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PubMed1996

UGA suppression by tRNACmCATrp occurs in diverse virus RNAs due to a limited influence of the codon context.

We have recently identified chloroplast and cytoplasmic tRNACmCATrp as the first natural UGA suppressor tRNAs in plants. The interaction of these tRNAs with UGA involves a Cm: A mismatch at the first anticodon position. We show here that tRNACmCATrp is incapable of misreading UAA and UAG codons in vitro, implying that unconventional base pairs are not tolerated in the middle anticodon position. Furthermore, we demonstrate that the ability of tRNACmCATrp to promote UGA read-through depends on a quite simple codon context. Part of the sequence surrounding the leaky UGA stop codon in tobacco rattle virus RNA-1 was subcloned into a zein reporter gene and read-through efficiency was measured by translation of RNA transcripts in wheat germ extract. A number of mutations in the codons adjacent to the UGA were introduced by site-directed mutagenesis. It was found that single nucleotide exchanges at either side of the UGA had little effect on read-through efficiency. A pronounced influence on suppression by tRNACmCATrp was seen only if 2 or 3 nt at the 3'-side of the UGA codon had been simultaneously replaced. As a consequence of the flexible codon context accepted by tRNACmCATrp, this tRNA is able to misread the UGA in a number of plant and animal viral RNAs that use translational read-through for expression of some of their genes.

Codon, Nonsense↗
PubMed1998

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates.

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.

Animals↗
PubMed2005

Novel mutations in the MEN1 gene in subjects with multiple endocrine neoplasia-1.

OBJECTIVE: To identify MEN1 gene mutations and characterize clinical manifestations in Chinese kindred with multiple endocrine neoplasia type 1 (MEN1) in Taiwan. PATIENTS AND METHODS: Eight unrelated subjects (one male and seven females, age range 26-70 years) with clinical manifestations of MEN1 were analysed. In addition, 45 relatives that included 10 affected (three males and seven females, age range 32-53 years) and 35 unaffected (17 males and 18 females, age range 15-80 years) subjects were evaluated. Genomic DNA extraction, polymerase chain reaction (PCR) and DNA sequence analysis were performed according to standard procedures. RESULTS: We identified heterozygous MEN1 gene mutations in all eight probands and 10 affected subjects as well as in 13 clinically asymptomatic relatives. Novel mutations included a missense mutation in a heterozygous mutation in exon 9 (GAC --> CAC) resulting in a substitution of aspartic acid by histidine at codon 418 (family 1); a nonsense mutation at codon 556 of exon 10 (GAG --> TAG) resulting in a stop codon and termination (family 2); a missense mutation in exon 2 (GGG --> GAG) causing the substitution of glycine by glutamic acid at codon 110 (family 3); and a deletion/insertion mutation in nucleotide 1200 of exon 8 resulting in frameshift and early termination (family 4). Affected subjects in families 5-7 shared the same C insertion at nucleotide 1650 of exon 10, similar to that previously described as a hotspot for mutation, and proband 8 had a previously described mutation in intron 4 of the MEN1 gene (IVS4-9 G --> A). We also found that 18 (58%) of our 31 MEN1 mutant carriers had clinical symptoms, whereas four (13%) had biochemical abnormalities without clinical symptoms, and nine (29%) were unaffected both clinically and biochemically. CONCLUSIONS: We have identified four novel mutations in the MEN1 gene in patients with MEN1 in Taiwan.

Adolescent↗
PubMed1994

An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli.

The 5' ends of many bacterial transcripts are important in determining mRNA stability. A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli. Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability. A stop codon in the 10th codon of lacZ increased mRNA stability. Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability.

Codon↗
PubMed1984

Mapping of the amino terminus of the H-1 parvovirus major capsid protein.

The amino terminus of VP2', the major capsid protein of the parvovirus H-1, was identified and mapped to the H-1 genome. The protein initiates at the start codon at nucleotide 2797 and is translated uninterrupted to the stop codon at nucleotide 4582. The primary sequence predicts a protein of 593 amino acids (65,500 daltons) with an amino acid composition which very closely matches the experimentally determined composition of the pure protein. The data suggest that the VP2' mRNA has a 5' leader sequence of ca. 650 bases and that protein translation initiates downstream from the sole splice junction.

