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D Koshland

Publications and source records attributed to D Koshland.

46 records · Page 3Linked to original sources
PubMed1989

The dynamics of chromosome movement in the budding yeast Saccharomyces cerevisiae.

Nuclear DNA movement in the yeast, Saccharomyces cerevisiae, was analyzed in live cells using digital imaging microscopy and corroborated by the analysis of nuclear DNA position in fixed cells. During anaphase, the replicated nuclear genomes initially separated at a rate of 1 micron/min. As the genomes separated, the rate of movement became discontinuous. In addition, the axis defined by the segregating genomes rotated relative to the cell surface. The similarity between these results and those previously obtained in higher eukaryotes suggest that the mechanism of anaphase movement may be highly conserved. Before chromosome separation, novel nuclear DNA movements were observed in cdc13, cdc16, and cdc23 cells but not in wild-type or cdc20 cells. These novel nuclear DNA movements correlated with variability in spindle position and length in cdc16 cells. Models for the mechanism of these movements and their induction by certain cdc mutants are discussed.

Cell Cycle↗
PubMed1987

The structure of sister minichromosome DNA before anaphase in Saccharomyces cerevisiae.

The role of DNA topology in holding sister chromatids together before anaphase was investigated by analyzing the structure of a small circular minichromosome in cell cycle (cdc) mutants of the yeast Saccharomyces cerevisiae. In the majority of cells arrested after S phase but before anaphase, sister minichromosome molecules are not topologically interlocked with each other. The analysis of the ploidy of minichromosomes in cells that are released from arrest demonstrates that the sister molecules are properly segregated when the cell cycle block is removed. Therefore, sister minichromosome molecules need not remain topologically interlocked until anaphase in order to be properly segregated, and topological interlocking of sister DNA molecules apparently is not the primary force holding sister chromatids together.

Anaphase↗
PubMed1987

A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae.

We have developed an assay in S. cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes. We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements. Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division. We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III. Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules. Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule. These results suggest plausible pathways for changes in chromosome number that accompany evolution.

Alleles↗
PubMed1985

Genetic analysis of the mitotic transmission of minichromosomes.

The fidelity of the mitotic transmission of minichromosomes in S. cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions. This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS 1) and a centromere (CEN3). The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome. However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions. We also use this assay to isolate and characterize mutations in ARS 1 and CEN3. The mutations in ARS 1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function. Finally, the phenotypes of the mutations in ARS 1 and CEN3 are consistent with their function in replication and segregation, respectively.

Base Sequence↗
PubMed1982

Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane.

Secretion of beta-lactamase was studied in Salmonella typhimurium infected with P22 phage carrying wild-type and mutant alleles of the structural gene. Cellular location of precursor and mature products of wild-type and temperature-sensitive and chain-terminating mutants was analyzed by cell fractionation and by trypsin accessibility in intact and lysed spheroplasts. The precursors of wild-type and all these mutants (none of which alter the signal peptide) are found sequestered within the cell, while all the mature forms have at least partially been translocated across the inner membrane. Thus most beta-lactamase molecules traverse the membrane after completion of their translation. It seems that the carboxyl terminus of beta-lactamase is not required for translocation across the inner membrane but is required for the protein to appear in the periplasm as a soluble species.

Amino Acid Sequence↗
PubMed1982

Diverse effects of mutations in the signal sequence on the secretion of beta-lactamase in Salmonella typhimurium.

Mutations in the beta-lactamase structural gene that alter the signal peptide were used to study secretion into the periplasm of Salmonella typhimurium. Processing and cellular location of mutant gene products were followed by pulse-chase and cell-fractionation experiments and by trypsin accessibility in intact and lysed spheroplasts. The precursor proteins examined never appear as a free species in the periplasm. Two of the signal-sequence mutants accumulate a precursor form that is trypsin-accessible in intact spheroplasts; the precursors synthesized by the remaining mutants resemble wild-type in that they remain trypsin-inaccessible. One of the latter mutants does produce mature protein, but at a very reduced rate. It thus appears that signal-sequence mutations can affect more than one step in the secretion process, and that processing of the signal peptide is not required for the protein to be translocated (at least partially) across the inner membrane.

Amino Acid Sequence↗
PubMed1980

Secretion of beta-lactamase requires the carboxy end of the protein.

Synthesis and secretion of beta-lactamase were studied in Salmonella typhimurium infected with P22 phage carrying the structural gene for beta-lactamase (the bla gene) in mutant or wild-type form. The wild-type gene was shown to specify two forms of beta-lactamase which differ in molecular weight by about 2500 daltons. This difference is consistent with removal, predicted on other grounds, of 23 amino-terminal residues (the "signal" sequence). All bla- mutants, including chain-terminating mutants lacking as much as 50% or as little as 10% of the protein, were apparently unaffected in this processing step. Pulse-chase experiments showed that more than 85% of the wild-type (as well as mutant) proteins are synthesized as complete overlength precursors before being processed to their mature forms. Virtually all the mature wild-type protein appears in the periplasmic space whereas a large fraction of the precursor appears in the cytoplasm. In contrast, both the precursor and processed forms of beta-lactamase proteins synthesized by chain-terminating mutants (including one which lacks only 10% of its residues from the carboxy end) are not secreted and apparently remain soluble in the cytoplasm. These results show that the carboxy-terminal amino acid sequence (at least) of beta-lactamase is essential to successful transport across the cytoplasmic membrane, and suggest that the presence (and probably also the act of removal) of the signal sequence does not suffice to ensure secretion.

Amino Acid Sequence↗
PubMed1980

Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.

Base Sequence↗