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PubMed1989

Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome.

Pulsed-field gel electrophoresis was used to screen temperature-sensitive mutants of vaccinia virus for the ability to convert replicated viral DNA into mature linear 185-kilobase hairpin-terminated genomes. Of 30 mutually noncomplementing mutants tested, 5 displayed a temperature-sensitive defect in the resolution of the telomere fusion configuration within concatemeric replicative intermediates, resulting in a failure to convert such intermediates to the linear monomeric genome. Adjacent genomic units in the concatemeric arrays generated in these mutants were arranged in both tandem and inverted orientations. The observation that four of the five mutants had a severe general defect in the synthesis of the late class of viral proteins suggests that at least one late protein is directly required to resolve the telomere fusion intermediate to hairpin termini. The identification of such telomere resolution proteins should be facilitated by genetic and molecular characterization of resolution-defective mutants, such as C63, in which late protein synthesis is not severely affected.

Animals↗
PubMed1995

The Hox11 gene is essential for cell survival during spleen development.

The HOX11 homeobox gene was identified via the translocation t(10;14) in T cell leukaemia. To determine the function of this gene in mice, null mutations were made using homologous recombination in ES cells to incorporate lacZ into the hox11 transcription unit. Production of beta-galactosidase from the recombinant hox11 allele in +/- mutants allowed identification of sites of hox11 expression which included the developing spleen. Newborn hox11 -/- mice exhibit asplenia. Spleen formation commences normally at E11.5 in hox11 -/- mutant embryos but the spleen anlage undergoes rapid and complete resorption between E12.5 and E13.5. Dying spleen cells exhibit molecular features of apoptosis, suggesting that programmed cell death is initiated at this stage of organ development in the absence of hox11 protein. Thus hox11 is not required to initiate spleen development but is essential for the survival of splenic precursors during organogenesis. This function for hox11 suggests that enhanced cell survival may result from the t(10;14) which activates HOX11 in T cell leukaemias, further strengthening the association between oncogene-induced cell survival and tumorigenesis.

Animals↗
PubMed2002

CP3 is involved in negative regulation of phytochrome A signalling in Arabidopsis.

Several mutants with altered phytochrome A (phyA) signalling have been identified in screenings under continuous far-red light (FR). The latter protocol could preclude the identification of mutants affected in the signalling pathway that operates even under transient phyA activation, compared to the high-irradiance response (HIR) pathway that requires continuous FR. Since some photomorphogenic mutants show shoot-height phenotypes, the screening was conducted on dwarf mutants of Arabidopsis thaliana (L.) Heynh. from the ABRC stocks grown under hourly FR pulses. The dwarf mutant cp3 (compacta 3) showed normal hypocotyl length and folded cotyledons in darkness but enhanced hypocotyl-growth inhibition and cotyledon unfolding under pulsed FR. The HIR and the response mediated by phyB were not affected. Under pulsed FR, seed germination and blocking of greening upon transfer to white light were enhanced in cp3. PHYA levels were normal in cp3. The phenotype under pulsed FR but not the adult phenotype required phyA. We propose that CP3 is involved in the negative regulation of the signalling pathway that saturates with transient activation of phyA.

Adaptation, Physiological↗
PubMed1993

T7 RNA polymerase mutants with altered promoter specificities.

The amino acid at position 748 in T7 RNA polymerase (RNAP) functions to discriminate base pairs at positions -10 and -11 in the promoter. We have constructed a series of T7 RNAP mutants having all possible amino acid substitutions at this position. Surprisingly, most (13/19) substitutions result in active RNAPs, and many of these exhibit altered promoter specificities. Identification of mutant RNAPs with altered specificities expands the repertoire of highly specific phage RNAPs that are available for use in phage RNAP-based transcription systems and highlights the complexity of sequence-specific DNA recognition.

Amino Acid Sequence↗
PubMed2005

PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells.

