cDNA was divided into two pools, and one of the following linkers containing recognition sites for BsmFI was ligated to each pool: Linker 1, 5' TTTGGATTTGCTGGTGCAGTACAACTAGGCTTAATAGGGACATG-3'(SEQ ID NO: 1).5'-TCCCTATTAAGCCTAGTTGTACTGCACCAGCAAATCC 15 [amino mod. C7]-3'(SEQ ID NO:2).; Linker 2,5' TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACATG-3'(SEQ ID NO:3), 5'-TCCCCGTACATCGTTAGAAGCTTGAATTCGAGCAG[amino mod. C7]-3' (SEQ ID NO:4). As BsmFI (Tagging Enzyme) cleaves 14 bp away from its recognition site, and 20 the NlaIII site overlaps the BsmFI site by I bp, a 15 bp SAGE tag was released with BsmFI. SAGE tag overhangs were filled-in with Klenow, and tags from the two pools were combined and ligated to each other. The ligation product was diluted and then amplified with PCR for 28 cycles with 5'-GGATTTGCTGGTGCAGTACA-3' (SEQ ID NO:5) and 5'-CTGCTCGAATrCAAGCTTCT-3'(SEQ ID NO:6), as primers. The 25 PCR product was analyzed by polyacrylamide gel electrophoresis (PAGE), and the PCR product containing two tags ligated tail to tail (ditag) was excised. The PCR product was then cleaved with NlalIl, and the band containing the ditags was excised and self-ligated. After ligation, the concatenated products were separated by PAGE and products between 500 bp and 2 kb were excised. These products were cloned into 30 the SphI site of pZero (Invitrogen). Colonies were screened for inserts by PCR with M13 forward and M13 reverse sequences located outside the cloning site as primers. 18 WO 00/77214 PCT/USOO/16223 PCR products from selected clones were sequenced with the TaqFS DyePrimer kits (Perkin Elmer) and analyzed using a 377 ABI automated sequencer (Perkin Elmer), following the manufacturer's protocol. Each successful sequencing reaction identified an average of 26 tags; given a 90% sequencing reaction success rate, this 5 corresponded to an average of about 850 tags per sequencing gel. SAGE data analysis Sequence files were analyzed by means of the SAGE program group (Velculescu et al., 1995), which identifies the anchoring enzyme site with the proper 10 spacing and extracts the two intervening tags and records them in a database. The 68,691 tags obtained contained 62,965 tags from unique ditags and 5,726 tags from repeated ditags. The latter were counted only once to eliminate potential PCR bias of the quantitation, as described (Velculescu et al., 1995). Of 62,965 tags, 2,332 tags corresponded to linker sequences, and were excluded from further analysis. Of the 15 remaining tags, 4,342 tags could not be assigned, and were likely due to sequencing errors (in the tags or in the yeast genomic sequence). If all of these were due to tag sequencing errors, this corresponds to a sequencing error rate of about 0.7% per base pair (for a 1Obp tag), not far from what we would have expected under our automated sequencing conditions. However, some unassigned tags had a much higher than 20 expected frequency of A's as the last five base pairs of the tag (5 of the 52 most abundant unassigned tags), suggesting that these tags were derived from transcripts containing anchoring enzyme sites within several base pairs from their polyA tails. Given the frequency of NlaIII sites in the genome (one in 309 base pairs), approximately 3% of transcripts were predicted to contain NlaIII sites within 10 bp of 25 their polyA tails. As very sparse data are available for yeast mRNA sequences and efforts to date have not been able to identify a highly conserved polyadenylation signal (Irniger and Braus, 1994; Zaret and Sherman, 1982), we used 14 bp of SAGE tags (i.e. the NIalI site plus the adjacent 10 bp) to search the yeast genome directly (yeast genome 30 sequence obtained from the Stanford yeast genome ftp site (genome-ftp.stanford.edu) on August 7, 1996). Because only coding regions are annotated in the yeast genome, 19 WO 00/77214 PCT/USOO/16223 and SAGE tags can be derived from 3' untranslated regions of genes, a SAGE tag was considered to correspond to a particular gene if it matched the ORF or the region 500 bp 3' of the ORF (locus names, gene names and ORF chromosomal coordinates were obtained from Stanford yeast genome ftp site, and ORF descriptions were obtained 5 from MIPS www site (http://www.mips.biochem.mpg.de/) on August 14, 1996). ORFs were considered genes with known functions if they were associated with a three letter gene name, while ORFs without such designations were considered uncharacterized. As expected, SAGE tags matched transcribed portions of the genome in a highly 10 non-random fashion, with 88% matching ORFs or their adjacent 3' regions in the correct orientation (chi-squared P value <10). In instances when more than one tag matched a particular ORF in the correct orientation, the abundance was calculated to be the sum of the matched tags. Tags that matched ORFs in the incorrect orientation were not used in abundance calculations. In instances when a tag matched more than 15 one region of the genome (for example an ORF and non-ORF region) only the matched ORF was considered. In some cases the 15th base of the tag could also be used to resolve ambiguities. For the identification of NORF genes, only tags were considered that matched portions of the genome that were further than 500 bp 3' of a previously identified ORF 20 and were observed at least two times in the SAGE libraries. 20 WO 00/77214 PCT/USOO/16223 tam CL -u -04 0mCI ~(~-M ~O 1111 WO 00/77214 PCT/USOO/16223 > ~ 0 > ~ u oao >08 M-0-1~ >00 -- 4~~ 0051 > > 0 1 8O--, > -0-fj (0>O G) 0 M 4000 -q C G) -j 0 ) O ) 00 0 00 -4-1-1 -- 4 0 :1 ]> (3) 0 G0c ffl0 J- n 0W 000 WW O o 0 0 -nc -< -5 O; c-ocm 0 a ;0 0 M ; ur 0r ; 0r 0; ;00 0 - W C~j0 0 aDd 5.ca r= 0 . CA (A = a(a= 4 Co 0 0) 0 UC g s s0 (- (D 00)0 D0 DrAc Fo 0 00 CD ( 03 ) 0 "9-46=9965 6o0 =r M (7 so L D :6 "= 0 =22= WO 00/77214 PCT/USOO/16223 00 > 0-4 -4-I ~ 0 -.4 0 o~~~l 00-10>0 1 M-l~ ~ ~ ~ 001 1 > 009 ~ ->- > ~ -9 o . - 0 -.