Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of -231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The yeast Saccharomyces cerevisiae has been the focus of intensive study as a model eukaryote. As part of this effort, an international program is under way to determine the nucleotide sequence of the 16 chromosomes that constitute its 13.5-Mbp nuclear genome. This endeavor will provide both a complete eukaryotic gene set and a reference set of experimentally amenable genes for comparison with those of other organisms. Currently, four yeast chromosomes have been sequenced (1-4); all have a high gene density, and a majority of the genes found are newly sequenced and of unknown function. Chromosome I is the smallest S. cerevisiae chromosome. It contains a DNA molecule that is only 231 kbp, making it the smallest known fully functional nuclear chromosome. This chromosome has been studied intensively, and mutants are available for a large number of its genes (5-7). Here we report the nucleotide sequence of chromosome I and describe several unusual features of its gene organization and chromosome structure as well as many newly discovered genes.** MATERIALS AND METHODS DNA Sources. Four sources of chromosome I DNA, all from S288C-derived yeast strains, were used to generate the tem-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.plates for DNA sequencing. These were the library of Riles et aL (8), a cosmid from the collection of Dujon (9), chromosome walking (10), and PCR amplified fragments of genomic DNA. DNA fragments, except those generated by PCR which were used directly, were subcloned into the Bluescript KS(+) plasmid from Stratagene prior to sequencing. All DNA sequencing was performed using double-stranded DNA templates.DNA Sequencing. Two methods were used for sequencing DNA templates: manual sequencing and machine-based sequencing with an Applied Biosystems sequencing machine (model 373A). Our manual sequencing used unidirectional nested deletions and was carried out as described (11, 12). For machine-based sequencing, three sets of templates were used: unidirectional nested deletions, PCR amplified chromosomal DNA, and, for the region spanning YAL062 to CDC24, cosmid DNA was shotgun cloned into Bluescript KS(+). In summary, the procedure for the Applied Biosystems machine (model 373A) used dye-labeled dideoxynucleotide terminators and a cycle sequencing kit (Prism Ready reaction dye terminator kit; Perkin-Elmer) and the protocol provided by the supplier. This method allowed us to process all four sequencing reactions in a single reaction tube. The cycle amplification reactions were performed with a Perkin-Elmer ...
Meiotic reciprocal recombination (crossing over) was examined in the outermost 60-80 kb of almost all Saccharomyces cerevisiae chromosomes. These sequences included both repetitive gene-poor subtelomeric heterochromatin-like regions and their adjacent unique gene-rich euchromatin-like regions. Subtelomeric sequences underwent very little crossing over, exhibiting approximately two-to threefold fewer crossovers per kilobase of DNA than the genomic average. Surprisingly, the adjacent euchromatic regions underwent crossing over at twice the average genomic rate and contained at least nine new recombination ''hot spots.'' These results prompted an analysis of existing genetic mapping data, which showed that meiotic reciprocal recombination rates were on average greater near chromosome ends exclusive of the subtelomeres. Thus, the distribution of crossovers in S. cerevisiae appears to resemble that found in several higher eukaryotes where the outermost chromosomal regions show increased crossing over.
Transcribed regions on a 42-kb segment of chromosome I from Saccharomyces cerevisiae were mapped.Polyadenylated transcripts corresponding to eight previously characterized genes (AL4K16, LTE1, CCR4, FUN30, FUN31, TPD3, DEPI, and CYS3) and eight new genes were identified. All transcripts were present at one to four copies per cell except for one which was significantly less abundant. This region has been sequenced, and the sizes, locations, and orientations of the transcripts were in nearly perfect agreement with the open reading frames. Disruptions in eight genes identified solely on the basis of a transcribed region, FUN38, FUN25, FUN26, FUN28, FUN30, FUN31, FUN33, and FUN34, indicated that all were nonessential for growth on rich medium at 30°C. Disruption of FUN30, a gene closely related to RAD16 and RAD54, surprisingly resulted in increased resistance to UV irradiation. No additional phenotypes, other than slow growth, were observed for all other mutants. The distribution of essential genes on chromosome I is discussed.
The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near the telomeres. DSB-prone regions were associated with all of the known "hot spots" for meiotic recombination on chromosomes I, III and VI.
Reciprocal recombination (chiasma formation) between homologs appears to be essential for promoting chromosome segregation at the first meiotic division. However, chiasmata that form near the ends of chromosomes are much less efficient at promoting segregation. To determine the frequency of reciprocal recombination near the end of a chromosome, genetic markers were inserted at approximately 7 kb intervals within the leftmost 30 kb of chromosome I from Saccharomyces cerevisiae. Analysis of recombination between the markers indicated that meiotic reciprocal recombination rates were much lower than on the rest of the chromosome and that rates increased with distance from the telomere. Thus, S. cerevisiae has evolved a mechanism that minimizes the occurrence of chiasmata that cannot promote meiotic segregation. Low rates of recombination were independent of the SIR2 and SIR3 gene products, suggesting that any mechanism for suppressing recombination was different from transcriptional repression due to a telomere position effect.
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