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PubMed1994

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.

Base Sequence↗

PubMed2004

Alternative splicing induced by nonsense mutations in the immunoglobulin mu VDJ exon is independent of truncation of the open reading frame.

In addition to triggering nonsense-mediated mRNA decay (NMD), premature translation-termination codons (PTCs) frequently induce alternative splicing, an observation referred to as nonsense-associated alternative splicing (NAS). In many cases, NAS is induced because the nonsense mutation alters a splicing signal, such as inactivating an exonic splicing enhancer. However, for a few genes, NAS was reported to be PTC specific, implying that a translation signal could influence splicing. Here, we investigated whether production of a previously undetected alternatively spliced transcript from immunoglobulin mu (Ig-mu) depends on premature termination of the open reading frame. We show that PTCs at different positions in the VDJ exon of an Ig-mu minigene activate usage of an alternative 3' splice site, generating an alternative transcript that lacks the initial PTC and a previously identified NMD-promoting element (NPE), but contains new PTCs because of a frame shift. Corroborating the importance of the NPE for maximal NMD response, the alternative transcript is only moderately down-regulated by NMD. We further demonstrate that NAS of Ig-mu minigene transcripts is not PTC specific. This finding, together with our results that contradict the previously reported frame dependence of TCR-beta NAS, challenges the idea that cells might possess mechanisms that would allow regulation of splice site selection in response to premature termination of the ORF.

Alternative Splicing↗

PubMed2001

Nonsense-mediated decay of mutant waxy mRNA in rice.

Two rice (Oryza sativa) waxy mutations of the Japonica background were shown to contain approximately 20% of the fully spliced mRNA relative to the wild type. Sequencing analysis of the entire waxy genes of the two mutants revealed the presence of premature translation termination codons in exon 2 and exon 7. These results indicated that the lower accumulation of fully spliced RNA in the mutants was caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. It is interesting that levels of RNA retaining intron 1 were not changed by premature nonsense codons, suggesting that splicing may be linked with NMD in plants, as previously found in mammalian cells. Measurements of the half-lives of waxy RNAs in transfected rice protoplasts indicated that the half-life of waxy RNA with a premature nonsense codon was 3.3 times shorter than that without a premature nonsense codon. Because the wild-type waxy transcripts, which are derived from the Wx(b) gene predominantly distributed among Japonica rice, have been shown to be less efficiently spliced and their alternative splicing has been documented, we examined whether these splicing properties influenced the efficiency of NMD. However, no effects were observed. These results established that NMD occurs in rice waxy RNA containing a premature nonsense codon.

Alternative Splicing↗

PubMed2001

The mtl genes and the mannitol-1-phosphate dehydrogenase from Klebsiella pneumoniae KAY2026.

The mtl operon of Klebsiella pneumoniae KAY2026 (formerly Aerobacter aerogenes 1033-5P14) was shown to contain as the promoter-proximal gene mtlA, encoding a D-mannitol-specific enzyme II transporter (IICBA(Mtl)). This gene is followed by mtlD, coding for a mannitol-1-phosphate dehydrogenase (MtlD, 382 amino acid residues), and mtlR (MtlR, 195 amino acid residues) coding for a putative repressor, gene mtlR overlaps the termination codon of mtlD. The DNA and protein sequences are highly similar to the corresponding genes (81% identical bp) and proteins (79-85% identical amino acids) of Escherichia coli K-12. A truncated form of MtlD lacking the 162 C-terminal amino acid residues still shows 10% dehydrogenase activity which may explain the controversy in the literature concerning the properties of mannitol-phosphate and other medium-length dehydrogenases.

Amino Acid Sequence↗

PubMed1987

Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content.

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.

Amino Acid Sequence↗

PubMed2000

Novel transcripts of fibroblast growth factor receptor 3 reveal aberrant splicing and activation of cryptic splice sequences in colorectal cancer.