- 1 C, -, 1 OOW11 vooo-I A 9 0 -1 -- 1--1 00 'M 23n WO 00/77214 PCT/USOO/16223 additional NORFs 'AGE Tag Seq. ID No. Chr Tag Pos Copies/cell 'GCGCAATTT 97 4 1108395 2 AAGTGATGA 98 7 593382 2 TGTTGAATT 99 10 608373 2 'AAGCAGTAA 100 3 155607 2 CATATGTTA 101 4 916112 2 CCTACACGG 102 6 223289 2 TAATTGGAC 103 10 392099 2 TCAGACAAA 104 14 687272 2 TATGAAAGA 105 15 81263 2 TTCGTTCTA 106 15 841970 2 GCAGGAGTT 107 16 188350 2 TCTATTAGG 108 2 418749 2 GGATTTCAG 109 4 1224930 2 AGATATAAT 110 5 52488 2 TGTTTTGGG 111 11 374761 2 ATTTTTAGT 112 11 508212 2 TGAAAAGAT 113 13 104160 2 AAGCCCATC 114 13 251273 2 GCGTCCTCA 115 15 832420 2 TTAGTTAAT 116 2 477623 2 TGGTAGCCA 117 3 56961 2 ATTAGACTA 118 3 162589 2 GTGACTCTT 119 4 1490879 2 GACTATAAG 120 5 251266 2 CT1TFTCAG 121 10 159213 2 TCATATAGT 122 13 158765 2 AACAAAGTG 123 13 171166 2 TGGGAAAGG 124 13 804600 2 ACTTTATAT 125 16 366449 2 ATACCAGCG 126 3 175540 1 CCTTGTATA 127 4 372624 1 GTACATTCA 128 5 67152 1 ATTTCTCTG 129 5 187462 1 AGTTGCTCC 130 7 317108 1 TAAGAAATC 131 7 836202 1 TTGGGCTAT 132 8 107992 1 AATGGTGAT 133 11 558686 1 TCATTTGGG 134 12 199358 1 TGAACTTTA 135 12 283720 1 CAGAAGGAG 136 13 652873 1 CGGTTACTA 137 15 803663 1 GATGAGAAG 138 15 1004369 1 AACCGTCCC 139 16 199141 1 CATTCATAC 140 2 164728 1 ATCTTTTTG 141 4 169784 1 TAGAATAAT 142 4 603508 1 TACGCTGTG 143 5 118089 1 ATATTAATT 144 6 64228 1 24 WO 00/77214 PCT/USOO/16223 3TTCTTGCCT 145 7 939579 1 ATATAGCTGC 146 10 181144 1 .CAAAAAAAA 147 11 91785 1 3AACTCCACA 148 11 94125 1 CTTCACTGC 149 11 374172 1 'ACATCATAA 150 11 625896 1 3AAGTATTGA 151 12 603999 1 GCGCGTATA - 152 13 206410 1 3GGTAGTACT 153 13 671730 1 AGTTTTGTC 154 15 33475 1 ,AATTCCTAC 155 1 172182 0.8 ~TTGATTTGA 156 2 46431 0.8 3GCTCTGGTT 157 2 414510 0.8 'AGAAATAGC 158 2 565130 0.8 TGTTATTT 159 2 616054 0.8 'GAAGTCAAA 160 2 680605 0.8 3TCTAGATAA 161 3 171584 0.8 GTCAAAATG 162 4 192750 0.8 3CGAGTAG 163 4 691301 0.8 3CTCCAATAG 164 4 1131020 0.8 TTATTTGAG 165 4 1237501 0.8 TTATATTGA 166 4 1401803 0.8 GGGTTGAAG 167 5 251266 0.8 kTTTTATTTG 168 5 447729 0.8 TCATAAAAA 169 5 548612 0.8 TATATAAAA 170 6 223182 0.8 oTACTTCTGC 171 8 34653 0.8 TAAGACAGT 172 10 227802 0.8 TCATAAGTT 173 10 471894 0.8 "AAATCTGAG 174 11 145617 0.8 ,TGGTAGAAA 175 11 151174 0.8 oACGTACACA 176 11 403208 0.8 'CAAGATCAA 177 11 425882 0.8 GCTTGTTCC 178 12 234966 0.8 ,ACATTCGTT 179 12 759953 0.8 oTTACATATA 180 12 789781 0.8 CTATAGCAA 181 13 228936 0.8 'CTTTCTGAA 182 13 297985 0.8 .CTTTAGAAT 183 13 777999 0.8 ATTAACACC 184 13 842122 0.8 ICGCAGGGGC 185 14 440984 0.8 GTTTATAAA 186 14 661710 0.8 AAAGTCATT 187 15 32081 0.8 TCGTAAACT 188 15 680625 0.8. TTTGGAGT 189 15 888343 0.8 kGGCATCTTG 190 16 250284 0.8 AATCAAAAC 191 16 453890 0.8 ATTGACGAA 192 16 560169 0.8 TGATGATTT 193 16 582360 0.8 'CTGT1T1TG 194 16 643476 0.8 11F AAAAA 195 1 101436 0.5 25 WO 00/77214 PCT/USOO/16223 AGTTTGATC 196 1 199848 0.5 kGCACCTATG 197 2 46913 0.5 GATTTATCC 198 2 418946 0.5 kCTGCATCTG 199 2 680860 0.5 'AAGTTAGGA 200 2 744770 0.5 TACCCAATT 201 3 29939 0.5 ACTTTGTAT 202 3 30056 0.5 'CGGCGGGTG 203 3 41645 0.5 AAATTGTTC 204 3 57108 0.5 CAAGTACTC 205 3 157855 0.5 ACTGTATGC 206 3 223882 0.5 'TATCGGCCA 207 3 278840 0.5 CAAGCCCAA 208 3 289917 0.5 'TACAGGGCT 209 4 93873 0.5 AGATCATCG 210 4 254851 0.5 7AACTCCTGG 211 4 340891 0.5 7AACGAGAAG 212 4 371850 0.5 1T1 AATAC 213 4 372058 0.5 CTCCAGTTG 214 4 381712 0.5 ATACGTTAC 215 4 471791 0.5 CGATTGGCT 216 4 509158 0.5 GTTTATAAG 217 4 521709 0.5 GTTTTCGTC 218 4 538839 0.5 CGAACCTCT 219 4 578702 0.5 CCACACACA 220 4 930972 0.5 CGTGCGTGC 221 4 1324367 0.5 TTCTTCAAC 222 5 116099 0.5 'CAAGTCTCG 223 5 159320 0.5 GAGCGAATT 224 5 207517 0.5 GTAGATTAT 225 5 280465 0.5 AAGTAGTT 226 5 286387 0.5 kCTTGGTATG 227 5 422942 0.5 TAATGTTAT 228 5 544523 0.5 ACACGCGCG 229 5 544555 0.5 ;GTCACTCCT 230 6 62983 0.5 AGTGATGAA 231 6 76141 0.5 TTATCTTGT 232 6 130327 0.5 kGTGATTGTT 233 6 256223 0.5 ICTTTGTTGT 234 7 72577 0.5 CATTGATTC 235 7 110590 0.5 TCACCGGAA 236 7 323655 0.5 CTATTCTGT 237 7 423957 0.5 3GGCCAACCC 238 7 433787 0.5 AAATATCTT 239 7 559397 0.5 AGTAGTAAC 240 7 622201 0.5 AGCGCACAA 241 7 735909 0.5 CGCTGTTTT 242 7 800300 0.5 GTAT1T17G 243 7 836202 0.5 'TAAACAAAG 244 7 836587 0.5 AGGAAGAAA 245 7 905046 0.5 3GAAAAATTA 246 7 958839 0.5 26 WO 00/77214 PCT/USOO/16223 TTGGATAGT 247 7 974754 0.5 GTTTGTGTA 248 8 202655 0.5 \GAAAAAAAC 249 8 386651 0.5 FAAAGTCCAG 250 8 518998 0.5 FAAGCAGATT 251 8 529129 0.5 ATGAGCATTT 252 9 97114 0.5 kGGTGCAAAA 253 9 229077 0.5 ~AACAAAGAG~ 254 10 628227 0.5 ,AATTGGCAA 255 10 721781 0.5 kCTCCCTGTA 256 11 93528 0.5 'TCTATTGAT 257 11 144281 0.5 3CTTTCCTTT 258 11 146665 0.5 kCCGCAAAGA 259 11 231872 0.5 TTGTTCAAA 260 12 230972 0.5 ATGTGCTGT 261 12 320426 0.5 3CAGATAGCG 262 12 341324 0.5 CTGACTTAG 263 12 368780 0.5 OCCGGATGTT .264 12 433912 0.5 )TAACGATTG 265 12 449917 0.5 IAATAACGAA 266 12 673851 0.5 CTGCTATrT 267 12 712476 0.5 iTTCTCTAGC 268 12 712712 0.5 ATCACCATC 269 12 794710 0.5 TGCACTTCT 270 12 806833 0.5 CTGTTTATG 271 12 867350 0.5 TGCTATATA 272 12 1017911 0.5 ACATTCTAA 273 13 95707 0.5 TCTTAGTTG 274 13 158970 0.5 CGAACACTT 275 13 278341 0.5 GCGCAAGTC 276 13 283795 0.5 TTTTCTTAA 277 13 363037 0.5 AAATGCATT 278 13 390802 0.5 AAATTGTGT 279 13 395599 0.5 ICAATACTAT 280 13 826521 0.5 GTGACGATG 281 14 60143 0.5 ACTGGTTTA 282 14 118854 0.5 TTTGACCTA 283 14 335512 0.5 GCGTTTGAT 284 14 478481 0.5 TCTGTTGCG 285 14 728251 0.5 AATTCAAAA 286 15 35952 0.5 TTGCTTGGT 287 15 242742 0.5 .GTT-TTCCTG 288 15 304813 0.5 TTAAAGATA 289 15 331453 0.5 AGGAGACAC 290 15 448624 0.5. TATATATCA 291 15 544530 0.5 'ATGGAATAG 292 15 571210 0.5 CGAGTCGAA 293 15 758202 0.5 AAAAAGAAA 294 15 882567 0.5 TTCCAGAAT 295 15 969884 0.5 GGACAATGT 296 15 970607 0.5 'GAATTAAGA 297 15 979894 0.5 27 WO 00/77214 PCT/USOO/16223 CTATATGTT 298 16 582230 0.5 ATATATCAT 299 16 589647 0.5 GAATTGATT 300 16 744406 0.5 ACTGTCTCC 301 16 824649 0.5 28 WO 00/77214 PCT/USOO/16223 coC -c )ra a ot nr t N r qr DML CD C). . .

