EP1190044A2 - Caracterisation du transcriptome d'une levure - Google Patents

Caracterisation du transcriptome d'une levure

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Publication number
EP1190044A2
EP1190044A2 EP00939837A EP00939837A EP1190044A2 EP 1190044 A2 EP1190044 A2 EP 1190044A2 EP 00939837 A EP00939837 A EP 00939837A EP 00939837 A EP00939837 A EP 00939837A EP 1190044 A2 EP1190044 A2 EP 1190044A2
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cont
norf
gene
expression
phase
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Victor Velculescu
Bert Vogelstein
Kenneth Kinzler
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Johns Hopkins University
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces

Definitions

  • 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 unrecognized genes.
  • transcriptome conveying the identity of each expressed gene and its level of expression for a defined population of cells.
  • 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.
  • transcriptome as defined above has not been characterized in any eukaryotic or prokaryotic organism, largely because of technological limitations. However, some general features of gene expression patterns were elucidated two decades ago through RNA-DNA hybridization measurements (Bishop et al, 1974; Hereford and Rosbash,
  • an isolated DNA molecule 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.
  • 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 phase, and G2/M.
  • a method for screening candidate antifi ⁇ ngal drugs 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 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.
  • a method for identifying human genes which are involved in cell cycle progression comprises the step of contacting human DNA with a probe which comprises at least 14 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.
  • 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-
  • 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 comprising a SAGE tag as shown in SEQ ID NOS:67-811.
  • 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 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.
  • Gray arrows indicate all potential SAGE tags (Nlalll sites) and black arrows indicate 3' most SAGE tags. The total number of tags observed for each 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 the basis of sequence information, and circles represent total genes.
  • FIG. 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 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 3 A, while data for Rot components were obtained from Hereford and Rosbash, 1977.
  • FIG. 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 abscissa and that from the - strand indicated below. Yellow bands at ends of the expanded chromosome represent telomeric regions that are undertranscribed (see text for details).
  • TDH2/3 , TEF 112 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 highly expressed in S-phase arrested cells.
  • Tag represents the 10 bp SAGE tag adjacent to the Nlalll site; Gene represents the gene or genes corresponding to a particular tag
  • 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 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.
  • Positive expression level indicates the tag is on the + strand of the chromosome; Negative expression level indicates the tag is on the - strand.
  • 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 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. New genes termed NORFs (not previously assigned open reading frames) have been found. They are uniquely identified by their SAGE tags.
  • 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 as determined by SAGE analysis.
  • Genes which have been found to have differential expression characteristics include: NORF N a 1, 2, 4, 5, 6, 17, 25, 27, TEF1/TEF2, EN02, ADH1, ADH2, PGK1, CUP1A/CUP1B, PYKl, YKL056C, YMR1 16C, YEL033W, YOR182C, YCR013C, 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.
  • 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-67-811.
  • Isolated DNA molecules which comprise yeast genes or coding sequences of yeast genes comprising SAGE tags as shown in SEQ ID NOS.37-
  • 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 isolated DNA molecules of the invention.
  • 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.
  • any DNA delivery techniques known in the 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. Antifungal drugs can be identified using yeast cells as described herein.
  • a differentially expressed NORF gene can be monitored by any means known in the art.
  • 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 as an antifungal agent.
  • Expression of more than one NORF gene can be monitored.
  • expression of 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 75, 100, 150, 250, 300, 350, 400, 450, or 500 or more NORF genes can be monitored in single or multiple assays.
  • differentially expressed genes are likely to be involved in cell cycle progression, it is likely that these genes are conserved among species.
