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PubMed1995

Stickler syndrome. A mutation in the nonhelical 3' end of type II procollagen gene.

BACKGROUND: All of the mutations in the type II procollagen (COL2A1) gene that have been identified in families affected with Stickler syndrome have been located primarily in the triple helical region of the gene. We report what we believe is the first premature stop codon in the globular C-propeptide region encoded by the COL2A1 gene, in a family affected with Stickler syndrome. DESIGN: Genomic DNA from affected and unaffected family members of this three-generation family was amplified using the polymerase chain reaction. The polymerase chain reaction products were directly sequenced for DNA analysis. RESULTS: Direct sequencing showed a single base deletion in exon 50, resulting in a premature stop codon in exon 51 in the globular C-propeptide of COL2A1 gene in all affected members. CONCLUSIONS: These results implicate premature stop codons as a common cause of Stickler syndrome. The location of this premature stop codon in the far end of the nonhelical 3' end of the gene indicates that a truncated C-propeptide of at least 84 amino acid residues is inadequate for the functional gene product.

Adolescent↗

PubMed1997

The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo.

A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.

Base Sequence↗

PubMed1994

Four novel mutant alleles of the arylsulfatase B gene in two patients with intermediate form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome).

Mucopolysaccharidosis type VI (MPSVI, Maroteaux-Lamy syndrome) is a lysosomal storage disease for which multiple clinical phenotypes have been described. A deficiency of the enzyme arylsulfatase B (ASB, N-acetylgalactosamine-4-sulfatase) is the cause of this autosomal recessively inherited disorder. The genotypes of two patients with an intermediate form of MPSVI have been determined by polymerase chain reaction (PCR) amplification of the entire open reading frame of the ASB gene and subsequent direct sequencing of both strands of the PCR fragments by an automated nonradioactive approach. In patient A, a C to T transition in allele I resulting in an exchange of the Arg codon 160 for a premature stop codon (R160*, exon 2), and a G to A transition in allele II leading to a Gln to Arg160 substitution (R160Q, exon 2) were detected. Patient B exhibited a 7-bp deletion in exon 1 of allele I resulting in a frame shift and a premature stop codon 33 triplets 3' of the site of deletion (delta G237-C243), and a C to T transition in allele II giving rise to a Trp to Arg152 substitution (R152W, exon 2). None of these four mutant alleles was present among 60 alleles of the ASB gene in unrelated controls, indicating that the former are not polymorphisms. These results emphasize the broad molecular heterogeneity of Maroteaux-Lamy syndrome and contribute to the establishment of a genotype/phenotype correlation in this disease.

Alleles↗

PubMed2004

Translation repression by GLD-1 protects its mRNA targets from nonsense-mediated mRNA decay in C. elegans.

Previously, we identified multiple in vivo mRNA targets of the maxi-KH/STAR domain protein GLD-1 by their ability to interact with GLD-1 in cytoplasmic extracts and, for all targets tested thus far, GLD-1 functions as a translational repressor. However, here we show that GLD-1 stabilizes the mRNAs of two targets, gna-2 (T23G11.2) and Y75B12B.1. gna-2 mRNA has two upstream open reading frames (uORF), resulting in two premature stop codons. We found that gna-2 mRNA is a naturally occurring mRNA target of nonsense-mediated mRNA decay (NMD) and that the binding of GLD-1 protects gna-2 mRNA from NMD, likely by repressing translation of the uORFs. Therefore, gna-2 mRNA comes under two posttranscriptional controls: (1) translation regulation by a specific translational repressor, GLD-1; and (2) uORF elicited regulation, mainly through NMD. As a result, these two posttranscriptional controls together provide precise temporal and spatial control of gene expression. Consistent with this novel mode of regulation, when GLD-1 mRNA targets acquire premature stop codon mutations, GLD-1 protects them from NMD. Analysis of several mRNA targets containing premature stop codons suggests that in translation repression, GLD-1 either represses ribosome assembly on the target mRNA, or subsequent ribosome elongation to the premature stop codon.

