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At least 307 records · Page 17Linked to original sources
PubMed1992

Familial apolipoprotein E deficiency and type III hyperlipoproteinemia due to a premature stop codon in the apolipoprotein E gene.

A kindred with apolipoprotein E deficiency and a truncated lower molecular weight apoE mutant, designated apoE-3Washington, has been identified. Gel electrophoresis demonstrated complete absence of the normal apoE isoproteins and the presence of a small quantity of a lower molecular weight apoE. Plasma apoE levels in the proband were approximately 4% of normal. This marked deficiency of apoE resulted in delayed uptake of chylomicron and very low density lipoprotein (VLDL) remnants by the liver, elevated plasma cholesterol levels, mild hypertriglyceridemia, and the development of type III hyperlipoproteinemia. Sequence analysis of the patient's apoE gene revealed a single nucleotide substitution of an A for a G, which converted amino acid 210 of the mature protein, tryptophan (TGG), to a premature chain termination codon (TAG), thus leading to the synthesis of a truncated E apolipoprotein of 209 amino acids with a molecular mass of 23.88 kDa. Northern blot analysis of differentiated monocyte-derived macrophages demonstrated a mutant mRNA indistinguishable in size from normal apoE mRNA. The nucleotide substitution also resulted in the formation of a new restriction site for Mae I. Using this enzyme we were able to establish that the proband is a homozygote and that her two offsprings are heterozygous for the epsilon-3Washington allele. These data demonstrate that the striking deficiency of apoE-3Washington results in a moderate form of type III hyperlipoproteinemia. The clinical presentation also suggests a dispensable role of apoE in the nervous system and in immunoregulation.

Apolipoproteins E↗
PubMed1994

Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa.

PURPOSE: To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein. METHODS: Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels. RESULTS: There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes. CONCLUSIONS: There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Adolescent↗
PubMed1998

Elimination of an HuIFN alpha 2b readthrough species, produced in Escherichia coli, by replacing its natural translational stop signal.

When human interferon-alpha 2b (HuIFN alpha 2b) was expressed intracellularly in Escherichia coli as insoluble aggregates, a HuIFN alpha 2b molecular species of high molecular weight was detected, even after immunoaffinity chromatography and characterized by mass spectrometry and automatic sequencing. This HuIFN alpha 2b species was synthesized by an inefficient reading of the UGA natural stop codon, stopping the translation at another UGA in frame placed 10 codons downstream of the HuIFN alpha 2b stop signal. To avoid this translational readthrough process the UGA termination codon was replaced by UAA, which is frequently used in highly expressed E. coli genes. Simultaneously, almost all the HuIFN alpha 2b gene 3' noncoding region was removed. Analysis by SDS-PAGE and enzyme-linked immunosorbent assay revealed the elimination of the undesired HuIFN alpha 2b molecular species and an almost twofold increase in the expression level. These results indicate that both factors, the stop codon used and the length of the transcription unit should be taken into account when the expression in E. coli of heterologous proteins is desired.

Base Sequence↗
PubMed1992

Prediction of gene structure.

We have developed a hierarchical rule base system for identifying genes in DNA sequences. Atomic sites (such as initiation codons, stop codons, acceptor sites and donor sites) are identified by a number of different methods and evaluated by a set of filters and rules chosen to maximize sensitivity; these are combined into higher-order gene elements (such as exons), evaluated, filtered and combined as equivalence classes into probable genes, which are evaluated and ranked. The system has been tested on an extensive collection of vertebrate genes smaller than 15,000 bases. Results obtained show that, on average, 88% of the predicted coding region for a transcription unit is actually coding, and 80% of the actual coding is correctly predicted. This will, in most applications, be sufficient for a search against protein sequence databases for the identification of probable gene function. In addition, the system provides a general test platform for both gene atomic site identification and the rules for their evaluation and assembly.

Algorithms↗
PubMed2002

Characterization of mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis.