-----------------------------------------------------------

C)M0 ' D U)U 0,) r D N mC DN MVL . . . . . -c a a) )C I-( % -0 0 C4 00- 0U 00M t , W r 0 D--0 -r:0-)C5C 50 C 6 0C4C : 04CC1"U D , O( 14C n) - o0 ~ % C.,- -- V- r) C) c e c CL 0 0 L -- - --- a --- --- --- oC0 10 F D D L c D0 0 00L0 0-< L) < )L D85oC)z <C 00~ ~ I-Hp<h 0 *0 0 000 CD < 0~ <0D 00 U) 1- 00 C mc I- a)C C0CNC V- co 0 CD~ v LC o - C ) U)COLO 0< ~ : I.~ I- 0 I o I I 0 <~ c 0 mma I,--- ------- M -------- CNCD ) -- Dfl- ~ ~ ~ ~ ~ ~ 1 w~ 'I- rl O N v 0 " n v w 29 WO 00/77214 PCT/USOO/16223 M- CN C'J ' 0 0 CN 0 ~ N 0 0 - 0 .. - ' a.) CU C D- C O0 -C NMI LO O Fl GO CD ~ cc CDM W) 1- <<o0000 Quo-<01 0< ~0 00 Q 0 '-C0 C O 1' * 0D U00D C) p L) OC~ 0 -C 6 (D !) I. . ? C) C < .(D <301 WO 00/77214 PCT/USOO/16223 ,It 0 oC )C4O -L )a oc Dc v n c)c o( 0 -0

I.

F-( I (D < C-) r * r(1) r0 (D < <1 WO 00/77214 PCT/USOO/16223 00 1 ' DT"IT o Go -I-m o a 640) vIn U)(D - c-o O>D 0-aaO OI-0000 MC COCaUCU)VGM V 4 -1V O D0 U)T U 0)e L6 <-< r <-O <CI0-I <0 1-0 or--ococDo~t ~*00cDO)< 0 < < F- < ( L) C) 0 <0 L) L) < (D co) 0 02 WO 00/77214 PCT/USOO/16223 00 .0 t6 I- < C) (D D <- C-) < << l C)< ( L)0 ( C) ~o< 000< 0L Mo O0c 00t 0 c -c n t o nmN-V-------------------N---------N--- N NN-oe MNL N C j 33 WO 00/77214 PCT/USOO/16223 C)OC) OaoCD i--oCD CD 0 a 0 e~j CD C 0 04 -C ------------------- 0 - -- - -- -- 00 < <) --- ------------------------------------ < F <D< ( v CD L Co CD C14 CV) C14 CN LO 34 C NC N WO 00/77214 PCT/USOO/16223 c Oc----------q------------ ---------4------------------------------- - CD C C4 C CD CD [ 0 ~ CN a [C%40 [ r [ [CD M- m- 0 D 0 o V[ [* C) a CD C- [ ,CNC C .[* .[e [. F . [. [ [ [. 0 H ~ O<) OOuO 0 000 [ [D [- , E E -E [ <~ ~ M ril 0 a o [ (D to [ CD N N co r- E - E CN ~ ~ 35 WO 00/77214 PCT/USOO/16223 ---------------------------------------------------- C .

----------------------------------------------------------

11 c I I 1 1 1 D f ( U 'n ) )"t00N o 0 D o a )0C lo00 C 0 o I)q -vt . na N w L 0 I- ~~ ~ ~ ~ r T.. V- 'rOO-ooooooo Q< LDO (D < oD<a r C < (D 0 < Z.) 36 WO 00/77214 PCT/USOO/16223 coCq1,0 0, m 0 0r q o C', o m m

-----------------------------------------------

CD I 'tC vP------- ------------------------------ M 0P- u)cq W Mr- M N ) aaC o maC 0 o4-W M 0 r N M V 0 0r-M M 0 -M M 0 C 0 0-)< <0C G 0 u~c LfC-) U 0 0 < < -~I -C U 0 V <<CDDt o(DcDOi8co0ul K< m ( --- m-m--------- w I-mw m1- DL 37 WO 00/77214 PCT/USOO/16223 C (DOO C) a 6 -o N9ccor-o0

--------------------------------------------

0 a o) I-~~ 000000000 0 0 < 00(0 0 0 c-c -( o 0 c m r- Ln r- N m O C') m-1m< v< V. 1-001I- I o0<00<38 WO 00/77214 PCT/USOO/16223 OC )C ~ -0 0 T -C C C4Ia T ( 0 mL a

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0 0 0 0 ~ F- 00D L) 0 < r- i(D * <C (D 0 (-)r 39 WO 00/77214 PCT/USOO/16223 [ C[ 0 [- o [ [ [ o m a- m [ [ [ [o 0 n [ [ OL a6[ E [