  • the 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 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 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 PCR assays, or for detection of gene expression for Southern or Northern blots or in situ hybridization.
  • 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, 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.
  • probes are present on an array so that multiple probes can 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 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.
  • one or more NORF probe(s) present on the array can be 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.
  • 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.
  • 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.
  • microtubule poison drugs such as vinblastin, taxol, vincristine, and taxotere can be used to generate gene expression profiles characteristic of microtubule poisons.
  • 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 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.
  • these genes are differentially expressed during the cell cycle.
  • 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 N a 1, 2, 4, 5, 6, 17, 25, or 27, TEF1ATEF2, EN02, ADH1, ADH2, PGK1,
  • CUP1A/CUP1B, PYK1, YKL056C, YMR116C, YEL033W, YOR182C, YCR013C, ribonucleotide reductase 2 and 4, and YJR085C can be used for monitoring alterations in gene expression.
  • any number of these genes such as 1, 2, 3, 4, 5, 10, 15, 20, 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 differentially expressed genes using nucleic acids which are immobilized on a solid support or in an array, such as the gene arrays described above.
  • genes particularly cell cycle genes, are likely to be conserved between yeast and mammals, including humans.
  • gene expression profiles characteristic 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 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.
  • a gene expression profile 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.
  • transcriptome The set of genes expressed from the yeast genome, herein called the transcriptome, using serial analysis of gene expression (SAGE). Analysis 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, 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.
  • SAGE serial analysis of gene expression
  • 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.
  • SAGE libraries were generated from yeast cells in three states: log phase,
  • the number of SAGE tags required to define a yeast transcriptome depends on the confidence level desired for detecting low abundance mRNA molecules. Assuming the previously derived estimate of 15,000 mRNA molecules per cell
  • SUP44/RPS4 was measured by hybridization at 75 +/- 10 copies/cell (Iyer and Struhl, 1996), in good 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 among these three states (see below), SAGE tags obtained from all libraries were combined and used to analyze global patterns of gene expression.
  • the 56,291 tags that precisely matched the yeast genome represented 4,665 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 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 physiological states examined and the large number of genes predicted solely on the basis of genomic sequence analysis.
  • 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 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 Murray, 1983), had very similar expression levels (5 and 6 copies per cell, respectively).
  • regions within 10 kb of telomeres appeared to be uniformly undertranscribed, containing on 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).
  • Table 1 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 EN02 (McAlister and Holland, 1982), PDC1 (Schmitt et al, 1983), PGK1 (Chambers et al, 1989), PYK1 (Nishizawa et al, 1989), and
  • ADHl (Denis et al, 1983), are known to be dramatically induced in the glucose-rich growth conditions used in this study.
  • glucose repressible genes such as the GAL1/GAL7/GAL10 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).
  • mating type a specific genes such as the a factor genes (MFA1, 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, 1982; Singh et al, 1983) were observed to be expressed at very low levels ( ⁇ 0.3 copies/cell).
  • MFA1, MFA2 Mating type alpha specific genes
  • STE2 alpha factor receptor
  • NORF5 one of the NORF genes was only expressed in S phase arrested cells and corresponded to the transcript whose abundance varied the most in 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).
  • G2/M arrested cells identified elevation of RBL2 and dynein light chain, both microtubule associated proteins (Archer et al, 1995 ; Dick et al, 1996).
  • Yeast genome intergenic regions were defined as regions outside annotated ORFs or the 500bp region downstream of annotated ORFs (yeast genome sequence 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 or adjacent to intergenic ORFs (lOObp upstream or 500bp downstream of the ORF)
  • 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 means that the tag is on the + strand of the chromosome; if negative, the tag is on the
