EP0970202A2 - Caracterisation du transcriptome de levure - Google Patents

Caracterisation du transcriptome de levure

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Publication number
EP0970202A2
EP0970202A2 EP98902680A EP98902680A EP0970202A2 EP 0970202 A2 EP0970202 A2 EP 0970202A2 EP 98902680 A EP98902680 A EP 98902680A EP 98902680 A EP98902680 A EP 98902680A EP 0970202 A2 EP0970202 A2 EP 0970202A2
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European Patent Office
Prior art keywords
gene
group
norf
phase
yeast
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EP98902680A
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German (de)
English (en)
Inventor
Victor E. Velculescu
Bert Vogelstein
Kenneth W. Kinzler
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Johns Hopkins University
School of Medicine of Johns Hopkins University
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Johns Hopkins University
School of Medicine of Johns Hopkins University
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Publication of EP0970202A2 publication Critical patent/EP0970202A2/fr
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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
    • C07K14/37—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.
  • the 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, 1977).
  • Another object of the invention is to provide a method for obtaining human homologs of the yeast genes which are involved in cell cycle progression. Another object of the invention is to provide probes for ascertaining phase in the cell cycle of a cell.
  • an isolated DNA molecule is provided. It comprises a yeast gene which is involved in cell cycle progression selected from the group of NORF genes identified in Table 3 or 4.
  • a method of using yeast 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 yeast gene which is involved in cell cycle progression selected from the differentially expressed genes identified in Tables 1, 2, 3 and 4.
  • a method for screening candidate antifungal drugs comprises the steps of: contacting a test substance with a yeast cell; monitoring expression of a yeast gene which is involved in cell cycle progression selected from the group of yeast genes identified in Tables 1, 2, 3 and 4, 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: hybridizing a probe comprising at least 14 contiguous nucleotides of a yeast gene which is differentially expressed between at least two phases selected from the group consisting of log phase, S phase, and
  • yeast gene is identified in Table 1, 2, 3, or 4.
  • isolated DNA molecules which comprise probes for ascertaining phase in the cell cycle of a cell, wherein the probe comprises at least 14 contiguous nucleotides of aNORF gene as identified in Table 3 or 4.
  • FIG. 1 Schematic of SAGE Method and Genome Analysis.
  • SAGE SAGE Method and Genome Analysis.
  • the type IIs 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 Nlalll site.
  • Automated sequencing of concatenated SAGE tags allowed the routine identification of about a thousand tags per 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 (NlaL ⁇ 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
  • 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).
  • Tag represents the 10 bp SAGE tag adjacent to the Nlalll 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
  • 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 ascertained transcripts.
  • 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.
  • NORFs hitherto unknown genes
  • 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 greater than 10%, 25%, 50%, or 100%.
  • the genes which have been found to have such differential expression characteristics are: NORF N ⁇ 1, 2, 4, 5, 6, 17, 25, 27, TEF1/TEF2, EN02, ADH1, ADH2, PGK1, CUPIA/CUPIB, PYK1, YKL056C, YMR116C, YEL033W, YOR182C, YCR013C, ribonucleotide reductase 2 and 4, and YJR085C.
  • the DNA molecules according to the invention can be genomic or cDNA. 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. Any technique for obtaining a DNA of known sequence may be used. 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, 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. Expression of a differentially expressed gene can be monitored by any means known in the art. When a test substance affects the expression of such a differentially expressed gene, it is a candidate drug for affecting the growth properties of fungi, and may be useful as an antifungal agent.
  • 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 genes identified by the present invention can be used to identify homologs in humans and other mammals. 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.
  • 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 as primers for PCR or for Southern or Northern blots.
  • Preferably the probes are anchored to a solid support. More preferably they 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 can contain more than 100, 500 or even 1,000 different probes in discrete locations.
  • 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, S phase arrested and G2/M phase arrested.
  • 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).
  • 56,291 tags (93%) precisely matched the yeast genome
  • 88 tags matched the mitochondrial genome
  • 91 tags matched the 2 micron plasmid.
  • 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 (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.
  • SUP44/RPS4 As an 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 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.
  • 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
  • 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
  • ORF Nonannotated ORF
  • RNA components defining cellular life.
  • POL1 and POLS those encoding enzymes required for DNA replication
  • NDCIO and SKPI kinetochore proteins
  • SKPI kinetochore proteins
  • chromosomal expression maps presents a cataloging of the expression level of genes, organized by their genomic positions. It is not surprising that gene expression is well balanced throughout the 16 chromosomes of S. cerevisiae. As most genes have independent regulatory elements, it would have been surprising to find a large number of physically adjacent genes that had similar high levels of expression. Of the few genes that were known to have coregulated divergent promoters, like the H3/H4 pair, SAGE data confirmed concordant levels of expression. For areas like telomere ends that are known to be transcriptionally suppressed, SAGE data corroborated low levels of expression.
  • 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. 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 genes. Tags which were derived from overlapping genes, or genes which have unusually long 3' untranslated regions may be misassigned. Increased availability of 3' UTR sequences in yeast mRNA molecules should help to resolve the ambiguities.
  • nocodazole 15ug/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).
  • Total yeast RNA was prepared using the hot phenol method as described (Leeds et al., 1991). mRNA was obtained using the MessageMaker Kit
  • PolyA RNA was converted to double- stranded cDNA with a BRL synthesis kit using the manufacturer's protocol except for the inclusion of primer biotin-5'-T lg -3'.
  • 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,
  • CTGCTCGAATTCAAGCTTCT-3* (SED ID NO:6), as primers.
  • the 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 NlalH, 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 M13 forward and M13 reverse sequences located outside the cloning site as primers. PCR products from selected clones were sequenced with the TaqFS
  • 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 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).
  • 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 1 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 (for Figure 2, Figure 3, and Figure 4).
  • 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. For Figure 4, only tags that matched the genome once were used.
  • TTGAACTACC YKL056C 58 strong similarity to human IgE-dependent histamine-releasing factor (21 K tumor protein)
  • TTCGGGTCAC YDR276C 56 strong similarity to Hordeum vuigare blt101 protein CCAGATATGA YIL093C
  • hypothetical protein TTTAAAATGG YMR116C 38 similarity to N crassa CPC2 protein GGTGTCGTTG YBR078W 34 strong similarity to sporulation specific Sps2p TACTCTTCGC YEL033W 33 hypothetical protein TGTAATTAAA YOR182C 26 homology to human ubiquitin-like protein/ ⁇ bosomal protein S30 GGAGATCTTG YCR013C 24 weak similarity to lepra B1496_F1_41 protein TCAAGAAGTT YER056AC 20 strong similarity to nbosomal protein L34 AAAAACTTTG YIL051C 18 strong similarity to YER0
  • Rbl2p a yeast protein that binds to beta-tubulin and participates in microtubule function in vivo. Cell 82, 425-434.
  • GAL3 carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL 10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases. Mol Cell Biol 8, 3439-3447.
  • yeast alpha-factor receptor structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res 13, 8463-8475.
  • WAF1 a potential mediator of p53 tumor suppression.
  • 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, 1418-1422.
  • MF alpha a putative alpha-factor precursor contains four tandem copies of mature alpha-factor.
  • 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.
  • Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone. Nucleic Acids Res 11, 4049-4063.

