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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↗
PubMed1985

Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains.

The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains.

DNA Restriction Enzymes↗
PubMed1992

Molecular cloning of the imidazoleglycerolphosphate dehydratase gene of Trichoderma harzianum by genetic complementation in Saccharomyces cerevisiae using a direct expression vector.

The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector. This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries. The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22,466 daltons. The predicted protein sequence showed 63.6%, 58.7%, and 38.4% identity respectively to the corresponding enzymes from S. cerevisiae, Pichia pastoris and E. coli. Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.

Amino Acid Sequence↗
PubMed1992

Physical analysis of murine albino deletions that disrupt liver-specific gene regulation or mesoderm development.

Complementation analyses of radiation-induced deletion mutations involving the albino (c) locus in Chromosome (Chr) 7 of the mouse have identified several loci, in addition to c, that have important roles in development. The "mesoderm-deficient" (msd) and "hepatocyte-specific developmental regulation-1" (hsdr-1) loci, which are proximal and tightly linked to c, are important in the formation of mesoderm and in the regulation of liver- and kidney-specific induction of various enzymes and proteins, respectively. Cloning deletion-breakpoint-fusion fragments caused by lethal albino deletions that genetically define the extents of the msd and hsdr-1 loci is one way of generating molecular probes for studying the gene(s) involved in these phenotypes. The distal breakpoints of five such deletions were positioned on a long-range (PFGE) map of approximately 1.7 Mb of wild-type DNA surrounding the c, D7Was12, and Emv-23 loci. In addition, the distal breakpoints of two viable albino deletions, which remove part of the tyrosinase gene and extend distally, were localized in the vicinity of the lethal deletion breakpoints. Therefore, the viable deletions can be exploited to generate additional DNA probes that should facilitate the isolation of breakpoint clones from chromosomes carrying lethal deletions defining hsdr-1 and msd.

Albinism↗
PubMed1990

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones.

Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines of Nicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes, ms1, ms2 and ms3, while no changes can be seen in the mitochondrial genome. However, differences were found between the in organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.

Cloning, Molecular↗
PubMed1991

Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade-A mutant deficient in this activity.

We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.

Amidophosphoribosyltransferase↗
PubMed1992

Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesis.

The mammalian CAD gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5' end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a complete CAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of the CAD cDNA was generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from a wild-type Chinese hamster ovary genomic cosmid library. Primary and secondary transfectants, which contain a limited number of cosmid sequences, have been shown to regain methotrexate sensitivity and to take up methotrexate. Furthermore, the DNA from three cosmid clones, isolated from a primary methotrexate-sensitive transfectant, after transfection rescued the methotrexate-resistant phenotype at a high frequency. Restriction endonuclease analysis of the DNA of these cosmid clones indicated that they overlapped extensively and shared two regions of Chinese hamster ovary DNA of 6.6 kb and 20.6 kb. These observations indicate that a gene involved in methotrexate uptake is contained in its entirety within one of these regions. This is the first report of the functional molecular cloning of a gene involved in methotrexate uptake. A general strategy is also described for screening large cosmid libraries from primary transfectants.

Animals↗
PubMed1977

Cold adapted variants of influenza A. II. Comparison of the genetic and biological properties of ts mutants and recombinants of the cold adapted A/AA/6/60 strain.

The genetic and biological properties of 13 recombinant influenza A clones derived at 25 degrees C from the A/AA/6/60-cold variant (by crosses with 4 different wild type strains) were compared with a set of 5-FU induced ts-mutants. The 5-FU mutants had previously been placed into 7 complementation-recombination groups; the A/AA/6/60-cold parent (PI-7) and the 12 cold recombinant clones which were ts were shown to share a lesion with only one of these groups. The parental strain and 5 recombinant clones were evaluated for replication in the lungs and nasal turbinates of hamsters. Each virus appeared to be attenuated; genetic stability correlated with the level of viral replication in the hamster lung, i.e., viruses which grew best showed a tendency to revert to the ts+ phenotype. Characterization of the ts+ revertants for the presence of the cold adaptation property revealed that these viruses exhibited a spectrum of cold adaptation properties. Two viruses, PI-7 (the parental cold variant) and the CR6 recombinant (A/Queensland/6/72) did not revert in either the lungs or nasal turbinates of hamsters.

Animals↗