A 2-microm DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae.

A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was developed. FRT DNA sequences were designed with different core mutations and subsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily eliminated from its integration site by in vivo site-specific recombination between the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombination between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombination between residual FRTs, is very low. In strains containing 2-microm ([cir+]) the site-specific reaction is catalysed by the endogenous Flp gene product, whereas in strains without 2-microm ([cir0]), the FLP gene is carried on the cassette, together with the marker gene. This system can be applied for haploid and diploid [cir+] and [cir0] strains.

The nucleotide sequence of Saccharomyces cerevisiae chromosome VII.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new. No correlation was found between G+C content and gene density along the chromosome, and their variations are random. Of the ORFs, 17% show high similarity to human proteins. Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S. cerevisiae genome. Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

Sequencing of a 40.5 kb fragment located on the left arm of chromosome VII from Saccharomyces cerevisiae.

The nucleotide sequence of a 40.5 kb DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae was determined and analysed. Twenty-eight open reading frames (ORFs) longer than 300 nucleotides were identified. Eight of the them correspond to the following known yeast genes: EMP24, GCN1, SPO8, COX13, CDC55, RPS26, COX4 and LSR1, also called GTS1. Twelve ORFs are new, among them eight show homology with other genes while four have no homology with any sequence in the databases. Eight additional ORFs are internal to or partially overlapping with other ORFs.

A putative helicase, the SUA5, PMR1, tRNALys1 genes and four open reading frames have been detected in the DNA sequence of an 8.8 kb fragment of the left arm of chromosome VII of Saccharomyces cerevisiae.

We report the sequence of an 8.8 kb segment of DNA from the left arm of chromosome VII of Saccharomyces cerevisiae. The sequence reveals seven open reading frames (ORFs) G1651, G1654, G1660, G1663, G1666, G1667 and G1669 greater than 100 amino acids in length and the tRNALys1 gene. ORF G1651 shows 100% identity with the ROK1 protein which is a putative RNA helicase of the 'DEAD box' protein family. ORF G1654 exhibits a motif highly conserved in ATP/GTP binding proteins generally referred to as 'P-loop'. From FastA analysis, G1660 and G1666 were found to be previously sequenced genes, respectively SUA5 and PMR1. The three other ORFs identified are partially (G1663) or completely (G1667 and G1669) overlapping with the PMR1 sequence on the complementary strand. This feature, together with their low codon adaptation indexes and the absence of significant homology with known proteins suggest that they do not correspond to real genes.

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination.

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.

The sequence of an 11.1 kb fragment on the left arm of Saccharomyces cerevisiae chromosome VII reveals six open reading frames including NSP49, KEM1 and four putative new genes.

We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins.

The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene.

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.