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Abstract

Cette invention concerne des gènes de levure qui sont exprimés de manière différentielle au cours du cycle cellulaire. On peut les utiliser pour étudier, modifier et surveiller le cycle cellulaire d'une cellule eucaryote; pour obtenir des homologues humains impliqués dans la régulation du cycle cellulaire, et pour identifier des agents antifongiques. On peut les former sous forme de réseaux sur des supports solides pour étudier un transcriptome de cellule dans diverses conditions.
EP98902680A 1997-01-23 1998-01-22 Caracterisation du transcriptome de levure Withdrawn EP0970202A2 (fr)

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PCT/US1998/001216 WO1998032847A2 (fr) 1997-01-23 1998-01-22 Caracterisation du transcriptome de levure

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US7504493B2 (en) 1997-01-23 2009-03-17 The John Hopkins University Characterization of the yeast transcriptome
JP2003516716A (ja) * 1999-06-16 2003-05-20 ザ ジョン ホプキンス ユニバーシティー 酵母トランスクリプトームの特徴付け
FR2821087B1 (fr) * 2001-02-16 2004-01-02 Centre Nat Rech Scient Procede d'analyse qualitative et quantitative d'une population d'acides nucleiques contenus dans un echantillon
DE10160660A1 (de) 2001-12-11 2003-06-18 Bayer Cropscience Ag Polypeptide zum Identifizieren von fungizid wirksamen Verbindungen
US20060147926A1 (en) 2002-11-25 2006-07-06 Emmert-Buck Michael R Method and apparatus for performing multiple simultaneous manipulations of biomolecules in a two-dimensional array
EP1771466B1 (fr) * 2004-07-23 2010-05-26 GE Healthcare UK Limited Marqueurs de phases du cycle cellulaire
EP3370744A4 (fr) * 2015-11-02 2019-04-17 Orig3N, Inc. Le blocage du cycle cellulaire améliore l'efficacité de production de cellules souches pluripotentes induites

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