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K R Thomas

Publications and source records attributed to K R Thomas.

At least 37 records · Page 2Linked to original sources
PubMed1991

Swaying is a mutant allele of the proto-oncogene Wnt-1.

Mice homozygous for the recessive mutation swaying (sw) are characterized by ataxia and hypertonia, attributed to the malformation of anterior regions of the cerebellum. We show that sw is a deletion of a single base pair from the proto-oncogene Wnt-1. The deletion is predicted to cause premature termination of translation, eliminating the carboxy-terminal half of the Wnt-1 protein. Histological examination shows that sw is phenotypically identical to a previously described wnt-1 mutation introduced into mice by gene targeting. Although both mutations in Wnt-1 disrupt primarily the development of the anterior cerebellum, they also exhibit a variability in expressivity such that rostrally adjacent structures in the midbrain and caudally adjacent structures in the posterior cerebellum can also be affected.

Alleles↗
PubMed1990

Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development.

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.

Animals↗
PubMed1990

Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination.

We demonstrate that the frequency of gene targeting is unaffected by the length of nonhomologous DNA transferred to a target chromosomal sequence. A result of this finding is that a much wider spectrum of designed genomic alterations is now feasible. As a first application, we inserted a 5.4-kilobase cassette of nonhomologous DNA into the int-2 locus in mouse embryo-derived stem cells by gene targeting. The inserted DNA contained a lacZ gene positioned to create an in-frame fusion with the int-2 protein-coding region. Upon differentiation of these cells to embryoid bodies, the int-2-lacZ fusion faithfully reproduced the expression pattern of int-2 RNA. This ability to target reporter genes, such as lacZ, to specific mouse loci, combined with the ability to move the tagged gene into different mutant backgrounds, may provide an ideal approach for analyzing interactions among genes that participate in a developmental network.

Animals↗
PubMed1988

Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.

Gene targeting--homologous recombination of DNA sequences residing in the chromosome with newly introduced DNA sequences--in mouse embryo-derived stem cells promises to provide a means to generate mice of any desired genotype. We describe a positive nd negative selection procedure that enriches 2,000-fold for those cells that contain a targeted mutation. The procedure was applied to the isolation of hprt- and int-2- mutants, but it should be applicable to any gene.

Animals↗
PubMed1988

Antigen-specific lymphocyte transformation induced by oocyst antigens of Eimeria bovis.

Lymphoproliferative responses against a preparation of Eimeria bovis antigens (EBAg) were measured in E. bovis-immune and naive animals. Optimal lymphocyte responsiveness could be measured after 7 days of culture in the presence of antigen at a cell concentration of 2 X 10(5) cells per well. The specificity of the reaction was confirmed by limiting dilution analysis. Whereas immune peripheral blood mononuclear cells responded to EBAg (f = 1/18,824), naive cells did not (f = 0). The helper function of cells proliferating in response to EBAg was investigated by raising T-cell lines and a clonal population derived from a line. The T-cell line showed an enhanced reactivity to EBAg by limiting dilution analysis (f = 1/256) and was interleukin-2 dependent. Limiting dilution analyses indicated at least two populations of cells: one that was interleukin-2 restricted and antigen dependent and another that was antigen independent. Supernatants from T-cell lines and the clone were analyzed for the production of lymphokines after antigen stimulation. Minimal amounts of interleukin-2 were produced. The T-cell line produced both gamma interferon (IFN-gamma) (750 U) and IFN-alpha (1,250 U), whereas the clone produced IFN-gamma (1,250 U) only. Short-term (4-day) stimulation of immune cells by EBAg induced the production of IFN-gamma (600 U) and a non-IFN macrophage-activating lymphokine. We conclude that this macrophage-activating lymphokine is only produced after short-term culture and that further culture of T cells results in the proliferation of other clones producing other factors (such as IFN).

Animals↗
PubMed1987

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.

Animals↗
PubMed1987

Complement component C5 is required for release of alveolar macrophage-derived neutrophil chemotactic activity.

The influx of neutrophils into the alveolar structures can be induced by stimulation of the resident lung phagocyte, the alveolar macrophage, to release a potent neutrophil chemoattractant(s). We hypothesized that the fifth component of complement (C5) on the cell surface may be required for activation of the alveolar macrophage to release neutrophil chemotactic activity. C5 was identified on guinea pig alveolar macrophages by epifluorescent microscopy, flow cytometry, and enzyme-linked immunoabsorbent assay of eluted macrophages. When cultured for 4 h with stimuli that induce the release of chemotactic activity or for 24 h without added stimuli, purified Fab' fragments of a goat anti-C5 antibody significantly inhibited the ability of macrophages to release chemotactic activity as determined by a blindwell chamber method (p less than 0.001, all comparisons). This inhibition of chemotactic activity was not detected when anti-C5 antibody was added after the culture period. In contrast, anti-C3 antibody had no inhibitory effect at 4 h or at 24 h (p greater than 0.2, all comparisons). Partial characterization of released chemotactic activity revealed it was of low molecular weight, partially lipid soluble, and not inhibited by C5a chemotactic factor inactivator. These studies suggest that C5 may have a regulatory role in the release of chemotactic activity by alveolar macrophages.

Animals↗
PubMed1987

Role of chemotactic factor inactivator in modulating alveolar macrophage-derived neutrophil chemotactic activity.

