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At least 1,675 records · Page 93Linked to original sources
PubMed1994

Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.

Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.

Amino Acid Sequence↗
PubMed1999

Mutational analysis of CvaA in the highly conserved domain of the membrane fusion protein family.

The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue-hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV.

ATP-Binding Cassette Transporters↗
PubMed2006

Mutation analysis of DARPP-32 as a candidate gene for schizophrenia.

Dopamine- and cAMP-regulated phosphoprotein of relative molecular mass 32kDa (DARPP-32) plays a pivotal role in the signal transduction of several neurotransmitters and neuromodulators that are implicated in the pathophysiology of a variety of neuropsychiatric disorders. A postmortem study reported a significantly reduced DARPP-32 expression in the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia, suggesting possible involvement of DARPP-32 in the pathophysiology of schizophrenia. Hence, DARPP-32 was considered as a candidate gene for schizophrenia in this study. We first systemically searched for mutations in the DARPP-32 gene in 50 Han Chinese patients with schizophrenia from Taiwan. Five molecular variants were identified, including a C-to-G substitution (g.-2036C>G) in the putative core promoter that obliterated a predictive AP-2 transcription factor binding site, a G deletion in the untranslated exon 2 (g.1238delG), a G-to-A and an A-to-G substitutions in intron 2 (IVS2+31G>A) and intron 6 (IVS6+32A>G), respectively, and a three-base pair deletion of AGA in exon 6 that resulted in deletion of a glutamate at codon 135 (E135del). Further SNP- and haplotype-based association study in 249 patients and 273 control subjects, however, did not detect association of these markers with schizophrenia. Hence, our results suggest that the reduced DARPP-32 protein in patients with schizophrenia is unlikely caused by mutations in the DARPP-32 gene itself and the DARPP-32 gene is also unlikely a major susceptibility gene for schizophrenia. Nevertheless, the identification of these molecular variants should help the study of gene regulation and structure-function relationship of DARPP-32, and the association study of DARPP-32 gene with other neuropsychiatric disorders.

Alleles↗
PubMed1995

Mutational analysis of influenza virus promoter elements in vivo.

RNA polymerase I transcription in vivo in transiently DNA-transfected cells has been used to express influenza virus vRNA molecules coding for chloramphenicol acetyltransferase (CAT) in an antisense orientation. Influenza virus superinfection provided viral RNA polymerase and other proteins required for transcriptional conversion of minus-strand vRNA into plus-strand viral mRNA molecules expressing CAT activity. This system has been used for analysis of the vRNA sequences which cooperatively constitute the vRNA promoter structure via nucleotide exchanges as well as deletions and insertions of both terminal segments. Several mutants caused greatly enhanced expression over wild-type levels, which was transmitted during serial passage of progeny virus. The data obtained for the mutations in various promoter elements support a model implicating double-stranded vRNA promoter structures in binding of viral polymerase, and in consecutive steps during initiation of RNA synthesis.

3T3 Cells↗
PubMed1993

[Mutation analysis of amyloid precursor protein in early-onset familial Alzheimer's disease].

Recently, three different mutations have been found at codon 717 of the amyloid precursor protein (APP) gene, changing the native valine to isoleucine, phenylalanine and glycine in some familial Alzheimer's disease (FAD) kindreds. More recently, some other mutations have also been reported at codons 670 and 671 (double mutation), and codon 692. As analysis of coding region except exon 16 and 17 has been insufficient in previous reports, we analyzed entire coding region of the APP gene of 6 Japanese early-onset FAD kindreds using automated sequencer. Three FAD families showed known 717 Val to Ile mutation, whereas no novel mutations were detected.

Age of Onset↗
PubMed2001

CDKNA2A mutation analysis, protein expression, and deletion mapping of chromosome 9p in conventional clear-cell renal carcinomas: evidence for a second tumor suppressor gene proximal to CDKN2A.

