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At least 811 records · Page 45Linked to original sources
PubMed1995

Mutational analysis of varicella-zoster virus major immediate-early protein IE62.

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.

Amino Acid Sequence↗
PubMed2004

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2.

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.

Amino Acid Sequence↗
PubMed2003

Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates.

The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence. Instead, the [4/5] asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop. The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex. The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry. The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.

Base Sequence↗
PubMed1994

Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha.

We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase [gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37] in microbody targeting. The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of various genetically altered watermelon MDH genes, whose protein products were localized by immunocytochemical techniques. It is shown that the presequence of gMDH is essential and sufficient for peroxisomal targeting; it can target the mature part of the mitochondrial MDH to microbodies, whereas deletion of the presequence results in accumulation of the mature form of gMDH in the cytosol. Alignment of the N termini of several peroxisomal proteins that are assumed to contain a peroxisomal targeting signal at the N terminus (PTS2) suggested the consensus seqence RL-X5-HL. A similar motif is present in the presequence of watermelon gMDH--namely, 10RI-X5-17HL. Mutational analysis revealed that substitutions of 10RI into DD or 17HL into DE destroyed the topogenic information, whereas substitutions of 25M into I and 26EE into LV did not. By combining our data with recent analyses of others on the presequences of mammalian thiolases, it is concluded that the peroxisomal targeting information of PTS2 is contained in the consensus sequence RL/I-X5-HL. In contrast to the higher plant and mammals, the Hansenula yeast peroxisomes seem to lack an enzyme capable of removing microbody presequences of higher eukaryotes.

Cell Compartmentation↗
PubMed2006

Mutation analysis of the short cytoplasmic domain of the cell-cell adhesion molecule CEACAM1 identifies residues that orchestrate actin binding and lumen formation.

CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe(454)) and lysine (Lys(456)) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe(454) at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr(457) and/or Ser(459) are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling.

Actins↗
PubMed2004

Acidophilic xylanase from Aureobasidium pullulans: efficient expression and secretion in Pichia pastoris and mutational analysis.

A yeast-like fungus Aureobasidium pullulans var. melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-1,4-beta-xylanase with an optimum pH of 2.0 [Ohta et al., J. Biosci. Bioeng., 92, 262-270 (2001)]. The xynI cDNA encoding the precursor protein (XynI) was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter. The 34 amino acid prepro-signal peptide of the A. pullulans XynI directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium. The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae alpha-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P. pastoris acid phosphatase. The N-terminal amino acid sequence and the apparent M(r) of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A. pullulans XynI signal sequence in P. pastoris. The three-dimensional model and mutational analysis of the xynI gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft, respectively, play a significant role in its low pH optimum.

Journal Article↗
PubMed2001

Novel posttranslational activation of the LYS2-encoded alpha-aminoadipate reductase for biosynthesis of lysine and site-directed mutational analysis of conserved amino acid residues in the activation domain of Candida albicans.

The alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi. The alpha-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The LYS2 and LYS5 genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of Candida albicans, a pathogenic yeast, in the presence of CoA and C. albicans lys2 mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type C. albicans CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of C. albicans.

Aldehyde Oxidoreductases↗
PubMed1998

Mutation analysis of the WT1 gene in myelodysplastic syndromes.

The WT1 tumor suppressor gene was examined for mutations in a panel of 44 patients with myelodysplastic syndromes (MDS) including acute myelogenous leukemias (AML) secondary to MDS, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and sequencing analysis. A WT1 mutation was detected in one out of 17 cases of AML secondary to MDS. This mutation exists upstream of the zinc finger region and is predicted to produce a truncated WT1 protein lacking the zinc finger region. No mutations were detected in 27 MDS patients who had not progressed to AML. This is the first report of analysis for WT1 mutations in a large number of MDS patients, suggesting that WT1 mutations are uncommon in MDS. Abnormalities in this gene may, however, contribute to a small proportion of cases showing progression from MDS into AML.

Amino Acid Sequence↗
PubMed2006

VLCAD deficiency: pitfalls in newborn screening and confirmation of diagnosis by mutation analysis.

We diagnosed six newborn babies with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) through newborn screening in three years in Victoria (prevalence rate: 1:31,500). We identified seven known and two new mutations in our patients (2/6 homozygotes; 4/6 compound heterozygotes). Blood samples taken at age 48-72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype-phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed2001

Correlation of mutational analysis to clinical features in Taiwanese patients with Gilbert's syndrome.

