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PubMed1994

The barley stripe mosaic virus gamma b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis.

Barley stripe mosaic virus contains seven genes, one of which specifies a 17-kD cysteine-rich protein, gamma b, that is known to affect virulence. To further characterize the role of gamma b in pathogenesis, we mutagenized sequences encoding amino acids within two clusters of cysteine and histidine residues in the cysteine-rich domain and a group of basic amino acids located between the clusters and determined the effects of these mutations on the symptom phenotype in barley. Three single amino acid substitutions in cluster 1 and two amino acid exchanges in the basic region caused bleached symptoms associated with pronounced elevations in accumulation of gamma b protein. In contrast, three single amino acid substitutions in cluster 2 and a mutation in the basic motif resulted in attenuated ("null") symptoms typical of those produced when the gamma b gene is deleted. Tissue infected with these "null" mutants accumulated slightly elevated amounts of the gamma b protein but significantly lower levels of coat protein and the putative movement protein beta b. Genetic complementation tests revealed that cluster 1 mutations are dominant over the wild-type gamma b gene, whereas those in cluster 2 are recessive. These results highlight the pivotal role of gamma b in pathogenesis and suggest that the two cysteine-rich clusters are functionally distinct and that they affect different aspects of disease development.

Amino Acid Sequence↗
PubMed1993

The isolation of mutagen-sensitive nuv mutants of Aspergillus nidulans and their effects on mitotic recombination.

More than 200 mutants of Aspergillus nidulans were isolated as hypersensitive to the monofunctional alkylating agent MNNG and/or UV-irradiation (designated nuv mutants). Of these, 23 were selected for further characterization. All were markedly hypersensitive to both MNNG and the quasi-UV-mimetic mutagen 4-NQO. The hypersensitive phenotype of each mutant was shown to result from mutation of a single gene. The nuv mutants exhibited a diverse range of growth responses on solid media containing various concentrations of MNNG or 4-NQO. This suggested that they represented many nonallelic mutations. Analysis to determine the dominance/recessiveness of the nuv mutations with respect to hypersensitivity revealed that most were fully recessive, although several appeared to be semidominant. A novel system to assay homologous mitotic recombination using simple plating tests was developed. The system was exploited to determine the effects of the nuv mutations on mitotic recombination. Of the 23 mutations tested, 10 caused a hypo-recombination phenotype and three a hyper-recombination phenotype, while 10 appeared to have no effect on recombination. The hypo-rec effect of one of the mutations, nuv-117, appeared to be semidominant. Transcomplementation analysis between seven of the nuv mutations defined at least six nonallelic loci.

4-Nitroquinoline-1-oxide↗
PubMed1998

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

3T3 Cells↗
PubMed2003

QTLs for tomato powdery mildew resistance (Oidium lycopersici) in Lycopersicon parviflorum G1.1601 co-localize with two qualitative powdery mildew resistance genes.

Tomato (Lycopersicon esculentum) is susceptible to the powdery mildew Oidium lycopersici, but several wild relatives such as Lycopersicon parviflorum G1.1601 are completely resistant. An F2 population from a cross of Lycopersicon esculentum cv. Moneymaker x Lycopersicon parviflorum G1.1601 was used to map the O. lycopersici resistance by using amplified fragment length polymorphism markers. The resistance was controlled by three quantitative trait loci (QTLs). Ol-qtl1 is on chromosome 6 in the same region as the Ol-1 locus, which is involved in a hypersensitive resistance response to O. lycopersici. Ol-qtl2 and Ol-qtl3 are located on chromosome 12, separated by 25 cM, in the vicinity of the Lv locus conferring resistance to another powdery mildew species, Leveillula taurica. The three QTLs, jointly explaining 68% of the phenotypic variation, were confirmed by testing F3 progenies. A set of polymerase chain reaction-based cleaved amplified polymorphic sequence and sequence characterized amplified region markers was generated for efficient monitoring of the target QTL genomic regions in marker assisted selection. The possible relationship between genes underlying major and partial resistance for tomato powdery mildew is discussed.