  • 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 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 transcriptomes from other organisms, should ultimately allow the generation of a minimal set of genes required for eukaryotic life.
  • SAGE analysis of yeast transcriptomes has several potential limitations. First, a small number of transcripts would be expected to lack an Nlalll site and therefore would not be detected by our analysis. Second, our analysis was limited to transcripts found at least as frequently as 0.3 copies per cell.
  • 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 - 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.
  • the analysis of the transcriptome in different cells and from different individuals should yield a wealth of information regarding gene function in normal, developmental, and disease states.
  • the source of transcripts for all experiments was S. cerevisiae strain YPH499 (MATa ura3-52 lys2-801 ade2-101 Ieu2- ⁇ l his3- ⁇ 200 trpl- ⁇ 63) (Sikorski and Hieter, 1989).
  • Logarithmically growing cells were obtained by growing yeast cells to early log phase (3 x 10 6 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.
  • hydroxyurea 0.1 M was added to early log phase cells, and the culture was incubated an additional 3.5 hours at 30°C.
  • nocodazole 15 ⁇ g/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). SAGE protocol
  • the cDNA was cleaved with Nlalll (Anchoring Enzyme). As Nlalll 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 Nlalll-based SAGE approach. After capture of the 3' cDNA fragments on 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 l i g at e d to each poo l : L i nker 1 , 5 ' -
  • TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGGGGACATG-3' (SEQ ID NO:3) , 5'-TCCCCGTACATCG AGAAGCTTGAATTCGAGCAG[amino mod. C7]-3' (SEQ ID NO:4).
  • PCR product containing two tags ligated tail to tail was excised.
  • the PCR product was then cleaved with Nlalll, 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 the Sphl site of pZero (Invitrogen). Colonies were screened for inserts by PCR with
  • Each successful sequencing reaction identified an average of 26 tags; given a 90% sequencing reaction success rate, this corresponded to an average of about 850 tags per sequencing gel.
  • Sequence files were analyzed by means of the SAGE program group (Velculescu etal, 1995), which identifies the anchoring enzyme site with the proper spacing and extracts the two intervening tags and records them in a database.
  • SAGE program group Velculescu etal, 1995
  • 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 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 lObp tag), not far from what we would have expected under our automated sequencing conditions.
  • yeast genome sequence obtained from the Stanford yeast genome ftp site (genome-ftp.stanford.edu) on August 7, 1996).
  • 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 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.
  • SAGE tags matched transcribed portions of the genome in a highly non-random fashion, with 88% matching ORFs or their adjacent 3' regions in the correct orientation (chi-squared P value ⁇ 10 '30 ).
  • 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.
  • a tag matched more than one region of the genome for example an ORF and non-ORF region
  • only the matched ORF was considered.
  • the 15th base of the tag could also be used to resolve ambiguities.
  • TACCACTCCT 9 EN02 YHR17 W 229 2-ph ⁇ 6-phoglycerat ⁇ d ⁇ tiydratase
  • TTGCCAGTCT 11 PDC1 YIR044C 207 pyruvat ⁇ decarbaxylase Isozyme 1 ⁇ . GGTGAAAACG 12 ADH1. ADH2 Y0L086C/YMR303C 182 alcohol dehy rooenas ⁇ I / II
  • TTGAACTACC 37 YKL056C 58 strong similarity to human IgE-dependent histamine-releasing factor (21 K tumor protein)
  • TTCGGGTCAC 38 YDR276C 56 strong similarity to Hordeum vulgare blt101 protein
  • CCAGATATGA 39 YIL093C 41 hypothetical protein ⁇ . TTTAAAATGG 40 YMR116C 38 similarity to ⁇ .crassa CPC2 protein

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Abstract

Cette invention concerne des gènes de levure exprimés de manière différentielle au cours du cycle cellulaire. On peut les utiliser pour étudier, modifier ou surveiller le cycle cellulaire d'une cellule eucaryote, pour obtenir des homologues humains intervenant dans la régulation du cycle cellulaire, et pour identifier des agents antifongiques ou autres classes de médicaments. On peut les constituer en réseaux sur des supports solides aux fins d'analyse d'un transcriptome de cellule dans diverses conditions.
EP00939837A 1999-06-16 2000-06-14 Caracterisation du transcriptome d'une levure Withdrawn EP1190044A2 (fr)

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US335032 1999-06-16
PCT/US2000/016223 WO2000077214A2 (fr) 1999-06-16 2000-06-14 Caracterisation du transcriptome d'une levure

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US8188255B2 (en) 2006-10-20 2012-05-29 Exiqon A/S Human microRNAs associated with cancer
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