+

C 0 L6 ------------ a ----- -- - - - - CD a a M a 0 tn Uvv .0I I 0 L) C- [ F- I< 3 C) C) <(D < () ( -r 0c ----------------- m -- ------- CR ~ ~ m r-a0L --- 0 --- 0c --- wm 40 WO 00/77214 PCT/USOO/16223 c: * ' c o0 , rv )C )0 o' 0( nc )v c ------------------------ D--------------------------- COO OO)CmCM OCO W w o N C C- -4------C)------------C------0r- MCD M n r, w0 Nm O r Ma O v nL n 0 0t OLOU)L 0 0D( DF < <)

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00<1 < < < 00C CD---------4----------------- -------- ------ ------------------ LOITCY 0 C v0)r v00 r, inC)CV C ) - I ( C rco 41 WO 00/77214 PCT/USOO/16223 LOCLO W w r 0'-0 N~~0.~.C4 0 0..................CD c-4o 0'- CDO - O 00 O ~ e,

-----------------------------------------

<4<< <0 L <- 00( I L Ml LnC o (D cn 0 tn 1-~ ~ ~ ~ ~ ~~C CD m- C-1 -1 -1 -1 1 -1 -1 - -D - - tn (D 1- 'T CNC0 m- CD 1- 1- 1-1 -1 -1 - -1 -1 -1 -1-1 -1 -1 m--------------------n L c -------- w I< < I 00mC M T c C0vvm00 nv - -- v ( 42 WO 00/77214 PCT/USOO/16223 * - -- - --- M - - t--------- CD [ -- [-------F ---- a - C 0 CD 000 a00 OD - [ E < r < 0- <

-

< 4 - 3 C) E* u 0 -E -E 43 WO 00/77214 PCT/USOO/16223 co0 vc or DO O-C)L C -' t m o o n o a 0 V-T-V- I r <-0 0D (D<.< d< 0 (D 00-- 00 0 00 00 < U) (D 44 WO 00/77214 PCT/USOO/16223 OOO OO O~O OO ~ OO OO 1..aO m Mu)-------------------------------------- --------- -------------------- IDC n"tI ) VU )q )L OC C%4( D C) -) V C 0 0 L6) LO ( o c D t c 0v-r _ - r - I- r _ r o G N -NNNN 0 <------------- < - - - - - < 45* D WO 00/77214 PCT/USOO/16223 C) (D co - 0----W-----------------------------------------------c C)C D0 N0C 1 )C 0 06 U l 0 0 ) C4C) L D r OCi0 N C)I f 0r O 0 1 0 (D D CD < < - - < <tI< D - 0 <00<<l 0 <-< (D (D 0 <o-I<l-CI <00L DF- 01- 00 D -0 0 a~~~~ ~ ~ 0 F!--<F ) Qu0( 46 WO 00/77214 PCT/USOO/16223 N C 0 I I < 00 00 <Uo<< <. 0 < (D a<00D ~ ~ CO < 15 < < (D a a 0 D Q -(. <<< <<1000. 00 0000D ------------------------ ------

----

< -- - - - -- - - - < - - -) C - - - - - - - - - - - - - -- C-) -. 47 WO 00/77214 PCT/USOO/16223 CO ---- D--D---------C-4-M-CD-v---CD-'-* 10 In '0-a------ --- ----------------------------------------------------- C [~~~~~~ [ C =[[ma LO~~ ~~ 0 [4 - 0 [ [ - [ . 0 6 a , : 0 C C Q.) (U~ L L' L"0UULL 0 0 < S I I [ E [ [ [ r[ i <e Io [r 00 i[ 000 .00 [it o 0 [ [ [ [ [ v m [ [o m 0 m 48 WO 00/77214 PCT/USOO/16223 LO mc . -00 0 ) C)c lM M q OL 0LO' DC 00 r- r -- r-- -- r----------------------------------------------------------------- F ~~~I f c66v6I I (D <( << <<0- 0 C0F - - U 0< D.)00 < (D< C < ---- --- 0 << OOO (DI~ 0 0 -) H< CQ 00C 00 (DHO HDo0p( (D HD 0 1(51 ( 0 0 a 0 1 ,I, III I 1 - H 'I (3D03 q N C N W0 W) V~ in) 0O 'T ~ cn) M) U U) (N 0 V) (N M0 M V (0 49 WO 00/77214 PCT/USOO/16223 C: 00v0 DM r---m-------------------------------------------m -- m m 00 4-~-W o N ' o c MC aaaa 01 r.T-T NNNNNN v0 v v~ v vq v v f i l fl0i lf l

-------------------------------------

F- < <) F-U<0 (D( () < 1K < <c.jc 0 r 0 oD (D <- OD < C)( <<00 D O < < C) UC-) U (D0 50 WO 00/77214 PCT/USOO/16223 CD a m N0 N- --- ----- 00 0 .I I L6 0 O0r F T.)V C- DC C-) 0 D( D( I -DF 0 < F- -Q O 0 0 l CD------ -------- -- <---- (D ~ ~ N-------------OCNcnLON T T - - - TI - MV LO IT q-coI vcoI-N -- v N n C MCN D51( WO 00/77214 PCT/USOO/16223 M CNMC SC oa o T"toI o o c0 0 0 < O 0 0 0 C V ) 0 () an. 9 E)6 a 0 11 a (D <000000DI0 0000 0000 000000000000000 52 WO 00/77214 PCT/USOO/16223 [D F F 04 Ni 0' 0 r ' [ F C -------------- 9CO.c ---- ------------ M----------------------L O5 - -, EO r- E r- w m

.

U' Ln - Lot O00L <00P i -I: <006 - F-ri<F i U E < C.) C) ( < ( D -- F-- F -- F F F ID Q Q LO~ ~ ~ F [n 1, 0 c v c 0 "- cn v [Dv C4 53 WO 00/77214 PCT/USOO/16223

----------------------------------------------------

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WO 00/77214 PCT/USOO/16223 Ei Ei.. .. .. . o~~~55 a 5 D 5 o 0 0 C 0000 555 55 500550CD 0 CD Q 0 55 5 CC LOd a) 0 D-0 0 0I CI-< <-C0 0h 0 0< 0 <~ <o << 00 CD CD~ 3 2 00 ) )0 C) 0 00 0 0 368 WO 00/77214 PCT/USOO/16223 it Uk 4t~ 4t 4t :t 4 4 55 a 5 a5S i5 -0 ca 50 -0 a a Ld a)5 5 00 0 5 05 5 0 B 04 VL Or CO~ 505 NMI )( 0 D - f O( ,0 ca ~ ~ ~ 4~~ r-~ 4k -r l l -r r I - r l -r-r -r l -r 00 F <-1 C))<I 0U(DO < C O)OOOO) OO0O1 F F 0 0 0 (.) 0 0 C) 0 o -)o0 Qo 0 0 C) C) 0o 0 uoC)(.5 L) 0 0 oo 369 WO 00/77214 PCT/USOO/16223 55 5 -~. .. .. .. .. -. . - . [~~~T [- [- [- [ [[ [ < < < ) [D [ [! a a 0 1 ) 00000 0 0 00CD0 00C 0 C)CD a 0000 a 0 050 5 370 WO 00/77214 PCT/USOO/16223 E S - CS555 5 .. .55 55555 05D50a055 D5 0 C) 0 oCD0 CD a C) a C