OBJECTIVES: Mutations in the promoter as well as in the coding region of the bilirubin UDP-glucuronosyltransferase gene (UGT1A1) have been found to be associated with Gilbert's syndrome. However, the genetic basis of Gilbert's syndrome in our population and correlation of these mutations to fasting serum bilirubin levels in patients with Gilbert's syndrome remain to be clarified. METHODS: We applied polymerase chain reaction-based direct-sequencing assays to examine mutations in UGT1A1 gene in 20 unrelated Gilbert's patients and in a family with Gilbert's syndrome. RESULTS: We studied three mutations that were previously reported to be associated with Gilbert's syndrome (i.e., the TATAA-box mutation, Gly71Arg, and Pro229Gln) in 20 patients with Gilbert's syndrome. Of the patients, 16, five, and six were found to have the TATAA-box, Gly71Arg and Pro229Gln mutations, respectively. Seven patients had simultaneous mutations both in the TATAA box and in the coding region. Of note, all six patients with Pro229Gln also had the TATAA-box mutation. Localization of Pro229Gln on the allele containing the TATAA-box mutation was demonstrated in a family with Gilbert's syndrome. The patients simultaneously heterozygous for both the TATAA-box mutation and Gly71Arg usually had serum bilirubin levels similar to those found in the patients homozygous for the TATAA-box mutation, but usually higher than those found in the patients heterozygous for the TATAA-box mutation alone. On the other hand, concurrence of Pro229Gln in patients with TATAA-box mutation or with Gly71Arg did not significantly affect serum bilirubin levels. CONCLUSIONS: The TATAA-box mutation and Gly71Arg are the major causes for Gilbert's syndrome in our population. Concurrence of mutations of Gly71Arg and TATAA-box usually exerts a synergistic effect on hyperbilirubinemia. Pro229Gln, which is regularly linked to the TATAA-box mutation, may not have a significant effect on serum bilirubin levels.

Adolescent↗
PubMed2005

Mutation analysis of Wilson disease in the Spanish population -- identification of a prevalent substitution and eight novel mutations in the ATP7B gene.

Wilson disease (WD) is a copper metabolism disorder characterized by hepatic and/or neurological damage. More than 200 mutations in the ATP7B gene causing this autosomal recessive defect have been reported. In certain populations, a high prevalence of particular mutations allows rapid screening and diagnosis of the disease. We identified the ATP7B alterations in Spanish patients with WD. Mutations in the ATP7B gene were analysed in a total of 64 individuals from 40 different WD families by PCR amplification, single-strand conformation polymorphism (SSCP) analysis and sequencing. Twenty-one different ATP7B gene mutations were identified, eight of which were novel. 74% of the disease alleles were characterized among the 40 unrelated probands. We identified a prevalent mutation in our population (Met645Arg), present in 55% of this 40 patients. The frequency of the remaining ATP7B alterations was low. In addition, 17 different polymorphic variants were found. There is remarkable allele heterogeneity in WD in the Spanish population. Nevertheless, SSCP screening for the most frequent mutations in our population is feasible and leads to the detection of about 74% of the mutated chromosomes. Molecular diagnosis of WD is very useful in clinical practice to confirm or support clinical suspicion.

Adenosine Triphosphatases↗
PubMed2001

Mutation analysis of a Mauritian hereditary breast cancer family reveals the BRCA2 6503deITT mutation previously found to recur in different ethnic populations.

Mauritius, a small island some 855 km off the east coast of Madagascar, has a multiethnic population of about 1.2 million with a high population density of about 611 per km(2). The recent industrialization of the island seems to have been accompanied, in less than 10 years, by an increase of at least 30% in breast cancer incidence. We have detected the BRCA2 6503delTT mutation in two sisters of the same family of Indian origin but living in Mauritius for at least five generations. This mutation has been found to recur in geographically diverse populations and haplotype analysis has shown a common ancestry. The haplotype of the mutation found in the Mauritian family differs from that found in other populations harbouring the same mutation, suggesting that the BRCA2 6503delTT mutation most likely arose independently.

BRCA2 Protein↗
PubMed2006

Mutation analysis of the paraplegin gene (SPG7) in patients with hereditary spastic paraplegia.

