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At least 73 records · Page 4Linked to original sources
PubMed1990

Genetic analysis of Aspergillus niger: isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group.

This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers, form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9 x 10(-5) (cnxG13 in linkage group I) to 2.1 x 10(-2) (cnxD6 in linkage group III). The mean frequency of haploid chlorate resistant segregants was 1.3 x 10(-3). The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD+ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia+ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 x 10(-3) and 2.0 x 10(-5) respectively.

Aspergillus niger↗
PubMed1991

Characterization of the Escherichia coli K12 gltS glutamate permease gene.

The gltS gene is known to encode a sodium-dependent, glutamate-specific permease. We have localized the Escherichia coli K12 gltS gene with respect to the spoT gene, sequenced it, and recombined a null insertion-deletion allele into the chromosome without loss of viability. The gltS null allele gives a Glt- phenotype, i.e. it abolishes the ability of a gltCc host to grow on glutamate as sole carbon and nitrogen source and also confers alpha-methylglutamate resistance. A multicopy plasmid expressing the gltS gene can reverse the Glt- phenotype of gltS- or wild-type strains while other plasmids show host-dependent complementation patterns. Induction of gltS gene overexpression under control of isopropyl-beta-D-thiogalactoside (IPTG)-inducible promoters severely inhibits growth. The GltS protein is deduced to be a 42425 dalton hydrophobic protein with 2 sets of 5 possible integral protein domains, each flanking a central hydrophilic, flexible region.

Alleles↗
PubMed1990

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants.

A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of beta-galactosidase alpha-complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.

Bacillus subtilis↗
PubMed1990

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli.

A system is described which enabled the selection of a heterologous lep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36,000 daltons, which is in agreement with the calculated molecular weight of 35,782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I genes.

Amino Acid Sequence↗
PubMed1992

Five novel elements involved in the regulation of mitosis in fission yeast.

Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 wee1ts cdc25ts triple mutant strains. These genes were designated wis1(+)-wis5+ for win suppressing, and do not correspond to win1+ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wis1+ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4+ appears to be a specific suppressor of the win1-1 mutation. wis2+ and wis3+ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1ts and cdc25ts, suggesting that the wis2+ and wis3+ products may interact with elements central to the mitotic control.

Cell Cycle↗
PubMed1975

Pyrimidine biosynthesis in Aspergillus nidulans: isolation and preliminary characterisation of auxotrophic mutants.

113 pyrimidine auxotrophs, unable to synthesise UMP have been selected in Aspergillus nidulans. These mutants can be classified by complementation into eight groups, and genetic analysis has shown that five loci are involved. One complex locus consists of the mutually complementing pyrA, pyrB and pyrC groups, as well as the cis-dominant pyrN group, members of which do not complement with members of the A, B or C groups. pyrA mutants have been shown to lack CPSase-ur, pyrB and pyrC mutants have been shown to lack ACTase, and pyrN to lack both these enzymes. This locus appears to code for products which form an enzyme aggregate. The four simple loci, as well as the complex loci have been located genetically, and distinguished from one another on the basis of accumulation of pyrimidine precursors in vivo. The synthesis of ACTase has been shown to subject to end-product repression.

Aspartate Carbamoyltransferase↗
PubMed1975

Genes coding for the structure of the acid phosphatases in Saccharomyces cerevisiae.

The phoE locus, one of the loci in which mutations lack the activity for repressible acid phosphatase, was found to be the structural gene for the enzyme by examining the enzymic characteristics of repressible acid phosphatase activity using cell extracts prepared from the leaky phoE mutants, the PHOE revertants and the PHOE recombinants between the different phoE mutants. Other evidence which strongly suggests that the phoC locus is coding for the constitutive acid phosphatase was obtained by a similar investigation. Although the phoC and phoE loci are tightly linked, they were separable by meiotic recombination.

Acid Phosphatase↗
PubMed1991

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification.

The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir- mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.

Amino Acid Sequence↗
PubMed1991

Closely linked lesions in a region of the X chromosome affect central and peripheral steps in gustatory processing in Drosophila.

We have analyzed a set of closely linked mutations on the X chromosome of Drosophila that lead to defects in gustatory behavior. The mutations map to a small region of the X chromosome between 10E1-4. Two distinct complementation groups, gustB and gustD, map to the ends of this region. These groups show complex complementation patterns with the mutations gustC and GT-1, which also map to this region. We describe the behavioral and electrophysiological properties of the mutants. These mutations affect peripheral receptor properties as well as more central processing steps in the gustatory pathway.

Animals↗
PubMed1978

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12- and P22 23- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L. can be turned on by superinfecting P22 24- 12- or P22 24- 23- mutants (and also not by P22 12- and P22 23-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12- or L virB 23- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.

DNA Replication↗
PubMed1976

Assembly of the mitochondrial membrane system. XVIII. Genetic loci on mitochondrial DNA involved in cytochrome b biosynthesis.

