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At least 1,693 records · Page 94Linked to original sources
PubMed1995

Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein.

Coronavirus gene expression involves proteolytic processing of the mRNA 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF 1b-encoded 100-kDa protein is proteolytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus 3C proteinase group (3C-like proteinase). In this report, the 3C-like proteinase has been further analysed by internal deletion of a 2.3-kb fragment between the 3C-like proteinase-encoding region and ORF 1b and by substitution mutations of its catalytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF 1a sequences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to the 100-kDa protein species. Site-directed mutagenesis studies have confirmed that the predicted nucleophilic cysteine residue (Cys2922) and a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 (His2820) are essential for the catalytic activity of the proteinase, and that the QS(G) dipeptide bonds are its target cleavage sites. Substitution mutations of the third component of the putative catalytic triad, the glutamic acid 2843 (Glu2843) residue, however, do not affect the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the 3C-like proteinase is capable of trans-cleavage of the 1a/1b polyprotein. These studies have confirmed the involvement of the 3C-like proteinase domain in processing of the 1a/1b polyprotein, the predicted catalytic centre of the proteinase, and its cleavage sites.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of the sequences at the termini of the Moloney murine leukemia virus DNA required for integration.

The insertion of retroviral DNA into the genome of the infected cell to form the integrated provirus requires the presence of short inverted repeat (IR) sequences at the viral DNA termini. To examine the sequence requirements at the IRs for integration of the Moloney murine leukemia virus, we generated a series of mutants with deletion and substitution mutations and tested their ability to replicate and form proviruses in vivo. The experiments did not detect evidence of a requirement for sequences outside the IRs, and only a very loose requirement for the IR sequences, with many point mutations well tolerated. Examination of the mutants suggests that bases very near the terminus and those approximately one turn of the DNA helix away from the terminus are important for recognition by the integrase function.

3T3 Cells↗
PubMed1994

Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek's disease virus.

The Marek's disease virus (MDV) glycoprotein B (gB) precursor, gp100, is proteolytically cleaved into two disulfide-linked subunits, gp60 and gp49. In the gB homologs of most other herpesviruses, a tetrapeptide, Arg-Xaa-Arg-Arg, is immediately upstream from the predicted cleavage site. We have investigated the specificity of the proteolytic cleavage in gp100 by introducing mutations within its predicted cleavage site (Arg-Leu-Arg-Arg) and expressed these mutants in recombinant fowlpox virus (FPV). The results show that all three Arg residues at the predicted cleavage site play an important role in the specific proteolytic cleavage of gp100. Furthermore, we demonstrated that the cleavage of gp100 is not necessary for transport of gB to the cell surface.

Amino Acid Sequence↗
PubMed2003

Mutational analysis of the transforming growth factor beta receptor type I gene in primary non-small cell lung cancer.

Transforming growth factor-beta receptor-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TGFbetaRI and flanking intron sequences from 53 primary non-small cell lung cancer (NSCLC) tissues were examined for alterations using SSCP and direct sequencing. No somatic point mutations other than two silent mutations and a polymorphism were found in the TGFbetaRI gene. The two silent mutations located at codon 344 (AAT to AAC) and codon 406 (TTA to CTA), respectively, and the polymorphism was at the 24th base of intron 7 (G to A). To investigate whether the presence of this polymorphism is associated with NSCLC, we determined its allele distribution in all the 53 carcinomas and 89 normal controls. Interestingly, we found that the subjects with homozygous genotype A/A displayed more than 3-fold increased risk of developing NSCLC than the common wild genotype G/G. As the first report, the present study showed that TGFbetaRI gene is not a frequent site of spontaneous mutational inactivation while the detected polymorphism is frequent in the pathogenesis of NSCLC.

Activin Receptors, Type I↗
PubMed1993

Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation, using TnphoA' elements.

All genes for phosphonate (Pn) utilization in Escherichia coli are in a large cluster of 14 genes named, in alphabetical order, phnC to phnP. Plasmids carrying these genes were mutagenized by using TnphoA'-1, and 43 mutants containing simple insertions were studied in detail. Their insertion sites were defined by restriction mapping and by DNA sequencing. One or more mutations in each phn gene was identified. In 23 mutants, expression of the TnphoA'-1 lacZ gene was phosphate starvation inducible. These mutants had TnphoA'-1 oriented in line behind the phnC promoter, i.e., in the + orientation. In 20 mutants, the TnphoA'-1 lacZ gene was expressed at a low basal level. These mutants had insertions in the opposite orientation. All 43 phn::TnphoA'-1 insertions were recombined onto the chromosome to test for mutational effects, and their structures on the chromosome were verified by DNA hybridization. Those in the + orientation were switched to TnphoA'-9, which has an outward promoter for expression of downstream genes. These insertions were tested for polar effects by measuring beta-glucuronidase synthesis from a uidA gene transcriptionally fused to the 3' end of the phnP gene. The results indicate the following: (i) the phnC-to-phnP gene cluster is an operon of 14 genes, and the phnC promoter is the sole psi promoter; (ii) three gene products (PhnC, PhnD, and PhnE) probably constitute a binding protein-dependent Pn transporter; (iii) seven gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, and PhnM) are required for catalysis and are likely to constitute a membrane-associated carbon-phosphorus (C-P) lyase; (iv) two gene products (PhnN and PhnP) are not absolutely required and may therefore be accessory proteins for the C-P lyase; and (v) two gene products (PhnF and PhnO) are not required for Pn use and may have a regulatory role because they have sequence similarities to regulatory proteins. The mechanism for breaking the C-P bond by a lyase is discussed in light of these results.