A nested reverse transcription-PCR analysis of FGFR3 from human colorectal carcinomas revealed novel mutant transcripts caused by aberrant splicing and activation of cryptic splice sequences. Two aberrantly spliced transcripts were detected with high frequency in 50% of 36 primary tumors and in 60% of 10 human colorectal cancer cell lines. Most transcripts used normal splice sites but skipped or included exons 8 and 9. Two mutant transcripts arose from cryptic splice donor sites in exon 7 that spliced to exon 10. The predicted translation products would exhibit frameshifts and a premature termination codon in exon 10. We propose that dysregulation of mRNA splicing frequently generates an aberrant FGFR3 transcript that may confer a selectable advantage on clones of cells in colorectal tumorigenesis.

Alternative Splicing↗

PubMed1994

Complex environment of nascent polypeptide chains.

Nascent polypeptides enter into high molecular weight complexes with other proteins during chain elongation in vitro and in vivo. The nature of these complexes was investigated using an in vitro translation system programmed with a single mRNA lacking a translational termination codon. Complexes containing nascent polypeptides (molecular mass < 20 kDa), the molecular chaperone hsp 73 and other unidentified proteins can be released from the translationally arrested polysomes by puromycin treatment. The apparent native molecular mass of the nascent chain binding complex was determined to be > 700 kDa by gel-filtration analysis. Complexes between the nascent polypeptide and at least hsp 73 appear to be sensitive to (disrupted by) ATP. The presence of ATP also dramatically alters the sensitivity of the nascent polypeptide chains to digestion by exogenous protease. Collectively, our data indicate that there may be a cytoplasmic machinery, including hsp 70, which comprises a nascent polypeptide chain binding complex.

Adenosine Triphosphate↗

PubMed1995

A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro.

Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-beta pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-beta transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-beta exons triggered a decrease in mRNA levels. Ptc-bearing TCR-beta transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the down-regulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-beta and beta-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.

Animals↗

PubMed1990

Molecular characterization of a new variant of hepatitis B virus in a persistently infected homosexual man.

Based on the diversity of nucleotide sequences of cloned hepatitis B virus DNA genomes, we have predicted possible replication of genetic variants of human hepatitis B virus. This prediction is exemplified by studies of a chronic carrier of HBsAg/adw2, who lacked anti-HBc but carried exceedingly high levels of hepatitis B virus DNA in serum. Molecular characterization of a number of clones revealed a restriction map that deviated significantly from the typical pattern of the adw2 subtype, especially around the EcoRI site commonly used as a reference point. Mutations appearing consistently in the precore and core regions included (a) mutation in the precore region resulting in a termination codon after the initiation codon, (b) mutation of the core initiation codon and (c) an inframe insert of 36 nucleotides in the precore region with a new initiation site for the core protein. The 36-nucleotide insertion resulted in a new core protein with 12 extra amino acids at its amino-terminal end. A few scattered point mutations were clustered in the amino-terminal half of the core gene. Although the core protein of this hepatitis B virus variant carried immunologically detectable HBcAg, the absence of a humoral immune response to HBcAg could have been caused by previous infection with human immunodeficiency virus. This naturally occurring human hepatitis B virus variant replicated efficiently without expressing the precore region, confirming previous observations made of the artificial mutants of duck hepatitis B virus.

Adult↗

PubMed2005

Novel PEX1 coding mutations and 5' UTR regulatory polymorphisms.

