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At least 865 records · Page 48Linked to original sources
PubMed1993

Using mitotic recombinant mutant clonal lymphocytes for physical mapping of polymorphic loci on the short arm of human chromosome 6.

Immunoselection has been used to identify human lymphocytes that have undergone spontaneous mutation resulting in the loss of expression of one of the codominant HLA-A alleles. Approximately 35% of such mutations are the consequence of mitotic recombination events. Mitotic recombination is the result of nonsister chromatid exchange that leaves the mutated cell homozygous for all loci distal to the crossover point. The location of the crossover has been regionalized for 99 independently derived mutant lymphocyte clones by identification of their loss or retention of heterozygosity at seven reference polymorphic loci on chromosome 6. If a polymorphic locus of unknown map position is studied in clones from this ordered set of mitotic recombinants, and clones that display loss of heterozygosity and retention of heterozygosity of the locus are observed, then the map position of the locus is between the appropriate reference loci of the ordered set. The newly mapped locus becomes a reference locus in turn. In this way the mitotic recombinant mutant clones can be used to generate an ordered set of crossover points with a theoretical resolution limited only by the number of mutants generated. In this paper such a set of mutants is used to refine or confirm the map position of eight polymorphic loci on chromosome 6.

Chromosome Mapping↗
PubMed1986

General nonchemotactic mutants of Caulobacter crescentus.

We have examined 35 mutants that have defects in general chemotaxis. Genetic analysis of these mutants resulted in the identification of at least eight che genes located at six different positions on the Caulobacter crescentus chromosome. The cheR, cheB and cheT genes appeared to be located in a three-gene cluster. Mutations in these three genes resulted in the inability of the flagellum to reverse the direction of rotation. Defects in the cheR gene resulted in a loss of the ability to methylate the methyl-accepting chemotaxis proteins. In vitro experiments showed that the lack of in vivo methylation in cheR mutants was due to the absence of methyltransferase activity. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. The specific defects responsible for the lack of a chemotactic response have not been determined for the other identified che genes.

Bacteria↗
PubMed1991

Isolation, characterisation and molecular cloning of new mutant alleles of the fission yeast p34cdc2+ protein kinase gene: identification of temperature-sensitive G2-arresting alleles.

The protein serine-threonine kinase p34cdc2+ plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. p34cdc2+ function is required both for the initiation of DNA replication and for entry into mitosis, and is also required for the initiation of the second meiotic nuclear division. Recent extensive analysis of p34cdc2+ homologue proteins in higher eukaryotes has demonstrated that p34cdc2+ function is likely to be conserved in all eukaryotic cells. Here we report the isolation and characterisation of five new temperature-sensitive alleles of the cdc2+ gene. All five have been cloned and sequenced, together with the meiotically defective cdc2-N22 allele, bringing the total of p34cdc2+ mutants cloned in this and previous reports to seventeen. The five temperature-sensitive alleles define four separate mutations within the p34cdc2+ protein sequence, two of which give rise to cell cycle arrest in G2 only, when shifted to the restrictive temperature. The nature of the mutation in each protein is described and possible implications for the structure and function of p34cdc2+ discussed.

Alleles↗
PubMed2003

Identification of an Arabidopsis mitochondrial succinate-fumarate translocator.

Complementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter-GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation.

Amino Acid Sequence↗
PubMed2004

Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms.

Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.

Bacterial Proteins↗
PubMed2000

Genetic analysis of the YDR1-BUR6 repressor complex reveals an intricate balance among transcriptional regulatory proteins in yeast.

A transcriptional repressor complex encoded by two essential genes, YDR1 and BUR6, was isolated from Saccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1. To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo. Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor. A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1(cs)) mutant led to the identification of recessive mutations in the SIN4 gene, which encodes a component of the SRB-MED complex. The sin4 alleles suppressed not only ydr1(cs) mutations but also bur6(cs) mutations. In contrast, deletion of the gal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1(cs) and bur6(cs) mutations, indicating that suppression is not due to general defects in the SRB-MED complex. Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of a ydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background. Biochemical analysis of the SRB-MED complex from the sin4 suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits. These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex.