3' Untranslated Regions↗

PubMed2006

TBG deficiency: description of two novel mutations associated with complete TBG deficiency and review of the literature.

Thyroxine-binding globulin (TBG) is the main thyroid hormone transport protein in serum. Inherited TBG defects lead to a complete (TBG-CD) or a partial (TBG-PD) deficiency and have a diagenic transmission, being clinically fully expressed only in hemizygous males and in homozygous females. In the present study, seven patients from two unrelated families with TBG-CD were studied and two novel TBG mutations were documented. In particular, a T insertion at the 5' donor splice site of exon 0, between nucleotides 2 and 3 at the beginning of intron 1 (g.IVS1+2_3insT) was found in one family and was named TBG-Milano. The other novel mutation is a T deletion at nucleotide 214 of exon 1, which leads to a frameshift at codon 50 with a premature stop codon at position 51 (c.214delT, P50fsX51) and was named TBG-Nikita. According to the X-linked transmission of the defect, females harboring the mutation showed a reduction in TBG levels with normal TSH and total thyroid hormone values at the lower limit of normal. Males harboring either TBG-Milano or TBG-Nikita, showed normal TSH values and low levels of total thyroid hormones and lacked TBG. In conclusion, we report two novel mutations of the TBG gene associated with a complete TBG defect. The first mutation lies at the 5' donor splice site of exon 0 and probably alters the start of translation, while the second is a single nucleotide deletion and leads to a premature stop codon.

Aged↗

PubMed1992

The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAs(Trp) with CmCA anticodon.

RNA-1 molecules from tobacco rattle virus (TRV) and pea early-browning virus (PEBV), two members of the tobravirus group, have recently been shown to contain internal, in-frame UGA termination codons which are suppressed in vitro. Our results suggest that a UGA stop codon also exists in RNA-1 of pepper ringspot virus (PRV), another tobravirus. UGA suppression may therefore be a universal feature of the expression of tobravirus genomes. We have isolated two natural suppressor tRNAs from uninfected tobacco plants on the basis of their ability to promote readthrough over the leaky UGA codon of TRV RNA-1 in a wheat germ extract depleted of endogenous mRNAs and tRNAs. Their amino acid acceptance and nucleotide sequences identify the two UGA-suppressor tRNAs as chloroplast (chl) and cytoplasmic (cyt) tryptophan-specific tRNAs with the anticodon CmCA. These are the first UGA suppressor tRNAs to be identified in plants. They have several interesting features. (i) Chl tRNA(Trp) suppresses the UGA stop codon more efficiently than cyt tRNA(Trp). (ii) Chl tRNA(Trp) contains an A24:U11 pair in the D-stem as does the mutated Escherichia coli UGA-suppressor tRNA(Trp) which is a more active suppressor than wild-type tRNA(Trp). (iii) The suppressor activity of chl tRNA(Trp) is dependent on the nucleotides surrounding the stop codon because it recognizes UGA in the TRV context but not the UGA in the beta-globin context.

Animals↗

PubMed2004

Nonsense-mediated and nonstop decay of ribosomal protein S19 mRNA in Diamond-Blackfan anemia.

Mutations in the ribosomal protein (RP)S19 gene have been found in about 25% of the cases of Diamond-Blackfan anemia (DBA), a rare congenital hypoplastic anemia that includes variable physical malformations. Various mutations have been identified in the RPS19 gene, but no investigations regarding the effect of these alterations on RPS19 mRNA levels have been performed. It is well established that mutated mRNA containing a premature stop codon (PTC) or lacking a stop codon can be rapidly degraded by specific mechanisms called nonsense mediated decay (NMD) and nonstop decay. To study the involvement of such mechanisms in DBA, we analyzed immortalized lymphoblastoid cells and primary fibroblasts from patients presenting different kinds of mutations in the RPS19 gene, generating allelic deletion, missense, nonsense, and nonstop messengers. We found that RPS19 mRNA levels are decreased in the cells with allelic deletion and, to a variable extent, also in all the cell lines with PTC or nonstop mutations. Further analysis showed that translation inhibition causes a stabilization of the mutated RPS19 mRNA. Our findings indicate that NMD and nonstop decay affect the expression of mutated RPS19 genes; this may help to clarify genotype-phenotype correlations in DBA.

