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PubMed2000

Thogoto virus matrix protein is encoded by a spliced mRNA.

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a segmented, negative-stranded RNA genome. In this study, we investigated the coding strategy of RNA segment 6 and found that it contains 956 nucleotides and codes for the matrix (M) protein. The full-length cDNA contains a single, long reading frame that lacks a stop codon but has coding capacity for a putative 35-kDa protein. In contrast, the M protein of THOV has an apparent molecular mass of 29 kDa as assessed by polyacrylamide gel electrophoresis. Therefore, we investigated the possibility of posttranscriptional processing of segment 6 transcripts by reverse transcription-PCR and identified a spliced mRNA that contains a stop codon and is translated into the 29-kDa M protein. Interestingly, the nontemplated UGA stop codon is generated by the splicing event itself. Thus, the unusual M coding strategy of THOV resembles that of Influenza C virus.

Amino Acid Sequence↗
PubMed2000

Protein requirements for assembly of virus-like particles of Junonia coenia densovirus in insect cells.

The coding sequences of four overlapping polypeptides starting at four different in-frame AUG codons and co-terminating at the stop codon of the cap gene of Junonia coenia densovirus (JcDNV) were inserted under the control of the p10 promoter of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate AcMNPV-VP1 (four polypeptides), AcMNPV-VP2 (three polypeptides), AcMNPV-VP3 (two polypeptides), and AcMNPV-VP4 (one polypeptide) recombinant viruses. In all cases, infection of Spodoptera frugiperda cells (Sf9) by each of the four recombinant viruses resulted in the production of virus-like particles (VLPs) 22-25 nm in diameter. The VLPs produced by the three recombinants AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-VP4 were abundant and contained three, two and one polypeptides, respectively. VP4, the shortest polypeptide, thus appears to be sufficient for assembly of VLPs morphologically similar to those formed with two to four polypeptides. The ratio of VPs did not appear to be critical for assembly of the particles. The polypeptide starting at the first AUG immediately downstream from the p10 promoter was always the most abundantly expressed in infected cells, regardless of the construct. In contrast, plaque-purified AcMNPV-VP1 recombinants were unstable and produced less than one-twentieth of the VLPs produced by the others. All VP transcripts started at the TAAG late motif of the p10 promoter and had a poly(A) tail 14 nt downstream of a poly(A) addition signal located 98 nucleotides downstream of the common stop codon. No significant transcription initiation inside the cap sequence of AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-PV4 was observed.

3' Untranslated Regions↗
PubMed1998

Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination.

Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 shows a sharp drop, as expected, at 30 bp downstream of the stop codon, presumably due to hairpin-forming sequences. Similar drops are observed for Haemophilus influenzae Rd, Bacillus subtilis and Chlamydia trachomatis, suggesting that these organisms also form hairpins at their transcription termination sites. On the other hand, 12 other prokaryotes- Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis PCC6803, Helicobacter pylori, Borrelia burgdorferi, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Aquifex aeolicus, Pyrococcus horikoshii, Mycobacterium tuberculosis and Treponema pallidum -show no apparent decrease in free energy value at the corresponding regions. This result suggests that these prokaryotes, or at least some of them, may never form hairpins at their transcription termination sites.

Base Composition↗
PubMed2002

SsrA-mediated protein tagging in the presence of miscoding drugs and its physiological role in Escherichia coli.

BACKGROUND: We have shown recently that read-through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho-independent terminator leads to SsrA-mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read-through of stop codons and SsrA-mediated protein tagging. RESULTS: Miscoding antibiotics caused translational read-through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA-mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read-through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3' end of the mRNA defined by the terminator hairpin. The SsrA-defective cells were more sensitive to the miscoding antibiotics compared to the wild-type cells. CONCLUSION: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics.

Aminoglycosides↗
PubMed2002

Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region.

The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.

Amino Acid Sequence↗
PubMed2006

Biological activity of hamster interferon-gamma is modulated by the carboxyl-terminal tail.