Biodegradation, Environmental↗
PubMed1999

Mutational analysis of target base flipping by the EcoRV adenine-N6 DNA methyltransferase.

DNA methyltransferases flip their target base out of the DNA helix. Here, we have investigated base flipping by wild-type EcoRV DNA methyltransferase (M.EcoRV) and five M.EcoRV variants (D193A, Y196A, S229A, W231R and Y258A). These variants bind to DNA and S-adenosylmethionine but have a severely reduced catalytic efficiency or are catalytically inactive. To measure base flipping three different assays were used, viz. analysis of the yields of photocrosslinking reactions between the enzymes and a substrate in which the target base is replaced by 5-iodouracil, analysis of the binding constants to substrates containing a mismatch base-pair at the target position and analysis of the salt dependence of specific complex formation. Our data show that the Y196A, W231R and Y258A variants are not able to stabilize a flipped target base, suggesting that the aromatic amino acid residues (Tyr196, Trp231 and Tyr258) are involved in hydrophobic interactions with the flipped base. The D193A variant behaves like wild-type M.EcoRV with respect to base flipping. The fact that this variant is catalytically inactive indicates that Asp193 has a function in chemical catalysis. The S229A variant can better flip modified bases but does not tightly lock the flipped base into the adenine-binding pocket, suggesting that Ser229 could form a contact to the flipped adenine.

Cross-Linking Reagents↗
PubMed1996

Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families.

The hereditary breast cancer gene BRCA2 was recently cloned and is believed to account for almost half of site-specific breast cancer families and the majority of male breast cancer families. We screened 49 site-specific breast cancer families for mutations in the BRCA2 gene using single strand conformation analysis (SSCA) followed by direct sequencing. We found mutations in eight families, including all four families with male breast cancer. The eight mutations were small deletions with the exception of a single nonsense mutation, an all were predicted to interrupt the BRCA2 coding sequence and to lead to a truncated protein product. Other factors which predicted the presence of a BRCA2 mutation included a case of breast cancer diagnosed at age 35 or below (P = 0.01) and a family history of pancreatic cancer (P = 0.03). Two mutations were seen twice, including a 8535delAG, which was detected in two French Canadian families. Our results suggest the possibility that the proportion of site-specific breast cancer families attributable to BRCA2 may be overestimated.

Adult↗
PubMed2003

Mutational analysis of a conserved signal-transducing element: the HAMP linker of the Escherichia coli nitrate sensor NarX.

The HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in the Escherichia coli nitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned the narX HAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for the narX(+) strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic alpha-helices have different roles in signal transduction.

Adenylyl Cyclases↗
PubMed1994

Mutation analysis of the Fanconi anemia gene FACC.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by a unique hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C (FACC) has recently been cloned. We have amplified FACC exons with their flanking intron sequences from genomic DNA from 174 racially and ethnically diverse families in the International Fanconi Anemia Registry and have screened for mutations by using SSCP analysis. We identified eight different variants in 32 families; three were detected in exon 1, one in exon 4, one in intron 4, two in exon 6, and one in exon 14. Two of the eight variants, in seven families, did not segregate with the disease allele in multiplex families, suggesting that these variants represented benign polymorphisms. Disease-associated mutations in FACC were detected in a total of 25 (14.4%) of 174 families screened. The most frequent mutations were IVS4 + 4 A-->T (intron 4; 12 families) and 322delG (exon 1; 9 families). Other, less common mutations include Q13X in exon 1, R185X and D195V in exon 6, and L554P in exon 14. The polymorphisms were S26F in exon 1 and G139E in exon 4. All patients in our study with 322delG, Q13X, R185X, and D195V are of northern or eastern European or southern Italian ancestry, and 18 of 19 have a mild form of the disease, while the 2 patients with L554P, both from the same family, have a severe phenotype. All 19 patients with IVS4 + 4 A-->T have Jewish ancestry and have a severe phenotype.

Base Sequence↗
PubMed1998

Mutational analysis of regulated exocytosis in Tetrahymena.

Genetic analysis of regulated exocytosis can be accomplished in ciliates, since mutants defective in stimulus-dependent secretion of dense-core vesicles can be identified. In Tetrahymena thermophila, secretion in wild-type cells can result in their encapsulation by the proteins released from vesicle cores. Cells with defects in secretion were isolated from mutagenized homozygous cells that were generated using a highly efficient method. Screening was based both on a visual assay for encapsulation, and on a novel panning step using differential centrifugation to take advantage of the selective mobility of mutants that fail to encapsulate upon stimulation. 18 mutants with defects in several ordered steps have been identified. Defects in a set of these could be localized to three stages: granule formation, transport to cell surface docking sites, and exocytosis itself. Mutants with defects in this last stage can be ordered into successive steps based on several criteria, including their responsiveness to multiple secretagogues and Ca2+ ionophores. The results of both somatic and genetic complementation on selected pairs also help to characterize the defective factors.

