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At least 793 records · Page 44Linked to original sources
PubMed2004

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor.

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.

Alleles↗
PubMed1979

Isolation and genetic analysis of nuclease halo (nuh) mutants of Neurospora crassa.

Nuclease halo (nuh) mutants of the ascomycete Neurospora crassa have been isolated which are characterized reduced release of deoxyribonuclease (DNase) activities from colonies grown on sorbose-containing agar media. To identify nuh mutants, mutagenized isolates were transferred to commercial DNase test agar, or grown on minimal medium and then overlayed with agar that contained heat-denatured DNA. DNase activity was visualized by acid precipitation which produced clear rings of digestion (haloes) around the colonies. To identify the number of genes in which mutations lead to reduced release of nuclease activity, eleven nuh mutants were checked for close linkage and linked pairs were tested for complementation. These mutants were assigned to eight genes, and all except one were mapped in six small regions of the Neurospora linkage maps. In addition, among a large number of existing mutants which were tested for nuclease haloes, two mutants were found that showed the Nuh phenotype, namely uvs-3 and uvs-6. One of the isolated nuh mutants was also found to be sensitive to UV and was mapped close to uvs-3; it may represent a new allele of this gene. As a first step towards identification of genuine nuclease mutants, extensively backcrossed strains of mutants from different genes have been assayed for nuclease activity with denatured DNA in extracts. A pronounced reduction, compared to wild type at the same stage of growth, was found in uvs-3 and also in nuh-3, a mutant that is not UV-sensitive.

Chromosome Mapping↗
PubMed2002

Cloning and characterization of a FAD-monooxygenase gene ( cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07.

A bacterium culture was isolated on the basis of its ability to degrade chloranilic acid, and was later identified as Pseudomonas putida (TQ07). Several transposon insertion mutants unable to degrade chloranilic acid were selected. The characterization of the site of insertion of one of these mutants led to the identification of the cadA gene encoding an enzyme with significant homology with FAD-monooxygenases involved in the degradation of aromatic and chloroaromatic compounds. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of "halo" formation (a zone of clearing color on agar plates around TQ07 colonies that degrade chloranilic acid) and degradation of chloranilic acid, unequivocally assigned cadA a function in the metabolism of this compound. We also found that most of the transposon insertion mutants unable to degrade chloranilic acid are clustered in a 10-kb region of the P. putidagenome that is encoded in a megaplasmid or in an unstable chromosomal region.

Amino Acid Sequence↗
PubMed1973

Genetic Control of the Transport of Hexose Phosphates in Escherichia coli: Mapping of the uhp Locus.

A number of mutations affecting the transport of hexose phosphates in Escherichia coli were ordered within the uhp locus. Three-point crosses by transduction or conjugation allowed the ordering of the alleles relative to the adjacent pyrE marker. The same linear map was obtained by both methods. This, combined with the regulatory properties of revertants of these mutants, allowed a tentative identification of two genes, one presumably coding for the transport system (uhpT) and the other(s) specifying a regulatory element (uhpR). The order of these is pyrE-uhpT-uhpR. Mutants exhibiting constitutive expression of the transport system were isolated. This behavior is genetically linked to the uhp locus, but more precise localization was not possible.

Journal Article↗
PubMed2001

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants.

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110-Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues.

Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1-, VirD2-dependent border-nicking activity in Escherichia coli harbouring a border-containing substrate plasmid. Analysis of the mutants led to the identification of a region from amino acids 45-60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA-binding domain. Deletion mutagenesis indicated that amino acids 2-16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5-27, completely inactivated VirD1.

Agrobacterium tumefaciens↗
PubMed1999

Human hepatitis B virus mutants: significance of molecular changes.

Human hepatitis B virus, the leading pathogen for hepatitis B, is a compact DNA virus with viral genes that largely overlap. An increasing number of mutations have emerged following human interventions such as vaccination and anti-viral therapy. While vaccine escape mutants are characterized by mutations on the antigenic hepatitis B surface antigen, those carrying mutations in other viral proteins are either resistant to anti-viral therapy or implicated in acute liver diseases. Molecular identification of these various mutants should shed new lights on the underlying mechanism of hepatitis B virus viral escape and resistance and provide helpful information on their effective eradication.

Antiviral Agents↗
PubMed1996

A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15.

A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.

Actins↗
PubMed2004

Identification of novel pathogenicity genes by PCR signature-tagged mutagenesis and related technologies.