Inactivation of tumor suppressor genes on chromosome 9p is considered a critical event in renal cell carcinoma pathogenesis. Alterations of CDKN2A on 9p21 have been reported in renal cancer cell lines, but their relevance for primary renal carcinomas is unclear. Loss of heterozygosity (LOH) was analyzed by using four polymorphic microsatellites at D9S970 (9p12-9p13), D9S171 (9p13), D9S1748 (9p21), and D9S156 (9p21) in 113 primary conventional clear-cell renal cell carcinomas (CRCCs). Allelic deletion was detected in 21 of 88 informative CRCCs (24%) with the highest rate of LOH being observed at D9S171 on 9p13 (20%). Chromosome 9p LOH was associated with short tumor-specific survival in stage pT3 RCC (P = 0.01). Fluorescence in situ hybridization analysis of 54 CRCCs revealed no homozygous CDKN2A deletions indicating that this mechanism of CDKN2A inactivation is rare in CRCC. Sequencing of 113 CRCCs showed that 13 tumors (12%) had a 24-bp deletion abrogating codons 4 through 11 of CDKN2A. Immunohistochemical CDKN2A expression was absent in normal renal tissue and was only detected in six of 382 CRCCs (1.5%) on a renal tumor microarray. These data suggest that CDKN2A alterations are present in a small subset of CRCCs and a second, yet unknown tumor suppressor gene proximal to the CDKN2A locus, may play a role in CRCC development.

Adenocarcinoma, Clear Cell↗
PubMed2001

Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae.

In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.

Alleles↗
PubMed1990

Mutational analysis of the HIV nef protein.

The nef protein is one of the regulatory proteins encoded by the human immunodeficiency virus (HIV). It has been shown to down-regulate viral transcription and displays features typical of some membrane-associated G proteins. To define the amino acid sequences involved in several of the properties of the nef protein, the corresponding gene has been submitted to a series of site-directed mutations. The mutants have been evaluated using various parameters which seem to correlate with the biological properties of nef. As previously observed for some signal transducing proteins, membrane localization and correct folding are critical for nef activity. Additionally, two domains, putative "P and G sites" which may be involved in nucleotide binding, have been identified.

Amino Acid Sequence↗
PubMed2006

Resistance mutational analysis of HIV type 1 subtype C among rural South African drug-naive patients prior to large-scale availability of antiretrovirals.

Baseline HIV-1 resistance data are important for resistance monitoring purposes especially in regions initiating large-scale antiretroviral treatment programs. We examined 40 protease and 35 reverse transcriptase amino acid sequences of HIV-1 subtype C from drug inexperienced patients from rural settings in South Africa for resistance mutations. Samples were collected between 2001 and 2004 prior to the availability of antiretrovirals through public health institutions. Ninety-five percent of patients had no major mutations in the protease gene, although substitutions M46L (2.5%) and G73S (2.5%), which according to the Stanford Genotypic Resistance Interpretation Algorithm are considered major mutations, were detected. In addition, a high prevalence of minor mutations was observed in the protease, with at least three minor resistance-associated mutations in 37% of the isolates. An isoleucine insertion at codon 37 was detected in one sequence. Most of the RT sequences were wild-type, although V118I (8.5%) and Y318F (5.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. Our data suggest that major resistance mutations among the drug-inexperienced population in South Africa may be rare, and routine resistance testing before the initiation of therapy in this initial stage of the treatment program may not be necessary.

Amino Acid Sequence↗
PubMed1993

Mutational analysis in a patient with a variant form of Gaucher disease caused by SAP-2 deficiency.

It is now clear that the lysosomal hydrolysis of sphingolipids requires both lysosomal enzymes and so-called sphingolipid activator proteins (SAPs). One gene, called prosaposin, codes for a precursor protein that is proteolytically cut into four putative SAPs. These four SAPs, of about 80 amino acids, share some structural features but differ somewhat in their specificity. Domain 3 of prosaposin mRNA contains the coding region for SAP-2, an activator of glucocerebrosidase. While most patients with Gaucher disease store glucosylceramide due to defects in glucocerebrosidase, a few patients store this lipid in the presence of normal enzyme levels. In this paper we describe the identification of a point mutation in domain 3 of a patient who died with this variant form of Gaucher disease. Polymerase chain reaction amplification was performed in the small amount of genomic DNA available using primers generated from the intronic sequence surrounding domain 3. The patient was found to have a T-to-G substitution at position 1144 (counting from the A of ATG initiation codon) in half of the M13 recombinant clones. This changes the codon for cysteine382 to glycine. His father and unaffected brother also had this mutation, but his mother did not. She was found to have half of the normal amount of mRNA for prosaposin in her cultured skin fibroblasts. Therefore, this child inherited a point mutation in domain 3 from his father and a deficiency of all four SAPs coded for by prosaposin from his mother.