Amino Acid Sequence↗
PubMed1992

Coding capacity determines in vivo accumulation of a defective RNA of clover yellow mosaic virus.

Naturally occurring defective RNAs (D RNAs) derived from the potexvirus clover yellow mosaic virus (CYMV) contain large internal deletions yet maintain a single open reading frame (ORF) representing the in-frame fusion of 5' and 3' terminal ORFs. Capped transcripts of the prototype 1.2-kb D RNA of CYMV were synthesized in vitro and used to inoculate broad bean plants. Progeny D RNA accumulated only if synthetic D RNA transcripts were coinoculated with CYMV RNA. Several experiments showed that helper-dependent accumulation of the D RNA in vivo depended on the maintenance of its encoded fusion ORF. (i) D RNAs with six-residue deletions introduced early in the fusion ORF accumulated, whereas those with four-residue out-of-frame deletions at the same sites were nonviable. (ii) Analysis of D RNAs containing termination codons at different locations showed that only the most 3' stop codon (maintaining over 93% of the fusion ORF) was permissive for D RNA accumulation. (iii) D RNAs with small in-frame deletions and insertions in their 3' coding regions were viable. (iv) Nonviable D RNAs containing disrupted fusion ORFs could not be complemented by the presence in the infection of a D RNA encoding a complete fusion ORF. Taken together, the results indicate that the process of translation, rather than the encoded product, modulates an event(s) which influences the propagation and/or accumulation of this RNA in vivo. This represents a unique requirement among plant virus D RNAs.

Base Sequence↗
PubMed2006

An asymmetric underlying rule in the assignment of codons: possible clue to a quick early evolution of the genetic code via successive binary choices.

Aminoacyl-tRNA synthetases (aaRSs) are responsible for creating the pool of correctly charged aminoacyl-tRNAs that are necessary for the translation of genetic information (mRNA) by the ribosome. Each aaRS belongs to either one of only two classes with two different mechanisms of aminoacylation, making use of either the 2'OH (Class I) or the 3'OH (Class II) of the terminal A76 of the tRNA and approaching the tRNA either from the minor groove (2'OH) or the major groove (3'OH). Here, an asymmetric pattern typical of differentiation is uncovered in the partition of the codon repertoire, as defined by the mechanism of aminoacylation of each corresponding tRNA. This pattern can be reproduced in a unique cascade of successive binary decisions that progressively reduces codon ambiguity. The deduced order of differentiation is manifestly driven by the reduction of translation errors. A simple rule can be defined, decoding each codon sequence in its binary class, thereby providing both the code and the key to decode it. Assuming that the partition into two mechanisms of tRNA aminoacylation is a relic that dates back to the invention of the genetic code in the RNA World, a model for the assignment of amino acids in the codon table can be derived. The model implies that the stop codon was always there, as the codon whose tRNA cannot be charged with any amino acid, and makes the prediction of an ultimate differentiation step, which is found to correspond to the codon assignment of the 22nd amino acid pyrrolysine in archaebacteria.

Amino Acyl-tRNA Synthetases↗
PubMed2005

Hermansky-Pudlak syndrome with a novel mutation.

We report a case of Hermansky-Pudlak syndrome (HPS) with a novel mutation in the HPS1 gene. This case showed oculocutaneous albinism and lysosomal ceroid accumulation, however platelet dysfunction was not observed. Histopathological findings of the biopsied lung tissue were compatible with HPS. Sequencing analysis showed the insertion of C in the codon 178 (739 bp) of the HPS1 gene forming a stop codon at codon 181. To the best of our knowledge, this is a novel HPS1 gene mutation.

Comorbidity↗
PubMed1998

Hb Tak confirmed by DNA analysis: not expressed as thalassemia in a Hb Tak/Hb E compound heterozygote.

Hemoglobin variants in which a frameshift results in chain elongation are unusual and also have a low population frequency. Hb Tak was previously characterized by amino acid analysis, and it was assumed to be due to an insertion of the dinucleotide CA into codon 146 [CAC-->CA(CA)C] which abolishes the normal stop codon at position 147. This insertion causes a frameshift which results in elongation of the beta chain by 11 amino acids. This variant has previously been described in a few Thai families. We report the DNA sequence of Hb Tak in an individual of Cambodian descent who is a Hb E/Hb Tak compound heterozygote. In contrast with extended variants of the alpha-globin chain that are expressed as alpha-thalassemias, the hematologic effect of Hb Tak/Hb E is a mild polycythemia. The combination of Hb Tak/Hb E is not expressed as a thalassemia.

