Search PubMed⌕ Search

Biomedical subjects

H J Whitfield

Publications and source records attributed to H J Whitfield.

35 records · Page 2Linked to original sources
PubMed1986

Coordinate developmental regulation of high and low molecular weight mRNAs for rat insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide that is thought to play a role in fetal growth and development. To study the hormonal and developmental regulation of IGF-II gene expression, we have isolated a cDNA clone for rat IGF-II (rIGF-II) from a 12S [1.2-kilobase-pair (kbp)] fraction of mRNA from a rat liver cell line (BRL-3A) that directs the cell-free synthesis of pre-pro-rIGF-II. In the present study, the rIGF-II probe was used to determine the size of IGF-II RNA. Surprisingly, in BRL-3A cells and in neonatal liver, the probe hybridized under stringent conditions 10-20 times more strongly to a larger (4 kbp) RNA than to 1.2-kbp RNA. The 4-kbp RNA is almost exclusively cytoplasmic and is colinear with a 551-base fragment of the rIGF-II cDNA insert containing coding and 3' noncoding regions. The 4-kbp and 1.2-kbp RNA species are regulated coordinately with developmental age, being high in liver from neonatal rats but not detectable in liver from older animals, suggesting that both IGF-II mRNA species arise from a single primary transcript by alternative RNA processing. Although oligodeoxynucleotide hybridization and S1 nuclease protection experiments suggest that the 4-kbp RNA contains an intact protein-coding region, fractions enriched in 4-kbp RNA do not direct the translation of pre-pro-rIGF-II in vitro. This may indicate that the 4-kbp RNA specifies an altered protein product that has not yet been recognized, or alternatively that it contains a normal protein-coding region but requires further RNA processing to be activated for translation.

Animals↗
PubMed1982

Increased hexose transport in Chinese hamster ovary cells resistant to 3-O-methyl-D-glucose.

3-O-Methyl-D-glucose-resistant mutants were selected from Chinese hamster ovary cells after mutagenesis with ethyl methanesulfonate. A mutant, MegR24, was isolated which was significantly more resistant than the parent to 3-O-methylglucose. Uptake of 50 microM D-glucose by metabolizing MegR24 cells was 2- to 3-fold higher than the parental cells in the absence or presence of 100 mM 3-O-methylglucose. A study of transport of D-[3H]glucose in ATP-depleted cells indicated an apparent Km for D-glucose transport that was 3-fold lower for the mutant (2.7 +/- 0.3 mM) than for the parent (8.9 +/- 1.0 mM). The apparent Km for transport of 3-O-methylglucose by the mutant (10.9 +/- 2.4 mM) was almost 2-fold lower than that of the parent (21.4 +/- 4.7 mM). The Vmax values for transport of D-glucose or 3-O-methylglucose by the mutant and parental cell lines were not significantly different. The MegR24 mutant also exhibited enhanced countertransport of D-[3H]glucose following preloading of ATP-depleted cells with 100 mM 3-O-methylglucose. Simple diffusion of hexoses as measured by L-glucose uptake was not altered in the mutant. These results suggest that the MegR24 hexose carrier has an increased affinity for transport of hexoses and that the resistance to the cytotoxic effects of 3-O-methylglucose exhibited by MegR24 is due to its ability to transport D-glucose 2- to 3-fold more efficiently than the parental strain.

3-O-Methylglucose↗
PubMed1982

Transcription termination factor rho from wild type and rho-111 strains of Salmonella typhimurium.

