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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↗
PubMed1976

Occurrence of a regulatory deficiency in purine biosynthesis among pur A mutants of Salmonella typhimurium.

A defect in the repression of the de novo purine biosynthetic enzymes was detected among purA mutants of Salmonella typhimurium. We suggest that the defect is caused by an altered purine regulation gene (purR) which affects the response level of at least five of the de novo enzymes to repression by excess adenine. Thus the unlinked genes controlling these enzymes constitute a regulation controlled wholly or in part by a purR gene product. The regulation of the guanine operon is regulated by some other mechanism independent of purR.

Adenine↗
PubMed1989

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination.

A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL. couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.

Dictyostelium↗
PubMed1989

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus.

The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic production in strains otherwise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.

Cloning, Molecular↗
PubMed1992

Complementation of the Saccharomyces cerevisiae srb1-1 mutation: an autoselection system for stable plasmid maintenance.

The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.

Cell Division↗
PubMed1991

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.

This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.

Aspergillus niger↗
PubMed1989

Isolation and characterization of dominant, pleiotropic drug-resistance mutants in Chlamydomonas reinhardtii.

Three independent pleiotropic drug-resistance (pdr) mutants were isolated by selecting for resistance to the anti-microtubule herbicides amiprophos-methyl (APM) and oryzalin (ORY). These three mutants and a previously isolated mutant, ani1 (anisomycin resistance), were semi-dominant in heterozygous diploids, and they displayed varying degrees of resistance to structurally and functionally unrelated inhibitors such as cycloheximide, cryptopleurine, emetine, atrazine, and nonidet P-40. Linkage analysis and genetic mapping suggested that three of the four mutants, including ani1, define a single locus, here named pdr1. The fourth mutant defined a new locus, pdr2, which is located on the left arm of linkage group VI. One pdr1 mutant exhibited unusual genetic interactions, including enhanced ts-lethality and synergistic increases in drug resistance, when combined with pdr2-1 and with herbicide-resistant alleles of three other genes.

Anisomycin↗
PubMed1985

Intragenic suppression of the temperature-sensitivity caused by a mutation in a gene controlling transcription (fit) in Escherichia coli.

Starting from a transcription-defective strain harbouring a temperature-sensitive mutation in the fit gene, a rifampicin-resistant, temperature-insensitive derivative has been isolated. Genetic analysis of this derivative demonstrated the presence of a second temperature-sensitive mutation in the same gene. The two mutations mutually suppress each other's phenotype. From cotransduction experiments, the fit gene has been mapped 0.32 min and 0.16 min clockwise from the aroD and pps loci, respectively, at 37.5 min on the linkage map. The mutants harbouring either or both of the fit mutations are defective in RNA synthesis at the non-permissive temperature. The fit gene product is suggested to function as an accessory transcription factor.

Escherichia coli↗
PubMed1977

Isolation of putrescine-requiring mutants of Neurospora crassa.

Two auxotrophs of Neurospora crassa have been isolated that give a positive growth response to putrescine, spermidine or spermine. One of the mutants is deficient in ornithine decarboxylase activity and has been designated put-I. Both mutants map on linkage group VR, fail to complement and are infertile when crossed to one another, indicating that they are probably alleles. A putrescine auxotroph is incapable of suppressing a pro-4 mutant. The isolation of the mutants confirms that putrescine is an essential factor the normal growth of the organism, and is synthesized via a single pathway in Neurospora.

Crosses, Genetic↗
PubMed1989

Cloning and physical characterization of linked lysine genes (lys4, lys15) of Saccharomyces cerevisiae.

The plasmid pSC4 which carries a 7.8 kb yeast DNA insert at the BamHI site of the Vector YEp13, complemented simultaneously MO-59-13c lys4, LU75 lys15 and LU32 lys4lys15 (double) mutations of Saccharomyces cerevisiae. The 1.9 kb BamHI-XbaI DNA insert of the subclone pSO51 complemented the LU75 lys15 mutation. The 2.8 kb Xhol-XhoI DNA insert of the pSO52 subclone, like pSC4, complemented all three mutations. The 1.9 kb BamHI-XbaI DNA and the 2.8 kb Xhol-XhoI DNA were 100 bp apart in the pSC4 DNA insert and exhibited no homology with each other upon Southern hybridization. The 1.9 kb BamHI-XbaI DNA insert exhibited homology with the pSC4 and pSO51 DNA as well as the genomic DNA of MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) when digested with appropriate restriction enzymes. The 2.8 kb XhoI-XhoI DNA insert exhibited homology with the pSC4 and pSO52 DNA as well as MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) genomic DNA, when digested with XhoI enzyme. The 2.8 kb DNA probe also hybridized with ply(A)+ RNA from RC1 and lys4+ transformant but not that from MO-59-13c lys4 mutant.

Blotting, Northern↗
PubMed1989

Adenine and pyrimidine genes of Aspergillus niger and evidence for a seventh linkage group.

Mutants of Aspergillus niger requiring adenine and one mutant requiring cytosine were isolated after low-dose mutagenesis and enrichment. In addition we had mutants of two genes involved in the pyrimidine biosynthesis isolated as 5-fluoro-orotic acid-resistant mutants. The fifteen adenine-less mutants could be placed in seven complementation groups. From each group a representative mutant was analyzed in order to determine the linkage group by analysis of the mutants in a heterozygous diploid carrying markers in six linkage groups. AdeF could not be assigned to any one of these linkage groups and proved to be linked to nicB, oliC and cnxC, none of which could be placed in a linkage group. Thus, conclusive evidence was obtained for a seventh linkage group. As pyrA was used as selection marker for transformation, we constructed a pyrA strain with a linked marker which can be used in the genetic analysis of transformations.

Adenine↗
PubMed1992

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos.

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early postimplantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.

Albinism↗