The attachment of pathogenic Neisseria species to human cells, in which type IV pili (Tfp) play a key but incompletely defined role, depends on the ability of these bacteria to establish contacts with the target cells but also interbacterial interactions. In an effort to improve our understanding of the molecular mechanisms of N. meningitidis adherence to human cells, we screened a collection of defined mutants for those presenting reduced attachment to a human cell line. Besides underscoring the central role of Tfp in this process, this analysis led to the identification of mutants interrupted in a novel gene termed pilX, that displayed an adherence as impaired as that of a non-piliated mutant but quantitatively and qualitatively unaltered fibres. Moreover, the pilX gene, which encodes a pilin-like protein that copurifies with Tfp fibres, was also found to be essential for bacterial aggregation. We provide here several piece of evidence suggesting that PilX has intrinsic aggregative but no adhesive properties and that the reduced numbers of adherent bacteria seen with a pilX mutant result from the absence of interbacterial interactions. These data extend the current model for Tfp-facilitated adherence of N. meningitidis to human cells by suggesting that the pili lead to an increase in net initial adherence primarily by mediating a cooperation between the bacteria, which is supported by the finding that a major effect on initial adherence could be observed in a wild-type (WT) genetic background after a mechanical removal of the bacterial aggregates.

Bacterial Adhesion↗
PubMed1992

Sensitive method for identification of minor hepatitis B mutant viruses.

There is increasing evidence for selection of hepatitis B virus (HBV) mutants during the natural course of infection, after immunization, and during interferon therapy. These mutants can prolong viral persistence, escape immune-mediated elimination, and influence the clinical sequelae of infection. Here we describe a sensitive and reliable method that allows detection of HBV mutants within a heterogeneous virus population. The method consists of amplification of HBV DNA with vector-HBV hybrid primers, efficient and enzyme-free cloning of the amplified DNA fragment, identification by hybridization with synthetic oligonucleotide probes, and sequencing of the cloned DNA fragments. We demonstrate that up to 10(5) HBV DNA fragments can be obtained in cloned form from a single amplification reaction, the ratio of HBV pre-C "wild-type" and mutant sequences of heterogeneous populations can be determined, and very minor virus subpopulations can be identified. The method can be applied to other viral or nonviral nucleic acid sequence variation studies and should help to resolve the role of mutant and wild-type viruses in pathogenesis and the forces driving their selection.

Base Sequence↗
PubMed1991

Isolation and characterization of Escherichia coli mutants with altered rates of deletion formation.

Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.

Base Sequence↗
PubMed1988

Engineering thermostability in subtilisin BPN' by in vitro mutagenesis.

A procedure has been developed for the isolation and identification of mutants of the bacterial serine protease, subtilisin, which exhibit enhanced thermostability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with a variety of chemical mutagens to introduce random mutations in the coding sequence. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermostability were selected by a simple plate assay procedure, which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. The identification and characterization of eight different stabilizing mutations are described. Several mutants containing various combinations of these stabilizing mutations were constructed by oligonucleotide-directed mutagenesis. Combining independent, stabilizing mutations in the same subtilisin molecule has resulted in an approximate multiplicative decrease in the rate of thermal inactivation. In this way, a variant of subtilisin has been constructed which is about 12-fold more stable than wild-type subtilisin, with no radical changes in the tertiary protein structure but rather minor, independent alterations in amino acid sequence. The ultimate goal in these studies is to be able to accurately predict where stabilizing changes can be made in a protein.

Amino Acid Sequence↗
PubMed1980

Targeted deletions of sequences from closed circular DNA.

Closed circular DNA interacts with complementary sequences of single-stranded DNA to form displacement loop (D loop) structures in vitro. The site of D-loop formation can be directed by using single-stranded DNA derived from a selected restriction fragment. Circular DNA containing a D loop can then be linearized by cleavage with endonuclease S1. This cleavage appears to remove a limited number of nucleotides from each strand of the circular DNA substrate. Incubation with polynucleotide ligase followed by propagation in vivo leads to circular DNA molecules that bear small, single deletions in the region of the single-stranded DNA sequence chosen for the formation of the D loops. We have utilized these manipulations of DNA to construct tetracycline-sensitive deletion mutants of plasmid pBR322. The level of mutagenesis obtained by the procedure is sufficiently high that selective growth and screening procedures are not necessary for the isolation or identification of mutants. The frequency, variety, and small size of the deletions obtained within the selected target regions present considerable advantage for genetic and biochemical analysis. The method is quite general in rationale and should be immediately applicable to phage and viruses having infectious circular DNA genomes or recombinant DNA species propagated in circular plasmid vectors.