Adolescent↗
PubMed1993

Mutational analysis of the Saccharomyces cerevisiae general regulatory factor CP1.

The Saccharomyces cerevisiae general regulatory factor CP1, a helix-loop-helix protein that binds the centromere DNA element I (CDEI) of yeast centromeres, is required in yeast for optimal centromere function and for methionine prototrophy. Mutant alleles of CEP1, the gene encoding CP1, were generated by linker insertion, 5'- and 3'-deletion, and random mutagenesis and assayed for DNA binding activity and their ability to confer CP1 function when expressed in yeast. A heterologous CDEI-binding protein, TFEB, was also tested for CP1 function. The results suggested that DNA binding is required for both biological functions of CP1 but is not sufficient. A direct and quantitative correlation was observed between the chromosome loss and nutritional (i.e., Met) phenotypes of strains carrying loss of function alleles, but qualitatively the chromosome loss phenotype was more sensitive to decreased CP1 expression. The data are consistent with a model in which CP1 performs the same general chromatin-related function at centromeres and MET gene promoters and is normally present in functional excess.

Alleles↗
PubMed2005

[IRF6 gene mutation analysis in a van Der Woude syndrome family in Henan province].

PURPOSE: To investigate IRF6 gene mutation in a van Der Woude syndrome (VWS) family in Henan province. METHODS: PCR and DNA sequencing was employed to detect the mutation of IRF6.Secondary construction transformation analysis was performed using PIX-Protein Identification software. RESULTS: A CGC>TGC(r.279c-->t) transversion of IRF6 was identified in condon 6, showing complete segregation with the disease phenotypes and was resulting in changes of the secondary constructure of IRF6. CONCLUSION: VWS is caused by mutations in IRF6 gene, and IRF6 is closely related to the development of lip, palate and tooth.

Abnormalities, Multiple↗
PubMed1996

Mutational analysis of the beta-subunit of yeast geranylgeranyl transferase I.

The gene CAL1 (also known as CDC43) of Saccharomyces cerevisiae encodes the beta subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded by cal1-1, and cdc43-2 to cdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. The cal1-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rho1p was observed in the cal1-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal1-1 is most efficiently suppressed by overproduction of Rho1p. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal1-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.

Alkyl and Aryl Transferases↗
PubMed2002

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase.

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.

Adenosine Triphosphate↗
PubMed1990

Mutational analysis identifies a new functional domain of the thyroid hormone receptor.

We studied the functional significance of a region of the thyroid hormone receptor ligand binding domain which is conserved in all members of the erbA superfamily. The homologous region of the glucocorticoid receptor has been implicated in the binding of heat shock protein 90, and deletions of the glucocorticoid receptor that include this region result in constitutive activity. Both deletion and point mutations were made in this area of the rat beta 1 thyroid hormone receptor (amino acids 286-305), and the functional consequences of these mutations were analyzed in JEG cells by transient cotransfection along with a T3 responsive reporter gene. All mutations studied resulted in significant inhibition of ligand-dependent transcriptional regulation without inducing significant constitutive activity. For some mutations, the lack of transcriptional response correlated with a diminished ability to bind ligand. However, point mutations of amino acids 288, 290, and 300 resulted in impaired transcriptional regulation despite wild type T3 binding affinity. In addition, mutations of these three amino acids failed to impair localization of the receptor to the nucleus or binding to DNA in vitro. Cotransfection of plasmids expressing the wild type and mutant T3 receptor proteins resulted in inhibition of the wild type T3 receptor function. We conclude that this region of the rat beta 1 T3 receptor is essential for full transcriptional activity, but this is not due to a role in T3 binding, DNA binding, or nuclear localization. We postulate that nuclear factors may need to bind to this region for full transcriptional activity.

Amino Acid Sequence↗
PubMed2006

Mutational analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA.

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3' interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA.

Bacterial Proteins↗