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PubMed2000

Mutations in KRIT1 in familial cerebral cavernous malformations.

OBJECTIVES: The recognition of six unrelated Hispanic-American families in which cerebral cavernous malformations (CCM) segregated as an autosomal dominant trait established a genetic basis for this disease. Linkage analysis subsequently identified locus heterogeneity with disease genes for CCM at chromosomal regions 7q, 7p, and 3q. Recently, mutations in KRIT1, a gene on 7q at the CCM1 locus, were identified in French and Hispanic-American families with CCM. This study confirms the identity the KRIT1 founder mutation in Hispanic-Americans and reports a novel KRIT1 mutation in a Caucasian family. METHODS: Oligonucleotide primers were designed to allow amplification of genomic DNA sequences from four Hispanic-American families and five non-Hispanic families for all 12 exons of the KRIT1 gene using the polymerase chain reaction (PCR). The amplified DNA was then screened using single strand conformation polymorphism analysis (SSCP) and sequencing. The expression pattern of KRIT1 was analyzed by Northern blotting. RESULTS: Analysis of the KRIT1 gene revealed a point mutation in exon 6 that predicts the substitution of a premature termination codon for glutamine at codon 248 in all four Hispanic-American families, confirming previous findings. SSCP analysis and sequencing revealed an 11 base pair duplication in exon 7 leading to a premature termination codon in one Caucasian family. Northern analysis demonstrated widespread expression of this gene, however, the highest level of expression was in the brain. CONCLUSION: The common KRIT1 mutation causing the majority of CCM in Hispanic-Americans has been identified and independently confirmed, allowing efficient presymptomatic molecular diagnosis. In keeping with prior results, both newly identified mutations create a premature termination codon and are predicted to initiate degradation of the mutant mRNA through the nonsense-mediated mRNA decay pathway. These data strongly suggest loss of function as the relevant patho-genetic mechanism.

Brain Neoplasms↗

PubMed2000

Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains.

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

Amino Acid Sequence↗

PubMed1994

Structure of the mouse arylsulfatase A gene and cDNA.

The murine arylsulfatase A (ARSA) gene and cDNA have been cloned and sequenced. The gene is 3.8 kb long and contains eight exons. All intron/exon splice junctions conform to the GT/AG consensus sequence. The genomic structure is similar to that of the human gene. One major RNA species of 3.2 kb is transcribed. This RNA species has a 5' untranslated region of 638 nucleotides and terminates in a region around nucleotide 700 downstream of the termination codon. In addition, a rare mRNA species terminating at a polyadenylation signal 135 nucleotides downstream of the termination codon has been found. A larger transcript of 4 kb can be detected in liver. The size difference is due to initiation of transcription 5' of the cap site of the 3.2-kb mRNA species. The entire ARSA cDNA has been cloned by PCR from reverse-transcribed RNA. The coding sequence has 1518 nucleotides and predicts a protein of 506 amino acids. The nucleotide as well as the amino acid sequence is highly conserved among humans and mice.

Amino Acid Sequence↗

PubMed2001

Emerging views on tmRNA-mediated protein tagging and ribosome rescue.

Transfer-messenger RNA (tmRNA), also known as SsrA or 10Sa RNA, is a bacterial ribonucleic acid that recycles 70S ribosomes stalled on problematic messenger RNAs (mRNAs) and also contributes to the degradation of incompletely synthesized peptides. tmRNA acts initially as transfer RNA (tRNA), being aminoacylated at its 3'-end by alanyl-tRNA synthetase, to add alanine to the stalled polypeptide chain. Resumption of translation ensues not on the mRNA on which the ribosomes were stalled but at an internal position in tmRNA. Termination soon occurs, tmRNA recruiting the appropriate termination factors allowing the release of the tagged protein that is subsequently recognized and degraded by specific cytoplasmic and periplasmic proteases, and permits ribosome recycling. Recent data suggest that tmRNA tags bacterial proteins in three other instances; when ribosomes stall at internal sites; during 'readthrough' of canonical termination codons; and when ribosomes are at the termination codon of intact messages. The importance of bacterial tmRNAs for survival, growth under stress, and pathogenesis is also discussed. Recent in vivo and in vitro studies have identified novel ligands of tmRNA. Based on the available experimental evidences, an updated model of tmRNA mediated protein tagging and ribosome rescue in bacteria is presented.

