Recombinational analysis of OXI1 mutants and preliminary analysis of their translation products in S. cerevisiae

Summary Recombinational analysis of oxil mutants was performed using a and a mutant strains with the same mitochondrial and nuclear backgrounds, derived from strain 777-3A.In spite of minor inconsistencies the overall map of oxi1 mutations can be constructed on the basis of wild-type recombinant frequencies in the two-point oxi1 ^{– –} x oxi1 ^– crosses. The frequencies of wild-type recombinants varied in a wide range from 0.003% to 16%, reaching the maximal values expected for unlinked mitochondria) markers. No distinct clusters of mutants were observed.The analysis of translation products of oxil mutants showed that all but one of the oxil mutants studied are connected with the conspicuous changes of the polypeptide band corresponding to subunit 11 of cytochrome c oxidase in electrophoresis on polyacrylamide gels. The exceptional G565 mutant showed no conspicuous change in subunit II, but lacked subunit I of cytochrome c oxidase.Various oxi1 mutants seemed to carry premature chain termination mutations. Most of them show a correlation between the length of the putative fragment of subunit II synthesized and the position on the genetic map. The direction of translation is from the V2 to the V60 mutation. The V2 mutation is proximal to cap and V60 proximal to the par locus.     

Recombinational analysis of oxi2 mutants and preliminary analysis of their translation products in S. cerevisiae

Summary Genetic and biochemical studies were performed with mutants allocated to the mitochondrial oxi2 gene.Recombinational analysis of 19 oxi2 mutants was performed using and a mutant strains derived from the same genetic background. The frequencies of wild-type recombinants in oxi2 ^– × oxi2 ^– crosses varied from 0.002 to 17%. The map of oxi2 mutations constructed on the basis of these frequencies shows many internal inconsistencies. In the course of rho ^– deletion mapping five classes of oxi2 mutations were distinguished. The results of deletion analysis are in agreement with those of recombinational mapping.The analysis of mitochondrial translation products by SDS-polyacrylamide electrophoresis of 20 oxi2 mutants shows that 17 of them are connected with conspicuous changes of 22 kd polypeptide band corresponding to subunit III of cytochrome oxidase. At least four of them carried instead of subunit III clearly visible significantly shorter polypeptides (12.8 to 20.1 kd). These were, most likely, shorter fragments of subunit III resulting from chain termination mutations. Colinearity was observed between the lenght of new polypeptides and the positions of the respective mutations on the recombinational map. These data confirm hat oxi2 encodes subunit III of cytochrome oxidase and suggest that translation of the oxi2 gene is in the direction from V303 to V273.     

Sporulation in mitochondrial OXI3 mutants of Saccharomyces cerevisiae

Summary By use of a set of mitochondrial oxi3 mutants (mit ^–, defective in cytochrome oxidase) we have shown that sporulation is possible at a very low level of respiration (below l% of the wild type respiration). A specific role for oxygen in biosynthesis during sporulation is suggested. Correlation of these results with the genetic map of the OXI3 region reveals that one group of mutants, mapping in the central part of the OXI3 region, is capable of sporulation.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe

Summary The three mutator strains ana ^r-8, ana ^r-14, and diu ^r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe. Abbreviations. MtDNA = mitochondrial DNA; S. pombe = Schizosaccharomyces pombe; cox1, cox2, and cox3 refer to the mt genes coding for the three subunits of cytochrome oxidase; ATPase 6 (oli2), ATPase 8 (aapl in Saccharomyces cerevisiae, urf a61 in HeLa) and ATPase 9 (olil) refer to the three respective subunits of ATP synthase; cob is thegene for apocytochrome b; urf a is the single intergenic unassigned reading frame in S. pombe; 1 rRNA and s rRNA refer to the large and small ribosomal RNA, respectively. Mut^– is a cytoplasmic mutator (the corresponding wild type allele is mut⁺). Mit^– are mitochondrially inherited respiratory deficient mutants with mitochondrial protein synthesis; RC = respiratory competent, RD = respiratory deficient.     

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae

Summary Ten nuclear suppressors (nam mutations) of the mitochondrial oxi1-V25 ochre mutation are characterized. They restore to some extent the functional form of cytochrome oxidase, as judged by the results of growth tests, cytochrome spectra, cytochrome oxidase activities, and electrophoresis of the products of mitochondrial translation. The nam mutants can suppress some mit ^– mutations mapping in four mitochondrial genes. They act on a number of chain-terminating mit ^– mutations. When grown on glycerol medium some double mutants nam _x-V25 show an increased sensitivity to paromomycin, while the growth of others is stimulated by the drug. The nam mutants are probably omnipotent suppressors resulting from mutations in nuclear gene(s) specifying mitoribosomal protein(s).     

Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants

We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase. DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations. All, except the missense mutant S157L, have impaired electron transfer and respiratory activity. Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized. In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced. In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable. A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes. Received: 20 January / 23 June 1998     

Glucosamine-resistant mutations in yeast affecting the glucose repression sensitivity of electron transport enzymes

Summary We have isolated two mutant strains, GSA^r-4 and GSA^r-5, which are able to grow on lactate in the presence of D-glucosamine. The glucosamine-resistant phenotype results from the cooperative effects of mutations in three loci, GAR1, GAR2 and GAR3. Both glucosamine resistant mutant strains were doubly mutant at gar1 gar2 (GSA^r-4) or gar1 gar3 (GSA^r-5). The mutants were also shown to exhibit glucose repression insensitive synthesis of NADH-cytochrome c reductase and cytochrome c oxidase. Glucose-repressible synthesis of the following enzymes was seen: succinic dehydrogenase, succinic: cytochrome c reductase, maltase (PNPGase), galactokinase, -galactokinase. The glucose-repression insensitivity segregates in association with the glucosamine resistance.     

Large-scale isolation of the two major polypeptides of the hepatitis B surface antigen (HBsAg) by gel filtration in 6 M guanidinium chloride

Purified hepatitis B surface antigen (HBsAg) of subtype ay was solubilized in guanidinium chloride and submitted to chormatography on Sepharose 4B in the presence of guanidinium chloride. The polypeptides P1 (M_r = 24,000) and P2 (M_r = 29,000) were eluted in the same fraction with a minor contaminant (M_r = 40,000). Large amounts of these two polypeptides were obtained in a single step. This technique which constitutes a method for large-scale purification of the P1 and P2 polypeptides should permit more complete characterization of the P1 and P2 polypeptides and of their antigenic determinants.     

Replacement of a conserved glycine residue in subunit II of cytochrome c oxidase interferes with protein function

In this paper we describe the isolation and characterization of a respiration-deficient yeast strain which is defective in the function of subunit II of cytochrome c oxidase. This strain, VC32, carries a mutation in the mitochondrial COX2 gene which converts a conserved glycine residue to arginine. The conserved glycine is in a region implicated as important for ligating the Cu_A redox center and for interaction with cytochrome c. We have also characterized five revertants of VC32 which have recovered respiratory function; all five were mapped to the mitochondrial genome. In three of the five revertants the wild-type glycine codon is restored, while in two of the five the mutant arginine codon is still present. These two strains are likely to possess alterations either in components of the mitochondrial translation machinery or in mitochondrially-encoded gene products that interact directly with subunit II to assemble an active oxidase complex.     

The polypeptides of canine distemper virus: Synthesis in infected cells and relatedness to the polypeptides of other morbilliviruses

The biochemical and immunological properties of the polypeptides of canine distemper virus (CDV), their synthesis and processing in infected cells, and their relatedness to the polypeptides of other morbilliviruses have been studied. CDV virions contain six major polypeptides which are analogous to those of measles virus (MV). These polypeptides with their estimated molecular weights (m_r) are: L (200,000); H (76,000); P (66,000); NP (58,000); F (62,000), which consists of two disulfide-linked polypeptides, F₁ (40,000) and F₂ (23,000); and M (34,000). The H, F₁, and F₂ polypeptides of CDV are glycosylated; the presence of carbohydrate on F₁ is in contrast to its absence on the F₁ of MV. The CDV F₂ has a larger apparent M_r than the MV F₂ (23,000 vs 12,000). The NP and P polypeptides of CDV are phosphorylated, and in pulse-chase experiments in CDV-labe;ed cells the P polypeptide was rapidly lost. In addition to the structural polypeptides, a putative nonstructural protein, NS (M_r 18,000), was found in CDV-infected cells. The polypeptide also turned over rapidly in pulse-chase experiments.The immunological relatedness of the polypeptides of MV and CDV and of two other morbilliviruses, rinderpest (RV) and a bovine encephalitis virus (107) was shown by immuno-precipitation of the viral polypeptides from CDV- and MV-infected cells with antisera against each of the four viruses. The only failure to exhibit reciprocal reactivity between CDV and MV was found with the H polypeptides, where only a one-way cross was found, i.e., MV antiserum precipitated all of the CDV polypeptides, whereas CDV antiserum precipitated all of the MV polypeptides except H. RV antiserum resembled that of MV; it precipitated all of the polypeptides of both MV and CDV, whereas 107 antiserum, like that of CDV, precipitated all of the CDV polypeptides and all of the MV polypeptides except H. These results indicate that these four morbilliviruses with different host ranges are antigenically closely related, with MV apparently more closely related to RV, and CDV to 107 virus. In spite of their antigenic similarities, the individual polypeptides of CDV and MV could be easily distinguished by peptide mapping. Some similarities were found in the internal polypeptides P, NP, and M, but very little in the surface glycoproteins, H and F₁.     