The Syrian golden hamster (Mesocricetus auratus) is highly susceptible to a number of intracellular pathogens. Interferon-gamma (IFN-gamma), the primary macrophage-activating cytokine, plays a key role in the host defense against intracellular pathogens. The hamster IFN-gamma cDNA encodes a 174 amino acid protein that has an additional 17 amino acids at the carboxyl-terminus compared to IFN-gamma of mice and rats. A homologous C-terminal tail is also found in other non-murine rodents. The biological activity of hamster IFN-gamma had not been investigated previously so we first demonstrated the activity of native IFN-gamma in assays of IFN-gamma-induced receptor signaling and antiviral activity against vesicular stomatitis virus. We then tested the hypothesis that the C-terminal tail of hamster IFN-gamma could influence its biological activity. A truncated hamster IFN-gamma, in which the C-terminal 17 aa were removed by insertion of a stop codon at the position corresponding to the stop codon in the mouse sequence, had approximately 10-fold greater activity than the full length protein when measured in the two bioassays. Polyclonal and monoclonal anti-hamster IFN-gamma antibodies specifically inhibited this biological activity. Collectively, these data indicate that this unique structural feature influences the biological activity of hamster IFN-gamma.

Animals↗
PubMed2002

Evolutionarily different RNA motifs and RNA-protein complexes to achieve selenoprotein synthesis.

A wealth of RNAs or RNA motifs are instrumental in controlling a variety of post-transcriptional or post-translational regulations. In this regard, selenocysteine incorporation in response to a redefined UGA stop codon certainly constitutes an intriguing and fascinating process. Translation elongation factors specialized for selenocysteine are needed to decode UGA selenocysteine codons. Discrimination between UGA selenocysteine and UGA stop codons also necessitates selenoprotein mRNA hairpins, called SECIS, that are internal to the coding frame in eubacteria or contained in the 3' untranslated regions in archaea/eukaryotes. This dichotomy leads to SECIS RNAs with distinct sequences and structures that tether the specialized translation elongation factor in a direct or indirect fashion, depending on the location of the SECIS RNA. The scope of this review is to bring a sharper focus on the SECIS RNA structures and SECIS RNA-protein complexes involved. Obviously, the examples described here highlight once again the versatility in form and function of RNA.

Base Sequence↗
PubMed1995

An alternative splicing site modifies the carboxyl-terminal trans-membrane domains of the Na+/Ca2+ exchanger.

The 6-kilobase (kb) cDNA of pTB11 clone and its 5' fragment of 3.7 kb encoding the canine heart Na+/Ca2+ exchanger (Nicoll, D.A., Longoni, S., and Philipson, K.D. (1990) Science 250, 562-565) were transiently expressed in 293 cells to investigate the role of the 3'-"untranslated" region. Both fragments yielded high levels of expressed protein that were well incorporated in the membranes. Cells expressing the 6-kb cDNA produced rearranged transcripts of smaller than expected size. A 120-kDa polypeptide was produced in cells expressing the modified exchanger, and Ca2+ uptake was higher in this type of transfected cells. A constant stretch of nucleotides located at the 3' end of the 6 kb cDNA was found to be connected, by alternative RNA splicing, to four different upstream sequence positions. The deduced hydrophobic sequence of the spliced-in exon could replace the IX or the XI trans-membrane domain of the exchanger protein in two spliced isoforms. The new exon sequence was not completely included in the pTB11 insert, i.e. these two products were artificially truncated. The RNA processing of these two alternative 5'-splicing sites also occurred in tissues, as shown by RNase protection analysis. In a third type of isoform the splicing took place downstream of the originally proposed stop codon, whereas in a fourth type a stop codon was introduced after the V hydrophobic segment in the large intracellular loop.

Alternative Splicing↗
PubMed2000

DGGE analysis of the coproporphyrinogen oxidase gene: two new mutations in DNA from Danish patients with hereditary coproporphyria.