Microbial pathogens possess a repertoire of virulence determinants that make unique contributions to bacterial fitness during infection. In this chapter, we focus on the recent progress and adaptations of signature-tagged mutagenesis (STM) by PCR instead of hybridization. This is a PCR-based STM mutation-based screening method using a population of bacterial mutants for the simultaneous identification of multiple virulence genes in microbial pathogens by negative selection. Modifications of STM developed in our laboratory have been applied to Pseudomonas aeruginosa PAO1. Screening of a collection of 6912 STM mutants in the rat chronic lung model of infection identified 214 P. aeruginosa STM mutants defective in virulence. For further studies, and to illustrate better the strategies that need to be utilized, we present detailed analysis of nine selected STM mutants. The data obtained indicate that in vivo, defects in virulence give a wide variety of phenotypes: defects in known virulence factors have been found, thereby validating the method; defects have also been found in orthologs with predicted functions, and in some genes whose functions cannot be predicted from databases. A general strategy and a simple scenario is discussed using the nine STM mutants selected for further characterization. PCR-based STM represent a genomics-based method for in vivo high-throughput screening of new virulence factors.

Bacteria↗
PubMed2001

Role of adenosine kinase in Saccharomyces cerevisiae: identification of the ADO1 gene and study of the mutant phenotypes.

Sequencing of the Saccharomyces cerevisiae genome revealed an open reading frame (YJR105w) encoding a putative protein highly similar to adenosine kinases from other species. Disruption of this gene (renamed ADO1) affected utilization of S-adenosyl methionine (AdoMet) as a purine source and resulted in a severe reduction of adenosine kinase activity in crude extracts. Furthermore, knock-out of ADO1 led to adenosine excretion in the medium and resistance to the toxic adenosine analogue cordycepin. From these data we conclude that ADO1 encodes yeast adenosine kinase. We also show that ADO1 does not play a major role in adenine utilization in yeast and we propose that the physiological role of adenosine kinase in S. cerevisiae could primarily be to recycle adenosine produced by the methyl cycle.

Adenosine↗
PubMed1989

Identification of T-cell receptor V beta deletion mutant mouse strain AU/ssJ (H-2q) which is resistant to collagen-induced arthritis.

Our laboratory is involved in investigating the role of T-cell receptor (Tcr) in collagen-induced arthritis (CIA). During these studies we found AU/ssJ (H-2q) mice to be resistant to CIA like SWR (H-2q), as compared with other H-2q strains with wild-type Tcr like DBA/1 and B10.Q. Upon screening with monoclonal antibodies F23.1 and KJ23a, AU/ssJ was found to be F23.1 negative (V beta 8 Tcr negative) and KJ23a positive (V beta 17a Tcr positive). Southern blot analysis on liver DNA using specific Tcr-V beta probes confirmed the deletion of V beta 8 gene family and also showed that AU/ssJ mice have deletions of V beta 9, V beta 13, V beta 12, and V beta 11 genes of Tcr. Further, these mice show a restriction fragment length polymorphism pattern with V beta 10, V beta 6, and V beta 17 probes similar to SWR mice as compared with B10 mice. Since SWR and AU/ssJ are from different backgrounds, these studies indicate that specific variable region beta chain genes of Tcr are crucial for susceptibility to CIA in mice. Furthermore, these studies identify an additional inbred strain which has also deleted 50% of its Tcr-V beta genes.

Animals↗
PubMed1994

Identification of mutation sites of a temperature-sensitive mutant of HCMV DNA polymerase activity.

The human cytomegalovirus (strain Towne) temperature-sensitive mutant ts 256 exhibits a virus specific DNA polymerase-negative phenotype. The position of the mutation of ts 256 was determined by three step marker-rescue assays to be within a 5.1 kb XbaI-BamHI fragment between map unit 0.33 and 0.35 in a HindIII-D fragment located in a long unique region. Nucleotide sequencing showed that the 5.1 kb fragment contained three open reading frames corresponding to those of the genes for UL52, UL53 and UL54 (DNA polymerase gene), respectively, of strain AD169. The functions of UL52 and UL53 are unknown. Comparison of the DNA sequences of the 5.1 kb fragments of the wild-type and ts 256 mutant revealed two base changes within UL53 and UL54, respectively, which result in amino acid substitutions. The mutation in the UL54 gene was located within a distinct conserved region VI common to alpha-like DNA polymerases, suggesting that this base change would be responsible for DNA negative phenotype.

Amino Acid Sequence↗
PubMed1989

Micrococcus luteus homolog of the Escherichia coli uvrA gene: identification of a mutation in the UV-sensitive mutant DB7.

Restriction fragments of Micrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in an Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with the E. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of the M. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of the E. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinicapyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to the E. coli UvrABC excinuclease.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines.

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.

DNA Fingerprinting↗
PubMed1993

Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directed mutagenesis.

Ser-49 located at the exposed surface loop of subtilisin J was replaced with Asp and Arg. Proteinase activity of the Asp-49 mutant was similar to that of wild-type, but the Arg mutant was inactivated. At 37 degrees C, mature subtilisin J protein of the Asp-49 mutant rapidly degraded, and specific breakdown products were accumulated. These proteins were analyzed by SDS-PAGE, and the N-terminal sequences were determined for the mature and deleted protein. We identified the autoproteolytic cleavage site in the mature Asp-49 mutant protein from sequencing data.

Amino Acid Sequence↗