We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma arginine deiminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370+/-50 microM. Its optimal temperature and pH were 41 degrees C and pH 6.4, respectively, and enzyme activity remained > or = 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the endotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

Animals↗
PubMed1996

Is the in-frame termination signal of the Escherichia coli release factor-2 frameshift site weakened by a particularly poor context?

The synthesis of release factor-2 (RF-2) in bacteria is regulated by a high efficiency +1 frameshifting event at an in-frame UGA stop codon. The stop codon does not specify the termination of synthesis efficiently because of several upstream stimulators for frameshifting. This study focusses on whether the particular context of the stop codon within the frameshift site of the Escherichia coli RF-2 mRNA contributes to the poor efficiency of termination. The context of UGA in this recoding site is rare at natural termination sites in E.coli genes. We have evaluated how the three nucleotides downstream from the stop codon (+4, +5 and +6 positions) in the native UGACUA sequence affect the competitiveness of the termination codon against the frameshifting event. Changing the C in the +4 position and, separately, the A in the +6 position significantly increase the termination signal strength at the frameshift site, whereas the nucleotide in the +5 position had little influence. The efficiency of particular termination signals as a function of the +4 or +6 nucleotides correlates with how often they occur at natural termination sites in E.coli; strong signals occur more frequently and weak signals are less common.

Base Sequence↗
PubMedDate not supplied

[Arrangement of the sense and terminating codons of the template in the A-segment of human ribosomes from photocrosslinking data withe oligonucleotide derivatives].

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3'-flanking sense codon or stop codon carrying a perfluoroarylazido group at G or U were used to study the position of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, UCC-recognizing tRNA(Phe) was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 or nucleotide A1825 in helix 44 close to the 3' end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem-loop segment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in various organisms.

Base Sequence↗
PubMed1999

Phenotyping of flavin-containing monooxygenase using caffeine metabolism and genotyping of FMO3 gene in a Korean population.

Flavin-containing monooxygenase (FMO) activity was determined in 82 Korean volunteers by taking molar concentration ratio of theobromine and caffeine present in the 1 h urine (between 4 and 5 h) samples collected after administration of a cup of coffee containing 110 mg of caffeine. Among 82 volunteers, there were 19 women and 63 men (30 smokers and 52 non-smokers). Volunteers were divided into two groups comprising low (0.53-2.99) and high (3.18-11.95) FMO activities separated by an antimode of 3.18. Peripheral bloods were sampled from these volunteers and their genomic DNAs were amplified by polymerase chain reaction with oligonucleotides designed from intronic sequences of human FMO3 gene. Comparing nucleotide sequences of the amplified FMO3 gene originating from randomly selected individuals with low and high FMO activities, nine point mutations were identified in the open reading frame sequences. Among these nine mutations, three FMO3 mutant types (FMO3/Stop148, Lys158 and Gly308) were selected and correlated with FMO activities observed in our Korean population. A rare FMO3/Stop148 mutant allele originating from FMO3/Gly148 occurred by substitution of G442T in exon 4 and yielded a premature TGA stop codon. The stop codon was detected in one individual having the second lowest FMO activity and he had the mutation in heterozygous state. In a pedigree study, he was found to have inherited the mutation from his mother who also had a heterozygous stop codon and equally low FMO activity. In our volunteers, two other common mutations were detected in exons 4 and 7. The one in exon 4 resulted from a G472A change eliminating a HinfI restriction site and produced an amino acid substitution from Glu158 to Lys. The other mutation in exon 7 resulted from an A923G change generating a DraII restriction site and produced a non-conservative replacement of Glu308 to Gly. Based on the secondary structure maps of FMO3 enzyme proteins for these two mutant types, FMO3/Gly308 mutation transformed the helix structure into a sheet shape and indicated that dysfunctional FMO3 may be produced. FMO3/Lys158 mutation did not alter the secondary structure. Approximately 80% of volunteers with homozygous and/or heterozygous mutations on either one or two of these mutations had low FMO activities. Thus, individuals with these FMO3 gene mutations may have defective metabolic activity for many clinically used drugs and dietary plant alkaloids which are oxidized primarily by hepatic FMO3.

