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Biomedical subjects

J C Stone

Publications and source records attributed to J C Stone.

At least 55 records · Page 3Linked to original sources
PubMed1989

The murine retinoblastoma homolog maps to chromosome 14 near Es-10.

Restriction fragment length variants have been exploited to map genetically Rb-1, the murine homolog of the human retinoblastoma gene. Rb-1 localized to mouse chromosome 14 on the basis of results from analysis of somatic cell hybrids. In an interspecific backcross involving Mus spretus, Rb-1 and the murine homolog of the human esterase D gene (ESD), which we refer to here as Esd, were inseparable. Furthermore, the strain distribution patterns of Rb-1 and Es-10 are the same in 31 of 32 recombinant inbred strains. Close linkage of the chromosome 14 morphological marker hairless (hr) to Rb-1 is also implied. These results localize Rb-1 on the mouse linkage map and provide close genetic markers to follow Rb-1 in somatic as well as in germline genetic experiments. Additionally, the results suggest that Es-10 is the murine homolog of ESD and provide further evidence for linkage conservation during mammalian evolution.

Animals↗
PubMed1988

p21-ras effector domain mutants constructed by "cassette" mutagenesis.

A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could not be altered, even conservatively, without a loss of function (codons 32 and 35); one which retained detectable biologic activity with conservative changes but which lost function with more drastic substitutions (codons 36 and 40); and one which retained function even with a nonconservative substitution (codon 39).

Amino Acid Sequence↗
PubMed1986

Stability of retrovirally transduced markers in a rat cell line.

A MoMLV-based retroviral vector capable of transmitting and expressing both the human hypoxanthine phosphoribosyltransferase (hprt) coding sequence and the Herpes simplex type 1 thymidine kinase (tk) gene has been constructed. After infection of a rat cell line, cell clones were selected on the basis of expressing both markers. They were subsequently found to contain a single provirus of the expected topology. The ease with which loss of expression of the markers can be monitored has allowed us to make observations on the stability of proviral genes. In particular, we have found indirect evidence of strong position effects on proviral gene expression by comparing the characteristic frequency of marker loss in different clonal proviral lines. Effects of the selection protocol on the apparent frequency of variants have also been noted. Finally, a combination of molecular and genetic observations lead us to invoke chromosome loss as the major factor influencing marker stability in this system.

Animals↗
PubMed1986

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

Animals↗
PubMed1985

Spontaneous temperature-sensitive mutations in bacteriophage T7.

Attempts to recover temperature-sensitive mutations affecting genes 13 and 14 (virion proteins) in bacteriophage T7 by analysis of amber revertants were confounded by the frequent occurrence of spontaneous temperature-sensitive mutations in other genes. These incidental temperature-sensitive mutations are physically distinct from but may be functionally related to genes 13 and 14, as shown by complementation and recombination studies. The possibility that these incidental temperature-sensitive mutations represent secondary-site suppressors of the pseudonormal suppressed amber products is discussed.

Genes↗
PubMed1985

Correspondence between immunological and functional domains in the transforming protein of Fujinami sarcoma virus.

Monoclonal antibodies reactive with either gag or fps portions of the wild-type Fujinami sarcoma virus transforming protein have been used to probe the structure of proteins encoded by mutant genomes constructed in vitro. The pattern of immunoreactivity suggests that the functional domain defined in genetic studies (Stone et al., Cell 37:549-558, 1984) corresponds to a discrete immunological domain in the native, wild-type Fujinami sarcoma virus protein. At least one mutation affecting both the structure and function of the proposed NH2-terminal fps-specific domain encodes a product with high specific activities in kinase assays. Furthermore, a cell line expressing high levels of this mutant protein is only moderately transformed. The striking correspondence between the immunological domain defined here and the functional domain inferred from the results of transfection experiments suggests that this non-kinase-specifying region constitutes a discrete structural as well as functional component of the viral protein.

Animals↗
PubMed1984

Plasmid-phage recombination in T7 infected Escherichia coli.

Recombination between genetically marked T7 bacteriophage and plasmids containing inserts of T7 DNA has been studied in order to gain some insight into the phage recombination process. The results suggest that plasmid-phage recombination requires the products of T7 genes 3 (endonuclease), 4 (DNA primase), 5 (DNA polymerase), and 6 (exonuclease), as has been demonstrated previously for phage-phage recombination. Plasmid replication does not compensate for a complete block in phage polymerase synthesis, suggesting a direct role for this enzyme in recombination, rather than an indirect role, by means of producing replicative structures that are recombinogenic. In most respects, plasmid-phage recombination appears to be similar to phage-phage recombination. The participation of two autonomous, structurally dissimilar, homologues, however, might render certain aspects of the recombination process more amenable to analysis. As examples, the characterization of an apparent marker effect and the demonstration of genetic heterozygotes among the products of plasmid-phage recombination are presented.