Bacterial Physiological Phenomena↗

PubMed1987

Nucleotide sequence analysis of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids.

RepFIC is a basic replicon of IncFI plasmid P307 which is located within a 3.09-kilobase SmaI fragment. The nucleotide sequence of this region has been determined and shown to be homologous with the RepFIIA replicon of IncFII plasmids. The two replicons share three homologous regions, HRI, HRII, and HRIII, which are flanked by two nonhomologous regions, NHRI and NHRII. A comparison of coding regions reveals that the two replicons have several features in common. RepFIC, like RepFIIA, codes for a repA2 protein with its amino-terminal codons in HRI and its carboxy-terminal codons in NHRI. Although the codons for the repA1 proteins are located in NHRII, the DNA region containing a putative promoter, ribosomal binding site, and initiation codons is located in HRII. This region also codes for an inc RNA. There are nine base-pair differences between the inc RNA of RepFIIA and that of RepFIC, and as a result, RepFIC and RepFIIA replicons are compatible. An EcoRI fragment from the F plasmid which shows homology with RepFIC of P307 has also been sequenced. This fragment contains only a portion of RepFIC, including the genes for the putative repA2 protein and inc RNA. The region coding for a putative repA1 protein is interrupted by the transposon Tn1000 and shows no homology with the repA1 region of RepFIIA and RepFIC of P307. Our comparative and structural analyses suggest that RepFIC and RepFIIA, although different, have a similar replication mechanism and thus can be assigned to the same replicon family, which we designate the RepFIIA family.

Base Sequence↗

PubMed1999

Epstein-barr virus nuclear antigen 1 sequences in endemic and sporadic Burkitt's lymphoma reflect virus strains prevalent in different geographic areas.

The Epstein-Barr virus (EBV) nuclear antigen EBNA1 is the only viral protein detectably expressed in virus genome-positive Burkitt's lymphoma (BL); recent work has suggested that viral strains with particular EBNA1 sequence changes are preferentially associated with this tumor and that, within a patient, the tumor-associated variant may have arisen de novo as a rare mutant of the dominant preexisting EBV strain (K. Bhatia, A. Raj, M. J. Gutierrez, J. G. Judde, G. Spangler, H. Venkatesh, and I. T. Magrath, Oncogene 13:177-181, 1996). In the present work we first study 12 BL patients and show that the virus strain in the tumor is identical in EBNA1 sequence and that it is matched at several other polymorphic loci to the dominant strain rescued in vitro from the patient's normal circulating B cells. We then analyze BL-associated virus strains from three different geographic areas (East Africa, Europe, and New Guinea) alongside virus isolates from geographically matched control donors by using sequence changes in two separate regions of the EBNA1 gene (N-terminal codons 1 to 60 and C-terminal codons 460 to 510) to identify the EBNA1 subtype of each virus. Different geographic areas displayed different spectra of EBNA1 subtypes, with only limited overlap between them; even type 2 virus strains, which tended to be more homogeneous than their type 1 counterparts, showed geographic differences at the EBNA1 locus. Most importantly, within any one area the EBNA1 subtypes associated with BL were also found to be prevalent in the general population. We therefore find no evidence that Burkitt lymphomagenesis involves a selection for EBV strains with particular EBNA1 sequence changes.

Africa, Eastern↗

PubMed1994

Alternatively spliced transcripts of the human complement C2 gene.

