Restriction endonuclease analysis of ribosomal DNA from Saccharomyces cerevisiae

Summary Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1970). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for DNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both and subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the subunit, beside the primary mutation affecting the subunit. The hypothetical subunit mutation seems to modify quantitatively the rifampicin resistance caused by the subunit mutation.     

Isolation and some properties of bacteriophage alpha3 gene J mutant

Summary To elucidate the in vivo function of the J gene of microvirid (isometric) phages, we isolated several strains carrying a double mutation in J and H genes from phage 3 and then constructed single mutants each having an amber codon in the J gene. The J mutants could not multiply in suppressor-less hosts and were deficient in single-stranded progeny DNA synthesis. Nucleotide sequences of the wild-type and mutant 3 J genes were analyzed to determine the mutation sites. The amino acid sequence of the J gene was also deduced from the nucleotide sequence and compared with those of X174 and G4.     

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Summary Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada ⁺) cells contained similar constitutive levels of O⁶-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada ⁺ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada ⁺, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - PNNG N-propyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MMS methyl methanesulphonate     

A Comparison of the Lipopolysaccharides and O-Specific Polysaccharides of Azospirillum brasilense Sp245 and Its Omegon-Km Mutants KM018 and KM252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas–liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units 2)--D-Rhap-(1 3)--D-Rhap-(1 3)--D-Rhap-(1 2)--D-Rhap-(1 2)--D-Rhap-(1 as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical composition.     

Degradation pathways from n-tridecane to α, ω-tridecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-tridecanedioic acid via n-tridecanoic acid and via ,-tridecanediol from n-tridecane in the mutant S 76 of Candida tropicalis was studied. It was found that resting cells of S 76 produced ,-tridecanediol from n-tridecane.With n-tridecanol as substrate, the ,-diol could also be detected. The mutant S 76 was able to produce ,-tridecanedioic acid using either n-tridecanol or n-tridecanoic acid as the sole carbon source. Quantitative changes in the concentration of -hydroxy tridecanoic acid and other intermediates were recorded during the formation of ,-dioic acid.The results confirm the existence of two metabolic pathways mentioned above in the course of ,-dioic acid formation from odd n-alkane in the mutant S 76 of C. tropicalis.     

Hereditary respiration deficiency in Saccharomycodes ludwigii

Saccharomycodes ludwigii, supposed to be petite-negative, gave rise to respiration-deficient mutants when acriflavine and ultraviolet irradiation, respectively, were applied to this yeast, strain IFO 1194. The frequency of such mutants was very low as compared with that in Saccharomyces cerevisiae and other petite-positive yeasts. Cytochrome composition was characterized by spectrophotometry at the temperature of liquid nitrogen. The respiratory mutants examined contained cytochrome c unaltered in quality and quantity. Cytochrome b was often present only in small amounts though never absent, while cytochrome a+a₃ was either present or absent. The respiratory mutants could form zygotes after conjugation with a wild-type culture of opposite mating type ( vs. a). The hybridization and segregation analysis of spore tetrads showed the inheritance of respiratory mutant character to be either Mendelian or non-Mendelian and similar to that of pet (nuclear) and rho^- (cytoplasmic) mutants, respectively, in Saccharomyces cerevisiae.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Increased osmotic tolerance of some Aspergilli isolated from ‘Usar’ (alkaline) soils — A possible indication of ecological specialization

Studies on the osmotic optima for growth of some Aspergilli isolated from Indian Usar (alkaline) soils have revealed the occurrence of four osmophilic and twenty-five other species which can be categorised as facultative tolerant on the basis of their osmotic concentration optima. In addition, the Usar soil strains possess a comparatively greater osmotic tolerance than their fertile soil counterparts which may be attributed to the relatively hypertonic conditions prevailing in Usar soils.     

The taxonomic significance of the trunk limbs of the chydoridae (Cladocera)

Summary The differences in the structure of the trunk limbs allow to outline three sections of Chydoridae (see table I and fig. 1), coinciding with the sections distinguished according to the structure of the head pores.

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Chydoridae (Cladocera)

Chydoridae (. ), , .

     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic ( and ) and regulatory () subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of free CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with and subunits of Xenopus CK2, with the subunit of D. rerio, and with Xl CK2A¹⁵⁶, which although inactive can bind tightly to CK2, and with Xl CK2E⁷⁵E⁷⁶, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2 or CK2E⁷⁵E⁷⁶ in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2, the activity increased further to 15–20-fold of the controls. Transfection of CK2 alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2A¹⁵⁶ yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 or CK2E⁷⁵E⁷⁶ with CK2A¹⁵⁶ caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 subunits. These results can be interpreted as meaning that CK2A¹⁵⁶ is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 subunit. Addition of recombinant CK2 to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 subunit is limiting in the extracts and that an excess of free CK2 has been produced in the transfected cells. Transfection of cells with CK2E⁷⁵E⁷⁶ results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2E⁷⁵E⁷⁶ to heparin increases considerably when it forms part of the holoenzyme CK2₂₂.     

An immunocytochemical investigation of non-neuronal enolase in cerebellum: a new astrocyte marker

Summary Immunohistochemical methods were used to study in the optical and electron microscopes the localization of the so-called non-neuronal enolase (the -isoenzyme) in adult mouse cerebellum. Three separate methods, including a novel modification involving sequential incubation with biotin-conjugated sheep anti-rabbit serum and avidin-conjugated peroxidase to reveal tissue-bound specific antibody, gave similar results. The enolase was found exclusively in astrocytes: astrocyte perikaryal cytoplasm and processes were heavily labelled and nuclei were frequently stained. The extensive network of astrocyte processes in the granular layer and white matter and the Bergmann fibres in the molecular layer are readily visualized by these methods. Neither neurones nor oligodendrocytes were found to be labelled. The results are discussed in relation to the possible existence of a hybrid form of the enolase and the biochemistry of cerebellar glial cells.     

Inheritance of the dwarf plant type in blackgram (Vigna mungo (L.) Hepper)

Summary The inheritance of the dwarf plant type was studied in blackgram (V. mungo (L.) Hepper). Type 9 has erect plant type with normal internode length. The mutant line, EMSD has reduced internode length. The F₁, F₂ and F₃ generations of a cross between Type 9 and EMSD and its reciprocal were studied. The extreme dwarf plant type appeared to be governed by a single recessive gene, dw ₁ dw ₁ with no cytoplasmic effect.Part of Ph.D. Thesis submitted by the first author     

Moraxellae: Identification

According to published reports, all strains ofMoraxella osloensis and Moraxella (Oligella) urethralis grew on a mineral-base medium supplemented with 0.015M (0.123%) sodium acetate, but not all alkalinized a mineral-base medium supplemented with 0.008% bromthymol blue and 0.2% sodium acetate. This seeming discrepancy was examined. The growth of most strains ofM. urethralis was inhibited by 0.008% bromthymol blue, and some by 0.2% anhydrous sodium acetate. All (42) strains ofM. osloensis andM. urethralis alkalinized a mineral-base medium supplemented with 0.1% sodium acetate trihydrate and 0.001% bromthymol blue or phenol red.     

Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli

Summary The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion GT, leading to amino acid substitution Val¹⁴⁶Phe. This mutational change marks the second domain of the subunit involved in rifampicin binding.