Alleles↗

PubMed1999

Characterization of the gene encoding the apoprotein of phytochrome B2 in tomato, and identification of molecular lesions in two mutant alleles.

The structure of the gene encoding the apoprotein of tomato phytochrome B2 (PHYB2) has been determined from genomic and cDNA sequences. The coding region is organized into four exons, like almost every other angiosperm phytochrome (phy). The deduced phyB2 apoprotein (PHYB2) consists of 1121 amino acids, with 82, 74 and 70% identity to tomato PHYB1, Arabidopsis PHYB, and Arabidopsis PHYD, respectively. In order to facilitate the identification of new mutants, we constructed a double mutant that is deficient in phyA and phyB1. When grown in white light, this mutant becomes only slightly taller than wild type and is similar in phenotype to the monogenic phyB1-deficient mutant. This double mutant has been used as the parent line for mutagenesis with gamma radiation. Several recessive mutants with long hypocotyls and reduced anthocyanin content were selected under white light and screened for mutations in PHYB2, PHYE and PHYF. Two of the triple-mutant lines, designated 55H and 70F, had elongated hypocotyls and fruit trusses, and pale immature fruits. Both belong to the same complementation group and both were found to have defects in PHYB2. Line 70F was found by Northern analysis to have a slightly larger PHYB2 transcript. Part or all of the intron between the second and third exons was found to be retained following RT-PCR of PHYB2 mRNA from line 70F. Three base substitutions were detected near the donor splice site for this intron, including a change from the consensus /GT to /GA at the 5' end of this intron. In every case, the C-terminal 164 amino acids of PHYB2 were replaced by 59 nonsense amino acids followed by a stop codon. Sequencing of PHYB2 from 55H revealed a single-nucleotide deletion near the end of the third exon, resulting in one incorrect codon followed immediately by a stop codon. The predicted mutant apoprotein in 55H is 90 residues shorter than wild-type PHYB2.

Alleles↗

PubMed1990

The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor.

Transforming growth factor beta 1 (TGF-beta 1) is proteolytically derived from the carboxyl terminus of a 390 amino acid precursor molecule termed pre-pro-TGF-beta 1. Previous studies have suggested that the pro piece of pre-pro-TGF-beta 1 may play an important role in the formation of an inactive, latent complex. These latent forms are thought to be important in the regulation of TGF-beta 1 activity. To understand this latent complex in more detail, we have expressed the pro domain of pre-pro-TGF-beta 1 in tissue culture cells independent of the mature growth factor. A stop codon was genetically engineered into the cDNA of pre-pro-TGF-beta 1 by changing the Arg-278 codon from CGA to the STOP codon TGA. The resulting protein is truncated just prior to the amino-terminal Ala residue of the mature growth factor. Transient expression studies and immunoblotting indicate that this pro piece is readily made and secreted by the COS-1 cells; the major form of the expressed pro piece, when analyzed by SDS-polyacrylamide gel electrophoresis, behaves as a disulfide-linked dimer (Mr 80,000). Bioassays, using mink lung indicator cells, reveal that the pro domain forms an inactive complex with exogenously added mature TGF-beta 1. Treatment of this complex with heat or acid results in the release of active TGF-beta 1, indicating an in vitro structure similar to natural, latent TGF-beta 1 complexes. The pro piece from TGF-beta 1 was also found to form latent structures with two closely related family members, TGF-beta 1.2 and TGF-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

KRAB zinc finger proteins: an analysis of the molecular mechanisms governing their increase in numbers and complexity during evolution.