ATP-Binding Cassette Transporters↗
PubMed1994

The PKU mouse project: its history, potential and implications.

To produce genetic-based animal models for the human disease phenylketonuria (PKU), we treated mice with the powerful germline mutagen ethylnitrosourea and screened the progeny of these animals for the symptom hyperphenylalaninemia (HPH). Six independent mutant strains have been produced to date that exhibit heritable HPH. The first mutation isolated was found to cause a reduced level of GTP-cyclohydrolase I activity and, as such, yields a model for tetrahydrobiopterin-dependent HPH. The next two mutations have yet to be fully characterized but cause syndromes that appear distinct from any PKU or HPH syndromes yet reported for humans and they are allelic. Next we isolated a mutation that caused a marked reduction in hepatic phenylalanine hydroxylase activity levels. The enzyme deficiency was not sufficient to cause a PKU syndrome but instead produced a mild HPH syndrome. This strain played an instrumental role, however, in the identification of two additional mutant strains that appear to model human PKU very accurately in the laboratory mouse. These latter strains have levels of HPH very similar to human PKU patients, exhibit a phenylalanine-dependent hypopigmentation, and have reproductive difficulties that resemble human maternal PKU.

Animals↗
PubMed2001

A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae.

A genome-wide screen of 4168 homozygous diploid yeast deletion strains has been performed to identify nonessential genes that participate in the bipolar budding pattern. By examining bud scar patterns representing the sites of previous cell divisions, 127 mutants representing three different phenotypes were found: unipolar, axial-like, and random. From this screen, 11 functional classes of known genes were identified, including those involved in actin-cytoskeleton organization, general bud site selection, cell polarity, vesicular transport, cell wall synthesis, protein modification, transcription, nuclear function, translation, and other functions. Four characterized genes that were not known previously to participate in bud site selection were also found to be important for the haploid axial budding pattern. In addition to known genes, we found 22 novel genes (20 are designated BUD13-BUD32) important for bud site selection. Deletion of one resulted in unipolar budding exclusively from the proximal pole, suggesting that this gene plays an important role in diploid distal budding. Mutations in 20 other novel BUD genes produced a random budding phenotype and one produced an axial-like budding defect. Several of the novel Bud proteins were fused to green fluorescence protein; two proteins were found to localize to sites of polarized cell growth (i.e., the bud tip in small budded cells and the neck in cells undergoing cytokinesis), similar to that postulated for the bipolar signals and proteins that target cell division site tags to their proper location in the cell. Four others localized to the nucleus, suggesting that they play a role in gene expression. The bipolar distal marker Bud8 was localized in a number of mutants; many showed an altered Bud8-green fluorescence protein localization pattern. Through the genome-wide identification and analysis of different mutants involved in bipolar bud site selection, an integrated pathway for this process is presented in which proximal and distal bud site selection tags are synthesized and localized at their appropriate poles, thereby directing growth at those sites. Genome-wide screens of defined collections of mutants hold significant promise for dissecting many biological processes in yeast.

Actins↗
PubMed1994

Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium.

As previously reported, guanine nucleotide regulation of adenylyl cyclase activity in the Dictyostelium mutant synag 7 can be restored in vitro by addition of a high-speed supernatant prepared from wild-type cells (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 15121-15125). We have designated this activity CRAC, for cytosolic regulator of adenylyl cyclase. Trypsinization of partially purified material demonstrated that this activity contains a protein. We report here a 50,000-fold purification of this protein using Q and S Sepharose fast flow and P11 cellulose chromatography, followed by sucrose gradient centrifugation and separation on SDS-polyacrylamide gel electrophoresis. Purification of wild-type and mutant supernatants in parallel allowed identification of an 88-kDa protein required for reconstituting activity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immunoblots of fractions from each purification step show that activity and the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a portion of the deduced N terminus of a gene, dagA, isolated from a mutant similar to synag 7. Expression of the dagA cDNA in synag 7 cells restores both the 88 kDa band and CRAC activity.

Adenylyl Cyclases↗
PubMed1996

Chromosomal localization of the neurological mouse mutations tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr).