The knowledge of at least 21 different mutations and several polymorphisms in the coproporphyrinogen oxidase (CPO) gene demonstrates that the molecular basis of hereditary coproporphyria is heterogeneous. We developed a DGGE-based assay for the analysis of exons 2 to 7, including 14-96 nucleotides of the flanking intronic sequences of the CPO gene. To render it suitable for the clinical diagnostic laboratory, we designed the assay to allow use of identical PCR conditions and the same DGGE gel for analyses of all the regions. Using this assay, and subsequent sequencing of gene regions containing interallelic variations, two novel mutations in the CPO gene were identified: a missense mutation (607G-->A), leading to the substitution of an alanine with a threonine, and a nonsense mutation (1281G-->A), giving rise to a stop codon 28 codons upstream to the wild-type stop codon.

Amino Acid Sequence↗
PubMed1998

Functionally null mutations in patients with the cblG-variant form of methionine synthase deficiency.

Methionine synthase (MS) catalyses the methylation of homocysteine to methionine and requires the vitamin B12 derivative, methylcobalamin, as cofactor. We and others have recently cloned cDNAs for MS and described mutations associated with the cblG complementation group that correspond to MS deficiency. A subset of cblG, known as "cblG variant," shows no detectable MS activity and failure of [57Co]CN cobalamin to incorporate into MS in patient fibroblasts. We report the mutations responsible for three cblG-variant patients, two of them siblings, who presented with neonatal seizures, severe developmental delay, and elevated plasma homocysteine. Cell lines from all three patients were negative by northern blotting, though trace MS mRNA could be detected by means of phosphorimage analysis. Reverse transcriptase-PCR, SSCP, and nucleotide sequence analysis revealed four mutations. All were functionally null, creating either a frameshift with a downstream stop codon or an insert containing an internal stop codon. Of the two mutations found in the siblings, one of them, intervening sequence (IVS)-166A-->G, generates a cryptic donor splice site at position -166 of an intron beginning after Leu113, resulting in a 165-bp insertion of intronic sequence at junction 339/340. The second is a 2-bp deletion, 2112delTC. Mutations in the third patient include a G-->A substitution, well within the intron after Lys203, which results in intronic inserts of 128 or 78 bp in the mRNA. The second mutation is a 1-bp insertion, 3378insA. We conclude that the absence of MS protein in these cblG variants is due to mutations causing premature translation termination and consequent mRNA instability.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2003

Mutations in the COL4A4 gene in thin basement membrane disease.

BACKGROUND: Patients with thin basement membrane disease (TBMD) are often from families where hematuria segregates with the COL4A3 and COL4A4 genes. These genes also are affected in autosomal recessive Alport syndrome. The aim of this study was to demonstrate COL4A4 mutations in TBMD. METHODS: Forty-eight unrelated individuals with TBMD who had no family members with autosomal recessive Alport syndrome were examined for COL4A4 mutations. The diagnosis of TBMD had been confirmed by renal biopsy (43/48, 90%) or by a family history of hematuria but without a renal biopsy (5/48, 10%). The 47 coding exons of COL4A4 were screened for mutations with the methods of enzyme mismatch cleavage or single stranded conformational polymorphism (SSCP) analysis, and exons that demonstrated electrophoretic abnormalities were sequenced. RESULTS: Nine variants that altered the coding sequences were identified. These were nonsense and frameshift mutations that resulted in stop codons (N = 3), and glycine (N = 3) and non-glycine missense variants (N = 3). Four intronic variants and three neutral polymorphisms were also detected. In total, four variants were considered 'pathogenic' principally because they resulted in stop codons or were not present in non-hematuric normal subjects. Three variants were considered 'possibly pathogenic' but two of these were each present in one of 46 non-hematuric normal subjects. CONCLUSIONS: Pathogenic COL4A4 mutations were demonstrated in three of the nine (33%) families in whom hematuria segregated with the COL4A3/COL4A4 locus. Two stop codons (R1377X and 2788/91delG) and a glycine substitution (G960R) resulted in hematuria in all 16 members who were tested from these three families. The S969X mutation described here in TBMD for the first time, as well as the R1377X mutation, also occur in autosomal recessive Alport syndrome.