------------------------------------

D CD 555D 555 5 5 5D a 0 DC D CD 0CD 0 CD CD 0 0 5 5 5 0 5 00D5 05D .4 0 NM L -M M 0 N m vL 0-f-r -r , -r , w O oc oc 0c oc oc <0 <0 <-0 Q 0~ <<<u <<1-<Q 0 < < 00 CDC00000000000000000000000000CD..C~ 371 WO 00/77214 PCT/USOO/16223 .. 5 .. . . . . E 55555 50-*Da 5 55 , ocoS C-5 55 -, - (D~~~~~ ~ ~ ~ r- CO -n C n'Tt t-w m 0 e MIT O01- CO CD CD 0 0 <0 < : S00C0>- D0C 0<h<0CD0 0 CD CD <<O0D0CD 1 C)CD0 0 0a C) 0 a 0 0 0 0 CD0 CD C 0 C)C) D 0 0 00 0 CDC) l C C 000000o00000~c~oooo3720 WO 00/77214 PCT/USOO/16223 S - [ CD 0 P1 >>>90 oooocooa 0D0 ID 00o050 0, C) v 0 ( - O v [ [ E * 6 [ 0 D co I-p-CDD 0 [ OOE E E F E [D E a a o a 0) - ) 00 E a C C 0 0 C). 0 0 373 WO 00/77214 PCT/USOO/16223 C C:)ooaoo5C Q55550 )0 0 5 5 5 0 0 0 5 05a

-----------------------------------------------------

4t t t 4 * 4 5500~~ ~~ I~5 I05 5 00 0 ..-------. ------------------------------- C6 C C C0 0 C 00 00 0C 4- 5 ;C 4t4t 4 0O O a 0 C I 0 00 F-- F CDC - --- -- - - - - - - - - ------------------------------------------------------ 00001--0374 WO 00/77214 PCT/USOO/16223 a C) > 0 > 5;5 ;5 ;5 C, CD CDO50 5055000 050 c0 000 ED 5- 5- Eooooaoa55 o coc >>>>C V LO 0 t- W 0 0 M V to 0SS ~(D (.) < 00OO J C J C < < <0<- 0<1<00 i0 Q0 00 <<<)L)L)0 0D<( D0 -C'D - 0 D( D()0 0 01- 0 0--< (D0 1D-D1D-i-DQ - 0 0 00 0 0 I- <<0 0 a 0 00a CD0 00 <0 C ) C D0 ( -CDC )0 0 0 0 C D-C CD0C ) C ) C C D( 0 375 WO 00/77214 PCT/USOO/16223 C5 5 00 00055050050q [ 05 [S ED 5 5 .. . .. ~OOO0O05555 500550 055005 0 ~ ~ ~ ( oo 5 0 05 05*5055or0 5 4- (D ILI CD 0 (D (D 0 0 D 0 1 376 WO 00/77214 PCT/USOO/16223 C c: 55 5 5 5 0 aCC : 0 C >5 QQQ~Q~o 0 a a o o CD a a55 0~o 00 O <U 0 0 ( o<r* ()p( ( U -( U, *0 0 0( C 0 0 0 000aa 0r : 0 0

---------------------------------------

WO 00/77214 PCT/USOO/16223 5i 55 o 5;3 ; 5o55 5 3; q5 5 O5?55 5OC)O C) Coo 0 C) a C5 -* o* a C c -0 a000000 a 0 c.) LO O D- 0M Nc ) cor-0 O0 ()C Nor)' . Df00 D F-0 <0D 0 0-C < <1 F~~ <~i-- eoj<ooI..

-----------------------------------

L) - 0 0O -0 - )0 0 (.9 () (D 00000 378 WO 00/77214 PCT/USOO/16223 > >> CooCD(D0 D 50 55 555 00 CD0000 0 0 0 0 0 5 5 0 5 0 5 S S o o d 5 5 5 5 5 5 0 C.) 04 ---- L ( ---------------------------------------------------------- ------ 00-0 000000U0 09 *8 OOOQU UQL) UU a0 0 079 WO 00/77214 PCT/USOO/16223 C)C)aC C)C C 0oo oo 000 00 0 000D 60 ~ ~ 00 5 5 0005 00500, 556 tfit E i E D, (s - -- -- -- - <<oooooooo Oo:)o ooooo 0 0 ' 0D 0 0 0 20 ' 0 0 0 0 0 CD ' 0 25F 0aZ 380 * t tc WO 00/77214 PCT/USOO/16223 500 55000C0000Da0C a~ o

---------------------------------------

t 4t t *k t 4t Q0 Q Q 000 Q Q Q 00000 a0050 0050CC (DC --------------------- ---------------------------------

F

< 00 m C- CD C 0 a0 a- 0 CD 0 CD)O<OOO<ID-00DC!) 381 WO 00/77214 PCT/USOO/16223 ES 55 5.. .. .. 5 .. 5 0 0 5 0 O D5 C ) 5 o 5o0 05 oC ) E E 0 a 5 CD O CD > CD 5o 55 >555>5 5 55 55 5 cn ~ CD I [o M [ a 0 [ 0 [ [ [ [ a N I 0 CI a [ a [ V- r- [ [ [ ~< < 000 1 <- < 0 0<I 0o 'D 0 0 (D (D (D 0000 a- 00 (D9C 0 (DC 0 0 (D 0 D D 0D [ E[[l | 0 0 V a [ [[[38 382 WO 00/77214 PCT/USOO/16223 0 C i i 5 ; >> >> > > Q 5 55 C) >0>>00 O 0-0 0 -- 0 500 <05o0ooo0 1< (8 << < < C: Ca DC 38 WO 00/77214 PCT/USOO/16223 > >5 >5 55 55 555 3 3 5 55 5 5 55555;5 55-- -.5 a05 55 5 CD a 0 5O 0 CDO55ao 5 ; D3 55 55 55 5; 55 5 0D c D ac O O 5 0 5 0 CD o56CD 0 23 C1 M )C - 0 ) C % f l C - W M aM V 0 C - 0 LOiCU) t O t) U)L) U 0 ( D ( D o ( o c D r - r -r l -r-r 0-V - I - T - T I f <-0 0D<0 < <000000<000000D0

<--------------------------------

00oo o'-. -o0 -000--00 001'-o0-CD

-------------------------------------------------

384 WO 00/77214 PCT/USOO/16223 0 0-0 05 i i i 55 3;oo S 55 Ca cla .0 0C 00 oca 55 555 0 DC 0 L P-- - - - - - - - - - - - - - - - - - - - co c o coCocoo C 41 4C4C NF 7F CD C (D CD Cl 0 a a a - - C0 0 Dc 0~ 0 a 0 00o1-1D a0a0 a0la 385 WO 00/77214 PCT/USOO/16223 - ) CV Co co co u V) n ) Cf CV) Qr) Qr) m ) m f m o m. n c)c)c)C CO0 n' )MMM0t n L dn o COC -( OU)L DI tr't0 V( )V MC Da C)a L 0 C o I N I nt N t N I --------------------------------------- _------- U- < 0.. 0< 00 (D C-) 0 0 D 0D 0- F- F- F C4% 00 - DC)0 - - - - M---------------- V-V- - - co CD m 0 D- LID) cvO~ 1w M a V N T . r OC Dr IN c - cn LO W- w.. IM.. w v v. . v IN -N v v Oto N F- I- m0 0a CN IN f- 00 a 0) - oN 0 n MN VN qN VN V VN VN VN (N LO U)l 386 WO 00/77214 PCT/USOO/16223 I I I -A C*4 C 04 m CC) c V) ) LO W) o~ < ('< < < 0<0 -0 0 <o o<0 0-~ 1I< I-ii- 0~000