Chromosome Mapping↗
PubMed1990

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Animals↗
PubMed1971

Allelic variability in comparative complementation confirming that the ade12-specified protein of yeast is bifunctional.

Mutants at the adel2 locus of Saccharomyces cerevisiae are deficient in adenylosuccinate synthetase and are also responsible for constitutive purine biosynthetic activity. Ninety-six alleles were tested for restoration of each of these functions by allelic complementation and the results from the two types of test of every allele pair in the two matrices were compared with each other. Either the reactions were consistent, both complementing or noncomplementing, or inconsistent, with either enzymatic or regulatory function restored. The frequency and distribution of inconsistent behavior accords with the expectation that the adel2 locus specifies a bifunctional protein serving independent roles in enzymatic activity and in regulation of pathway activity. In a parallel experiment and analysis the addition of 1.0 m KCl increases the overall frequency of positive complementation, but skews the distribution of inconsistency sharply toward restoration of the regulatory function.

Adenine↗
PubMed1970

ICR-induced frameshift mutations in the histidine operon of Salmonella.

Both the acridine half-mustard, ICR191, and the nonalkylating azaacridine derivative, ICR364-OH, induce three classes of frameshift mutations in the histidine operon of Salmonella typhimurium. (i) One class is completely stable in reversion tests and is presumed to represent deletion of one or a few critical nucleotide pairs or two nearby frameshifts. One extended deletion was found out of 11 stable mutations. (ii) Of two spontaneously reverting classes which also are considered to predominantly involve base deletions, one is unaffected in reversion with ICR191, nitrosoguanidine, and diethylsulfate, and the other is induced to revert with ICR191. (iii) A third class, considered to predominantly involve base additions, responds in reversion tests with ICR191 as well as with nitrosoguanidine and diethylsulfate. Other investigators have shown that one mutant of this class is a "plus" frameshift and that nitrosoguanidine acts in reversion to delete a guanine plus cytosine base pair. Although such plus frameshifts are found with high frequency among mutations selected from acridine-treated bacteria or when strong selection pressure is applied for their detection in reversion tests, data from this laboratory indicate that this class of plus frameshifts is rare among mutations derived spontaneously or after treatment with a variety of other mutagens. Finally, we demonstrate that the alkylating ICR191 and the nonalkylating ICR364-OH preferentially cause mutations in different chromosome regions and that their spectra of activity only partially overlap that found for spontaneous frameshift mutations.

Acridines↗
PubMed1998

The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus.

3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB, leuC and leuD genes of an extreme thermophile, Thermus thermophilus, was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli. The order was the leuC, leuD, and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron-sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved.

3-Isopropylmalate Dehydrogenase↗
PubMed1983

Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus.

Thirty-nine new temperature-sensitive mutants of vaccinia virus have been isolated, expanding a previously reported collection of mutants (R. C. Condit and A. Motyczka, Virology 113, 224-241, 1981) to a total of 65. The 65 mutants have been assigned to 32 complementation groups, based primarily on a qualitative spot test described previously (Condit and Motyczka, 1981). Representatives of each complementation group have been assayed for DNA and protein synthesis at the nonpermissive temperature, revealing one new DNA-negative complementation group, three new groups which contain mutants defective in late protein synthesis, and ten new groups containing mutants which synthesize DNA and protein in a normal fashion. Marker rescue has been achieved with 29 of the 65 mutants using cloned DNA fragments from wild-type virus. These 29 mutants together represent 20 of the 32 complementation groups. A preliminary physical map of the mutants is presented.

DNA, Viral↗
PubMed1986

Expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants.

Transformed cell lines derived from murine C127 cells were constructed that express the influenza virus RNA-dependent RNA polymerase proteins (PA, PB1, and PB2). Cell lines that express only one or all three of the proteins were tested for their ability to complement temperature-sensitive viral mutants incubated at the nonpermissive temperature. Two cell lines were isolated that express all three polymerase genes and complement the growth of PB2 temperature-sensitive mutants at the nonpermissive temperature. One of these lines also complemented PA temperature-sensitive mutants. The viral titers obtained in these two cell lines were 12-fold to 1000-fold higher than the viral titers obtained upon growth of the corresponding temperature-sensitive mutant in C127 cells at the nonpermissive temperature.

