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At least 883 records · Page 49Linked to original sources
PubMed1989

Identification of a single nucleotide change in the hypoxanthine-guanine phosphoribosyltransferase gene (HPRTYale) responsible for Lesch-Nyhan syndrome.

Complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) causes the Lesch-Nyhan syndrome. Previous characterization of a mutant form of HPRT, HPRTYale, from a subject with the Lesch-Nyhan syndrome revealed normal mRNA and protein concentrations, no residual catalytic activity, and cathodal migration upon PAGE. We have cloned and sequenced HPRTYale cDNA. The nucleotide sequence of full-length HPRTYale cDNA revealed a single nucleotide substitution compared with normal HPRT cDNA: G----C at nucleotide position 211. This transversion predicts substitution of arginine for glycine at amino acid position 71, explaining the cathodal migration of HPRTYale. Chou-Fasman secondary structure analysis predicts a change in the probability of beta-turn formation in the region containing the mutation. Inclusion of the bulky arginine side chain in place of glycine probably disrupts protein folding as well. Cloning mutant forms of cDNA allows identification of specific mutations, provides insight into mutational mechanisms, and facilitates structure-function analysis of mutant proteins.

DNA↗
PubMed1990

Disruption of formin-encoding transcripts in two mutant limb deformity alleles.

The recent identification of a gene residing at the mouse limb deformity (ld) locus permits us to test the hypothesis that disruption of this gene is responsible for an inherited anomaly affecting embryonic pattern formation. The gene gives rise to alternatively processed messenger RNAs that can be translated as a family of related protein products, termed the formins. We have now analysed transcripts from this gene in four independently isolated mutant alleles. In two of these, the ldHd allele (created by insertion of a transgene) and the ldIn2 allele (created by a translocation-inversion involving mouse chromosomes 2 and 17), a common subset of ld transcripts is abolished, but others are apparently unaltered. The correlation of altered transcripts in two independent ld mutants strongly supports the notion that one or more altered formins is responsible for the observed phenotype. That the defect is limited to the limb and kidney, despite expression of ld mRNA in other unaffected organs, suggests that these mutant alleles represent only partial loss of ld function.

Alleles↗
PubMed2004

Rapid chromosomal assignment of medaka mutants by bulked segregant analysis.

Genetic screens in medaka are leading to the identification of an increasing number of unique mutant phenotypes. However, so far only a few genes responsible for these phenotypes have been characterized. Furthermore, no protocols using a systematic positional cloning strategy have been developed to determine the implicated genes. The PCR-based bulked segregant analysis is a fast and reliable tool to accomplish the initial steps of the positional cloning of a mutation. Here we describe the selection of a panel of genetic markers that, evenly distributed over the 24 chromosomes of medaka, provide a full coverage of the compact medaka genome (800 Mb) when used in bulked segregant analysis. The reference panel, which consists of 48 EST-derived markers, is anchored to a collection of more than 2000 polymorphic markers, thus facilitating a rapid transition from chromosomal assignment to fine mapping of the mutants. More importantly, since most of the genetic screens have been performed in the inbred Cab strain (derived from the Southern population), the selection of markers included in this panel was intended to optimize the recognition of polymorphisms between Cab and the polymorphic inbred mapping strain Kaga. Here we present a reliable mapping panel, confirmed both by the assignment of the locus responsible for the medaka mutation eyeless/Rx3 to chromosome 12, and by the analysis of its resolution power using representative markers.

Animals↗
PubMed1998

Two novel mutations of the arylsulfatase B gene in two Italian patients with severe form of mucopolysaccharidosis. Mutations in brief no. 127. Online.

Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome, is a autosomal recessive disorder, due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsufatase B, ASB: EC 3.1.6.12). Three classical forms of the disease have been differentiated: severe, intermediate, mild. Mutational analysis of the ASB gene resulted in the identification of 30 ASB mutant alleles, each of which was found to be unique among unrelated patients, demonstrating a broad molecular heterogeneity of the disease. In this communication we present two novel mutant alleles in two severely affected subjects. Both alterations, the missense mutation G302R and the nonsense Q456X, were found in homozygosity and were confirmed by amplification refractory mutation system (ARMS) or restriction analysis. The missense G302R mutation concerns an amino acid which may be of special importance to the polypeptide, since 302 position is completely conserved in all the eukaryotic sulfatases aligned so far; the nonsense mutation Q456X leads to the translation of a putative mutant ASB protein lacking the last 78 amino acids with a loss of the 8 kD mature polypeptide, one of the two peptides generated by intralysosomal proteolytic processing of the 64kD precursor.

