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Biomedical subjects

D Botstein

Publications and source records attributed to D Botstein.

At least 253 records · Page 14Linked to original sources
PubMed1978

Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda.

A number of independent insertions into bacteriophage lambda of the translocatable tetracycline-resistance element Tn10 have been isolated and characterized. The physical positions and relative orientations of several such insertions were determined. Two independent insertions appear to lie in the same orientation at or very near the same site in the cI gene, and two more lie in opposite orientations at or near the same position in or near the rex gene. Insertions in or near genes cI, rex, and cIII have been characterized genetically for their effects on expression of nearby genes. Tn10 appears to exert a polar effect on expression of distal genes when it is inserted within an operon, even when expression of that operon is under the influence of lambda N-function. In addition, Tn10 insertions in rex appear to influence in some way expression of an "upstream" gene, cI. Lambda derivatives carrying Tn10 give rise to spontaneously occurring, tetracycline-sensitive deletions at high frequencies. It is likely that formation of these deletions is promoted in some way by the Tn10 element. Lambda::Tn10 phages carrying a Tn10 element that has undergone several successive cycles of translocation since its first isolation and characterization have been analyzed. The results confirm that Tn10 often retains its physical and functional integrity during many cycles of translocation. Lambda derivatives carrying Tn10 have been used to generate insertions of Tn10 in the chromosome of Escherichia coli. This process is independent of recA function, and seems to be quite analogous to the translocation of Tn10 in Salmonella typhimurium as studied previously.

Coliphages↗
PubMed1977

Simple Mendelian inheritance of the reiterated ribosomal DNA of yeast.

A diploid strain of yeast (Saccharomyces cerevisiae) was found to be heterozygous for two forms of the highly repetitious ribosomal DNA. These forms could be distinguished by the pattern of fragments produced after digestion with the site-specific restriction endonuclease EcoRI. The mode of inheritance of ribosomal DNA was determined by tetrad analysis. Of 14 tetrads analyzed, 12 clearly showed the ribosomal DNA forms segregating as a single Mendelian unit. The simplest interpretation of this result is that all of the approximately 100 copies of the ribosomal DNA genes of the yeast cell are located on one chromosome and that meiotic recombination within these genes is suppressed. Two of the 14 tetrads showed the segregation patterns expected as the result of mitotic recombination within the ribosomal DNA.

Base Sequence↗
PubMed1976

Structure and assembly of the capsid of bacteriophage P22.

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.

Capsid↗
PubMed1976

Yeast suppressors of UAA and UAG nonsense codons work efficiently in vitro via tRNA.

A cell-free protein-synthesizing system, containing an S-100 fraction from yeast, ribosomal subunits from Krebs ascites cells, and ribosome initiation factors from rabbit reticulocytes, translates yeast, adenovirus, and rabbit globin messenger RNAs and the RNA from bacteriophage Qbeta. An amber mutation in the Qbeta synthetase gene is suppressed in vitro if the S-100 fraction s from yeast strains carrying amber suppressor mutations. Suppressor SUP6-2 gives 16% suppression, and the recessive lethal suppressor RL-1 gives 50% suppression. Extracts from strain FM6, which has the ochre suppressor SUP4-1, give a longer protein product from the normal synthetase gene at Qbeta with an efficiency of 63%. This implies that UAA is the terminator for the synthetase gene, and that synthesis of this read through protein can be used as an assay for ochre suppression. Suppression in each of these cases is mediated by tRNA, since pufified tRNA is the only fraction from suppressing strains that is required in an otherwise nonsuppressing cell-free system.

Bacteriophages↗
PubMed1976

Specialized transduction by bacteriophage P22 in Salmonella typhimurium: genetic and physical structure of the transducing genomes and the prophage attachment site.

P22pro-1 and P22pro-3 are specialized transducing derivatives of phage P22 that carry the proA and proB genes of Salmonella typhimurium. These genes lie immediately adjacent to the prophage attachment site on the bacterial chromosome. By examining DNA heteroduplexes in the electron microscope, we found that DNA molecules from P22pro-1 and P22pro-3 each contain a substitution which adds length to the composite genome making the intracellular replicated genome too long to fit into a single phage particle. In this respect, and in many of their biological properties, the proline-transducing phages resemble P22Tc-10, another specialized transducing phage with an oversize, intracellular replicated genome which carries a tetracycline-resistance determinant from an R-factor.--Unlike P22Tc-10, however, P22pro-1 and P22pro-3 fail to integrate normally during lysogenizing infections, even when provided with all known integration functions. These results suggest that the proline substitutions have created a defect in the phage attachment site and suggest that the Campbell model for the formation of specialized transducing phages is applicable to phage P22 with the additional feature that oversize genomes can be produced and propagated.--A physical and genetic map of the P22 genome near the prophage attachment site was constructed which shows that the insertion from the R-factor in P22Tc-10 is not at the attachment site: it is therefore unlikely that P22Tc-10 was formed in an abnormal prophage excision event as envisioned in the Campbell model, but was instead the result of a direct translocation from the R-plasmid to P22.

Chromosome Mapping↗
PubMed1975

Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins.

An analysis of revertants of missense mutants in phage P22 has shown: (i) New temperature-sensitive (TS) and cold-sensitive (CS) phenotypes are often acquired concomitant with reversion. (ii) In many cases, these new phenotypes are due to second-site mutations (suppressors) that correct the original defect. (iii) Sometimes the suppressor mutation is not in the same gene as the original mutation. (iv) Extragenic suppressors are almost always in genes whose products are known to interact physically with the original gene products. (v) The suppressor mutations typically retain their TS or CS phenotypes when crossed into wild-type genetic backgrounds. (vi) Some TS and CS mutants derived by reversion can themselves be reverted to produce additional mutations. We have shown that genetic reversion of missense mutants can be of value in producing new temperature-sensitive and cold-sensitive mutations affecting related functions. We suggest that our approach can be extended to organisms with large genomes.

Chromosome Mapping↗
PubMed1975

Recessive lethal amber suppressors in yeast.

Recessive lethal amber suppressor mutations have been isolated in a diploid strain of Saccharomyces cerevisiae. Diploids carrying these suppressors upon sporulation yield asci with only two live spores, both lacking the suppressor. At least two classes of recessive lethal suppressors exist. Aneuploid strains carrying one wild type and one suppressor locus have been isolated and used in mapping studies; one suppressor maps on chromosome III, the other does not.

Aneuploidy↗