Transcription termination factor rho was purified to near homogeneity from the wild type and temperature-sensitive rho-111 mutant strains of Salmonella typhimurium. Each protein had identical physical properties with respect to native and subunit molecular weight, elution from ion-exchange columns, and poly(C)-dependent ATPase specific activity at 30 degrees C. The mutant protein exhibited a thermolabile poly(C)-dependent ATPase activity. The transcription termination and nascent RNA-dependent ATPase activities associated with the purified wild type S. typhimurium rho protein were not present in the mutant protein. Binding studies demonstrated that the stability of the rho-111:poly(C) complex was significantly more sensitive to ionic strength and temperature than that of the rho +: poly(C) complex. This result suggests that the altered activities of the mutant protein are due to its decreased ability to participate in a specific interaction with RNA which is insensitive to ionic strength. The rho-111 mutation resulted in a 20- to 30-fold elevation in the level of the mutant protein, indicating that rho biosynthesis in S. typhimurium is autogenously regulated. Therefore, defective molecular interactions between the mutant rho protein and RNA might account for the absence of transcription termination in vitro, and the polarity suppressor phenotype and defective autogenous regulation of rho biosynthesis in vivo.

Adenosine Triphosphatases↗
PubMed1982

Association of adhesive, invasive, and virulent phenotypes of Salmonella typhimurium with autonomous 60-megadalton plasmids.

The plasmid DNA content of six invasive and adhesive strains of Salmonella typhimurium was determined, and all six strains (CR8500 [S850], CR6600 [TML], W118, NY, PR, and S2204) were found to harbor at least one plasmid equivalent in size to the 60-megadalton plasmid ("cryptic" plasmid), pSLT, which is normally resident in S. typhimurium strain LT2. The role of such 60-megadalton plasmids in the adhesive and invasive properties of strain CR6600, a commonly encountered salmonella pathogen that produces type 1 fimbriae, and strain CR8500, a representative FIRN biotype which does not produce type 1 fimbriae, was studied further by obtaining derivatives of these strains that no longer harbored an autonomous 60-megadalton plasmid. Strains CR6260 and CR6190 and strains CR8100 and CR8353, which were "cured" derivatives of strains CR6600 and CR8500, respectively, were significantly less adhesive and invasive in the HeLa cell test. A 53.5-megadalton colicin plasmid harbored by strain CR6600 did not detectably influence these properties. Additionally, strain CR6260 was avirulent, and strain CR8100 was 1,000 to 10,000-fold less virulent for orally infected mice as compared with their respective parental strains. Significantly, the virulence of strain CR8100 correlated with tissue colonization by bacteria that exhibited autonomous copies of a 60-megadalton plasmid. We propose that this plasmid exists in both autonomous and integrated states and that the in vivo environment selects for bacteria with autonomous plasmid copies which can express the virulent phenotype, thus enabling such strains to survive the defense mechanisms of the host.

Adhesiveness↗
PubMed1981

Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity.

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-111 mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-111-bearing strain is the consequence of a single mutation. The rho-111 mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-111 mutation did not alter in vivo expression of either the tryptophan or histidine operons.

Adenosine Triphosphatases↗
PubMed1977

Physical characterization of a plasmid cointegrate containing an F'his gnd element and the Salmonella typhimurium LT2 cryptic plasmid.

A recombinant plasmid (pAS19) isolated from a derivative of Salmonella typhimurium LT2, containing the strain LT2 cryptic plasmid and an F'his gnd element, has been physically characterized. The pAS19 plasmid contour length equals the sum of the contour lengths of the cryptic plasmid and F'his gnd element. Deoxyribonucleic acid (DNA)-DNA hybridization experiments demonstrated that whereas the pAS19 plasmid exhibits extensive DNA homology with both the cryptic plasmid and the F'his gnd element, there is little DNA homology between these latter two plasmids. The DNA fragmentation pattern of the pAS19 plasmid produced by the restriction endonuclease R-EcoRI is consistent with that expected for a composite plasmid cointegrate containing most, if not all, of the DNA sequences present in its two component plasmids.

Animals↗
PubMed1974

Evidence for a composite state of an F'his, gnd element and a cryptic plasmid in a derivative of Salmonella typhimurium LT2.