Zellweger syndrome and its milder variants--neonatal adrenoleukodystrophy and infantile Refsum disease--comprise a clinical continuum of diseases referred to as the Zellweger spectrum. Mutations in the PEX1 gene, which consists of 24 exons and encodes a AAA ATPase protein required for peroxisomal protein import, account for approximately two-thirds of the known Zellweger spectrum patient mutations. In this paper, we report on four novel PEX1 mutations and two polymorphisms in an Australasian cohort. Two of the mutations--c.1108_1109insA and c.2391_2392delTC--that lead to the introduction of a premature termination codon in exons 5 and 14, respectively, are associated with the severe Zellweger phenotype. One patient with a milder disease phenotype was a compound heterozygote for two missense mutations (I989T and R998Q), both affecting amino acids in the second, C-terminal AAA domain of the protein. PTS1 protein import levels in cultured skin fibroblasts from this patient were almost 20% of normal control levels. We have also characterized two co-segregating polymorphisms in the 5' UTR of the PEX1 gene. Based on reporter assays, the c.-137T>C polymorphism leads to reduced PEX1 expression, whereas the c.-53C>G polymorphism leads to increased expression. When present together, these regulatory polymorphisms lead to near-normal PEX1 expression. Altered PEX1 expression due to the presence of either the c.-137T>C or the c.-53C>G variant could impact on residual PEX1 function if another co-allelic mutation was present which did not completely abolish PEX1 function. It also follows that the presence of polymorphisms in the PEX1 promoter region could have implications for patients with mutations in other PEX proteins known to interact with PEX1, such as PEX6. Thus, although not deleterious in control individuals, these polymorphisms could contribute to phenotypic heterogeneity among Zellweger spectrum patients.

5' Untranslated Regions↗

PubMed2000

A novel two base pair deletion in the factor V gene associated with severe factor V deficiency.

We studied a family in which the proband, a 13-year-old boy, had unmeasurable plasma levels of coagulation factor V antigen and activity. Clinical symptoms were severe, with several episodes of haemorrhages in the mucosal tracts (gastrointestinal, nose and urinary) and recurrent haemarthroses that caused permanent arthropathy. Sequence analysis of the factor V gene demonstrated the presence of a novel 2 base pair (bp) homozygous deletion in exon 13 at positions 2833-2834. This mutation, present in the heterozygous state in the asymptomatic mother and absent in the healthy brother, introduced a frameshift and a premature stop at codon 900. This would predict the synthesis of a truncated factor V molecule, lacking part of the B domain and the complete light chain. Because of the existence of a surveillance mechanism that selectively recognizes and degrades mRNA molecules carrying premature termination codons, we analysed the relative abundance of mutant vs. wild-type mRNA molecules in the platelets of the heterozygous proband's mother. The mutant mRNA was significantly reduced in amount (mutant/wild-type ratio 0.35). This is the first reported mutation in the factor V gene causing severe factor V deficiency, the effect of which was quantitatively analysed at mRNA level.

Adolescent↗

PubMed1991

Cloning and characterization of human placental catechol-O-methyltransferase cDNA.

Catechol-O-methyltransferase (COMT) cDNA clones were isolated from a human placental cDNA library using synthetic oligonucleotides as probes. All four positive clones isolated contained an open reading frame, which potentially coded for a 24.4-kD polypeptide, presumably corresponding to the cytoplasmic form of the COMT (S-COMT). In addition to the S-COMT sequences, two of the clones carried extensions in the 5' end, which potentially coded for a 50-amino-acid peptide extending the S-COMT reading frame. This sequence contained a stretch of signal sequence-like hydrophobic amino acids in its amino terminus. The deduced human COMT polypeptide had 80% similarity with the previously characterized rat COMT. Expression of one of the cDNA clones in human K-562 cells resulted in cell clones with 3- to 10-fold increased COMT activity. Cell-free translation of transcripts synthesized in vitro from one of the short cDNAs yielded a 26-kD product, similar in size to human S-COMT. Translation of transcripts from one of the long cDNAs gave 30-kD and 26-kD polypeptides, suggesting translation initiation from two different AUG initiation codons. The 30-kD protein, but not the 25-kD protein, associated with microsomal membranes in translation lysates. A potential polyadenylation signal AATTAA was detected in the 3' ends of two of the clones 265 nucleotides downstream from the COMT translation termination codon. RNA blotting on human placental RNA revealed a 1.5-kb-long COMT-specific transcript. DNA analysis suggested that human, as well as rat, canine and monkey cells have one gene for COMT.

