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PubMed1996

Characterization of six mutations in exon 37 of neurofibromatosis type 1 gene.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders, with an incidence of 1 in 3,000. We screened a total of 320 unrelated NF1 patients for mutations in exon 37 of the NF1 gene. Six independent mutations were identified, of which three are novel, and these include a recurrent nonsense mutation identified in 2 unrelated patients at codon 2281 (G2281X), a 1-bp insertion (6791 ins A) resulting in a change of TAG (tyrosine) to a TAA (stop codon), and a 3-bp deletion (6839 del TAC) which generated a frameshift. Another recurrent nonsense mutation, Y2264X, which was detected in 2 unrelated patients in this study, was also previously reported in 2 NF1 individuals. All the mutations were identified within a contiguous 49-bp sequence. Further studies are warranted to support the notion that this region of the gene contains highly mutable sequences.

Adult↗
PubMed1994

Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis.

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.

Amino Acid Sequence↗
PubMed1992

A vector, pSHT, for the expression and secretion of protein domains in mammalian cells.

A phagemid (pSHT) containing the pUC and M13 ori sequences was constructed to facilitate the expression of partial cDNAs or of sequences encoding mammalian membrane- and secretory-protein domains. It provides a start codon and signal sequence flanked upstream by the simian virus 40 and bacteriophage T7 promoters and downstream by cloning sites, stop codons in all three frames, splicing and polyadenylation signals.

Animals↗
PubMed2006

Novel single-base deletional mutation in major intrinsic protein (MIP) in autosomal dominant cataract.

PURPOSE: To further elucidate the cataract phenotype, and identify the gene and mutation for autosomal dominant cataract (ADC) in an American family of European descent (ADC2) by sequencing the major intrinsic protein gene (MIP), a candidate based on linkage to chromosome 12q13. DESIGN: Observational case series and laboratory experimental study. METHODS: We examined two at-risk individuals in ADC2. We PCR-amplified and sequenced all four exons and all intron-exon boundaries of the MIP gene from genomic and cloned DNA in affected members to confirm one variant as the putative mutation. RESULTS: We found a novel single deletion of nucleotide (nt) 3223 (within codon 235) in exon four, causing a frameshift that alters 41 of 45 subsequent amino acids and creates a premature stop codon. CONCLUSIONS: We identified a novel single base pair deletion in the MIP gene and conclude that it is a pathogenic sequence alteration.

Aquaporins↗
PubMed2004

Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.

Aspartate Kinase↗
PubMed1999

Structure of the human VIPR2 gene for vasoactive intestinal peptide receptor type 2.

The VPAC(2) (vasoactive intestinal peptide (VIP)(2)) receptor is a seven-transmembrane spanning G protein-coupled receptor which responds similarly to VIP and pituitary adenylate cyclase activating polypeptide (PACAP) in stimulating cAMP production. Recently, we reported the localisation of the human VPAC(2) receptor gene (VIPR2) to chromosome 7q36.3 (Mackay, M. et al. (1996) Genomics 37, 345-353). Here, we describe the characterisation of the VIPR2 gene structure and promoter region. The VIPR2 gene is encoded by 13 exons, the initiator codon of the 438 amino acid open reading frame is located in exon 1 and the termination signal and a poly-adenylation signal sequence are located in exon 13. The 5' untranslated region extends 187 bp upstream of the initiator codon and is extremely GC-rich (80%). The poly-adenylation signal is located 2416 bp downstream of the stop codon. Intron sizes range from 68 bp (intron 11) to 45 kb (intron 4) and the human gene spans 117 kb.

5' Untranslated Regions↗
PubMed2000

Bernard-Soulier syndrome in a patient doubly heterozygous for two frameshift mutations in the glycoprotein ib alpha gene.