Animals↗
PubMed1993

[Direct genetic diagnosis in Huntington's chorea].

Huntington's disease is a late manifesting autosomal dominant neurodegenerative disorder. It is characterized by motor disturbance, loss of cognitive functions and psychiatric manifestations. Recently, the disease causing mutation, an unstable DNA sequence in the coding region of the Huntington gene on chromosome 4p, has been identified. A trinucleotide (CAG) repeat is expanded over the normal range and can easily be detected by standard laboratory methods. Accurate genetic testing can now be offered in clinically questionable cases and to subjects at risk for Huntington's disease. Furthermore, there seems to be a correlation between the size of the expanded CAG repeat and the age of onset in affected individuals. We have investigated more than 130 individuals from different affected families and illustrate the advantages and the clinical application of the new method.

Adult↗
PubMed1971

Specific suppression of mutations in genes 46 and 47 by das, a new class of mutations in bacteriophage T4D.

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.

Carbon Isotopes↗
PubMed1985

Temperature-sensitive mutations causing reversible paralysis in Caenorhabditis elegans.

A method has been developed for the isolation of temperature-dependent paralytic mutants of the nematode Caenorhabditis elegans, based on a screening procedure using short-time exposure to 30 degrees C. Of ten mutants isolated, eight lose their motilities between 30 degrees C and 33 degrees C without prominent changes in body forms. The other two strains that are mainly described in this report are accompanied by alterations in body forms. One mutation, cn101, is recessive and an allele of cha-1. The cn101 mutant shows reversible paralysis at 30 degrees, accompanied by a hypercontracted and coiled body form. At the restrictive temperature, the strain is resistant to all tested inhibitors of acetylcholinesterase (AChE). Another mutation, designated mah-2 (cn110), is a sex-linked semidominant that is mapped as 0.6 map units left of dpy-6. The cn110 mutant is rapidly paralyzed at the restrictive temperature and has a straight and rigid body form; the mutant rapidly recovers when the temperature is lowered. No disorganization of the muscle structure was detected by polarized light and electron microscopic inspection.

Alleles↗
PubMed1980

Genetic and biochemical studies on glutamate-pyruvate transaminase from Drosophila melanogaster.

We have used electrophoretic variants of glutamate-pyruvate transaminase (GPT, E.C., 2.6.1.2) in Drosophila melanogaster to genetically map the structural gene to position 42.6 on the X chromosome. By pseudodominance tests over several deficiencies we have localized it cytogenetically to the interval 11Fl-2 to 12Al-2. The sedimentation constant (s20,w) of the native enzyme was determined in sucrose density gradients to be 5.9 and the native molecular weight approximately 87,000. The similarity in physical properties to mammalian enzymes suggests that the enzyme may also be dimeric in D. melanogaster.

Alanine Transaminase↗
PubMed2001

Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors.

pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors. In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli. pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers. These vectors could be introduced into S. suis, E. coli, Salmonella typhimurium, S. pneumoniae, and S. equi ssp. equi by electrotransformation. The recA gene was cloned from S. suis and sequenced, and this information was used in the construction of a recA mutant of S. suis. Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S. suis and E. coli recA mutants compared with the parental strains. These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S. suis and E. coli. Moreover, cloning of the functional S. suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S. suis but not in E. coli. These results showed that pSET vectors are useful tools for cloning and analyzing S. suis genes in S. suis strains directly.

Base Sequence↗
PubMed1976

Assembly of the mitochondrial membrane system. XIX. Genetic characterization of mit- mutants with deficiencies in cytochrome oxidase and coenzyme qh2-cytochrome c reductase.