We have refined the map positions and identified molecular markers for three neurological mutations in the mouse, tottering (tg), Purkinje cell degeneration (pcd), and nervous (nr). These mutations were localized using simple sequence length polymorphisms between the mouse strain on which the mutation arose and the inbred strain onto which the mutation was bred. This approach to mutation mapping is generalizable to any mutant that has been backcrossed for several generations. The tg mutation was localized to the 1.1 cM region of chromosome 8 distal to simple sequence repeat (SSR) D8Mit103 and proximal to SSRs D8Mit79, D8Mit105, and D8Mit283. The pcd locus was mapped to the 5 cM interval of chromosome 13 between SSRs D13Mit139 and D13Mit67, and the nr locus was mapped between SSRs D8Mit155 and D8Mit18, a 5.6 cM region of chromosome 8. For each mutation, several SSRs distinguishing mutant from wild type chromosomes were identified within these regions. The definition of molecular markers distinguishing mutant from wild type alleles makes possible for the first time identification of tg, pcd, and nr mutants prior to behavioral manifestation of the mutant genotype. Thus, developmental studies of these mutants designed to describe or dissect the biochemical basis of the induction of the mutant phenotype are now feasible.

Alleles↗
PubMed2006

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea.

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Alkylating Agents↗
PubMed1994

Phenotype of the fission yeast cell cycle regulatory mutant pim1-46 is suppressed by a tobacco cDNA encoding a small, Ran-like GTP-binding protein.

Mutations in which the onset of mitosis is uncoupled from the completion of DNA replication has recently been described. Characterization of these mutants led to the identification of Pim1/Spi1 in fission yeast and RCC1/Ran proteins in mammalian cells. Their Saccharomyces cerevisae homologues, the MTR1/CNR1 proteins, appear to be involved in controlling RNA metabolism and transport. Here the isolation and partial characterization of plant cDNA clones which encode proteins homologous to the mammalian/fission yeast/budding yeast Ran/Spi/CNR proteins are reported. Higher plants appear to contain more than one gene per haploid genome which codes for Ran proteins. These genes are expressed in different plant tissues, including root tips and stems, known to contain mitotically active cells. The tobacco Ran-like proteins, like their mammalian and yeast homologues, are soluble proteins which are found in the cytoplasm and in the nucleus. In addition, it has been shown that overexpression of the tobacco Nt-Ran-A1 cDNA suppressed the phenotype of the temperature-sensitive fission yeast pim1-46 mutant. These results suggest that the plant Ran genes can be functionally equivalent to the mammalian/fission yeast/budding yeast Ran/Spi/CNR genes and that they may play a role: (i) in maintaining a coordinated cell cycle; (ii) in controlling RNA metabolism and transport in higher plants; and/or (iii) in protein import into the nucleus.

Amino Acid Sequence↗
PubMed1983

Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants.

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TK-negative nonsense mutants.

DNA Restriction Enzymes↗
PubMed1984

Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene.

Three classes of HSV-1(F) mutants expressing a resistance phenotype to two highly potent-type common monoclonal antibodies, H126-5 and H233, to glycoprotein B (gB) were selected. Class 1 mutants, selected for resistance to neutralization from nonmutagenized virus stocks, expressed a gB which reacted in biotin-avidin-enhanced surface immunoassays and in immune precipitation tests with the selecting antibodies. Class 2 and 3 mutants were selected for nonreactivity in the biotin-avidin-enhanced surface immunoassay from BUdR-mutagenized, preneutralized virus stocks, but differ in that the selecting antibodies immune precipitated the gB of Class 2 but not that of Class 3. Mutants expressing a resistance phenotype to one monoclonal antibody (H126-5 or H233) invariably retained reactivity in all tests with the heterologous antibody, and recombinants resistant to both antibodies were produced by cotransfection of intact DNA of one mutant with a cloned DNA fragment from another mutant. Class 1 mutations were mapped by marker transfer to a 1734-bp DNA fragment. Class 2 and 3 mutations were mapped to a region defined by a maximum of 377 bp and a minimum of 46 bp, in a biotin-avidin-enhanced surface immunoassay with a panel of DNA fragments of HSV-1(F) BamHI G carrying staggered deletions across the region encoding gB. This region does not overlap the neutralizing antibody determinant site mapped by T.C. Holland, R.M. Sandri-Goldin, L.E. Holland, S.D. Marlin, M. Levine, and J. Glorioso (1983, J. Virol. 46, 649-652) and is located 3' to the ts lesion of HSV-1(HFEM)tsB5 and 5' to the syn3 locus of that virus. It was concluded that (i) inasmuch as the biotin-avidin-enhanced surface immunoassay does not destroy the virus contained in the plaque, it is a rapid and convenient method for both identification and selection of mutants reactive and nonreactive to specific monoclonal antibodies. (ii) gB may contain multiple domains carrying epitopic sites of neutralizing monoclonal antibodies. (iii) The resistance phenotype may arise from mutations which alter the conformation or the amino acid sequence of the epitope. These mutations might be differentiable on the basis of reactivity of mutated gB with selecting monoclonal antibody in nondenaturing and denaturing environments, respectively.