The stimulated alveolar macrophage is a potent source of neutrophil chemotactic activity. The release of this chemotactic activity can be inhibited by pretreating alveolar macrophages with anti-C5 antibody. We hypothesized that C5a, a fragment cleaved from C5 when C5 is activated, might activate the alveolar macrophage to release neutrophil chemotactic activity and that chemotactic factor inactivator, a serum inhibitor of C5a, could decrease this release. Activated complement components including C5a were found to stimulate guinea pig macrophages to release chemotactic activity into their culture supernatants at levels that were significantly higher than the chemotactic activity of C5a alone (P less than 0.001). Chemotactic factor inactivator was found to cause a marked reduction in the chemotactic activity released by macrophages stimulated with phagocytic and nonphagocytic stimuli (P less than 0.001, all comparisons). These data indicate that C5a can stimulate alveolar macrophages to release chemotactic activity in vitro, and that chemotactic factor inactivator may play a role in modulating this process.

Aminopeptidases↗
PubMed1986

High frequency targeting of genes to specific sites in the mammalian genome.

We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected.

Alleles↗
PubMed1985

Rapid enzyme system for the identification of pathogenic Neisseria spp.

Gonochek II is a combination of three enzyme substrates in one tube which will give a rapid identification (30 min) of those pathogenic Neisseria spp. which can be isolated on Thayer Martin or similar selective media. Eighty isolates were tested by Gonochek II and a carbohydrate utilization method; total agreement was achieved between the two methods.

Aminopeptidases↗
PubMed1983

Flexibility in RNA priming of Okazaki pieces at the E. coli replication fork.

We present results which suggest considerable flexibility in the RNA priming of Okazaki pieces at the E. coli replication fork. Using film lysates on cellophane discs, we have identified RNA at the 5' ends of Okazaki pieces. All four ribonucleotides are found to be present at the RNA-DNA junction if all four ribonucleoside triphosphates are used. However, if only ATP, or ATP and GTP are used, then only 2' (3')AMP, or 2' (3')AMP and 2' (3')GMP are found at the RNA-DNA junction. A nearest neighbor analysis of RNA associated with Okazaki pieces using alpha 32P-CTP as a probe shows a similar dependence of nearest neighbor composition on the ribonucleoside triphosphate composition of the incubation mixture. Thus, the nucleotide composition of the RNA primers at the ends of Okazaki pieces varies as a function of the ribonucleoside triphosphates available.

DNA↗
PubMed1983

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.

Animals↗
PubMed1980

The effect of calcium on protoplast release from species of Aspergillus.

The effect of various osmotica and lytic enzyme treatments, on the release of stable protoplasts from Aspergillus niger and two strains of Aspergillus fumigatus, has been investigated. Most osmotica supported protoplast release at a concentration of 0.6 M except for CaCl2 and Ca(NO3)2. Supplementation of 0.6 M KCl with 0.2 M CaCl2 enhanced release from both species, but depended upon strain of organism, concentration of osmoticum and enzyme system employed. More protoplasts were released at low concentrations of KCl and/or when chitinase was present in the digestion medium. This enhancement effect was concerned with the Ca2+ ion since addition of Ca(NO3)2 also gave a higher yield and EDTA severely depressed protoplast release. Further investigations suggested that the effect of Ca2+ was not primarily acting as a supplementary osmoticum or concerned with protoplast membrane stabilization. Experiments using 45CaCl2 showed that the enhancement effect was probably due to Ca2+ affecting chitinase activity.

Aspergillus fumigatus↗
PubMed1979

The effect of beta-glucuronidase and chitinase on the cell wall of Aspergillus niger and Aspergillus fumigatus.

The effects of beta-glucuronidase and chitinase have been tested on the hydrolysis of the cell walls of the economically important fungi, Aspergillus niger and Aspergillus fumigatus. The extent of wall hydrolysis was measured by assaying for total reducing sugars, N-acetyl sugars and protoplast production. Maximum reducing sugar release was attained after 40 min incubation, both with beta-glucuronidase supplemented with chitinase and beta-glucuronidase alone, whereas N-acetyl sugar release reached a maximum at 80 min incubation. beta-Glucuronidase was effective in releasing protoplasts from both species of Aspergillus. This release was enhanced by adding chitinase to the incubation medium at 0 and 20 min, but with addition at 60, 80 and 100 min increase in protoplast yield was much reduced. The results of re-incubation experiments with chitinase suggest that this enzyme may in some way be inhibited during the later stages of incubation. Pronase used in combination with beta-glucuronidase slightly enhanced protoplast release.

Aspergillus fumigatus↗
PubMed1978

Processivity of DNA exonucleases.

A homopolymer system has been developed to examine the digestion strategies of DNA exonucleases. Escherichia coli exonuclease I and lambda-exonuclease, are processive enzymes. However, T7 exonuclease, spleen exonuclease, E. coli exonuclease III, the 3' leads to 5'-exonuclease of T4 DNA polymerase, and both the 3' leads to 5' and the 5' leads to 3' activity of E. coli DNA polymerase I dissociate frequently from the substrate during the course of digestion. Regions of duplex DNA are a dissociation signal for exonuclease I.

Animals↗
PubMed1976

Preparation and properties of a national reference endotoxin.

A large pool of refined endotoxin was prepared from Escherichia coli O113 by extraction with hot acqueous phenol. It was characterized chemically and biologically and will be available for a reference standard designated as reference endotoxin EC.

Animals↗