A method designed to select mutants constitutive for expression of the histidine operon has been applied to a Salmonella typhimurium LT2 strain containing an F'his,gnd element and a cryptic plasmid. One of the mutants isolated, strain AA0019, has not only increased levels of histidinol phosphate phosphatase (hisB), but also increased levels of gluconate-6-phosphate dehydrogenase (gnd). Ultracentrifugation studies of extrachromosomal deoxyribonucleic acid (DNA) isolated from strain AA0019 revealed the presence of a single species of covalently closed circular (CCC) DNA that sedimented more rapidly through neutral and alkaline sucrose gradients than any of its possible plasmid precursors. From neutral sucrose gradients, sedimentation coefficients of 130, 100, and 86S were derived, corresponding to the CCC DNA of the large plasmid in strain AA0019, the F'his,gnd element and the cryptic LT2 plasmid, respectively. An Escherichia coli plasmid-free strain that upon mating had received the large 130S plasmid also contained 86S and 100S CCC DNA components. A histidine-requiring derivative of strain AA0019 obtained after acridine orange treatment retained the cryptic plasmid DNA. Apparently, the large plasmid in strain AA0019 consists of the F'his,gnd element and the cryptic LT2 plasmid of the parental strain.

Acridines↗
PubMed1973

Isolation and characterization of a mutant of Salmonella typhimurium deficient in a major deoxyribonucleic acid polymerase activity.

A mutant of Salmonella typhimurium strain LT2 that is deficient in a major deoxyribonucleic acid (DNA) polymerase activity has been isolated and characterized. This mutant resembles the pol mutants of E. coli in that it has low DNA polymerase activity and it is sensitive to methyl methane sulfonate as well as ultraviolet irradiation. Revertants selected for methyl methane sulfonate resistance are no longer sensitive to ultraviolet irradiation and contain normal DNA polymerase levels. No direct role in replication can be ascribed to this polymerase activity since cells grow well in its absence. In addition, the LT2 plasmid has been shown to exist in the mutant strain.

Alkanesulfonates↗
PubMed1968

Procedure for identifying nonsense mutations.

A method has been devised for the rapid identification of nonsense mutations (UAG, UAA, UGA codons) in Salmonella. The mutations to be tested are reverted, and the revertants are replica-printed onto lactose plates spread with lawns of tester strains. These tester strains contain F' lac episomes with nonsense mutations in the episomal Z gene. The revertants are infected with the episome from the tester strain lawn. Because S. typhimurium is unable to ferment lactose, only those revertants which have nonsense suppressors are able to grow on lactose. If colonies appear on the lactose plate, it may be concluded that the original strain carries a nonsense mutation, since nonsense suppressors suppress the mutant phenotype.

Bacteriological Techniques↗
PubMedDate not supplied

Isolation of a cDNA clone encoding rat insulin-like growth factor-II precursor.

Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic polypeptides of relative molecular mass (Mr) approximately 7,500 isolated from human plasma each containing four peptide domains in a single chain and identical at more than 60% of their amino acid loci. The B- and A-domains of the IGFs are approximately 40% identical to the B- and A-chains of human insulin. IGF-I and IGF-II have similar in vitro biological activities and receptor reactivity, but are immunologically distinct. IGF-I appears to mediate the effects of growth hormone on cartilage to promote skeletal growth whereas IGF-II may have a special role in fetal development and in the central nervous system. To investigate the in vivo role of IGF-II, we have studied IGF-II biosynthesis in the BRL-3A rat liver cell line. BRL-3A cells synthesize and secrete a 7,484 Mr protein 93% identical to human IGF-II and representing rat IGF-II (rIGF-II). Rat IGF-II is synthesized as a approximately 22,000 Mr prepro-rIGF-II (ref. 12) from 12 S poly(A)+mRNA. In addition, approximately 20,000 Mr pro-rIGF-II has been identified in lysates of biosynthetically labelled intact BRL-3A cells. We report here the isolation of an almost complete cDNA clone for rIGF-II. Our results indicate that pro-rIGF-II is synthesized as a 156 amino acid peptide precursor (17,619 Mr) containing mature rIGF-II 1-67 at its amino-terminus and an 89-residue carboxy-terminal peptide extension.

Amino Acid Sequence↗