Amino Acid Sequence↗
PubMed2005

Decoding the decoding region: analysis of eukaryotic release factor (eRF1) stop codon-binding residues.

Peptide synthesis in eukaryotes terminates when eukaryotic release factor 1 (eRF1) binds to an mRNA stop codon and occupies the ribosomal A site. Domain 1 of the eRF1 protein has been implicated in stop codon recognition in a number of experimental studies. In order to further pinpoint the residues of this protein involved in stop codon recognition, we sequenced and compared eRF1 genes from a variety of ciliated protozoan species. We then performed a series of computational analyses to evaluate the conservation, accessibility, and structural environment of each amino acid located in domain 1. With this new dataset and methodology, we were able to identify eight specific amino acid sites important for stop codon recognition and also to propose a set of cooperative paired substitutions that may underlie stop codon reassignment. Our results are more consistent with current experimental data than previously described models.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis).

A genomic clone encoding manganese-containing superoxide dismutase (SOD; EC 1.15.1.1) was isolated from a Hevea brasiliensis genomic library made in lambda phage EMBL3 by using a heterologous cDNA probe of MnSOD from Nicotiana plumbaginifolia. The nucleotide sequence of 4968 bp from the genomic clone was determined. Based on the putative translation initiation codon and stop codon, PCR primers were designed and utilized for cloning the full-length cDNA from total mRNA. Of the two distinct cDNAs of MnSOD isolated, MnSOD-A has a perfect match with exons of the nuclear gene, while MnSOD-B has a 90.2% homology and is 6 nucleotides longer than MnSOD-A in the putative transit peptide region. The nuclear gene comprises 6 exons and 5 introns, giving a total length of 3211 bp. The sequences of 1400 bp upstream of the initiation codon and 320 bp downstream of the stop codon were also determined. Southern analysis of genomic DNA from Hevea probed with a genomic fragment indicated there are at least two genes of MnSOD in Hevea. Northern blot analysis showed that MnSOD transcripts were present in all tissues examined (leaf, petiole, root, latex, callus) with young leaves showing the highest levels in intact plants. The transcript level in embryogenic callus was nearly 50-fold higher than in mature leaves. In addition, transcripts of MnSOD could be induced 3- to 5-fold in response to sucrose, ethephon and Murashige-Skoog salts.

Amino Acid Sequence↗
PubMed1996

The translational stop signal: codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, approximately 40,000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor 3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.

Base Sequence↗
PubMed2005

Empirical codon substitution matrix.

BACKGROUND: Codon substitution probabilities are used in many types of molecular evolution studies such as determining Ka/Ks ratios, creating ancestral DNA sequences or aligning coding DNA. Until the recent dramatic increase in genomic data enabled construction of empirical matrices, researchers relied on parameterized models of codon evolution. Here we present the first empirical codon substitution matrix entirely built from alignments of coding sequences from vertebrate DNA and thus provide an alternative to parameterized models of codon evolution. RESULTS: A set of 17,502 alignments of orthologous sequences from five vertebrate genomes yielded 8.3 million aligned codons from which the number of substitutions between codons were counted. From this data, both a probability matrix and a matrix of similarity scores were computed. They are 64 x 64 matrices describing the substitutions between all codons. Substitutions from sense codons to stop codons are not considered, resulting in block diagonal matrices consisting of 61 x 61 entries for the sense codons and 3 x 3 entries for the stop codons. CONCLUSION: The amount of genomic data currently available allowed for the construction of an empirical codon substitution matrix. However, more sequence data is still needed to construct matrices from different subsets of DNA, specific to kingdoms, evolutionary distance or different amount of synonymous change. Codon mutation matrices have advantages for alignments up to medium evolutionary distances and for usages that require DNA such as ancestral reconstruction of DNA sequences and the calculation of Ka/Ks ratios.

