The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.
We have identified the gene DYNI, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (Mr 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain ofDictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. CeUl Biol. 119, 1597Biol. 119, -1604 with 40% identity in the putative motor domain. Disruption of DYNI causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast.Nuclear and cytoplasmic microtubules in the yeast Saccharomyces cerevisiae participate in several well-defined cellular processes that include the segregation of chromosomes and the migration of the nucleus during mitosis as well as the migration and fusion of nuclei in karyogamy (1). These processes are believed to involve microtubule-based motor enzymes, and recently several genes containing sequence regions with homology to the motor domain characteristic of the kinesin superfamily have been identified in S. cerevisiae and other fungi (2-8).The cytoplasmic form ofthe microtubule-associated motor dynein has ATPase activity and moves toward the minus ends of microtubules in in vitro gliding assays (9). Cytoplasmic dynein exists in a wide variety of eukaryotic cells, including Dictyostelium (10, 11), and indirect evidence has suggested that it plays a major role in the movement of chromosomes toward the minus ends of spindle microtubules at the anaphase stage of mitosis (12, 13), as well as in the retrograde transport of organelles in nerve axons (14)(15)(16) Transformations were done with the lithium acetate method (18). Synchronization of yeast cultures was achieved by treating with a mating factor as described by Berlin et al (19).PCR Primers. Oligonucleotide primers were designed from the deduced amino acid sequence of the sea urchin dynein ,B heavy chain (20, 21) in regions that were conserved between sea urchin dynein isoforms (22). The nucleotide sequences of the two degenerate primers that were used to PCR-amplify pDLP1 from yeast genomic DNA are 5'-CCTGCTGGNAC-NGGNAARAC-3' (sense strand, targeting amino acidsequence PAGTGKT) and 5'-TACCCIGGRTTCATIGTDA-TRAA-3' (antisense strand, targeting amino acid-sequence FITMNPG).DNA Sequencing. Nucleotide sequencing of the original probe, pDLP1, and other restriction fragments in the vicinity of the putative hydrolytic ATP-binding site was done by subcloning into M13mpl8/mp19 vectors and using universal M13 primers and a Sequenase 2.0 DNA sequencing kit (United States Biochemical).The nucleotide sequence of the complete gene was determined as part of the ongoing project to sequence the entire chromosome XI in yeast (strain S288C). Relevant cosmids were soni...
The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.
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