Alleles↗
PubMed1994

A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria.

Phycobilisomes are the multiprotein complexes predominantly responsible for harvesting light energy in cyanobacteria and some eukaryotic algae. When the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient, the phycobilisomes are specifically and rapidly degraded. Degradation may be either partial (after phosphorus deprivation) or complete (after sulfur or nitrogen deprivation). We have developed a visual screen to obtain mutants unable to degrade their phycobilisomes upon nutrient starvation. Complementation of one of these mutants led to the identification of a gene, designated nblA, that encodes a 59 amino acid polypeptide essential for phycobilisome degradation. Transcription of nblA increases dramatically in sulfur- or nitrogen-deprived cells and moderately in phosphorus-deprived cells. Using the phosphorus-regulated alkaline phosphatase (phoA) promoter as a tool, we engineered constructs from which we could control the expression of either sense or antisense nblA. Increased expression of sense nbLA caused complete phycobilisome degradation during phosphorus deprivation, while expression of antisense nblA prevented phycobilisome degradation. Hence, nblA is necessary, and may be sufficient, for the degradation of phycobilisomes under adverse environmental conditions. Further investigation of the mechanism by which nblA causes phycobilisome destruction may reveal general principles that govern the specificity of macromolecular complex degradation.

Alkaline Phosphatase↗
PubMed1994

The binding epitopes of neurotrophin-3 to its receptors trkC and gp75 and the design of a multifunctional human neurotrophin.

Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin-3 (NT-3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta-strand bundle and in contrast to NGF does not make use of non-conserved loops and the six N-terminal residues. The gp75 epitope is dominated by loop residues and the C-terminus of NT-3. A novel rapid biological screening procedure allowed the identification of NT-3 mutants that are able to signal efficiently through the non-preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT-3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF-, BDNF- and NT-3-dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.

Amino Acid Sequence↗
PubMed2006

Alternative chaperone machinery may compensate for calreticulin/calnexin deficiency in Caenorhabditis elegans.

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin [crt-1(jh101)]- and calnexin [cnx-1(nr2009)]-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, we analyzed the proteome of crt-1(jh101) mutants and [crt-1(jh101);cnx-1(nr2009)] double mutants. When grown at 20 degrees C, we found that five proteins were up-regulated and two proteins were down-regulated in crt-1(jh101) mutants; nine proteins were up-regulated and five proteins were down-regulated in [crt-1(jh101);cnx-1(nr2009)] double mutants. In addition, elevation of the cultivation temperature to 25 degrees C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin.

Animals↗
PubMed2000

A GDP-fucose-protected, pyridoxal-5'-Phosphate/NaBH(4)-sensitive lys residue common to human alpha1-->3Fucosyltransferases corresponds to Lys(300) in FucT-IV.

Human alpha1-->3/4fucosyltransferases (FucTs) contain a common essential pyridoxal-5'-phosphate(PLP)/NaBH(4) reactive, GDP-fucose-protectable Lys. For identification, site-directed mutants at lysines of FucT-IV and -VII were prepared and tested. Non conserved lysine mutants K119Y and K394Q were similar to wild-type FucT-IV. However, mutants of conserved lysines K228R and K300R were distinct. The specific activity of K228R was 2- to 3-fold lower but retained K(m) values for donor and acceptor substrates as wild-type FucT-IV. The specific activity of K300R was reduced over 400-fold with an apparent K(m) for GDP-fucose over 200 microM. FucT-VII mutants K169R and K240R (equivalent to K228R and K300R for FucT-IV, respectively) were inactive. No change in PLP/NaBH(4) sensitivity occurred with K119Y, K228R, and K394Q compared to wild-type FucT-IV. These and previous results (A. L. Sherwood, A. T. Nguyen, J. M. Whitaker, B. A. Macher, M. R. Stroud, and E. H. Holmes, J. Biol. Chem. 273, 25256-25260, 1998) demonstrate that of three conserved lysines in FucT-IV, two (Lys(228) and Lys(283)) are not involved in substrate binding but perhaps in catalysis. The third site, Lys(300), is involved in GDP-fucose binding and PLP/NaBH(4) inactivation.