Adult↗
PubMed2004

Truncation of the mismatch repair protein PMS2 during the neoplastic transformation of human breast epithelial cells in vitro.

Alterations in known mismatch repair (MMR) genes have been found in many cancers, such as in hereditary non-polyposis colorectal cancer syndrome (HNPCC), in addition to specific oncogenes and tumor suppressor gene abnormalities. We have reported mutations in the MMR genes hMSH2, hPMS2 and hMSH6 in human breast epithelial cell lines (HBEC). In hPMS2 we found a non-sense mutation in codon number 471 by a change of glutamine (CAG) by stop codon (TAG). We also observed that the number of alterations in the MMR genes were increased during the process of cell transformation and tumorigenesis. In the present study in order to determine the role of those mutations in the hPMS2 protein activity, we used western blotting, protein truncation test and MMR activity assay employing HBEC cells. In the transformed and tumor cell lines we found a premature hPMS2 protein truncated by western blot and also we confirmed this truncation using the protein truncation test. In the MCF-10F cells only the wild-type hPMS2 protein was detected. The functionality of the hPMS2-truncated protein was confirmed by DNA heteroduplex assay using specific mispaired bases in mutated M13mp2 phages. DNA repair activity was proficient in MCF-10F cells, partially deficient in the transformed cells, and totally deficient in the tumor cell lines. In order to test whether the MMR deficiency was due to an hPMS2 defect, we used the wild-type heterodimer hPMS2-hMLH1 in the in vitro reaction of DNA heteroduplex assay; we have found that addition of the hPMS2 wild-type increased the MMR repair efficiency by more than 40% in tumor cell lines. Our results indicate that a protein-truncating mutation in the hPMS2 gene had occurred during the process of cell transformation and tumorigenesis and that truncation is related to a lack of function of this gene.

Adenosine Triphosphatases↗
PubMed2003

Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein.

Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3' and 5' end of the KCTIP1 gene were obtained using the SMART RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3'end of this gene was obtained using the GSP1 primer, and a 690 bp fragment corresponding to the 5' end of this gene was obtained using the GSP2 primer. Two primers that flank the putative open reading frame (ORF) were designed to obtain the cDNA containing the complete ORF by RACE PCR reaction. The full-length cDNA of KCTIP1, containing a 756 bp open reading frame (ORF), was approximately 1.1 kb; the start codon was located at the nucleotides of 99-101 and stop codon at the nucleotides of 855-857 followed by a poly (A) tail. The KCTIP1 cDNA sequence in this research was released in GenBank with accession number AF521135. Using ExPASy Proteomics tools provided by EMBL, the isoelectric point and MWt of KCTIP1 are estimated as 5.77 and 26.3 kD respectively. Transmembrane prediction analysis revealed the deduced KCTIP1 protein sequence contains six transmembrane regions at amino acid residues of 20 - 42, 57 - 79, 86 - 108, 113 - 135, 142 - 164 and 217 - 239. Two highly conserved asparagine-proline-alanine (NPA) motifs were located at 85 - 87 and 199 - 201 amino acid residues respectively. KCTIP1 is also predicted to contain the Cys residue (Cys 118) that are shown to confer Hg-sensitivity in Arabidopsis gamma-TIP and delta-TIP. Similarity analysis showed that KCTIP1 shared 77% - 79% amino acid sequence identity with the TIPs from Vitis berlandieri, Brassica oleracea and Arabidopsis thaliana. Expression analyses indicated that KCTIP1 had different expression among species of Mangroves. Expressions of KCTIP1 in Kandelia candel, Rhizophora apoculata and Ceriops tagal were suppressed by salt, and were insensitive to salt stress in unknown species of Mangroves. Previous studied showed that salt conditions might result in large and rapid changes in extracellular water potential and serious disturbance to the cytoplasm. In order to compensate for this imbalance, the relative contribution of water channels to flow across the root could thus vary. K. candel is a species that is native to intertial zone of tropical and subtropical coast and is well-adapted to salt conditions. The coordinated down-regulation of aquaporins in this plant may decrease membrane water permeability and thus increase the cellular water conserva- tion during periods of salt stress. The results reported here are consistent with the postulated roles for tonoplast water channels in regulating the hydraulic permeability of the vacuolar membranes and in adjusting the water homeostasis of the protoplasm under various physiological conditions. The identification of KCTIP1 as one of salt-responsive genes implies that intracellular osmotic equilibration is a part of salt-tolerant mechanisms in Mangroves.