Amino Acid Sequence↗
PubMed2003

A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations.

A genomewide screen of a collection of 4,847 yeast gene deletion mutants was carried out to identify the genes required for suppressing mutations in the CAN1 forward-mutation assay. The primary screens and subsequent analysis allowed (i) identification of 18 known mutator mutants, providing a solid means for checking the efficiency of the screen, and (ii) identification of a number of genes not known previously to be involved in suppressing mutations. Among the previously uncharacterized mutation-suppressing genes were six genes of unknown function including four (CSM2, SHU2, SHU1, and YLR376c) encoding proteins that interact with each other and promote resistance to killing by methyl methanesulfonate, one gene (EGL1) previously identified as suppressing Ty1 mobility and recombination between repeated sequences, and one gene (YLR154c) that was not associated with any known processes. In addition, five genes (TSA1, SOD1, LYS7, SKN7, and YAP1) implicated in the oxidative-stress responses were found to play a significant role in mutation suppression. Furthermore, TSA1, which encodes thioredoxin peroxidase, was found to strongly suppress gross chromosomal rearrangements. These results provide a global view of the nonessential genes involved in preventing mutagenesis. Study of such genes should provide useful clues in identification of human genes potentially involved in cancer predisposition and in understanding their mechanisms of action.

Base Sequence↗
PubMed1999

Molecular characterization of the nitrite-reducing system of Staphylococcus carnosus.

Characterization of a nitrite reductase-negative Staphylococcus carnosus Tn917 mutant led to the identification of the nir operon, which encodes NirBD, the dissimilatory NADH-dependent nitrite reductase; SirA, the putative oxidase and chelatase, and SirB, the uroporphyrinogen III methylase, both of which are necessary for biosynthesis of the siroheme prosthetic group; and NirR, which revealed no convincing similarity to proteins with known functions. We suggest that NirR is essential for nir promoter activity. In the absence of NirR, a weak promoter upstream of sirA seems to drive transcription of sirA, nirB, nirD, and sirB in the stationary-growth phase. In primer extension experiments one predominant and several weaker transcription start sites were identified in the nir promoter region. Northern blot analyses indicated that anaerobiosis and nitrite are induction factors of the nir operon: cells grown aerobically with nitrite revealed small amounts of full-length transcript whereas cells grown anaerobically with or without nitrite showed large amounts of full-length transcript. Although a transcript is detectable, no nitrite reduction occurs in cells grown aerobically with nitrite, indicating an additional oxygen-controlled step at the level of translation, enzyme folding, assembly, or insertion of prosthetic groups. The nitrite-reducing activity expressed during anaerobiosis is switched off reversibly when the oxygen tension increases, most likely due to competition for electrons with the aerobic respiratory chain. Another gene, nirC, is located upstream of the nir operon. nirC encodes a putative integral membrane-spanning protein of unknown function. A nirC mutant showed no distinct phenotype.

Aerobiosis↗
PubMed2001

Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein.

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

Amino Acid Sequence↗
PubMed2003

The identification of candidate genes for a reverse genetic analysis of development and function in the Arabidopsis gynoecium.

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.

Arabidopsis↗