Conversion of 5′-Methylthioadenosine into S-adenosylmethionine by yeast cells

5′-Methylthio[U-¹⁴C]adenosine was used as a culture supplement for Candida utilitis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The U-¹⁴C)-labeled adenine of 5′-methylthio[U-¹⁴C]adenosine did not contribute to the labeling of the amino acid component of the sulfonium compound.     

Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.     

Reversal of malignant transformation induced by the oncogenic virus SV40. I. Induction of reversal to normal type for the contact inhibition trait

The possibility of induction by the oncogenic DNA-containing virus SV40 of reversions to normal phenotype as regards contact inhibition ("flat" revertants), was studied in spontaneously transformed chinese hamster fibroblasts. Negative selection was used for detection of revertants. The method adopted allowed to study the mutagenic activity of the virus, while excluding its transforming effect. In all experiments the frequency of revertants after infection exceeded that in control series. The value of induction varied from 1.2 to 28.4 X 10(-6). The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) known to increase the frequency of mutations induced by carcinogens in vitro, displayed no enhancing effect on the frequency of revertants induced by SV40. The lack of enhancement of virus-induced reversions after TPA treatment might be explained by the lack of the transforming effect of SV40 in the system studied. Some of the normal "flat" colonies were T-antigen positive, i. e. the viral oncogene was expressed. The role of mutations induced by SV40 in cellular genes controlling malignancy is discussed.     

Molecular analysis of G+C-rich upstream sequences regulating transcription of the human carbonic anhydrase II gene.

The upstream promoter sequences of the human carbonic anhydrase II (CA II) gene have been studied by 5' deletion analysis. Promoter activity was assayed by transfection and chloramphenicol acetyltransferase assay in both human HeLa cells and murine L cells. This investigation showed that the CA II promoter is comparable in activity to that of the simian virus 40 early-region promoter and enhancer and that the CA II upstream sequences exert a different pattern of control in the two cell lines.     

Estradiol and estrogen receptor-dependent stabilization of a minivitellogenin mRNA lacking 5,100 nucleotides of coding sequence.

We have developed a transfection assay to investigate the estrogen-mediated stabilization of cytoplasmic vitellogenin mRNA. A minivitellogenin (MV5) gene containing the 5' and 3' untranslated and coding regions but lacking 5,075 nucleotides of internal coding sequence was constructed. Cotransfection of the MV5 plasmid and a Xenopus estrogen receptor expression plasmid into Xenopus liver tissue culture cells yielded a 529-nucleotide MV5 mRNA, which was specifically stabilized by estrogen. MV5 mRNA exhibited the increased stability indicative of positive regulation when the estradiol-estrogen receptor complex was present and was not destabilized by unliganded estrogen receptor. Transfected estrogen receptor, estradiol, and 529 nucleotides of the 5,604-nucleotide vitellogenin B1 mRNA were sufficient for stabilization.     

A long range restriction map of the distal human X chromosome short arm around the steroid sulfatase locus.

The distal short arm of the human X chromosome is of interest because it contains genes which escape X chromosome inactivation and because it is subject to frequent deletions in human patients. The steroid sulfatase gene has been particularly well studied as an example of a gene which escapes X inactivation and which is included in a number of these deletion events. For these reasons a physical map of the region around the STS gene would be of interest. We have constructed a rare cutting enzyme map of this area and have determined the position of several nearby markers with respect to STS. We have also oriented the 5' and 3' ends of the STS gene on this map and have determined the centromeric and telomeric portions of the region. Finally, we have shown that this map can be used to locate deletion breakpoints in STS deficient patients.     

Biosynthesis and control of beta-lactam antibiotics: the early steps in the "classical" tripeptide pathway

The interaction between growth and secondary metabolism develops from physiological responses of the producer organism to its environment. Nutrients are channelled into primary growth processes or into secondary processes such as antibiotic biosynthesis by a variety of metabolic controls, the nature of which has been extensively studied in organisms producing beta-lactam antibiotics via the tripeptide, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. In the following article we review the early stages of beta-lactam biosynthesis in fungi and actinomycetes, keeping in mind the regulation of primary pathways that provide the amino acid precursors of this group of antibiotics, as well as the regulation of the secondary pathway itself. Of special importance to organisms engaging in secondary metabolism are the control mechanisms that suppress the nonessential process during rapid growth but allow secondary metabolic genes to be expressed and resources to be diverted when environmental factors generate the appropriate biochemical signals.     

Cloning of cDNAs for human phosphoribosylpyrophosphate synthetases 1 and 2 and X chromosome localization of PRPS1 and PRPS2 genes

Cloned cDNAs representing the entire, homologous (80%) translated sequences of human phosphoribosylpyrophosphate synthetase (PRS) 1 and PRS 2 cDNAs were utilized as probes to localize the corresponding human PRPS1 and PRPS2 genes, previously reported to be X chromosome linked. PRPS1 and PRPS2 loci mapped to the intervals Xq22-q24 and Xp22.2-p22.3, respectively, using a combination of in situ chromosomal hybridization and human x rodent somatic cell panel genomic DNA hybridization analyses. A PRPS1-related gene or pseudogene (PRPS1L2) was also identified using in situ chromosomal hybridization at 9q33-q34. Human HPRT and PRPS1 loci are not closely linked. Despite marked cDNA and deduced amino acid sequence homology, human PRS 1 and PRS 2 isoforms are encoded by genes widely separated on the X chromosome.