-------------------------

01- (D a)14 C' N CN CN 04 N ' M ' M ' M~ M Nt IT (N ( V N IV IT T V N 'T (N) (O (N (N (n -D( D 0 ------------- --- ----------------- --------------------------- - r- CD Mr- M I V 0 w 0N M Nfl a 0387( WO 00/77214 PCT/USOO/16223 to r-- 0-----------------T--O---- M00N-T---- M 0N C%00(000CD( c6 c C 4 ~ ~ ~ ~ D 0OI Cl009oC 9 <ao 1 a) - 00 O0 O0 TC mC - l .1 on-c ,- 0o ( (1 0 0 0 i DO 0 o r <) 0)' ( I - 0) 0 1 0 (0 <0( 0( J0 0 rN IN( N(N~ 0 < 0 C) Q o (D <( o----------------------- ------------- (NNNN(Nr 0---------------------------------------

------------------------------

(D 0 D - - 388 WO 00/77214 PCT/USOO/16223 000 0 0 o00 0 "C )( Y )C) C 0coCD 0 CDCDC) D 0 m m m Ie co m CV Ie Cf CV Cf I nC)m m N IT IT q I I to D 0 -- n O n 'T V U' r-LoU)V) L I) .) O O O L L L L 9 a9c9 9 (o9 9 9 6 6 6 9 (n 0 0 o 0 e <-< ca U((D r (D F- I D (D(D - - 1 " X (,9V kFl

---------------------------------------

389ou" WO 00/77214 PCT/USOO/16223 C'4 o -T LOCD ------- 0 -------------- D to If 0) U) Ij co io n o O TV 1 ul0a)C N N Ci LOl LO0 D r- r- M~ M~ M~ M 0a C., W W V it-a- a CD D (D OD co co 0 r_ a )0 c) f )C coI-( D( Y cr -0 N( V TVVVVq LC) LoUl------------------) ~WCD0~CD1 31 10C. rODr- I UOURCD 390 WO 00/77214 PCT/USOO/16223 ---- -1---- - -- 1--- - -- -c~dc I, -c O 0 0 L - i. -V-0 0 CD D C 0 C OC C 0C) D co 6 :a << oo (D E 001: O o c F- (1--D C) 0 00 0 00 M<CD NMi.-- O0 '' J- 0 V- 0 -0M INM0VM CDC) 1 C - M0T-VM 0 0V0 00a o)a)m 0 ) O 00 Mt, MCNa VM - 14(It- 0391VC1 WO 00/77214 PCT/USOO/16223 m m D m Lo- - - - - -o - - - D 4FI m D0a , n r qU)0 0N00r N n0 r oL oNN o00Vp V 0 o o Lo r NI JOOPP<<MLm urfl <~~

-----------------------------------------

392< WO 00/77214 PCT/USOO/16223 fo r-00 O o r-Nc? o O O O OO0O) c)'TV) w0 MO MO 0 0 0 o0 - - T-L r

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co U) m 0 N m Lo 0 0 Lo99 T '7 04 M - - - - - - - - - -- - I- - M-- - - - - o q M L l o~~~o 0000000 1 c600 ~ <1-< I-< N M W l- l- M MLO N M W L W 0 M 0 -r oMt I o00C Vt M 40 l)toLncocoCDw o ww r w9 o o3930)c WO 00/77214 PCT/USOO/16223 .TL D.-------NC - L , C DqV-Nc)q O0r- Oc w- w - -- - - - - - - - - - -M-- - L 0 L 000 0 NNNNNt c LnL - ----------------------------------------- W 0 V MC') CDMm 0t r-a0 .4cO O< r0000000000-------------------- < < < <(D C C) D 0 39-4 WO 00/77214 PCT/USOO/16223 *6-o0666996' oco-C LOC mL IZc 4- r.- aLo c Lo n qP- LO w- N-- - - - - - - - - - - - C- C 00 (D 0 0' < < I (D U U u L) U (9 L) r C < )uL ( )L < << C) ( (DF- -- ( . . 0) ) c) ' c) D D 0 0 c N0 1-39MM5 WO 00/77214 PCT/USOO/16223 oo ~ ~ ~ C co0 00 0c o00c D0 )0 )a) 0MCD0)000 00 00C 0 0 LO U) L LOLo O L LOLO O L LOLO O L LOLO o L LO C ( D D c c w t 9 99 666o -- LO ILO Lo) U) m m '0 NJ~ U DC -C)C)CJC '1 CO '- CD - * 0 <<<<<..Di -0 00 <<0 o 0 I- 500 000 0 0 << 0<<0 0~o< Cl) 0110 01 < _oo 0000<-- 0000~o r-co -D C ) % C C D W 0 0 r- T 0D r ' T 2 0C C4 v MO 0) CD a (M M ) Im N C r I, ') C D- '4D' (D0 0) DmC m ~0cn V'IT0V 0 0 N m0C 396 WO 00/77214 PCT/USOO/16223 CO 0 0 N C) VIn C r-CO ) C - N M 1 LO(D - CO0) 4 C) L C) c CNN C' CNN C4 N 4 N(N c) M M M C ., too o ~ ~ ~ ~ qr 00 0 0 0-- < 0 k~00 F- 0 0 0~H 00 ~ ~ ~ ~ ~ ~ - I-F0-<~0000 0 .o o~ o. r-c) to UD U, U, U U U, (D 0)L ( T- N O -~~ V ) C) 9 ' 0 W) v~ r ~ ~- ..- r- a) ,- (nLON NN (N r C 40 C . .N) cn (*CN 00 C4V -M -- ) CnC)q o 0 0 0L - - -- - - - - - -- f)m o c) 04(D(DIT0 m 4 o C) ) ococe ( 0 L - 0 39V(D7 WO 00/77214 PCT/USOO/16223 ( 0CD CD co co (D (D (D CD q q 6 6 6 6 6OC qCo D r '-...OC 00 0OV"(0 O0 O Oc D0 cn r-'-- C r- F CDa U 0C 0 ooo( 0<<< CD I.- C I o N (! 0C)c C o Da)C 0 o om0 l D0 ) to( )0r-r ()-f T-288 M9C T-CC M< 0 MF A Tc 0000 " -CDC % c 0C C) <<<00a 0 DOLOO m) m-- cn 000 v0<<0 V Dt I-r - l -r-C na (0 < << 0< <1 <o < < < (D D (0D~ < a0~- Lg r F << D OM 0CD0O << <C. C)< I D ; <000r( oIu < F . < OF-~ J JC cts ( 0 < DC 0 < - -- - < (Fl--- --- Q---2 3980 WO 00/77214 PCT/USOO/16223 (0 (D CD W (i co (D -l P- P- r- P- P-I-r-r-r T-~~~ 9-r 9-. L~oa 0 o 0vi onOoo r rl D -0 -IT mmo w wmrcm O0 l eL 0 ' MmJ 4-l l - I r L - r - r - r - L Lo------------------------------------ -- V--- ----- 8nL i &~ 31( < (D c o 0 oc I*i Jr-PP 0< < ,:c < < 0 - ,J 0 C ~O~O~O)O) C.) J a~EDN 1'0 (D ( C)(,D(D (D 0 1)(D399 WO 00/77214 PCT/USOO/16223 (D (D LD (DCD-------- - -- ----------- -- --------------------------- -- m m 0t , I 0U o o1 )1 T- I- T- a 09 9 9 00 0 0 c 0 CD ejcC to 0 (D - w w c (D < L)C D <o <(OO <- 0 dJ 00 000