To investigate the possible presence of alternatively spliced C2 gene transcripts, we amplified mRNA from HepG2 cells by reverse transcription-PCR using primers derived from the 5' and 3' untranslated regions of the C2 mRNA. Cloning of the resulting products revealed the presence of four novel C2 mRNA size variants. Nucleotide sequencing indicated that the variant mRNAs were probably derived through differential splicing of C2 gene transcripts. Specifically, nucleotide sequence deletions in the four variant mRNAs could be attributed to splicing out of: 1) exons 2 and 3; 2) exon 3; 3) exon 17; and 4) exons 6 and 7 and the 5' region of exon 18. The results were confirmed by RNase protection assays using HepG2 mRNA. Inspection of the nucleotide and the deduced amino acid sequences indicated that in the first two variants the alternative splicing did not affect the C2 open reading frame. In the other two variants, frameshifts in exon 18 resulted in termination codons up- or downstream of the authentic termination codon. All four variants C2 mRNAs were capable of encoding truncated C2 proteins, and were detected by reverse transcription-PCR not only in HepG2 cells but also in human liver, U937, and U105-MG cells. The latter analyses indicated the presence of an additional C2 mRNA variant lacking the region encoded by exon 6.

Alternative Splicing↗

PubMed1996

Developmental genetic analysis of troponin T mutations in striated and nonstriated muscle cells of Caenorhabditis elegans.

We have been investigating a set of genes, collectively called mups, that are essential to striated body wall muscle cell positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat-sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(up1), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail. The mup-2 mutations lie within these cDNA sequences: mup-2(up1) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intracellular Ca2+. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles.

Alleles↗

PubMed1985

Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2.

Escherichia coli peptide chain release factors are proteins that direct the termination of translation in response to specific peptide chain termination codons. The mechanisms of codon recognition and peptidyl-tRNA hydrolysis are unknown. We have characterized the genes encoding release factor 1 (RF-1) and release factor 2 (RF-2) to study the structure-function relationships of the proteins and their regulation in the bacterium. In this report, we present the gene structure of RF-1 and RF-2, and a partial peptide sequence of RF-2. RF-1 and RF-2 are highly homologous in their primary structure. In addition, an in-frame premature opal (UGA) termination codon is located within the RF-2 coding region at amino acid position 26. This region of the protein was sequenced by automated Edman degradation to confirm the predicted reading frame, and a second independent isolate of the RF-2 gene was identified and sequenced to confirm the DNA sequence. These results imply that a frameshift occurs prior to the premature termination codon, thus allowing for translation of RF-2 to be completed. This may represent a mechanism of translational control of RF-2 expression. An alternative possible means of translational regulation is discussed.

Amino Acid Sequence↗

PubMed1990

Heterogeneity of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein encoded by the hepatitis delta virus (HDV). Two HDAg species of different sizes have been detected in the sera and livers of the infected humans, chimpanzees, and woodchucks, even though only one RNA species was previously identified in most of the HDV strains. To study HDAg heterogeneity, we took advantage of the fact that a single base mutation at nucleotide 1015 (C to U), which results in an amber termination codon in the HDAg open reading frame (ORF), eliminates a unique Ncol restriction enzyme site. We screened various HDV cDNA clones and detected sequence heterogeneity of the HDAg-coding region on the basis of the presence or absence of the Ncol site. Five delta hepatitis patients were examined. In every patient, two types of HDAg-coding sequence were detected at nucleotide 1015: one which contains a C and results in an ORF encoding a delta antigen of 214 amino acids, and the other which possesses a U and results in an amber termination codon and a truncated HDAg species of 195 amino acids. The in vitro translation products of these two ORFs comigrated with the two HDAg species from the patient's plasma on SDS polyacrylamide gels. Polymerase chain reaction (PCR) amplification of the HDV RNA from some patients' sera and subsequent sequencing showed several additional mutations in the HDAg-coding region. These mutations are independent of the C or U nucleotide change at the site of the amber termination codon.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Viral↗

PubMed1999

gamma-Glutamyl transferase (GGT) deficiency in the GGTenu1 mouse results from a single point mutation that leads to a stop codon in the first coding exon of GGT mRNA.

