Biomedical subjects
Publications and source records attributed to E Balzi.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.
In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 [McCusker and Haber, Genetics 119 (1988a) 303-315]. The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain. The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from scl1+ possesses a putative secretory signal peptide. The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of scl1+ demonstrates that it is an essential gene.
The Saccharomyces cerevisiae gene PDR1, responsible for pleiotropic drug resistance, was isolated from a genomic DNA cosmid library by hybridization to the flanking LEU1 gene, followed by subcloning the drug-sensitive phenotype into the transformed pdr1-1, pdr1-2, and pdr1-3 drug-resistant mutants. A RNA molecule of 3.5 kilobases was identified as the PDR1 transcript. The nucleotide sequence of the complementing DNA fragment contained a 3192-nucleotide open reading frame. Disruption of the pdr1 and PDR1 genes restored or increased drug sensitivity. Analysis of the PDR1 deduced amino acid sequence revealed several homologies to four different regulatory proteins involved in the control of gene expression, including a cysteine-rich motif suggested to be a metal-binding domain for DNA recognition. A model is proposed of a general transcriptional control by PDR1 of several target genes encoding proteins from plasma, mitochondria, and possibly other permeability barriers.
In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H+-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to map the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5kb HindIII fragment was subcloned and it restored both Leu+ and Pma+ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5' end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.
A 5.45-kb fragment containing the 5' end of the ribosomal RNA transcriptional unit from the fission yeast Schizosaccharomyces pombe was cloned in the yeast-E. coli shuttle vector YEp13. The transcription start point was mapped by R looping and S1 nuclease protection. The sequence of the entire external transcribed spacer (ETS) and its flanking regions was determined. Comparison of the sequence around the transcription start point with those of four budding yeasts (Saccharomycetoideae) reveals a consensus sequence from position -9 to -4 from the start. This sequence is likely to be an important element of the promoter for yeast RNA polymerase I (Pol.I). Comparison of all known Pol.I promoter sequences reveals a strong bias for nucleotides (nt) at several positions between -16 and +10. These nt may have a critical role in the transcription initiation process. The S. pombe ETS, which comprises 1355 bp, is significantly longer than those of the budding yeasts and lacks any significant sequence homology with the Saccharomyces cerevisiae ETS. R-loop analysis reveals a putative processing site within the ETS of S. pombe.
Cocultures of nylon wool-separated, non-adherent (T enriched) mouse spleen cells with mitomycin C-treated adherent cells resulted in the production of antiviral activity in the culture supernatants. This antiviral activity was defined as interferon gamma (IFN-gamma) because of its lability at pH 2 and the susceptibility to inactivation by anti mouse IFN-gamma antibodies. Treatment of non-adherent cells with anti-Thy 1.2 or anti-Lyt 1.2 antibody plus complement abolished IFN production. In contrast, anti-Lyt 2.2 plus complement treatment had no effect on IFN production. Abrogation of IFN production was also seen when anti-Ia antibodies were added. IFN could be observed in the SMLR, regardless of the H-2 haplotype of the mouse strains with the exception of NZB mice. Addition of hydrophilic polymers such as polyethyleneglycol or DEAE-dextran enhanced IFN production.
Mouse immune interferon was produced from a T-lymphoma cell line, L12-R4, upon stimulation with phorbol myristic acetate at a concentration of 2 x 10(-7) M/ml. The crude interferon, concentrated by precipitation with (NH4)2SO4 at concentration of 80%, was sequentially chromatographed on two sorbents, namely Sephacryl S-200 and Phenyl-Sepharose CL-4B. With molecular sieve chromatography L12-R4 interferon was observed to have an apparent molecular weight of 45.000 d, while from its chromatographic behaviour on Phenyl-Sepharose CL-4B interferon appeared to be highly hydrophobic like other mammalian interferons. The L12-R4 tumor cells therefore provide a unique source of immune interferon for purification and may represent an useful model for studying the molecular mechanisms involved in T cell differentiation leading to immune interferon production.