Nineteen mutants of S. cerevisiae exhibiting a double deficiency in cytochrome oxidase and coenzyme QH2-cytochrome c reductase (also cytochrome b deficient) have been studied. The mutants have been crossed to a set of rho- tester strains with different segments of mitochondrial DNA. The mutants have also been crossed to mit- testers with defined genetic lesions. In addition, crosses were performed with a respiratory competent strain to ascertain whether mitotic and meiotic segregants could be isolated with only one of the two enzymatic deficiencies. The rho- testers allowed the doubly deficient mutants to be separated into two classes. Mutants in class 1 were not restored by any of the rho- testers and appeared to have separate mutations, one in cytochrome oxidase and the other in cytochrome b. Mutants in class 2 were restored by a set of rho- clones whose retained segments of mitochondrial DNA contained the cytochrome b but not the cytochrome oxidase loci. These appeared to behave as single hit mutations. Further studies, however, indicated that both class 1 and class 2 mutants carried separate mutations in two different loci. Mitotic and meiotic segregants with a single enzymatic deficiency could be isolated. In a number of strains, the mutations were mapped in known cytochrome oxidase and cytochrome b loci. The apparent discrepancy of the rho- tests for the class 2 mutants was shown to be probably due to a high unstability in one of the mutations. It has been concluded that all the doubly deficient strains carry two mutations in previously described cytochrome oxidase and cytochrome b loci. This conclusion argues against the existence of a single gene on mitochondrial DNA that controls the biosynthesis of the two respiratory enzymes.

Cell Division↗
PubMed2005

A rapid method for retrovirus-mediated identification of complementation groups in Fanconi anemia patients.

Fanconi anemia (FA) is a rare autosomal recessive disorder that results from mutations in at least 11 different genes. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intragenic variations, emphasizing the importance of identifying the complementation groups. In the present study we have employed bicistronic retrovirus vectors that coexpress FA-specific cDNAs for complementation groups A, C, F, and G, together with the enhanced green fluorescence protein (EGFP), allowing for specific analysis of transduced EGFP+ cells within bulk cultures by flow cytometry. In addition, the assay relies on the correction of the characteristic FA-associated G2/M arrest after treatment of cells with DNA-damaging agents, which is analyzed by flow cytometry. Results obtained with this assay matched the complementation groups known for 12 control lymphoblast cell lines tested. We report here the results obtained for 48 FA patients with unknown complementation groups using this new assay. Complementation groups were identified for 24 patients. We have identified mutations in the genes corresponding to the assigned complementation group in 23 samples. This assay has now been established in a standardized fashion for complementation assignments in FA patients and the subsequent directing of rapid mutation analysis in those patients.

Cell Line↗
PubMed2004

Skeletal defects in ringelschwanz mutant mice reveal that Lrp6 is required for proper somitogenesis and osteogenesis.

Here, we present evidence that Lrp6, a coreceptor for Wnt ligands, is required for the normal formation of somites and bones. By positional cloning, we demonstrate that a novel spontaneous mutation ringelschwanz (rs) in the mouse is caused by a point mutation in Lrp6, leading to an amino acid substitution of tryptophan for the evolutionarily conserved residue arginine at codon 886 (R886W). We show that rs is a hypomorphic Lrp6 allele by a genetic complementation test with Lrp6-null mice, and that the mutated protein cannot efficiently transduce signals through the Wnt/beta-catenin pathway. Homozygous rs mice, many of which are remarkably viable, exhibit a combination of multiple Wnt-deficient phenotypes, including dysmorphologies of the axial skeleton, digits and the neural tube. The establishment of the anteroposterior somite compartments, the epithelialization of nascent somites, and the formation of segment borders are disturbed in rs mutants, leading to a characteristic form of vertebral malformations, similar to dysmorphologies in individuals suffering from spondylocostal dysostosis. Marker expression study suggests that Lrp6 is required for the crosstalk between the Wnt and notch-delta signaling pathways during somitogenesis. Furthermore, the Lrp6 dysfunction in rs leads to delayed ossification at birth and to a low bone mass phenotype in adults. Together, we propose that Lrp6 is one of the key genetic components for the pathogenesis of vertebral segmentation defects and of osteoporosis in humans.

Aging↗