A mitochondrial frameshift suppressor maps in the tRNASer-var1 region of the mitochondrial genome of the yeast S. cerevisiae

Summary A polypeptide chain-terminating mutation (M5631) previously has been shown to be a +1T insertion in the yeast mitochondrial gene oxi1, coding for subunit II of the cytochrome c oxidase. A spontaneously arisen frameshift suppressor (mfs-1) that is mitochondrially inherited suppresses this mutation to a considerable extent. The suppressor mutation was mapped by genetic and molecular analyses in the mitochondrial tRNA^Ser-var1 region of the mitochondrial genome of the yeast S. cerevisiae. Genetic analyses show that the suppressor mfs-1 does not suppress other known mitochondrial frameshift mutations, or missense and nonsense mutations.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

Analysis of a DNA segment from rat liver mitochondria containing the genes for the cytochrome oxidase subunits I,II and III,ATPase subunit 6, and several tRNA genes

Summary The sequence of a 6.24 kb DNA segment of the mitochondrial genome from rat liver has been determined. It comprises several genes coding for mitochondrial protein subunits and five tRNA genes in the following order: cytochrome oxidase subunit I — tRNA _(UCN) ^Ser —tRNA^Asp — cytochrome oxidase subunit II — tRNALys —ATPase subunit — cytochrome oxidase subunit III —tRNA^Gly — potential open reading frame — tRNA^Arg —two potential open reading frames. The tRNA genes were detected by a computer search programme. The assignments for the protein coding sequences were made through comparison with known sequences, mainly from the yeast mitochondrial proteins (e.g. Bonitz et al. 1980). Our data are discussed with regard to the features of gene arrangement, codon usage, and tRNA structure in mammalian mitochondria (Anderson et al. 1981).Abbreviations COX I, COX II, COX III mitochondrial cytochrome oxidase subunits I, II, and III - ATPase mitochondrial ATPase subunit 6 - U.R.F. unidentified reading frame (Anderson et al. 1981). Other abbreviations follow IUB-IUPAC conventions.     

Formaldehyde polymers, 25. Copolymerization of 6,8-dimethyl-4-oxo-5-chromanylmethyl acrylate with styrene,methyl methacrylate,and vinyl chloride

The free radical copolymerizations of 6,8-dimethyl-4-oxo-5-chromanylmethyl acrylate (DCA) with styrene (St), methyl methacrylate (MMA), and vinyl chloride (VC) by the solution polymerization method were studied. For copolymerization of DCA(M₁) with St(M₂), the reactivity ratios were r₁=0,32, r₂=0,70; with MMA(M₂) they were r₁=0,43, r₂=2,00; and with VC(M₂) they were r₁=4,00, r₂=0,08. The Q and e values for DCA were 0,54 and 0,42, respectively. It was also shown that the chromanone skeleton acts as UV-absorber, and inhibits photodegradation of the copolymers.     

Polypeptide synthesis induced inNicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus

Summary Infection ofNicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M_r and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M_r of RRV-specific polypeptides ranged from 210 000 to 18 000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.     

Radical copolymerization of cyclic acetals derived from acrolein, 1. Copolymerization with acrylonitrile and α-chloroacrylonitrile

Copolymerizations of 2-vinyl-1,3-dioxane ( 1a ), 5,5-bis(hydroxymethyl)-2-vinyl-1,3-dioxane ( 1b ), and 5,5-dimethyl-2-vinyl-1,3-dioxane ( 1c ) with acrylonitrile (AN) and α-chloroacrylonitrile (CPAN) in N,N-dimethylformamide (DMF) were investigated. For the AN(M₁)/ 1a (M₂), AN(M₁)/ 1b (M₂), and AN (M₁)/ 1c (M₂) pairs, the copolymerization parameters r₁ are 5,2, 3,7, and 3,9, respectively. For the CPAN (M₁)/ 1a (M₂), CPAN (M₁)/ 1b (M₂), and CPAN (M₁)/ 1c (M₂) pairs, the r₁ values are 7,95, 7,80, and 8,90, respectively. In all cases the r₂ value is approximately zero. A clearly different influence of the monomer feed composition on the copolymerization rate is observed when the reaction takes place in the absence of solvent compared with that made in DMF. The explanation is related with the existence of associations between acrylonitrile or x-acrylonitrile and the vinyl-dioxane compound.     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

Laminin production by murine melanoma cells: possible involvement in cell motility

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [³⁵S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [³⁵S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the M _r=950kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the M _r=400kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (M _r =200kD) and as a disulfide-linked B dimer (M _r=400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.