Animals↗
PubMed2002

New human sodium/glucose cotransporter gene (KST1): identification, characterization, and mutation analysis in ICCA (infantile convulsions and choreoathetosis) and BFIC (benign familial infantile convulsions) families.

Cotransporters represent a major class of proteins that make use of ion gradients to drive active transport of substrate into cells. A new human gene, KST1, encoding a member of the sodium/glucose cotransporter family, was identified onto human chromosome 16p12-p11. This genomic region contains a major gene responsible for a syndrome of infantile convulsions and paroxysmal dyskinesia (ICCA syndrome), inherited as an autosomal dominant trait, as well as for benign familial infantile convulsions (BFIC). The entire coding sequence of the human KST1 gene was determined using a combination of methods including in silico comparison of its rabbit orthologous DNA complementary to RNA (cDNA) to the corresponding human genomic sequences, reverse transcription-polymerase chain reaction on human brain RNA, 5' and 3' rapid amplification of cDNA ends. The gene is divided into 16 exons and the predicted protein of 675 amino acids contains 14 transmembrane domains. It shares significant homology to the sodium-glucose transporter 1 cotransporter proteins. An alternatively spliced transcript resulting from the skipping of exon 6 led to a predicted protein lacking the 4th transmembrane domain. As ion transporters are good candidates for a large variety of human diseases, including paroxysmal disorders, a mutation search was performed in four families with ICCA or BFIC syndromes. No pathogenic mutation was found, although several polymorphic variants with amino acids exchanges were identified. Due to its broad expression in human tissues, the human KST1 gene could be involved in several other diseases mapped to human chromosome 16p12-p11.

Amino Acid Sequence↗
PubMed2002

Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18.

Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of two DR alpha residues involved in dimers of HLA-DR molecules.

Crystallographic analysis of HLA-DR1 molecules reveals a "dimer of dimers" with two reciprocal salt bridges between Glu 88 and Lys 111 of the two DR alpha chains. To determine whether these amino acids are critical for Ag presentation, we generated a panel of human B cell transfectants expressing DR alpha chains with mutations at residues 88, 111, or both. The mutant DR alpha chains, paired with endogenous DR3 beta chain, form cell surface dimers that retain epitopes recognized by a panel of anti-DR3 Abs. Replacement of Glu 88 with Ala (88A) selectively eliminates the ability to activate an alloreactive (anti-DR3) T cell clone. Mutant DR molecules with Lys substituted for Glu 88 (88K) fail to activate an alloreactive, an Ag-specific, and a peptide-specific T cell line. The DR alpha 88 mutants bind an exogenously supplied DR3-specific peptide and the mutant DR molecules migrate as dimers on SDS-PAGE, implying that their defective Ag presentation is not due to an inability to bind antigenic peptides. In contrast, substitution of Lys 111 with either Ala (111A) or Glu (111E) does not abrogate Ag presentation. Further, the defect introduced by Glu 88 to Lys mutation (88K) is not overcome by compensatory Lys to Glu mutation at position 111 (111E). Taken together, these results indicate an important functional or structural role for position 88 of the DR alpha chain, but argue against a requirement for interaction between DR alpha 88 and 111 during Ag-specific T cell stimulation.

Antigen Presentation↗
PubMed1993

Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1.

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.

Base Sequence↗
PubMed1994

Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis.

Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.

Base Sequence↗
PubMed2002

Mutation analysis of synapsin III gene in schizophrenia.

Synapsin III is a new synapsin family gene with the putative function of synaptogenesis regulation and neurotransmitter release in the brain. The gene was mapped to 22q12-q13, a schizophrenia susceptible region gene as suggested by several linkage studies. Hence, the synapsin III gene is considered a candidate gene of schizophrenia. We systematically sequenced the protein coding and 5'-promoter regions of the synapsin III gene to look for mutations in 62 Han Chinese schizophrenic patients from Taiwan with positive family history. Further case-control association study was performed among 163 patients and 151 controls using the genetic polymorphic markers identified from these 62 patients. Three single nucleotide polymorphisms (SNPs) were identified: g.-631C > G and g.-196G>A at 5'-promoter region, and g.69G>A at exon 1. Besides, no other mutations were identified in these patients. The g.69G>A polymorphism does not alter the amino acid threonine at codon 23 (ACG>ACA). Further case-control association studies also did not find significant differences of genotype or allele frequency distributions of these three polymorphisms between 163 patients and 151 non-psychotic comparison individuals. Hence, our data are not in favor of a large effect of synapsin III gene in the pathogenesis of schizophrenia.

Asian People↗
PubMed1996

Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.

Acetophenones↗