Krüppel-related zinc finger proteins, with 564 members in the human genome, probably constitute the largest individual family of transcription factors in mammals. Approximately 30% of these proteins carry a potent repressor domain called the Krüppel associated box (KRAB). Depending on the structure of the KRAB domain, these proteins have been further divided into three subfamilies (A + B, A + b, and A only). In addition, some KRAB zinc finger proteins contain another conserved motif called SCAN. To study their molecular evolution, an extensive comparative analysis of a large panel of KRAB zinc finger genes was performed. The results show that both the KRAB A + b and the KRAB A subfamilies have their origin in a single member or a few closely related members of the KRAB A + B family. The KRAB A + B family is also the most prevalent among the KRAB zinc finger genes. Furthermore, we show that internal duplications of individual zinc finger motifs or blocks of several zinc finger motifs have occurred quite frequently within this gene family. However, zinc finger motifs are also frequently lost from the open reading frame, either by functional inactivation by point mutations or by the introduction of a stop codon. The introduction of a stop codon causes the exclusion of part of the zinc finger region from the coding region and the formation of graveyards of degenerate zinc finger motifs in the 3'-untranslated region of these genes. Earlier reports have shown that duplications of zinc finger genes commonly occur throughout evolution. We show that there is a relatively low degree of sequence conservation of the zinc finger motifs after these duplications. In many cases this may cause altered binding specificities of the transcription factors encoded by these genes. The repetitive nature of the zinc finger region and the structural flexibility within the zinc finger motif make these proteins highly adaptable. These factors may have been of major importance for their massive expansion in both number and complexity during metazoan evolution.

Algorithms↗

PubMed2004

A case of Rett syndrome from Ukraine--clinical diagnosis confirmed by mutation analysis of the MECP2 gene.

Rett syndrome (RTT) is an X-linked disorder caused by mutations in the methyl-CpG-binding protein 2 gene (MECP2). The incidence is 1:10,000-1:15,000 females worldwide. To date, the mutational spectrum of MECP2 in the Ukrainian population is not known. Here we present first Ukrainian girl with classic clinical signs of RTT, in whom mutation of MECP2 gene was detected. Total genomic DNA was extracted from a dry blood spot using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. Genomic DNA was used to amplify coding sequence and exon/intron borders of MECP2 gene. Products were examined by restriction analysis and automatic direct sequencing. The sequencing analysis of our patient revealed a small deletion of 4 bases AAAG at position 856-859 in exon 4 of MECP2 gene (856-859del4). This mutation leads to a frameshift (K286fs) and a premature stop codon. The creation of premature stop codon results in synthesis of truncated MeCP2 protein. Localization of the mutation into the transcription repression domain (TRD) probably affects the function of MECP2 protein in the process of transcriptional repression. To our knowledge this is the first case from Ukraine, in whom clinical diagnosis of RTT was confirmed by mutation analysis of MECP2 gene. Mutation analyses of further patients are needed to establish the spectrum of MECP2 mutations in the Ukrainian population. (Tab. 1, Fig. 3, Ref. 22.)

Chromosomal Proteins, Non-Histone↗

PubMed2001

Genetic variability of respiratory syncytial virus subgroup B strain isolated during the last 20 years from the same region in Japan: existence of time-dependent linear genetic drifts.

The genetic variability of 32 respiratory syncytial virus (RSV) sub-group B isolates from a single community in Japan during the 20 years from 1980 to 1999 was determined. Two variable regions of the attachment (G) protein gene were amplified by reverse transcriptase-polymerase chain reaction amplification and their products were sequenced directly. Phylogenetic analysis of nucleotide sequences revealed seven distinct branches in which strains isolated during seasons of close proximity were located: however, isolates from the same season were often in plural branches. There was a tendency for recent isolates to lie at the end of each branch and these linear evolutionary changes were typically represented in a branch containing nine strains isolated during 6 seasons from '80 to '86. Three kinds of usages of stop codons were confirmed and isolates located in each branch used the same stop codons. These observations suggest that there are multiple subgroup B lineages co-circulating and that each lineage strain may exhibit linear evolutionary genetic drifts in order to survive over successive epidemics within the same population, although it has a conserved uniform G protein length with the use of the same stop codon.

