Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.     

Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase.

Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis.     

An immunodiffusion activity stain for yeast histidinol dehydrogenase.

A procedure is described for detecting enzymatically active yeast histidinol dehydrogenase-antibody complexes in immunodiffusion agar. The method employs a coupled dye system, consisting of a tetrazolium salt and a phenazine methosulfate intermediate, that produces an insoluble formazan and stains active precipitin lines red. The specificity of the reaction is indicated by its dependence on substrate and by its dependence on an intact HIS4C region, based on observations with mutant forms of the yeast HIS4 multifunctional protein.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

γ-Glutamylamine cyclotransferase

Summary -Glutamylamine cyclotransferase, an enzyme found in a number of animal tissues and cells, catalyzes the conversion of -(L--glutamyl)-L-lysine to free lysine and 5-oxo-L-proline as well as the release of free amines and the formation of 5-oxo-L-proline from a variety of other L--glutamylamines. Among its substrates are both the mono- and di--glutamyl derivatives of putrescine, spermidine and spermine, and a derivative of -(L--glutamyl)-L-lysine in which both the -amino group and the carboxyl group of the lysine moiety are blocked. The enzyme does not act on most -glutamyl--amino acids, nor is it active toward the -lysyl derivatives of L-aspartic acid or D-glutamic acid. Derivatives of -(L--glutamyl)-L-lysine in which the -amino or the -carboxyl function of the glutamyl moiety is blocked also do not serve as substrates. The specificity of -glutamylamine cyclotransferase is in accordance with the proposal that it functions biologically in the latter stages of the catabolism of products of the action of transglutaminases. Some suggestions as to the manner in which -glutamylamine cyclotransferase serves this function are made based on present knowledge of protein degradation.     

Use of in situ hybridization to investigate the regulation of hippocampal corticosteroid receptors by monoamines.

The hippocampus receives major noradrenergic and serotoninergic (5-HT) innervations which interact with corticosteroid-sensitive cells. However, the subregional localization of these actions and the corticosteroid receptor types involved have not been defined and current ligand binding techniques for estimating corticosteroid receptors are hampered by several methodological limitations. We have developed in situ hybridization histochemical techniques to allow specific and sensitive estimation of glucocorticoid (GR) and mineralocorticoid receptor (MR) mRNA expression in rat hippocampus. Investigation of the effects of 5,7-dihydroxytryptamine lesions of 5-HT neurons showed significantly reduced GR and MR mRNA expression in some hippocampal subregions. Both abnormal 5-HT neurotransmission and excessive corticosteroid secretion are associated with major affective disorders, particularly depression. The crucial interaction between these two systems may occur, at least in part, at the level of regulation of hippocampal corticosteroid receptor expression.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.     

The use of azidoarylimidoesters in RNA-protein cross-linking studies with Escherichia coli ribosomes

A series of related hetero-bifunctional RNA-protein cross-linking reagents has been prepared, carrying an imidoester or N-hydroxysuccinimide ester function at one end of the molecule, and a phenylazido function at the other. These compounds have been applied to RNA-protein cross-linking studies with ribosomal subunits, and one of them, p-azido-phenylacetic imidoester, has proved to be a particularly useful reagent for this purpose. The reagent first reacts specifically with protein amino groups, and subsequent photolysis of the azide group leads to cross-linking to the RNA in yields of up to 8% of the total protein. The whole reaction takes place under very mild conditions in aqueous solution.The individual proteins concerned in the cross-links have been identified by two-dimensional gel electrophoresis, and the existence of a covalent cross-link was confirmed by the isolation by two different methods of protein-oligonucleotide complexes carrying a ³²P label. Although most of the ribosomal proteins could be cross-linked to their corresponding ribosomal RNA within the individual subunits, RNA-protein cross-links at the ribosomal subunit interface were only detectable in vanishingly small amounts.The advantages of this type of genuine hetero-bifunctional reagent in RNA-protein cross-linking studies are discussed.     

The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide.

The his4 region of yeast encodes the information for the third (phosphoribosyl-AMP cyclohydrolase), second (phosphoribosyl-ATP pyrophosphohydrolase), and tenth (histidinol dehydrogenase) steps in the histidine biosynthetic pathway. These three activities co-purify with a single protein which has a subunit molecular weight of 95,000 (95,000 protein), as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Extracts of yeast strains which carry nonsense or deletion mutations in various portions of the his4 region, purified in parallel by affinity chromatography on AMP-agarose columns, were examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. All such mutant extracts examined were found to lack the 95,000 protein found in a strain carrying a wild type his4 allele. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride, during the purification of the trifunctional enzyme prevented the degradation of the 95,000 protein to polypeptides of lower molecular weight. Monospecific antibody prepared against the 95,000 protein removed all three of the activities specified by his4 from solution; active 95,000 protein was recovered in the resuspended immunoprecipitates. All this evidence shows that the product of the his4 region is a trifunctional, 95,000-dalton protein. Preliminary evidence from two-dimensional gel electrophoresis, NH2-terminal analysis, and gel filtration column chromatography indicates that the native trifunctional enzyme is a dimer of identical 95,000-dalton subunits.     

Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH.

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347--352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213--217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.