Amino Acid Sequence↗
PubMed1988

Clinical and genetic heterogeneity in black patients with homozygous beta-thalassemia from the southeastern United States.

The presence of various substitutions and deletions resulting in beta-thalassemia was studied in 19 black patients with homozygous beta-thalassemia and in numerous relatives; all patients were from Georgia, South Carolina, and Alabama. Methodology included gene mapping, amplification of genomic DNA with Taq polymerase, identification of known nucleotide substitutions or a single nucleotide deletion through hybridization with synthetic oligonucleotides, cloning and sequencing of a beta-globin gene, and sequencing of amplified genomic DNA. Of the 38 chromosomes tested, 21 (55%) had the A----G substitution at nt -29, eight (21%) had the C----T substitution at nt -88, three (8%) had the substitution at codon 24, while one each of the following abnormalities were also detected: frameshift at codon 6, a C----A mutation at nt 848 of the beta IVS-II (new), an A----T mutation at codon 61 (new), a deletion of 1.35 kilobases including the 5' end of beta, a Ggamma(Agamma delta beta)(0)-thalassemia, and one thalassemia determinant that remained unidentified. The C----A mutation at nt 848 of IVS-II occurred at a position 3 nucleotides 5' to the third exon, adjacent to the invariant AG dinucleotide of the acceptor sequence. The A----T mutation in codon 61 (AAG----TAG) resulted in the creation of a stop codon and thus in beta(0)-thalassemia. The various mutations occurred on chromosomes with different haplotypes; however, chromosomes with a specific mutation but with different haplotypes belonged to one specific framework, which suggested that crossovers were responsible for these different types. Hemoglobin (Hb) F levels were generally high (55% to 75% with 98.5% in one patient with beta(0)/beta(0)); a few patients with specific haplotypes and an alpha-thalassemia-2 heterozygosity had a lower Hb F level. The Ggamma in the Hb F was consistently high when the C----T mutation occurred at nt -158 to the Cap site of the Ggamma-globin gene; seven patients with +/+ at this site had an average Ggamma of 73.8%, eight patients with +/- had 64.8%, and one patient with -/- had 34.2%. Variations in hematologic values and in Hb F, Ggamma, and Hb A2 levels of relatives with a beta-thalassemia heterozygosity depended to some extent on the types of mutations or deletions and on the haplotypes of the chromosomes with the beta-thalassemia determinant.

Adolescent↗
PubMed1988

Molecular cloning and analysis of P. 69, a vir-controlled protein from Bordetella pertussis.

The cloning sequencing and analysis of an important antigenic component of Bordetella pertussis is described. The gene for P.69, in common with a variety of other so called "virulence" genes, (e.g., adenylate cyclase (AC), pertussis toxin (PT) and filamentous haemagglutinin (FHA)), is under control of the vir locus. The protein P.69 is externally localised on cells and protein preparations are protective as judged by the mouse intra-cerebral challenge test. The gene encoding the P.69 antigen was isolated by hybridization of mixed oligonucleotide probes against B. pertussis genomic DNA. These oligonucleotides were designed from the protein sequence data obtained from a cyanogen bromide digest of the P.69 protein. DNA sequence analysis reveals a G:C rich gene capable of encoding a protein of 910 amino acids and Mr of 93478, whose likely promoter and ribosome binding sites show little homology to their E.coli counterpart. In common with some of the genes in the PT operon the sequence 5'-CCTGG-3' was found 5' to the ATG initiation codon. At the 3', end 29 bases after the TAA stop codon, the sequence 5'GTTTTTCCT-3' was found in an equivalent position to the same sequence in the PT operon. Examination of the protein sequence reveals two regions with directly repeated elements (GGAVP)3(GGFGP)2 and (PQP)5.

Amino Acid Sequence↗
PubMed1994

Novel germline mutation of the p53 tumor suppressor gene in a child with incidentally discovered adrenal cortical carcinoma.