Adolescent↗
PubMed1996

Expression and characterization of a modified flavin-containing monooxygenase 4 from humans.

The inability to obtain flavin-containing monooxygenase 4 (FMO4) in heterologous systems has hampered efforts to characterize this isoform of the FMO gene family. Neither the human nor the rabbit ortholog of FMO4, each of which has been cloned and sequenced, has been expressed. Attempts to achieve expression of FMO4 have been made with Escherichia coli, baculovirus, yeast, and COS systems. The cDNAs encoding FMO4 have extended coding regions compared with those encoding other FMO isoforms. The derived amino acid sequences of FMO1, -2, -3, and -5 from all species examined contain about the same number of residues (531-535 residues), whereas the derived sequences of human and rabbit FMO4 contain 558 and 555 residues, respectively. We have investigated whether the elongation of the FMO4 coding region is related to the inability to achieve expression. The cDNA encoding human FMO4 has been modified by a single base change that introduces a stop codon at the consensus position. This modification allows for expression in E. coli. Lack of expression of intact FMO4 is caused by a problem that occurs following transcription, a problem that is overcome completely by relocation of the stop codon 81 bases to 5' of its normal position. Truncated FMO4 is expressed as an active enzyme with characteristics typical of an FMO isoform. Possible functional changes resulting from altering the 3'-end of an FMO were investigated with human FMO3. Elongation of the coding region of the FMO3 cDNA to the next available stop codon (FMO3*) resulted in the expression of an enzyme with properties very similar to those of unmodified FMO3. Elongation of FMO3 lowered the level of expression in E. coli but did not eliminate it. As with FMO4, the difference in expression levels between FMO3 and elongated FMO3 (FMO3*) appears to be related to translation rather than transcription. The functional characteristics of FMO3 and FMO3* are not significantly different.

Amino Acid Sequence↗
PubMedDate not supplied

Conservation of an open-reading frame as an element affecting 5' splice site selection.

Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5(') and 3(') splice sites. Evidently, the compliance of a given sequence with the consensus 5(') splice site sequence is not sufficient to define it as a functional 5(') splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5(') splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5(') splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5(') splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10490 latent 5(') splice sites. The utilization of 10045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein.

Algorithms↗
PubMed2001

Increased transcription levels induce higher mutation rates in a hypermutating cell line.

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.

Animals↗
PubMed1994

Mutations in helix 34 of Escherichia coli 16 S ribosomal RNA have multiple effects on ribosome function and synthesis.

Helix 34 of E. coli 16 S rRNA (1046 to 1067 and 1189 to 1211) has been proposed to participate directly in the termination of translation at UGA stop codons. We have constructed mutations in this helix in plasmid-encoded rDNA to explore the specific functional roles of the sequence UCAUCA (1199 to 1204) and a secondary structure also involving positions 1054 and 1057-1058. The rRNA mutations were analyzed for their effects on in vivo translational accuracy (stop codon readthrough and frameshifting) as well as growth rate, ribosome synthesis and incorporation into polysomes. Mutations at positions 1054, 1057, 1058, 1199 and 1200 had significant effects on translational accuracy, causing non-specific readthrough of all three stop codons as well as enhanced +1 and -1 frameshifting. Mutations at 1202 and 1203, however, had no effect. The incorporation of deleterious mutant subunits into 70 S ribosomes and polysomes was severely reduced and was associated with a slower growth rate and increased synthesis of host-encoded ribosomes. These data support the proposal that helix 34 is an essential component of the decoding center of the 30 S ribosomal subunit and is not restricted in function to UGA-codon specific termination.

Base Sequence↗
PubMed2005

Genetic interactions between [PSI+] and nonstop mRNA decay affect phenotypic variation.

