Biomedical subjects

V L Funanage

Publications and source records attributed to V L Funanage.

26 records · Page 2Linked to original sources

PubMed1986

Localization of Chinese hamster dihydrofolate reductase gene to band p23 of chromosome 2.

It has been shown that gamma irradiation causes extensive deletions at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells. We have analyzed seventeen DHFR-negative (DHFR-) mutants of CHO cells for cytogenetic alterations involving the dhfr locus on chromosome 2. Five DHFR- mutants contained the same large deletion [del(2)(p16p23)] in the short arm of chromosome 2. This deletion comprised about 18% of the short arm and was estimated to be 41,000 kb in length. Four other DHFR- mutants contained smaller deletions of about 9200 kb. One of these mutants had a partial deletion of bands 2p22 and 2p23, whereas the others showed deletion of band 2p23. Inversions of chromosome 2 were seen in two other DHFR- mutants. An analysis of the breakpoints involved in these cytogenetic alterations indicates that the hamster dhfr gene resides in band p23 of chromosome number 2.

Animals↗

PubMed1985

Coregulation of dihydrofolate reductase and thymidylate synthase B in bacillus subtilis.

A 2.0-kb fragment of Bacillus subtilis 168 chromosomal DNA has been shown to contain both the dihydrofolate reductase (dfrA) and thymidylate synthase B (thyB) genes. In addition to the close proximity of dfrA and thyB, the expression of these genes seems to be regulated coordinately. Mutations that map near or within the dfrA gene resulted in coordinate increases in both dihydrofolate reductase and thymidylate synthase B activities. Also, when trimethoprim, a specific inhibitor of dihydrofolate reductase and thymidylate synthase B activities. Also, when trimethoprim, a specific inhibitor of dihydrofolate reductase, was added to growing cells, both dihydrofolate reductase and thymidylate synthase B activities increased coordinately.

Bacillus subtilis↗

PubMed1984

Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5.

Cells from a dihydrofolate reductase-deficient Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11----q22 region.

Animals↗

PubMed1982

Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.

Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-K1. Three independently isolated mutants, DRB6 DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-K1 cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 microM, 300 microM DRB was required to inhibit 60% of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRB-sensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 microM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 microM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR-10) in DRB10 and confirmed that the drbR-10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and alpha-amanitin-resistant cell lines grew in medium containing both DRB and alpha-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.

Amanitins↗

PubMed1978

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.

Amino Acids↗

PubMed1977

Characterization of Salmonella typhimurium mutants with altered glutamine synthetase activity.

A number of glutamine auxotrophs of Salmonella typhimurium were isolated and characterized genetically. Three of the mutations appear to be closely linked and are complemented by episomes carrying the glnA region of Escherichia coli. The lesions in these strains are approximately 20% linked by P1 transduction with a mutation in the rha gene, but are unlinked to ilv. Another mutation causing glutamine auxotrophy in strain JB674 is genetically distinct from the others. Strain JB674 grown in glucose medium containing ammonia as the nitrogen source has reduced levels of glutamine synthetase that is more adenylylated than in the parent strain, suggesting that the enzyme can not be deadenylylated normally. The lesion causing glutamine auxotrophy in JB674 lies in the region corresponding to the glnB and glnE genes affecting glutamine synthetase modification in Klebsiella areogenes. Four Gln+ revertants of JB674 have glutamine synthetase activities 4 to 6 fold higher than normal. One mutation causing this increased enzyme synthesis has been shown by three-factor crosses with the glnA mutations to lie near or within the glnA gene.

Chromosome Mapping↗

PubMedDate not supplied

Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy.

Myotonic dystrophy (DM), an autosomal dominant neuromuscular disease, is associated with expansion of a polymorphic (CTG)n repeat in the 3'-untranslated region of the DM protein kinase (DMPK) gene. The repeat expansion results in decreased levels of DMPK mRNA and protein, but the mechanism for this decreased expression is unknown. Loss of a nuclease-hypersensitive site in the region of the repeat expansion has been observed in muscle and skin fibroblasts from DM patients, indicating a change in local chromatin structure. This change in chromatin structure has been proposed as a mechanism whereby the expression of DMPK and neighboring genes, sine oculis homeobox (Drosophila) homolog 5 (SIX5) and dystrophia myotonica-containing WD repeat motif (DMWD), might be affected. We have developed a polymerase chain reaction (PCR)-based method to assay the chromatin sensitivity of the region adjacent to the repeat expansion in somatic cell hybrids carrying either normal or affected DMPK alleles and show that hybrids carrying expanded alleles exhibit decreased sensitivity to PvuII digestion in this region. Semiquantitative multiplex reverse transcriptase PCR (RT/PCR) assays of gene expression from the chromosomes carrying the expanded alleles showed marked reduction of DMPK mRNA, partial inhibition of SIX5 expression from a congenital DM chromosome, and no reduction of DMWD mRNA. Nested RT/PCR analysis of DMPK mRNA from somatic cell hybrids carrying the repeat expansions revealed that most of the DMPK transcripts expressed from the expanded alleles lacked exons 13 and 14, whereas full-length transcripts were expressed predominantly from the normal alleles. These results suggest that the CTG repeat expansion leads to a decrease in DMPK mRNA levels by affecting splicing at the 3' end of the DMPK pre-mRNA transcript.

Cells, Cultured↗

PubMedDate not supplied

Cloning and mapping of the dihydrofolate reductase gene of Bacillus subtilis.

The structural gene for dihydrofolate reductase (dfrA) from the Bacillus subtilis 168 chromosome has been cloned, along with the thyB gene, on a 4.5-kb insert contained on chimeric plasmid pER1. The presence of the dfrA gene on pER1 was demonstrated by showing that: (i) transformation of Escherichia coli strains RUE10(Thy-) and RUE11(Thy+) with pER1 resulted in a 60 to 130-fold increase in dihydrofolate reductase (DFRase) activity with a turnover number characteristic of that of B. subtilis and (ii) pER1-mediated transformation of trimethoprim-resistant E. coli strain D05, which overproduced a DFRase with a decreased affinity for trimethoprim, resulted in a 41-fold increase in DFRase activity with an affinity for trimethoprim similar to that of the B. subtilis enzyme. The dfrA gene was mapped to the 200 degrees region of the B. subtilis chromosome, and the gene order was established as thyB dfrA ilvA. Furthermore, the dfrA gene was shown to be linked closely (95-99% cotransformation) to the thyB gene.

Bacillus subtilis↗