PURPOSE: We report a case of adrenal cortical carcinoma in an infant, which was incidentally discovered by renal sonography after a urinary tract infection. The previous death of a sibling after rhabdomyosarcoma in infancy prompted a search for a heritable p53 tumor suppressor gene mutation in this family. PATIENTS AND METHODS: Starting with frozen adrenal carcinoma tissue, polymerase chain reaction (PCR) amplification followed by direct sequencing of exons 4-8 of p53 was used to search for a mutation. When a mutation was identified in exon 6 of the tumor p53 sequence, PCR amplification and direct sequencing of exon 6 alone was then performed on DNA from peripheral blood lymphocytes (PBLs) of all immediate family members to determine whether a germline mutation was present. A different set of primers was used by a second laboratory at our institution to independently confirm the presence of the mutation in the adrenal carcinoma and in paraffin-embedded rhabdomyosarcoma tissue of the deceased sibling. RESULTS: A C-to-T transition was identified at a CpG site in codon 196 resulting in a change from arginine to a stop codon (CGA to TGA). The identical mutation, present as the sole p53 allele in the tumor DNA samples and in the heterozygous state with wild type p53 allele in DNA from PBLs (germline), was found in the adrenal carcinoma, the rhabdomyosarcoma, and the PBLs of the tumor-bearing child and her healthy father and 5-year-old brother. This nonsense mutation of p53 has never before been reported in the germline. The extended pedigree showed only one known additional cancer. CONCLUSIONS: A novel germline p53 mutation was identified by investigation of a sibling pair with cancers associated with the Li-Fraumeni syndrome in a family with an otherwise negative history for cancer. The implications of this case for identification of carriers of p53 germline mutations and their clinical management are discussed.

Adrenal Cortex Neoplasms↗
PubMed1993

Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins.

Two different isoproteins are encoded by the apolipoprotein (apo) B gene, apoB-48 and apoB-100. ApoB-48, core component of intestinally derived chylomicrons, has an accelerated plasma turnover as compared with the full-length protein apoB-100. A posttranscriptional modification of the apoB mRNA by conversion of cytidine into uridine at nucleotide position 6666 changes the genomically encoded glutamine codon CAA at amino acid residue 2153 into a translational stop codon UAA. This mRNA editing explains the formation of the truncated isoform apoB-48. In the present investigation editing of apoB mRNA in liver and intestine from 12 different mammalian species was measured by a quantitative primer extension analysis of reverse-transcribed and polymerase chain reaction- (PCR) amplified apoB mRNA in order to determine whether i) editing of apoB mRNA is generally restricted to the intestine or may also be found in the liver of other species than rodents, and ii) hepatic expression of apoB mRNA editing influences lipoprotein concentrations in plasma. Intestinal apoB mRNA was edited at high levels in all species, 40% in sheep, 73% in horse, 82% in pig, 84% in dog, 84% in cat, 87% in guinea pig, 88% in rat, 89% in mouse, and > 90% in human, monkey, cow, and rabbit. In liver apoB mRNA was edited to 18% in dog, to 43% in horse, to 62% in rat, and to 70% in mouse. Low levels of editing below 1% were detected in liver of rabbit and guinea pig. In contrast, hepatic apoB mRNA from human, monkey, pig, cow, sheep, and cat liver was not edited. The results of the primer extension analysis were confirmed by cloning and sequencing of the PCR products from dog, horse, cat, guinea pig, sheep, and cow for all of which the apoB cDNA sequence had not been established by previous investigations. Primer extension analysis of apoB mRNA from dog intestine and dog liver indicated C/U editing at C6655 in addition to C6666. Cloning and sequencing of apoB cDNA from dog liver and intestine confirmed additional C/U editing at C6655 which changes ACA for threonine at amino acid residue 2149 into AUA for isoleucine. Synthesis and secretion of apoB-48-containing lipoproteins from liver was demonstrated by pulse labeling of freshly isolated horse hepatocytes and immunoprecipitation with apoB-specific antibodies or density gradient ultracentrifugation. The concentrations of VLDL, LDL, and HDL in all species were determined after fractionation by density gradient ultracentrifugation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1998

Systematic mutation screening of the pro-opiomelanocortin gene: identification of several genetic variants including three different insertions, one nonsense and two missense point mutations in probands of different weight extremes.