DNA Primase↗
PubMed1984

Identification of functional regions in the transforming protein of Fujinami sarcoma virus by in-phase insertion mutagenesis.

A novel mutagenesis procedure based on the insertion of a hexameric nucleotide sequence into Rsa I restriction sites of cloned DNA has been applied to a copy of the Fujinami sarcoma virus (FSV) genome with the aim of identifying functional regions in the transforming protein. Mutations specifying peptide insertions in both the NH2- and the COOH-terminal fps-specific portions of the transforming protein reduce or abolish the capacity of the genome to induce transformed foci in rat-2 cells. Insertion of multiple copies of the hexamer into one central position in the oncogene results in dislocation of the NH2- and COOH-terminal regions in the primary structure, but has no inhibitory effect on focus induction. Taken together, the results imply that both the NH2- and COOH-terminal fps-specific portions of the FSV oncogene product possess determinants which function in fibroblast transformation, and that cooperation of these two regions is not sensitive to their separation in the primary structure.

Animals↗
PubMed1983

A recombinant DNA plasmid which inhibits bacteriophage T7 reproduction in escherichia coli.

Escherichia coli plasmids carrying a DNA restriction fragment corresponding to the extreme right end of the T7 bacteriophage genome render cells incompetent to support reproduction of wild-type T7. Processing of intracellular concatemeric phage DNA and cell lysis are perturbed by one such plasmid, pRS148. The results are discussed with reference to the possibility that the right end of the T7 genome encodes a lysis-related function.

DNA Replication↗
PubMed1983

The characterization of a mutant affecting DNA metabolism in the development of D. melanogaster.

Using a "single-fly" nucleic acid hybridization method, we have surveyed a collection of D. melanogaster strains in search of variants which affect DNA complementary to the polypyrimidine sequence corresponding to one strand of the 1.705 satellite. Hybridization of labelled polypyrimidine probe to polypurine sequence in nucleic acid extracts of single flies, followed by thermal chromatography over hydroxyapatite led to the identification of one variant. The strain Cy/M(2)S2(10) produced excess hybrid, much of which had low thermal stability. A developmental analysis of the low-melt hybrid phenotype showed that certain tissues, in particular the ovaries, were affected. In addition to the biochemical phenotype, the break down of nurse cell nuclei in Cy/M(2)S2(10) ovaries during oocyte maturation was abnormal. A genetic analysis demonstrated that both the biochemical and cytological phenotypes were the consequences of a single recessive mutation in the DNase-1 gene on chromosome III. Studies with purified DNA demonstrated that the low-melt hybrid phenotype resulted from the accumulation of low molecular weight DNA complementary to the polypyrimidine probe.

Animals↗
PubMed1982

Filicidal ring-Y chromosomes in D. melanogaster.

Ring-Y chromosomes are recovered infrequently from crosses of ring-Y-bearing males to females of certain strains (OSTER 1964). Experiments described here have unveiled a diverse class of genes that exert a maternal effect on the behavior during cleavage of these "filicidal" ring chromosomes. Cytological observations of inviable embryos have revealed that the ring-Y chromosome causes gross disorganization of the cleavage nuclei. This inviability may be equivalent to the "dominant lethality" attributed to unstable ring-X chromosomes (HINTON 1955; PASZTOR 1971). Mapping studies indicate that no single region of the normal Y is solely responsible for the unusual behavior of ring-Y chromosomes.

Animals↗
PubMed1975

Alkali lability of bacteriophage phi W-14 DNA.

The molecular weight of bacteriophage phi W-14 DNA, determined by velocity sedimentation in neutral sucrose gradients, was 92 +/- 6 X 10(6). The DNA showed marked fragmentation in alkaline sucrose gradients. This fragmentation was not a consequence of preexisting single-strand interruptions in the DNA, since thermal denaturation of DNA yielded intact single strands. The alpha-putrescinylthymine groups in phi W-14 DNA appeared to be labile; some, or parts of some, of these groups were cleaved from the DNA in alkali.

Bacteriophages↗