Amino Acid Sequence↗
PubMed1998

Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis.

The activity of the developmental transcription factor sigmaF in Bacillus subtilis is governed by a switch involving the dual function protein SpoIIAB. SpoIIAB is an antisigma factor that forms complexes with sigmaF and with an alternative partner protein SpoIIAA. SpoIIAB is also a protein kinase that can inactivate SpoIIAA by phosphorylating it on a serine residue. We sought to identify amino acids in SpoIIAB that are involved in the formation of the SpoIIAB-SpoIIAA complex by screening for mutants that were defective in the activation of sigmaF. This genetic screen, in combination with biochemical analysis and the construction of loss-of-side-chain (alanine substitution) mutants, led to the identification of amino acid side-chains in the N-terminal region of SpoIIAB that could contact SpoIIAA. Unexpectedly, the same amino acid side-chains (R20 and N50) that appear to touch SpoIIAA are required for binding to, and may represent sites of contact with, sigmaF. We propose that the N-terminal region of SpoIIAB forms a binding surface that is responsible for the formation of both the SpoIIAB-SpoIIAA and the SpoIIAB-sigmaF complexes, and that in some cases the same amino acid side-chains contact both partner proteins. N50 is also the defining residue of a region of amino acid sequence homology known as the N-box that is shared by SpoIIAB and related serine protein kinases, as well as by members of a mechanistically dissimilar family of protein kinases that undergo autophosphorylation at a histidine residue. We discuss the implications of this finding for the mechanism of histidine autophosphorylation.

Alanine↗
PubMed1999

Bacteriophage PRD1 contains a labile receptor-binding structure at each vertex.

Bacteriophage PRD1 is a membrane-containing virus with an unexpected similarity to adenovirus. We mutagenized unassigned PRD1 genes to identify minor capsid proteins that could be structural or functional analogs to adenovirus proteins. We report here the identification of an amber mutant, sus525, in an essential PRD1 gene XXXI. The gene was cloned and the gene product was overexpressed and purified to near homogeneity. Analytical ultracentrifugation and gel filtration showed that P31 is a homopentamer of about 70 kDa. The protein was shown to be accessible on the virion surface and its absence in the sus525 particles led to the deficiency of two other viral coat proteins, protein P5 and the adsorption protein P2. Cryo-electron microscopy and image reconstruction of the sus525 particles indicate that these proteins are located on the capsid vertices, because in these particles the entire vertex structure was missing along with the peripentonal major capsid protein P3 trimers. Sus525 particles package DNA effectively but loose it upon purification. All of the PRD1 vertex structures are labile and potentially capable of mediating DNA delivery; this is in contrast to other dsDNA phages which employ a single vertex for packaging and delivery. We propose that this arises from a symmetry mismatch between protein P2 and the pentameric P31 in analogy to that between the adenovirus penton base and the receptor-binding spike.

Adenoviridae↗
PubMed2000

The correlation of genotype and phenotype in Portuguese hyperphenylalaninemic patients.

To understand the basis for the clinical heterogeneity of phenylalanine hydroxylase deficiency among Portuguese hyperphenylalaninemic patients, genotype-phenotype correlations were established. A group of 61 patients was completely genotyped, leading to the identification of 20 different mutant alleles in 36 different genotypic combinations, including a mutant allele not reported previously. The severity of those mutations found within this hyperphenylalaninemic population, which have not been previously expressed in vitro, were assessed. The results obtained by the present study exhibit a strong correlation between the predicted residual enzyme activity, as deduced from the genotype of the patients, and the biochemical phenotype represented by the diagnostic parameters (phenylalanine levels before the beginning of treatment and the dietary phenylalanine tolerance). It was observed that only a judicious follow-up and compliance with the appropriate diet permits the correct assessment of the clinical phenotype of the patients. Additionally, based upon the correlation observed between genotypes and diagnostic parameters, it was possible to predict the potential residual enzyme activity of those mutations (identified in our patients) which have not yet been studied in vitro.