Pro-opiomelanocortin (POMC) is the precursor of melanocortins (adrenocorticotropin: ACTH, beta-endorphin, beta-lipotropin: beta-LPH, corticotropin like intermediate peptide, alpha-, beta- and gamma-melanocyte-stimulating hormone: alpha-, beta- and gamma-MSH) some of which act in the brain to reduce food intake and are potential mediators of leptin action. Recently, three different mutations in the POMC gene (POMC) were identified in two unrelated children that lead to early-onset extreme obesity, adrenal insufficiency, and red hair pigmentation. In the present study we systematically screened the coding region of POMC in 96 extremely obese children and adolescents, 60 healthy underweight individuals and 46 patients with anorexia nervosa (AN) and identified several variants. a) A 9 and an 18 base pair insertion (9bp and 18bp: AGC AGC GGC and AGC AGC GGC AGC AGC GGC, respectively, between codon 73 and 74; 1,2). These in-frame variants lead to the insertion of three or six amino acids (Ser-Ser-Gly; Ser-Ser-Gly-Ser-Ser-Gly) carboxy-terminal to gamma-MSH. Frequencies of the 9bp insertion allele varied between 3 and 5% among the different study groups (Pearson's chi2 P>0.5). b) Both an out-of-frame 6 bp insertion (within codon 176: GGG CCC) leading to the insertion of two amino acids (Arg-Ala) and a premature stop-codon (G-7316-T: Glu-180-Stop) within the gamma-LPH sequence were maternally inherited in an obese female proband. This proband inherited another missense mutation from her father (Glu-188-Gly). c) A missense mutation (G-7016-A; Asp-80-Asn) was observed in a single patient with AN who also harboured the 9bp insertion on a paternally derived haplotype. d) The allelic co-occurence of two silent mutations (C-6982-T and C-7285-T) was detected in two obese subjects. e) Two further silent mutations (C-3832-T; C-7111-G) were detected in an underweight and an obese subject, respectively. We conclude that the POMC gene harbors several different polymorphisms and mutations, none of which can readily be associated with the phenotypes under study.

Adolescent↗
PubMed1993

Overproduction of soluble trichodiene synthase from Fusarium sporotrichioides in Escherichia coli.

Trichodiene synthase is a sesquiterpene cyclase isolated from various fungal species which catalyzes the cyclization of farnesyl diphosphate (FPP) to trichodiene. The trichodiene synthase gene (Tox5) of Fusarium sporotrichioides has previously been cloned and expressed as 0.05-0.1% of total cell protein in Escherichia coli. We have used polymerase chain reaction to amplify the trichodiene coding sequence carried on the plasmid pTS56-1. The resulting DNA, carrying a BamHI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon as well as a HindIII site adjacent to the translational stop codon, was inserted into the corresponding sites of the expression vector pLM1. The latter vector carried the promoter and translational leader sequence from T7 gene 10 and the E. coli rmBT1T2 tandem transcription terminator. This construct was cloned into E. coli BL21 (DE3). The resulting transformants, when induced with isopropyl beta-D-thiogalactoside, produced trichodiene synthase as 20-30% of total soluble protein. The recombinant synthase, which could be purified five-fold to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and gel filtration on Superose 12, was identical to native protein in steady-state kinetic parameters and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the expected MENFP N-terminal sequence.

Base Sequence↗
PubMed2003

A novel mutation in exon 5 of the glucokinase gene in an Argentinian family with maturity onset diabetes of the young.

Maturity onset diabetes of the young (MODY) is caused by mutations in at least six different genes, including the glucokinase gene (MODY 2) and genes encoding the tissue-specific transcription factors (MODY 1 and MODY 3-6). To determine the presence of mutations in MODY 2 in four members of a family who have the clinical characteristics of MODY, we performed polymerase chain reaction and single strand conformation polymorphism screening, followed by DNA sequencing. We found a novel mutation which consisted of the deletion of a cytosine in the position 2 of the exon 5 codon 168. This mutation produced a frame shift which determines a stop codon at position 203 in exon 6. The identification of a mutation in glucokinase gene and transcription factor genes in patients with early-onset diabetes confirms the diagnosis of MODY and has important implications for clinical management.

Adult↗