Amino Acid Sequence↗

PubMed2003

Genetic diversity: frameshift mechanisms alter coding of a gene (Epstein-Barr virus LF3 gene) that contains multiple 102-base-pair direct sequence repeats.

Frameshift mutations provide recognized mechanisms for changing the coding potential of an organism. Here, multiple frameshifts are identified in repetitive sequences within an Epstein-Barr virus unspliced early gene, LF3, which is associated with the viral replicative cycle and also transcriptionally expressed in many virally associated tumors. On the DNA strand encoding LF3, there are three open reading frames, only one of which contains an initiation codon. Most (>95%) of the gene consists of numerous (>20, varying with cell source) GC-rich copies of a 102-bp direct repeat (called IR 4) flanked by small unique sequences. LF3 may express a protein if its initiation and termination codons reside in the same reading frame, but this is not always the case. Frameshifting events, occurring in short runs of pyrimidines (mainly C residues) in the repeats, give rise to mutations which may provide a mechanism for escape of an LF3 function from host surveillance. Sequence studies link these frameshifts to DNA replication errors. Notably, the number of sites in LF3 at which such mutations can occur permits a very large amount of diversity in this gene. Our data also suggest a second degeneracy mechanism within the protein itself, which influences its stability and may reflect a host defense mechanism. LF3 thus provides a potentially important model for studying the quest for supremacy between a virus and its host.

Amino Acid Sequence↗

PubMed1999

Mechanisms of mRNA surveillance in eukaryotes.

A conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs. A defining aspect of aberrant transcripts is that the spatial relationship between the termination codon and specific downstream sequence information has been altered. A key, yet unknown, feature of the mRNA surveillance system is how this spatial relationship is assessed in individual transcripts. Two views have emerged to describe how discrimination between proper and improper termination might occur. In the first view, a surveillance complex assembles onto the mRNA after translation termination, and scans the mRNA in a 3' to 5' direction for a limited distance. If specific downstream sequence information is encountered during this scanning, then the surveillance complex targets the transcript for rapid decay. An alternate view suggests that the downstream sequence information influences how translation termination occurs. This view encompasses several ideas including: (a) The architecture of the mRNP can alter the rate of key steps in translation termination; (b) the discrimination between a proper and improper termination occurs via an internal, Upf1-dependent, timing mechanism; and (c) proper termination results in the restructuring of the mRNP to a form that promotes mRNA stability. This proposed model for mRNA surveillance is similar to other systems of kinetic proofreading that monitor the accuracy of other biogenic processes such as translation and spliceosome assembly.

Animals↗

PubMed1991

Characterization of an operon encoding succinyl-CoA synthetase and malate dehydrogenase from Thermus flavus AT-62 and its expression in Escherichia coli.

An open reading frame (ORF) was found upstream of the mdh gene in Thermus flavus by computer-aided analysis. It was identified as the gene encoding the alpha subunit of succinyl-CoA synthetase (SCS) and termed scsA. Nucleotide sequencing of a further upstream region revealed the presence of another ORF, corresponding to the sequence of the beta subunit of SCS. The latter gene was termed scsB. The scsB gene was found to contain an unusual translational initiation codon, TTG. S1 nuclease mapping indicates that transcription starts at the nucleotide at position--31 upstream of the initiation codon of the beta gene. The scsB and scsA genes along with the mdh gene appear to form an operon and are most likely co-transcribed in this order, because the intercistronic regions between them are very short; in fact, the termination codon of scsB overlaps the initiation codon of scsA. A stretch characteristic of the--10 region of a typical prokaryotic promoter was found upstream of scsB, whereas no sequence characteristic of a typical--35 region was present. Escherichia coli harboring a plasmid containing scsA and scsB did not produce thermostable SCS activity, even when a synthetic promoter for E. coli was attached. However, when an inverted repeat present in front of scsB, which covers the putative ribosome-binding site and is capable of forming a stable stem-loop structure, was altered by site-directed mutagenesis, overproduction of heat-stable SCS was observed.