Adolescent↗
PubMed1995

The Janus protein tyrosine kinases in hematopoietic cytokine signaling.

Cytokines regulate cellular functions through their interaction with members of the cytokine receptor superfamily. These receptors couple ligand binding to induction of tyrosine phosphorylation through their association with the Janus protein tyrosine kinases (Jaks). Receptor dimerization induces Jak homo- or hetero-associations, autophosphorylation and activation of Jak catalytic activity. The activated Jaks are hypothesized to phosphorylate the receptors as well as a number of proteins involved in cellular signaling including SHC, the p85 subunit of PI 3-kinase, Vav and members of the Signal Transducers and Activators of Transcription (Stats) family of transcription factors. Receptor phosphorylation also allows association of hematopoietic cell phosphatase with the receptor complex, resulting in its down-regulation. Receptor mutants have allowed the identification of specific domains that are required for each function and their correlation with biological responses.

Cytokines↗
PubMed1978

Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae.

Three new genes nifM, nifI and nifN have been mapped in the nif gene cluster of Klebsiella pneumoniae and a fourth gene nifJ has been confirmed as being a separate cistron. Polar nif mutations were obtained by transposition of Tn7 to plasmid pRD1, and of Tn5 and Tn10 to plasmid pMF100, a derivative of pRD1. Complementation analysis of the nif::Tn mutants led to the identification of at least six transcriptional units: nifB; nifA; nifJ; nifH, nifD and nifK; nifE and nifI; nifN, nifM and nifF. Biochemical and genetic evidence suggest that the three genes nifH, nifD and nifK, which are probably the structural genes for nitrogenase, belong to the same operon and are transcribed from nifH to nifK. A polypeptide with a molecular weight of approximately 120,000 is presumed to be the nifJ product.

Chromosome Mapping↗
PubMed1994

Identification of the iron-carbonyl stretch in distal histidine mutants of carbonmonoxymyoglobin.

Soret-excitation resonance Raman (RR) spectra are reported for six distal histidine mutants of carbonmonoxymyoglobin including H64A, H64V, H64L, H64I, H64W, and H64W/L29F. Based on 13CO isotope shifts, the iron-carbonyl stretching vibrations are unambiguously identified. The correct assignment of these modes eliminates the differences in the conformational substate occupations predicted by the RR versus IR data.

Binding Sites↗
PubMed1993

Amplification of human APO(a) kringle 4-37 from blood lymphocyte DNA.

We have been able to amplify the lysine binding pocket region of human apo(a) kringle type 5 starting from the DNA isolated from peripheral blood lymphocytes. This development now permits the identification of Lp(a) mutants that by lacking their ability to bind to lysine/fibrin would have a lesser thrombogenic potential.

Amino Acid Sequence↗
PubMed1990

Protein kinase C and tumor promoters.

The complexity of PKC has made it difficult to define the precise biochemical processes associated with the various PKC-related cellular responses observed. In the past year, we have seen progress in complementary approaches that are helping to solve the puzzles. These include purification and characterization of individual isozymes, expression of normal and mutant PKCs, immunolocalization, and identification of specific activators and inhibitors. All of these will be useful in identifying the primary targets of PKC phosphorylation and their function.

Animals↗
PubMed2005

Recognition of antimicrobial peptides by a bacterial sensor kinase.

PhoQ is a membrane bound sensor kinase important for the pathogenesis of a number of Gram-negative bacterial species. PhoQ and its cognate response regulator PhoP constitute a signal-transduction cascade that controls inducible resistance to host antimicrobial peptides. We show that enzymatic activity of Salmonella typhimurium PhoQ is directly activated by antimicrobial peptides. A highly acidic surface of the PhoQ sensor domain participates in both divalent-cation and antimicrobial-peptide binding as a first step in signal transduction across the bacterial membrane. Identification of PhoQ signaling mutants, binding studies with the PhoQ sensor domain, and structural analysis of this domain can be incorporated into a model in which antimicrobial peptides displace divalent cations from PhoQ metal binding sites to initiate signal transduction. Our findings reveal a molecular mechanism by which bacteria sense small innate immune molecules to initiate a transcriptional program that promotes bacterial virulence.

Adaptation, Physiological↗