GGTenul, a recently described genetic murine model of gamma-glutamyl transferase (GGT) deficiency, was induced by the point mutagen N-ethyl-N-nitrosourea and is inherited as an autosomal recessive trait. The phenotype of systemic GGT deficiency suggested a mutation site within the cDNA coding region which is common in all GGT transcripts. To identify this site, total lung and kidney RNA was isolated from normal and mutant mice, amplified by RT-PCR using GGT-specific primers, cloned as two overlapping approximately 1 kb GGT cDNA fragments, sequenced and compared with that in the literature. A single base pair substitution was identified in the coding region at position 237, where thymidine became adenine, and this mutation replaced a leucine codon, TTG, with a termination codon, TAG. This mutation site was confirmed in mutant genomic DNA by PCR using primers that flanked the predicted site and spanned the intron between the common GGT non-coding exon and the first GGT coding exon. This PCR product was sequenced directly with the secondary 3' PCR primer, the mutation site identified and the protocol then utilized to genotype animals. In addition to this mutation, the steady-state level of GGT mRNA in mutant kidney is reduced 3-fold compared with the control. Heterodimeric GGT protein is not detectable by western blot in either whole kidney homogenate or a microsomal membrane fraction. The steady-state mRNA level of gamma-glutatmyl cysteinyl synthetase was unchanged in mutant mice compared with normal, but that of heme oxygenase-1 and Cu,Zn-SOD was induced 4- and 3-fold, respectively. Hence, the GGTenul mouse model of GGT deficiency results from a single point mutation in the first coding exon of GGT mRNA and the resulting impairment in glutathione turnover induces oxidative stress in the kidney.

Animals↗

PubMed1992

Readthrough suppression in the mammalian type C retroviruses and what it has taught us.

Mammalian type C retroviruses use translational suppression to synthesize the enzymes which function in virus replication. The UAG termination codon at the end of the coding region for the viral core proteins is translated as glutamine at a frequency of approximately 5%, allowing synthesis of the enzymes as part of a large fusion protein. This unusual mechanism has several benefits for the virus: first, it modulates the relative levels of synthesis of the core proteins and the enzymes. This is essential for the proper assembly of the virus particle, since the fusion protein alone is apparently unable to assemble into particles. Second, the presence of the core protein moiety in the fusion protein probably provides a mechanism for targeting the enzymes to the virus particle. The mechanism of the suppression phenomenon is now under investigation. Recent studies have revealed that suppression in the viral context is dependent upon a complex cis-acting signal in the viral mRNA, including a pseudoknot beginning 9 nucleotides 3' of the termination codon. In addition, studies with viral mutants have shown that UAA and UGA, like UAG, are efficiently suppressed in the presence of this signal, and have identified the amino acids used in the suppression of these termination codons in reticulocyte lysates. In several cases, this analysis revealed the existence of previously unknown suppressor tRNAs. One important question which has not been answered is whether the suppression mechanism used by the virus has a parallel in the synthesis of host proteins.

Base Sequence↗

PubMed1993

Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA.

Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA. The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured. With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition. The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence. The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage. The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state. Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal.

Base Sequence↗

PubMed1971

Mammalian peptide chain termination. II. Codon specificity and GTPase activity of release factor.

In vitro peptide chain termination with release factor preparations from rabbit reticulocytes, guinea pig liver, or Chinese hamster liver is directed with UAAA, UAGA, or UGAA, suggesting that UAA, UAG, and UGA are terminator condons for mammalian cells. Purified release factor from rabbit reticulocytes has ribosomaldependent GTPase activity, which is stimulated by UAAA. GTP hydrolysis appears requisite for in vitro peptide chain termination in mammals.

Animals↗

PubMed1993

The translational termination signal database.

The Translational Termination Database (TransTerm) consists of the immediate context sequences around the natural termination codons from 45 organisms, and summary tables. The influence of termination codon context on their effectivness as stop signals has been widely documented. The SPECIES--TRI.DAT table shows trinucleotide stop codon usage in each organism and for comparison the occurrence of these sequences in the noncoding region. The SPECIES--TETRA.DAT table contains is a similar table of tetranucleotide stop signal usage. The database is available from EMBL.

Animals↗

PubMed1986

High rate of somatic point mutation in vitro in and near the variable-region segment of an immunoglobulin heavy chain gene.

The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment.

Base Composition↗

PubMed1998

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Alleles↗

PubMed1993

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.

Arginine↗