L)

M ------------- t- T- 0 - q I- V 00 00 0 -U C 0v f -0 00ML , oo CYD o1 CWr cl I UT-0011MMM 400 WO 00/77214 PCT/USOO/16223 C' - -m or I'(- Q r* 0 0 0 00 C)( n cn t O 0) T-- 'r- U- O(N V Ta V(D"r 1 C40 -r <.0 I0 000 401 WO 00/77214 PCT/USOO/16223 00 ) :) -N---V-- --------------------- ----------------- 66V6OL - )U l0 )t)V D( D( 0CD( DC -r -I l

------------------------------------------------

ci CD C 000 Ce D0 L f - T( nL lU)0 oc oC o<00 <-<<< <1 00 F-0 1-1 00 <1o 000 o ~00 L-) 00 00000009 0o <00 q- L) ) 0 0-D c< (D0 <<0 00 0 0(D~ 00< D<01-0-0 F ~ - 0 D0 DDF- - - ---- --- -- L) < D F < D L < < U) v D MM 0 0 wa 0 tn inw 0O0 0 ) oo CN 0r-LOw r- ww m co -'J0L 0 C OL 0)00 CN N (DN 4 D O T r ) O L G f,(0M D0 0 -r qq~~1 0) C41-0 1Cf 4C -0 ) )0 Nrr-r N Y)0)0 CY c) V - o(Dto(DI' r f-r-r-r402CDC WO 00/77214 PCT/USOO/16223 r- rl- --- - - -- P- r- r-wc 0 C) C) c\ r r-0a CDo (DO OLOiOq N F m0 q.v v r N 00 00 00to 00 0< u. 6) 6) U)( ~ (DO(D<ODOC8)~ 0 0 <r (DO 0 0 0 < <,O< 00 o( CD Ci0 0L)c) ar-m0m00 00 Naa 1-q 00w-( ot -a0 NNvw' C) 0IT tU)Nf-oT DC C)C - I - - - - - - - - - - - - - - - - - - - - - - - - - - o L LO (0 L co a C4 ( C) ) r-CD 43 00 0 e 4c WO 00/77214 PCT/USOO/16223 0o00 00 o0'-00' L-oo r- - . c) Co cs, LOC4C-)N N( (D 0-c0 CD qTw w- C) v vCoD 0 O c f r- i - 0i i V i r ' 404 WO 00/77214 PCT/USOO/16223 References Archer, J. E., Vega, L. R., and Solomon, F. (1995). Rbl2p, a yeast protein that binds to beta-tubulin and participates in microtubule function in vivo. Cell 82, 425 434. 5 Bajwa, W., Torchia, T. E., and Hopper, J. E. (1988). Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL 1, GAL2, GAL7, GAL10, GAL80, and MELI; encoded protein strikingly similar to yeast and Escherichia coli galactokinases. Mol Cell Biol 8, 3439-3447. Basrai, M. A., Kingsbury, J., Koshland, D., Spencer, F., and Hieter, P. (1996). 10 Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae. Mol Cell Biol 16, 2838-2847. Bishop, J. 0., Morton, J. G., Rosbash, M., and Richardson, M. (1974). Three abundance classes in HeLa cell messenger RNA. Nature 250, 199-204. Burkholder, A. C., and Hartwell, L. H. (1985). The yeast alpha-factor receptor: 15 structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res 13, 8463-8475. Chambers, A., Tsang, J. S., Stanway, C., Kingsman, A. J., and Kingsman, S. M. (1989). Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP 1. Mol Cell Biol 9, 5516-5524. 20 Denis, C. L., Ferguson, J., and Young, E. T. (1983). mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on a nonfermentable carbon source. J Biol Chem 258, 1165-1171. Dick, T., Surana, U., and Chia, W. (1996). Molecular and genetic characterization of SLCI, a putative Saccharomyces cerevisiae homolog of the 25 metazoan cytoplasmic dynein light chain. Mol Gen Genet 251, 38-43. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825. Elledge, S. J., and Davis, R. W. (1989). DNA damage induction of 30 ribonucleotide reductase. Mol Cell Biol 9, 4932-4940. Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., 405 WO 00/77214 PCT/USOO/16223 Galibert, F., Hoheisel, J.D., Jacq, C., Johnston, M., Louis, E.J., Mewes, H.W., Murakami, Y., Philippsen, P., Tettelin, H., and Oliver, S.G. (1996). Life with 6000 genes. Science 274, 546-567. Gottschling, D. E., Aparicio, 0. M., Billington, B. L., and Zakian, V. A. (1990). 5 Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751-762. Hagen, D. C., McCaffrey, G., and Sprague, G. F., Jr. (1986). Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. Proc Natl Acad Sci U S A 83, 10 1418-1422. Hereford, L. M., and Rosbash, M. (1977). Number and distribution of polyadenylated RNA sequences in yeast. Cell 10, 453-462. Irniger, S., and Braus, G. H. (1994). Saturation mutagenesis of a polyadenylation signal reveals a hexanucleotide element essential for mRNA 3' end formation in 15 Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 91, 257-261. Iyer, V., and Struhl, K. (1996). Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93, 5208-5212. Kurjan, J., and Herskowitz, I. (1982). Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha 20 factor. Cell 30, 933-943. Leeds, P., Peltz, S. W., Jacobson, A., and Culbertson, M. R. (1991). The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev 5, 230 3-2314. Lewin, B. (1980). Gene Expression 2, (New York, New York: John Wiley and 25 Sons), pp. 694-727. McAlister, L., and Holland, M. J. (1982). Targeted deletion of a yeast enolase structural gene. Identification and isolation of yeast enolase isozymes. J Biol Chem 257, 7181-7188. Michaelis, S., and Herskowitz, 1. (1988). The a-factor pheromone of 30 Saccharomyces cerevisiae is essential for mating. Mol Cell Biol 8, 1309-1318. Mushegian, A. R., and Koonin, E. V. (1996). A minimal gene set for cellular life 406 WO 00/77214 PCT/USOO/16223 derived by comparison of complete bacterial genomes. Proc. Natl. Acad. Sci. USA 93, 10268-10273. Nguyen, C., Rocha, D., Granjeaud, S., Baldit, M., Bernard, K., Naquet, P., and Jordan, B. R. (1995). Differential gene expression in the murine thymus assayed by 5 quantitative hybridization of arrayed cDNA clones. Genomics 29, 207-216. Nishizawa, M., Araki, R., and Teranishi, Y. (1989). Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae. Mol Cell Biol 9, 442-451. Renauld, H., Aparicio, 0. M., Zierath, P. D., Billington, B. L., Chhablani, S. K., 10 and Gottschling, D. E. (1993). Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage. Genes Dev 7, 1133-1145. Rose, M. D., Winston, F., and P. Hieter. (1990). Methods in Yeast Genetics. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 177. 15 Schena, M., Shalon, D., Davis, R. W., and Brown, P. 0. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467-470. Schmitt, H. D., Ciriacy, M., and Zimmermann, F. K. (1983). The synthesis of yeast pyruvate decarboxylase is regulated by large variations in the messenger RNA 20 level. Mol Gen Genet 192, 247-252. Sikorski, R. S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27. Singh, A., Chen, E. Y., Lugovoy, J. M., Chang, C. N., Hitzeman, R. A., and 25 Seeburg, P. H. (1983). Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone. Nucleic Acids Res 11, 4049-4063. Smith, M. M., and Murray, K. (1983). Yeast H3 and H4 histone messenger RNAs are transcribed from two non-allelic gene sets. J Mol Biol 169, 641-661. St John, T. P., and Davis, R. W. (1979). Isolation of galactose-inducible DNA 30 sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell 16, 443-452. 407 WO 00/77214 PCT/USOO/16223 Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270, 484-487. Zaret, K. S., and Sherman, F. (1982). DNA sequence required for efficient transcription termination in yeast. Cell 28, 563-573. 408