Amino Acid Sequence↗

PubMed1989

Characterization of the human parathyroid hormone-like peptide gene. Functional and evolutionary aspects.

The single-copy gene coding for the human parathyroid hormone-like peptide was isolated from a human placental genomic library. The gene spans 13 kilobases and contains seven exons. Exons I and II encode 5'-noncoding regions; each has its own transcription initiation site, and the two promoters are separated by over 1000 base pairs of genomic DNA. Exon III encodes the prepro-coding region, and exon IV encodes the mature peptide sequence. At the end of exon IV the splice site interrupts codon 139 of the mature peptide. Exon V, which is contiguous with exon IV, encodes a stop codon and a 3'-noncoding region. Exon VI encodes 34 additional amino acids, a stop codon, and a second 3'-noncoding region. Exon VII encodes two extra amino acids, a stop codon, and a third 3'-noncoding region. This genomic organization reveals how the multiple human parathyroid hormone-like peptide RNA transcripts, which have been observed, arise by both alternative splicing out of exons and use of multiple promoters. The mRNAs, which can potentially be formed from the primary transcript of this gene, could have one of three different carboxyl-terminal coding regions. The use of different exons to encode the different functional domains, 5'-noncoding region, pre-pro-coding region, and mature peptide region is identical to the organization of the human parathyroid hormone gene. This strongly suggests a common evolutionary origin of the two genes.

Amino Acid Sequence↗

PubMed2005

Invariant amino acids essential for decoding function of polypeptide release factor eRF1.

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125-131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.

Amino Acid Sequence↗

PubMed2003

A cryo-electron microscopic study of ribosome-bound termination factor RF2.

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.

Binding Sites↗

PubMed2005

Cloning and sequence analysis of putative glyceraldehyde-3-phosphate dehydrogenase gene from Monascus purpureus KCCM11832.

Using a synthetic oligonucleotide probe, glyceraldehyde-3-phosphate dehydrogenase gene (gpd1) was cloned from Monascus purpureus KCCM11832. The 2834 bp EcoRV-HindIII region harbored 1183 bp 5'-UTR containing such regulatory elements as CT box, common in fungal gpd's, and gpd box previously found exclusively in Aspergillus gpd's. Full-length cDNA was cloned by PCR, and its sequence was determined. Transcription starting point was located 88 bp upstream from start codon. Polyadenylation signal sequence occurred 201 bp downstream from stop codon. Region from start codon ATG to stop codon TAA including introns showed 62 approximately 69% nucleotide sequence identity to those of Aspergillus gpd's. Significant bias in third position, with pyrimidines favored over purines, was observed in codon usage. The deduced amino acid sequence had 81 approximately 85% identity to Aspergillus gpd's. Monascus purpureus GPD was located at the same clade with Aspergillus GPD's.

Amino Acid Sequence↗

PubMed1995

Clinical relevance of mutations in the precore genome of the hepatitis B virus.

A stop codon in the precore genome of the hepatitis B virus (HBV) in anti-HBe positive HBV carriers may be associated with a more progressive form of HBV infection. Earlier studies, however, were mainly performed in patients from the Mediterranean area who had severe infection. The aim of this study was to evaluate the prevalence of precore mutants in an unselected population living in northern Europe. Twenty of 42 of these patients are infected predominantly with a virus strain, which has the typical stop codon in the precore genome, characterised by a mutation at base 83. In six patients there was an additional G to A mutation at base 86 of the precore genome. Statistical analysis showed no difference between the patients with or without a stop codon in the precore genome. When patients with a double mutation at base 83 and 86 of the precore genome were compared with the other anti-HBe positive HBV carriers, however, the corresponding clinical data were worse. Therefore we suggest, that it is not the stop codon in the precore gene itself, but the occurrence of a double mutation at bases 83 and 86, which is associated with a more severe course of disease in anti-HBe positive HBV carriers.

Adult↗

PubMed2004

Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus.

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

Animals↗