Amino Acid Sequence↗

PubMed1981

Diversity of germ-line immunoglobulin VH genes.

The sequences of four embryonic mouse immunoglobulin VH genes have been compared. All genes end at codon 98 and code for a hydrophobic signal peptide of 19 residues interrupted at codon -4 by an intron of 83 base pairs. Substitutions occur in all gene segments but at a significantly higher frequency in the hypervariable regions. The data suggest an evolutionary basis for the diversity of immunoglobulin genes. Divergence resulted also in a termination codon in two of the genes, suggesting that part of the V gene repertoire cannot be expressed unless some correction mechanism is available.

Animals↗

PubMed1992

Molecular cloning and partial characterization of a novel collagen chain, alpha 1(XVI), consisting of repetitive collagenous domains and cysteine-containing non-collagenous segments.

In an effort to identify new members of the collagen family, we screened a human placenta cDNA library with a collagenous probe. A novel 3.7 kb cDNA was identified encoding an open reading frame of 1,186 amino acids and containing a termination codon. The predicted polypeptide consists of 9 repetitive collagenous (stretches of Gly-X-Y) and several non-collagenous segments. Two cysteinyl residues separated by two amino acid residues (Cys-X-X-Cys) are regularly located in the N-terminal region of each non-collagenous segment. The deduced amino acid sequence described above is distinct from those of known types of collagen. Therefore, this novel collagen chain is designated alpha 1(XVI). Northern blot analysis revealed an alpha 1(XVI) mRNA of 5.2 kb, indicating that the overlapping cDNA clones isolated in this study covered nearly three-fourths of the mRNA. As a tool for further study on the expression of type XVI collagen, we prepared an antibody against the nonadecapeptide CFLSLERPRAEEARGDNSE, derived from the putative translation product of the cDNA. In immunoblot analysis, the antibody recognized a 160 kDa protein, which was bacterial collagenase-sensitive. Immunohistochemical stainings of human placental tissues with anti-peptide antibody revealed a positive reaction with amnion, the membranous tissue lining the amniotic cavity. The gene of alpha 1(XVI) chain, COL16A1, is mapped on the short arm of human chromosome 1 (1p13-p34).

Amino Acid Sequence↗

PubMed1995

Human cytomegalovirus UL102 gene.

We have identified and characterized the transcript for the human cytomegalovirus (HCMV) UL102 gene. The UL102 gene product is proposed to encode the primase-associated factor. The primase-associated factor is one of the three components of the helicase-primase complex, along with UL105 (helicase) and UL70 (primase). In order to characterize the UL102 transcription unit we used single-stranded antisense RNA probes to identify an abundant 2.7-kb transcript originating from the UL102 region. This transcript can be initially detected at 24 h postinfection and in the presence of phosphonoformic acid but not in the presence of cycloheximide. A 2.7-kb cDNA clone containing this transcript was isolated from a 72-h HCMV (strain Towne) cDNA library. Sequence analysis of this clone revealed a continuous unspliced transcript between the region of UL101X and UL102; the only in-frame translational stop codon is 2,619 bp downstream from the first ATG in the message. Genome sequencing of the UL102 region from strains AD169 and Towne revealed that the UL101X stop codon TAA was actually TAC and that the cDNA and genomic sequences were in agreement. The cDNA clone starts 5 nucleotides (nt) upstream of the UL101X ATG, continues through the putative ATG of UL102, and ends 97 nt downstream of the putative termination codon of the UL102 open reading frame. Primer extension analysis indicated a transcriptional start site 23 nt upstream of the UL101X open reading frame.

Amino Acid Sequence↗