Chromosome Deletion↗
PubMed1968

Analysis of sporulation mutants. II. Mutants blocked in the citric acid cycle.

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.

Acetates↗
PubMed2001

Molecular cloning and characterization of dystrophin and Dp71, two products of the Duchenne Muscular Dystrophy gene, in zebrafish.

Dystrophin, the protein responsible for Duchenne Muscular Dystrophy (DMD), plays a critical role in the maintenance of the muscle membrane integrity. There are several forms of dystrophin derived from the DMD gene by alternative promoter usage. In addition to full-length dystrophin (Dp427), four shorter transcripts have been identified: Dp260, Dp140, Dp116 and Dp71. The functional role played by the different products of the DMD gene is not yet determined. To get insight into the function of dystrophin and related products, we have investigated the presence of dystrophin in zebrafish. This choice takes advantage of large-scale mutagenesis screens in zebrafish, which have led to the identification of several mutants with motility defects. The identification and characterization of the genes affected by these mutations is likely to provide relevant information for the understanding of the molecular mechanisms of muscle development and function. Two cDNA clones encoding the homologues of dystrophin and Dp71 in zebrafish were identified and characterized. Both transcripts exhibit a high degree of sequence homology with the dystrophin and Dp71 proteins described in higher vertebrates. In addition, three alternative spliced transcripts that occur at the C-terminal end of the zebrafish DMD gene have been identified. These transcripts exhibit different patterns of tissue expression. We have also determined the chromosomal localization of dystrophin on the radiation hybrid map of the zebrafish genome. Our results indicate that the dystrophin gene is localized to linkage group one. Altogether, these results give new insights on the physiological role played by dystrophin and related proteins, and provide new tools for the identification of mutated genes associated with muscle defects in zebrafish.

Alternative Splicing↗
PubMed1994

string of pearls encodes Drosophila ribosomal protein S2, has Minute-like characteristics, and is required during oogenesis.

The first allele of string of pearls (sop) was isolated as a recessive female sterile mutant in a P element enhancer trap screen. Oogenesis in homozygous sop females arrests at approximately stage 5. In addition, homozygous flies of both sexes have Minute-like characteristics that include reduced bristles, delayed development and larval lethality. sop maps to 30D/E on chromosome 2L and encodes the Drosophila homolog of eukaryotic ribosomal protein S2. The gene is present in a single copy in the Drosophila genome and the level of mRNA present in mutant animals is reduced. The identification of a mutant allele that blocks development at a mid-stage of oogenesis may indicate that sop has a specific developmental role during oogenesis in addition to its general role in protein synthesis as a component of the small ribosomal subunit.

Alleles↗
PubMed1998

Dissecting embryonic and seedling morphogenesis in Arabidopsis by promoter trap insertional mutagenesis.

Development can be considered to comprise the co-ordinated regulation of patterning at different levels: patterning of cells to form tissues, patterning of tissues to form organs, and patterning of organs to generate the characteristic architecture of the organism. These processes are expected, in turn, to be mediated by the precise spatial and temporal regulation of patterns of gene expression during development, which depend on appropriate signalling mechanisms. In order to investigate molecular events of morphogenesis in plants, we have utilized a system of promoter trap insertional mutagenesis in Arabidopsis, to generate both phenotypic mutants and gene fusions that represent markers useful in studying the regulation of patterning. A screen of transgenic seedlings containing a T-DNA promoter trap has led to the identification of mutants defective in seedling shape and embryonic development, and of GUS fusion genes that are expressed in spatially restricted patterns. Mutants have been crossed with marker lines expressing cell type-specific GUS activities, to investigate their cellular organization. For example, the POLARIS marker gene is expressed in the embryonic and seedling root tip. When crossed with hydra, which lacks an embryonic root, and with emb30, which lacks both embryonic and seedling roots, it is nevertheless expressed in the correct relative position, and we hypothesize that it represents a novel marker of root positional information, independent of root morphogenesis.

Arabidopsis↗
PubMed1994

maf1 mutation alters the subcellular localization of the Mod5 protein in yeast.