1. Fourteen cytoplasmic mutants of Saccharomyces cerevisiae with a specific deficiency of cytochrome b have been studied. The mutations have been shown to occur in two separate genetic loci, COB 1 and COB 2. These loci can be distinguished by mit- X mit- crosses. Pairwise crosses of cytochrome b mutants belonging to different loci yield 4-6% wild type recombinants corresponding to recombinational frequencies of 8-12%. In intra-locus crosses, the recombinational frequencies range from 1% to less than 0.01%. The two loci can also be distinguished by mit- X rho- crosses. Twenty rho- testers have been isolated of which ten preferentially restore mutations in COB 1 and ten others in COB 2. 2. The COB 1 and COB 2 loci have been localized on mitochondrial DNA between the two antibiotic resistance loci OLI 1 and OLI 2 in the order OLI 2-COB 2-COB 1-OLI 1. The results of mit- X mit- and mit- X rho- crosses have also been used to map the cytochrome b mutations relative to each other. The maps obtained by the two independent methods are in good agreement. 3. Mutations in COB 1 have been found to be linked to the OLI1 locus in some but not in other strains of S. cervisiae. This evidence suggests that there may be a spacer region between the two loci whose length varies from strain to strain. 4. Two mutations in COB 2 have been found to cause a loss of a mitochondrial translation product corresponding to the cytochrome b apoprotein. Instead of the wild type protein the mutants have a new low-molecular weight product which is probably a fragment of cytochrome b. The fact that the mutations revert suggests that they are nonsense mutations in the structural gene of cytochrome b.

Anti-Bacterial Agents↗
PubMed1976

Mutagen sensitivity of Drosophila melanogaster. III. X-linked loci governing sensitivity to methyl methanesulfonate.

Twenty-eight X-linked, recessive mutations of Drosophila melanogaster conferring enhanced sensitivity to the monofunctional alkylating agent, methyl methanesulfonate, have been recoered and assigned to five complementation groups. These groups can be distinguished on the basis of map location and variations in the pattern of mutagen sensitivity. Allelism of members of one complementation group with the previously described meiotic mutant, mei-41, (Baker and Carpenter, 1972) as well as the frequent appearance of female infertility with mutagen sensitivity suggests associated defects in meiotic chromosome behavior or early embryogenesis. Examination of the mutagen sensitivity of double mutants has led to the formulation of a working model of DNA repair for this organism. Studies of a similar nature (Boyd et al., 1976) have identified five additional X chromosome complementation groups, suggesting that the genome of Drosophila melanogaster may contain many loci involved with mutagen sensitivity. The continued isolation and characterization of conditional mutants of this type promises future insights into the mechanisms of DNA replication, DNA repair and recombination in this complex higher eucaryote.

Animals↗
PubMed1975

Heterokaryons in the analysis of genes and gene regulation.

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.

Animals↗
PubMed1979

A two-year-old patient with an atypical expression of GM1-beta-galactosidase deficiency: biochemical, immunological, and cell genetic studies.

Cultured skin fibroblasts from a 2-year-old boy with an atypical form of beta-galactosidase deficiency have been studied. With the artificial substrate 4-methylumbelliferyl-beta-D-galactopyranoside, 5--15% residual activity was found in fibroblasts from this patient. Most of this activity was in the monomeric A form of the enzyme, very little in the multimeric B form. Km value, pH profile, and heat lability of the mutant enzyme were similar to those of beta-galactosidase from control fibroblasts. Immunological studies showed that the mutant enzyme cross-reacted with an antiserum raised against human liver beta-galactosidase, but the catalytic activity per unit antigenic activity was lower than normal. It was demonstrated by somatic cell hybridization that the gene mutation in this patient is different from that in patients with type 1 or type 2 GM1-gangliosidosis. No genetic complementation was found after fusion of fibroblasts from this patient with those from two other clinical variants of GM1-gangliosidosis formerly designated type 3 and adult type 4.

Cells, Cultured↗
PubMed1991

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph.

Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA- auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35,854, predicted from the deduced amino acid sequence, was similar to the 38,000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.

Amino Acid Sequence↗
PubMed1991

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.

Cell Separation↗
PubMed1976

Hypoxanthine-guanine phosphoribosyl transferase deficiency.

In man congential lack of enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan snydrome. The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation HG-PRT deficiency and neurological dysfunction. But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described. Patients with partial HG-PRT defiency have clinically distinctive findings. Sometimes mild neurological abnormalities are observed. Because of marked overproduction of ric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency. There is considerable molecular heterogeneity in HG-PRT deficiency in man. Mutant ebnzymes may exhibit different kinetic and electrophoretic properties, indicating that hterwe might be a mutation on the structural gene coding for HG-PRT. Lack of HG-PRT disturbs purine interconversions profoundly. In addition to an important function of HG-PRT in the uptake of the purine hypoxantine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT...

Animals↗
PubMed1990

Genetic localization of a series of genes affecting glucose oxidase levels in Aspergillus niger.

A number of mutants of Aspergillus niger, affected in glucose oxidase (GOX) expression, are described. The overproducing mutants could be classified into seven complementation groups whereas two glucose oxidase-negative complementation groups were recognized. These nine gox loci were assigned to linkage groups using master strains with marked chromosomes. Three gox loci are in linkage group II, one is in III, two are in V and two are in linkage group VII. One weak glucose oxidase-overproducing mutant could not be assigned to one of the linkage groups. These genetically well characterized mutants will be used in a strain improvement program based on genetic recombination.

Aspergillus niger↗