BACKGROUND: Mutations in the SPG7 gene, which encodes paraplegin, are responsible for an autosomal recessive hereditary spastic paraplegia (HSP). OBJECTIVE: To screen the SPG7 gene in a large population of HSP families compatible with autosomal recessive transmission. METHODS: The authors analyzed 136 probands with pure or complex HSP for mutations in the SPG7 using denaturation high-performance liquid chromatography and direct sequencing. RESULTS: The authors identified 47 variants including 6 mutations, 27 polymorphisms, and 14 changes with unknown effects. In one family from Morocco, compound c.850_851delTTinsC and c.1742_1744delTGG heterozygous mutations were shown to be causative. This family had complex HSP with cerebellar impairment. Progression of the disease was rapid, resulting in a severe disease after 8 years of duration. Also detected were 20 families with one heterozygous mutation that was not found in a large control population. The mutations produced highly defective proteins in four of these families, suggesting that they were probably causative. Direct sequencing of all exons and reverse transcription PCR experiments demonstrated the absence of a second mutation. However, the p.Ala510Val missense substitution previously described as a polymorphism was shown to be significantly associated with HSP, suggesting that it had a functional effect. CONCLUSION: SPG7 mutations account for less than 5% of hereditary spastic paraplegia (HSP) families compatible with autosomal recessive inheritance. Cerebellar signs or cerebellar atrophy on brain imaging were the most frequent additional features in patients with SPG7 HSP. Rare nucleotide variants in SPG7 are frequent, complicating routine diagnosis.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2005

Mutation analysis of the HDAC 1, 2, 8 and CDKL5 genes in Rett syndrome patients without mutations in MECP2.

Mutations in the MECP2 gene are found in only 80% of patients with Rett syndrome (RTT). Therefore other genes have to be involved in the pathogenesis of RTT. By using our defined diagnostic criteria we first re-evaluated 50 girls with possible RTT in whom the sequencing of the MECP2 gene had not revealed any mutations. Only 15 of theses patients fulfilled all criteria for RTT and could be considered to have classical RTT. In eight of these, further molecular analyses revealed large deletions of the MECP2 gene. In the remaining seven girls we then analyzed the genes HDAC1, HDAC2, and HDAC8 that encode for the histone deacetylases 1, 2, and 8 which interact with MeCP2 and are essential for its function. Although these histone deacetylase genes have been considered as good candidate genes for RTT our molecular analysis of these genes did not detect any mutations. Because recently mutations in CDKL5 were reported in patients with RTT, we included this gene in our analysis but failed to detect any mutations. We conclude that only a subgroup of girls with possible RTT and no detectable mutation in the sequencing of the MECP2 gene do really have classical RTT. In many of those large MECP2 gene deletions can be detected by further analysis. The genes HDAC1, HDAC2, and HDAC8 do not seem to play a role in the pathogenesis of RTT and at least in our subgroup no mutations in the CDKL5 gene were detected.

Cell Cycle Proteins↗
PubMed1998

Mutation analysis of the PTEN/MMAC1 gene in lung cancer.

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.

Carcinoma, Small Cell↗
PubMed2005

Mutational analysis of hMsh6 in Israeli HNPCC and HNPCC-like families.

Germline mutations in DNA mismatch repair (DNA-MMR) genes, mainly hMlh1 and hMsh2, underlie Hereditary Non-Polyposis Colorectal Cancer (HNPCC). Germline hMSH6 gene mutations have been reported in a small subset of HNPCC families. In the present study, ethnically diverse individuals with HNPCC and HNPCC-like features were genotyped for hMsh6 germline mutations using exon-specific PCR, DGGE, and DNA sequencing. The study encompassed 92 individuals representing 88 unrelated families who were previously analyzed for Msh2 and Mlh1 mutations: Jewish Ashkenazim (n = 44), non-Ashkenazim (n = 27), Israeli Moslem-Arab (n = 15), Druze (n=3), and Cypriot non-Jews (n = 3). Of the study population, 71 had colon cancer (CRC), mean age at diagnosis was 50.9+/-13.2 years (range 16-73 years), 5 had endometrial cancer (two with concurrent CRC), (mean 43.6+/-3.26 years, range 38-45 years), and unaffected individuals (n = 18) were first degree relatives within HNPCC families and were genotyped at a mean age of 48.3+/-11.7 years (range 30-69 years). Of the 92 individuals analyzed, none showed a truncating hMsh6 mutation, and 6 (6.6%) harbored one of three germline missense mutations: a previously reported one (V878A), and two novel mutations (V509A, S227I). The pathogenic significance of these three missense mutations is yet unclear. In addition, 5 polymorphisms were detected, 2 of which were novel. We conclude that the rate of pathogenic hMsh6 mutations in HNPCC families of Jewish and Mediterranean origin is low, and that mutations in other genes probably account for the phenotype in these families.

Adult↗
PubMed2006

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure.

Mutational screening of the bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes in a population with premature ovarian failure (POF) identified no new mutations. However, three single nucleotide polymorphisms in the BMP15 gene, two in the 5' untranslated region (31T>G and 71C>G) and another in exon 1 (387G>A), were found to be common in both POF and control groups.

Adult↗