Two forms of Mod5p, a tRNA modification enzyme, are found in three intracellular compartments, mitochondria, cytoplasm and nucleus, but are encoded by a single MOD5 gene. The two forms of the enzyme, Mod5p-I and Mod5p-II differ at the N-termini and are produced by initiation of translation at different start codons. Mod5p-I does contain a mitochondrial targeting signal and is distributed between mitochondria and cytoplasm, whereas Mod5p-II is found in the cytosol and nucleus (Boguta, M., et al. 1994, Mol. Cell. Biol. 14, 2298-2306). In the present work mutants which mislocalize the Mod5p-I enzyme were isolated. The screen was based on a correlation between the amount of cytosolic protein and the efficiency of tRNA mediated suppression. Identification of mutants is possible because a red pigment accumulates in the cells unable to suppress an ade2-1 nonsense allele. The maf1 mutant, with an altered intracellular localization of the Mod5p-I protein, was isolated. Immunofluorescence data suggest that the mutation causes mislocalization of the Mod5p-I to the nucleus.

Antimicrobial Cationic Peptides↗
PubMed2004

Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold.

Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a approximately 42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.

Animals↗
PubMed1994

Analysis of in vivo mutation in exon 8 of the rat hprt gene.

We have analyzed mutations in exon 8 of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in T-lymphocytes from the spleens of ethylnitrosourea-treated female rats. Presumptive hprt- mutants were isolated by clonal growth in the presence of 6-thioguanine. DNA from 6-thioguanine-resistant colonies was amplified by the polymerase chain reaction using intronic primers flanking hprt exon 8. The identification of mutant sequences and the separation of mutant DNA from contaminating wild-type DNA was accomplished by denaturing gradient gel electrophoresis. Of 118 clones analyzed, 19 contained mutations and DNA sequence analysis identified eight unique sequence alterations. We also used single-strand conformation polymorphism analysis to screen for mutations in the same fragment of the hprt gene. This analysis was less successful than denaturing gradient gel electrophoresis in detecting the eight unique mutations. The procedures described here may represent a useful approach for studying the mechanisms of in vivo mutation.

Animals↗
PubMed1998

Identification of point mutations in mixtures by capillary electrophoresis hybridization.

We have developed a rapid method for unambiguous identification and mutant fraction determination of individual mutants in mixtures of DNA sequence variants each differing by one or a few nucleotides. This method has applications to such diverse areas as interpretation of mutational spectra, screening of populations for polymorphisms and identification of species in environmental mixtures. In our approach, a mixture of unknown sequences labeled with a fluorescent dye is combined with a set of predetermined sequences (standards) representing the variants to be assayed. Labeling the standards with another dye allows the two sets of variants to be measured independently. Using constant denaturing capillary electrophoresis, the sequence variants are separated as individual peaks on the basis of differential melting equilibria. The unknown sequence variants are initially identified based on co-migration with particular standards. This preliminary identification is verified by hybridization of the unknown variants with the co-migrating standards within the capillary. We demonstrate the use of capillary electrophoresis hybridization to dissect complex mutational spectra of human cells in culture.

Animals↗
PubMed1986

Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions.

We examined bovine papillomavirus type 1 (BPV-1) DNAs mutated in the E2 open reading frame (ORF) to determine their ability (i) to transform C127 cells and (ii) to remain extrachromosomal in transfected cells. Results obtained with deletion mutants and insertion mutants containing a linker with translational termination codons in all possible reading frames indicated that an E2 ORF gene product(s) is necessary for efficient transformation, as well as viral plasmid replication and maintenance in the context of the full BPV-1 genome. Complementation assays in which mutant BPV-1 DNAs were transfected into cell lines expressing some viral functions from integrated BPV-1 cDNAs demonstrated that the E2 ORF product, when provided in trans, could allow BPV-1 E2 mutants to remain extrachromosomal. The E2 function could also augment transformation of some, but not all, BPV-1 E2 mutants, allowing identification of another region of BPV-1 involved in cellular transformation. It is likely that the role of the BPV-1 E2 product(s) in transformation and plasmid maintenance is indirect. A BPV-1 mutant altered in the E5 ORF is transformation defective and unable to replicate as a stable plasmid in C127 cells.

Animals↗