Amino Acid Substitution↗
PubMed1985

Pea cytochrome oxidase subunit II gene has no intron and generates two mRNA transcripts with different 5'-termini.

We have isolated and sequenced the cytochrome oxidase subunit II (COII) gene from pea mitochondria. The coding sequence (777 bp) shows over 90% homology to the COII genes from three monocotyledonous plants (rice, maize and wheat) and one dicotyledonous plant (Oenothera berteriana). Several codons are deleted, however, in the pea COII gene. Of interest is the deletion in pea of the last three codons, including the stop codon, found at the 3' end of the other four COII genes. Instead, a new stop codon has been created due to a single-base substitution at the 13th bp downstream from the position of the original stop codon. This pea gene does not contain an intron which is found in all three monocots. Two distinct 5' termini of the pea COII transcripts have been identified by S1 nuclease mapping, one at 285 bp (site I) and the other at 302 bp (site II) upstream from the ATG codon. They are located at two identical sites within nearly perfect direct repeats. Transcripts with the 5' end corresponding to site I occur five time more frequently than those with the 5' end corresponding to site II. Both transcripts have the same 3' terminus which has been mapped to be at 193 to 195 bp downstream from the stop codon.

Base Sequence↗
PubMed1997

Characterization of a mutant recombinant S100 protein using electrospray ionization mass spectrometry.

Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC). Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting. The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected. One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified. The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10. The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation. The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10. Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon. Another stop codon (TGA) located 27 base pairs downstream halts translation. The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass. Readthrough occurs in strains of E. coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.

Amino Acid Sequence↗
PubMed1998

[Low frequency of precore stop mutation in patients with dual infection of hepatitis B and C viruses].

In order to study the mutation of hepatitis B virus(HBV) precore gene in patients with dual infections of HBV and HCV and its possible clinical significance, the precore stop codon mutation (stop 28) was analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism(RFLP) in patients with chronic liver disease, which were 25 cases with HBV DNA and HCV RNA positive(group A) and 31 cases with HBsAg and HBV DNA positive but anti-HCV and HCV RNA negative (group B). The results showed that the serum HBV DNA positive rate by 1st PCR in patients with dual infections(group A, 16%) was significantly lower than that in patients with HBV infection alone (group B, 65%), the stop 28 positive rate was also significantly lower (28%, versus 68%). The results suggest that the low frequency of precore stop codon mutant in patients with HBV and HCV dual infections was possibly due to low replication of HBV.

Adolescent↗
PubMed1985

Primary structure of the succinyl-CoA synthetase of Escherichia coli.

The primary structure of the succinyl-CoA synthetase of Escherichia coli has been deduced from the nucleotide sequence of a 2451-base-pair segment of DNA containing the corresponding sucC (beta subunit) and sucD (alpha subunit) genes. The genes are located at one end of a gene cluster that encodes several citric acid cycle enzymes: gltA-sdhCDAB-sucABCD; gltA, citrate synthase; sdh, succinate dehydrogenase; sucA and sucB, the dehydrogenase (E1) and succinyltransferase (E2) components of the 2-oxoglutarate dehydrogenase complex. The sucC and sucD genes are separated from the sucA and sucB genes by a 273-base-pair segment containing four palindromic units, but they appear to be expressed from a sucABCD read-through transcript that extends from the suc promoter to a potential rho-independent terminator at the distal end of sucD. The stop codon of the sucC gene overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling of the sucC and sucD genes. The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon), and it encodes a polypeptide of Mr 41 390 corresponding to the beta subunit of succinyl-CoA synthetase. The sucD gene comprises 864 base pairs (288 codons, excluding the start and stop codons), and it encodes a product of Mr 29 644, corresponding to the alpha subunit of succinyl-CoA synthetase. The alpha subunit contains a 12-residue amino acid sequence that is identical with the histidine peptide previously isolated from the phosphoenzyme. This sequence forms part of one of the two potential nucleotide binding sites detected in the alpha subunit.

Amino Acid Sequence↗