We report here the genetic basis of Bernard-Soulier syndrome in a compound heterozygote for two mutant glycoprotein (GP) Ib alpha alleles. One allele contained a novel four base-pair deletion (TGAG) that eliminated the last base of the codon for Ser39 (AGT) and the entire codon for Glu40 (GAG), causing a reading frame shift that yielded a stretch of 51 amino acids before a premature stop codon. The other allele also contained a frame-shift mutation, caused by deletion of the last two bases of the codon for Tyr492 (TAT). This allele produced a truncated glycoprotein Ib alpha that, although not expressed on the surface of the patient's platelets, was detectable in the plasma. The second allele has been identified previously by our group and other investigators as the cause of Bernard-Soulier syndrome in patients of northern European ancestry. This allele carried a haplotype identical to those of the previously reported cases, with the following polymorphic markers: two tandem repeats in the VNTR region, C at nucleotide -5 from the ATG start codon and a substitution of G for A in the third base for codon Arg342. These findings suggest that this particular Bernard-Soulier mutation occurred once on the background of a rare haplotype and has spread throughout the northern European population.

Bernard-Soulier Syndrome↗
PubMed1989

Characterization of single base substitutions in edited apolipoprotein B transcripts.

Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.

Animals↗
PubMed2005

Antibiotic-induced expression of a cryptic cpb2 gene in equine beta2-toxigenic Clostridium perfringens.

The cpb2 gene of beta2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non-porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full beta2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant beta2-toxin showed the absence of expression of the beta2-toxin in equine and the other non-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the beta2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by beta2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.

Amino Acid Sequence↗
PubMed2006

Identification of nine new HLA class I alleles in volunteers from the Singapore stem cell donor registries.

Based on unusual probe hybridization patterns, two human leukocyte antigen (HLA)-A, five HLA-B, and two HLA-C alleles (A*240208, A*3211Q; B*1822, B*3938Q, B*4606, B*4607N, B*5139; Cw*0321, Cw*0734) were identified in individuals from the Singapore Bone Marrow Donor Program and Singapore Cord Blood Bank. Eight of the nine alleles encode amino acid substitutions altering the antigen-binding region including three alleles with changes altering a cysteine at codon 164 (A*3211Q, B*3938Q, B*4607N). This substitution either eliminates a key disulfide bond or results in a stop codon, both likely affecting the expression of the HLA molecules. Only one allele (A*240208) carries a synonymous substitution.

Alleles↗
PubMed1991

Role of translation of the pheA leader peptide coding region in attenuation regulation of the Escherichia coli pheA gene.

In Escherichia coli, the expression of the pheA gene is regulated by attenuation of transcription. To study the molecular details of pheA attenuation, we introduced mutations in the pheA leader peptide coding region and analyzed their effects by using pheA promoter-lacZ gene transcription fusions (pheAp-lacZ). Mutations in the ribosome-binding site (pheAe1213) or in the translation initiation codon (pheAe24) of the pheA leader peptide coding region resulted in superattenuation of pheA expression. However, the presence of a stop codon upstream to the tandem phenylalanine codons (pheAe3334) led to an increase in the basal-level expression of pheA. This increase was further enhanced in the presence of prfA release factor mutant. The level of pheA expression in all three mutants was the same when cells were starved for phenylalanine. These results demonstrate that efficient translation of the pheA leader peptide coding region and the position of the ribosome on the leader transcript play decisive roles in the attenuation regulation of pheA.

Base Sequence↗
PubMed1994

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana.

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Arabidopsis↗
PubMed1992

Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs.

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.

Amino Acid Sequence↗
PubMed1990

Saturation site-directed mutagenesis of thymidylate synthase.

We have subjected 12 different codons of a synthetic Lactobacillus casei thymidylate synthase (TS) gene to saturation site-directed mutagenesis to create amino acid "replacement sets" at each of those positions. The target residues were chosen because they are highly conserved and because they are important for the structure and function of the protein as indicated by solution and structural studies. The mutagenesis procedure involved excision of a fragment of the synthetic gene containing the target codon, followed by its replacement with a mixture of oligonucleotides which code for all 20 amino acids and the amber stop codon. TS mutants were identified by DNA sequencing, and catalytically active mutants were identified by genetic complementation using a Thy- strain of Escherichia coli. Only 3 of the 12 target amino acids examined were essential for TS activity; and of the 125 total mutants identified, 57 were catalytically active. These results point to a high degree of plasticity of TS in accommodating function with structural change.

Amino Acid Sequence↗
PubMed1998

The herpes simplex virus type 1 U(L)17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA.

