Search citation statements
Paper Sections
Citation Types
Year Published
Publication Types
Relationship
Authors
Journals
We have developed a new procedure for easy and rapid identification of autonomously replicating sequences (ARSs) and have applied it to the analysis of chromosome V of Saccharomyces cerevisiae. The procedure makes use of the ordered λ phage clone bank of this chromosome that we have constructed, and includes transposition of a mini‐transposon and selection of transposon‐containing derivatives, isolation of their DNA and circularization at their cos‐ends, transformation of yeast cells with the circularized DNA, and scoring transformation frequency. The transposon used was derived from Tn5supF, contained the yeast LEU2 gene, and was placed, together with the hyperactive transposase gene, on a mini‐F plasmid for stable maintenance in Escherichia coli K‐12. Sixteen regions of chromosome V showing ARS activity were identified, of which 12 were newly found in this work. Thus, the procedure will be useful for systematic genomic scale analysis of ARSs in yeast and related organisms in which ordered clone banks have been established. The average distance between adjacent ARS‐containing regions was approximately 40 kb. Two‐dimensional gel electrophoretic analysis of chromosome replication indicated that one of the newly identified ARSs was functional as an actual in situ replication origin, at least under the conditions employed.
We have developed a new procedure for easy and rapid identification of autonomously replicating sequences (ARSs) and have applied it to the analysis of chromosome V of Saccharomyces cerevisiae. The procedure makes use of the ordered λ phage clone bank of this chromosome that we have constructed, and includes transposition of a mini‐transposon and selection of transposon‐containing derivatives, isolation of their DNA and circularization at their cos‐ends, transformation of yeast cells with the circularized DNA, and scoring transformation frequency. The transposon used was derived from Tn5supF, contained the yeast LEU2 gene, and was placed, together with the hyperactive transposase gene, on a mini‐F plasmid for stable maintenance in Escherichia coli K‐12. Sixteen regions of chromosome V showing ARS activity were identified, of which 12 were newly found in this work. Thus, the procedure will be useful for systematic genomic scale analysis of ARSs in yeast and related organisms in which ordered clone banks have been established. The average distance between adjacent ARS‐containing regions was approximately 40 kb. Two‐dimensional gel electrophoretic analysis of chromosome replication indicated that one of the newly identified ARSs was functional as an actual in situ replication origin, at least under the conditions employed.
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 ...
Most of the 97 transcripts of the genes on chromosome VI of Saccharomyces cerevisiae that were identified by a series of Northern hybridization experiments (Yoshikawa and Isono, Nucl. Acids Res., 19, 1189-1195, 1991) have been correlated with the open reading frames (ORFs) deduced from the nucleotide sequence data of this chromosome (Murakami et al., Nature Genet., 10, 261-268, 1995). This was performed by comparing the experimentally constructed physical map and the one produced from the nucleotide sequence data, as well as the sizes and positions of observed transcripts and those of sequenced ORFs. Thus, 75 ORFs of chromosome VI were correlated uniquely with the corresponding transcripts and 3 ORFs with two transcripts of different sizes. Comparing the relative abundance levels of individual transcripts with that of the RPO41 transcript, highly expressed genes of chromosome VI were found to be located almost exclusively on the Crick strand. Based on the correlation between the abundance level of the experimentally identified transcripts and the codon adaptation indices of the corresponding gene, the genes on chromosome VI of S. cerevisiae were classified into three groups. The data thus provides information concerning their chromosomal locations as well as their likely levels of expression in vegetatively growing cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.