Yeast strains can reversibly interconvert between [PSI+] and [psi-] states. The [PSI+] state is caused by a prion form of the translation termination factor eRF3. The [PSI+] state causes read-through at stop codons and can lead to phenotypic variation, although the molecular mechanisms causing those phenotypic changes remain unknown. We identify an interaction between [PSI+]-induced phenotypic variation and defects in nonstop mRNA decay. Nonstop mRNA decay is triggered when a ribosome reaches the 3' end of the transcript. In contrast, we observed little interaction between [PSI+]-induced phenotypic variation and defects in nonsense-mediated decay, which lead to suppression of premature stop codons. These results suggest that at least some of the phenotypic effects of [PSI+] may be due to read-through of "normal" stop codons, thereby producing extended proteins. Moreover, these observations suggest that nonstop mRNA decay may limit [PSI+]-induced phenotypic variation. Such a process would allow periodic sampling of the 3' UTR, which can diverge rapidly, for novel and beneficial protein extensions.

Blotting, Northern↗
PubMed1998

Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis.

Class III multidrug resistance (MDR) P-glycoproteins (P-gp), mdr2 in mice and MDR3 in man, mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte. Mice with a disrupted mdr2 gene completely lack biliary phospholipid excretion and develop progressive liver disease, characterized histologically by portal inflammation, proliferation of the bile duct epithelium, and fibrosis. This disease phenotype is very similar to a subtype of progressive familial intrahepatic cholestasis, hallmarked by a high serum gamma-glutamyltransferase (gamma-GT) activity. We report immunohistochemistry for MDR3 P-gp, reverse transcription-coupled PCR sequence analysis, and genomic DNA analysis of MDR3 from two progressive familial intrahepatic cholestasis patients with high serum gamma-GT. Canalicular staining for MDR3 P-gp was negative in liver tissue of both patients. Reverse transcription-coupled PCR sequencing of the first patient's sequence demonstrated a homozygous 7-bp deletion, starting at codon 132, which results in a frameshift and introduces a stop codon 29 codons downstream. The second patient is homozygous for a nonsense mutation in codon 957 (C --> T) that introduces a stop codon (TGA). Our results demonstrate that mutations in the human MDR3 gene lead to progressive familial intrahepatic cholestasis with high serum gamma-GT. The histopathological picture in these patients is very similar to that in the corresponding mdr2(-/-) mouse, in which mdr2 P-gp deficiency induces complete absence of phospholipid in bile.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1993

Domain organization of penicillin-binding protein 5 from Escherichia coli analysed by C-terminal truncation.

The structural organization of penicillin-binding protein (PBP) 5 was investigated by C-terminal truncation. Compared with other low-M(r) penicillin-interacting proteins, PBP5 carries a C-terminal extension of about 100 amino acids. The sites for introduction of stop codons were chosen on the basis of the established three-dimensional structure of the Streptomyces albus G beta-lactamase [Dideberg, Charlier, Wéry, Dehottay, Dusart, Erpicum, Frère and Ghuysen (1987) Biochem. J. 245, 911-913] and comparative hydrophobic cluster analysis [Gaboriaud, Bissery, Bencheritt and Mornon (1987) FEBS Lett. 224, 149-155]. Two stop codons were introduced at positions Ile-354 or Val-348 to construct an optimized soluble form of PBP5 for crystallization purposes. The newly constructed soluble and enzymically active form (PBP5s353) was isolated by dye-affinity chromatography and gave rise to small crystals. Another two stop codons were introduced at positions Arg-261 or Ala-276 to determine the minimal enzymically active 'core protein'. The truncated form (PBP5s275), missing the entire C-terminal extension, showed unaltered penicillin-binding characteristics and a catalytic-centre activity 40% that of PBP5s353 + 9 using bisacetyl-L-Lys-D-Ala-D-Ala as substrate. This protein, however was more susceptible to proteolytic degradation, which might indicate a role of the C-terminal portion in stabilizing the protein.

Amino Acid Sequence↗