Claims

1. An isolated DNA molecule comprising a coding sequence of a yeast gene selected from the group of NORF genes comprising a SAGE tag as shown in SEQ ID NOS:67-81 1. 5

2. The isolated DNA molecule of claim 1 which is involved in cell cycle progression.

3. The isolated DNA molecule of claim 2 wherein expression of the NORF gene varies by at least 10% between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M. 10

4. The isolated DNA molecule of claim 2 wherein expression of the NORF gene varies by at least 25% between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

5. The isolated DNA molecule of claim 2 wherein expression of the NORF gene varies by at least 50% between any two phases of the cell cycle selected from the 15 group consisting of: log phase, S phase, and G2/M.

6. The isolated DNA molecule of claim 2 wherein expression of the NORF gene varies by at least 100% between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

7. The isolated DNA molecule of claim 2 wherein expression of the NORF 20 gene varies by a statistically significant difference (greater than 95% confidence level) between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

8. The isolated DNA molecule of claim 7 wherein the NORF gene is selected from the group consisting of NORF N 2 1, 2, 4, 5, 6, 17, 25, and 27. 25

9. The isolated DNA molecule of claim 2 wherein the NORF gene is not expressed in at least one phase of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

10. The isolated DNA molecule of claim I which is genomic.

11. The isolated DNA molecule of claim 1 which is cDNA. 30

12. A method of using NORF genes to affect the cell cycle, comprising the 409 WO 00/77214 PCT/USOO/16223 step of: administering to a cell an isolated DNA molecule comprising a coding sequence of a NORF gene whose expression varies by at least 10% between any two phases of the cell cycle selected from the group consisting of log phase, S phase, and 5 G2/M.

13. The method of claim 12 wherein the cell is a yeast cell.

14. The method of claim 12 wherein the cell is a fungal cell.

15. The method of claim 12 wherein the cell is a mammalian cell.

16. The method of claim 12 wherein the NORF gene is selected from the 10 group consisting of NORF N 2 1, 2, 4, 5, 6, 17, 25, and 27.

17. A method for screening candidate antifungal drugs, comprising the steps of: contacting a test substance with a yeast cell; monitoring expression of a NORF gene whose expression varies by at 15 least 10% between any two phases of the cell cycle selected from the group consisting of log -phase, S phase, and G2/M, wherein a test substance which modifies the expression of the yeast'gene is a candidate antifungal drug.

18. The method of claim 17 wherein the NORF gene is selected from the group consisting of NORF N 2 1, 2, 4, 5, 6, 17, 25, and 27. 20

19. A method for identifying human genes which are involved in cell cycle progression, comprising the steps of: contacting human DNA with a probe which comprises at least 10 contiguous nucleotides of a NORF gene whose expression varies by at least 10% between any two phases of the cell cycle selected from the group consisting of log 25 phase, S phase, and G2/M phase, wherein a human DNA sequence which hybridizes to the probe is identified as a sequence of a candidate human gene which is involved in cell cycle progression.

20. The method of claim 19 wherein the NORF gene is selected from the group consisting of NORF N 2 1, 2, 4, 5, 6, 17, 25, and 27. 30

21. A probe comprising at least 14 contiguous nucleotides of a NORF gene comprising a SAGE tag as shown in SEQ ID NOS:67-81 1. 410 WO 00/77214 PCT/USOO/16223

22. The probe of claim 21 wherein expression of the NORF gene varies by at least 10% between any two phases of a cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

23. The probe of claim 22 wherein expression of the NORF gene varies by 5 at least 25% between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

24. The probe of claim 22 wherein expression of the NORF gene varies by at least 50% between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M. 10

25. The probe of claim 22 wherein expression of the NORF gene varies by at least 100% between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M.

26. The probe of claim 22 wherein the NORF gene is not expressed in at least one phase of the cell cycle selected from the group consisting of: log phase, S 15 phase, and G2/M.

27. The probe of claim 22 wherein expression of the NORF gene varies by a statistically significant difference (greater than 95% confidence level) between any two phases of the cell cycle selected from the group consisting of: log phase, S phase, and G2/M. 20

28. The probe of claim 22 wherein the gene is selected from the group consisting of NORF N 2 1, 2, 4, 5, 6, 17, 25, and 27.

29. The method of claim 17 wherein said step of monitoring expression is performed using nucleic acid molecules which are immobilized on a solid support.

30. The method of claim 29 wherein the nucleic acid molecules are in on 25 array.

31. The method of claim 19 wherein a probe which comprises a portion of the NORF gene is in an array on a solid support.

32. An array of probes on a solid support wherein at least one probe comprises at least 14 contiguous nucleotides of a NORF gene comprising a SAGE tag 30 as shown in SEQ ID NOS:67-81 1.

33. The array of claim 32 wherein the at least one NORF gene is involved 411 WO 00/77214 PCT/USOO/16223 in cell cycle progression.

34. The array of claim 32 wherein the NORF gene is selected from the group consisting of NORF No. 1, 2, 4, 5, 6, 17, 25, and 27.

35. The array of claim 32 which comprises at least 100 probes of distinct 5 sequence.

36. The array of claim 32 which comprises at least 500 probes of distinct sequence.

37. The array of claim 32 which comprises at least 1,000 probes of distinct sequence. 10

38. A method of identifying a candidate drug as a member of a class of drugs having a characteristic effect on gene expression in a yeast. cell, comprising the steps of: contacting a yeast cell with a candidate drug; and monitoring expression in the yeast cell of at least one NORF gene 15 whose expression is affected by the class of drugs, wherein detection of a difference in expression of the at least one NORF gene in the yeast cell relative to expression in the absence of the candidate drug identifies the candidate drug as a member of the class of drugs.

39. The method of claim 38 wherein the step of monitoring expression is 20 performed using nucleic acid molecules which are immobilized on a solid support.

40. The method of claim 39 wherein the nucleic acid molecules are in an array.

41. The method of claim 38 wherein expression of two or more NORF genes is monitored. 25

42. The probe of claim 21 which is immobilized on a solid support. 412