Previous studies have suggested that the U(L)17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U(L)17 open reading frame [HSV-1(deltaU(L)17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5' end of the U(L)17 open reading frame [HSV-1(U(L)17-stop)] were plaque purified on engineered cell lines containing the U(L)17 gene. A virus derived from HSV-1(U(L)17-stop) but containing a restored U(L)17 gene was also constructed and was designated HSV-1(U(L)17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(deltaU(L)17) nor HSV-1(U(L)17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(deltaU(L)17) or HSV-1(U(L)17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(deltaU(L)17) compared to wild-type virus show no detectable differences. These data indicate that the U(L)17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U(L)17 gene product, an anti-U(L)17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent Mr 77,000 and weakly with a protein of apparent Mr 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U(L)17 protein. We therefore conclude that the U(L)17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

Animals↗
PubMed1997

Mutations in purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase deficiency is an inherited disease of purine metabolism characterized clinically as combined immunodeficiency. The molecular defects have been published for 4 different alleles in 3 patients. We report four new mutations including two amino acid substitutions, A174P and G190V, a single codon deletion, delta I129, and a point mutation in intron 3 which leads to aberrant splicing and creation of a premature stop codon in exon 4 (286-18G-->A). Of the previously reported mutations, E89K was found in one additional patient, and R234P was found in 3 unrelated patients, making R234P the most common mutation reported to date in this disease.

Alleles↗
PubMed2005

Fetal type IV glycogen storage disease: clinical, enzymatic, and genetic data of a pure muscular form with variable and early antenatal manifestations in the same family.

We report on a family of three consecutive fetuses affected by type IV glycogen storage disease (GSD IV). In all cases, cervical cystic hygroma was observed on the 12-week-ultrasound examination. During the second trimester, fetal hydrops developed in the first pregnancy whereas fetal akinesia appeared in the second pregnancy. The diagnosis was suggested by microscopic examination of fetal tissues showing characteristic inclusions exclusively in striated fibers, then confirmed by enzymatic studies on frozen muscle. Antenatal diagnosis was performed on the third and fourth pregnancies: cervical cystic hygroma and low glycogen branching enzyme (GBE) activity on chorionic villi sample (CVS) were detected in the third pregnancy whereas ultrasound findings were normal and GBE activity within normal range on CVS in the fourth pregnancy. Molecular analysis showed that the mother was heterozygous for a c.1471G > C mutation in exon 12, leading to the replacement of an alanine by a tyrosine at codon 491 (p.A491T); the father was heterozygous for a c.895G > T mutation in exon 7, leading to the creation of a stop codon at position 299 (p.G299X). GSD IV has to be considered in a context of cervical cystic hygroma with normal karyotype, particularly when second trimester hydrops or akinesia develop. Enzymatic analysis of GBE must be performed on CVS or amniotic cells to confirm the diagnosis. Characteristic intracellular inclusions are specific to the disease and should be recognized, even in macerated tissues after fetal death. Genetic analysis of the GBE gene may help to shed some light on the puzzling diversity of GSD IV phenotypes.

Adult↗
PubMed1997

Copper/zinc superoxide dismutase 1 and sporadic amyotrophic lateral sclerosis: analysis of 155 cases and identification of a novel insertion mutation.

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the brain and spinal cord and leading to death within 5 years of symptom onset. The great majority of ALS cases are sporadic, with the familial form (FALS) representing fewer than 10% of all cases. Mutations in the copper/zinc superoxide dismutase 1 (SOD-1) gene have previously been identified as the underlying cause of approximately 20% of FALS cases. As the familial and sporadic forms of the disease are clinically similar, we have sought to determine whether such mutations in SOD-1 underlie any sporadic ALS cases. We have screened 155 sporadic cases by single-strand conformation polymorphism and have identified 4 sporadic cases that possess point mutations in exon 4 of the SOD-1 gene. Two of these mutations are identical to those previously reported in FALS cases. One mutation is novel, resulting in a frameshift at Val118 due to the replacement of G (first base in the last codon of exon 4) by AAAAC. This mutation results in a truncated SOD-1 protein due to the introduction of a stop codon three residues into exon 5.

Alleles↗