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PubMed1989

Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior.

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.

Base Sequence↗

PubMed2006

A myocilin Gln368STOP homozygote does not exhibit a more severe glaucoma phenotype than heterozygous cases.

PURPOSE: To describe the phenotype of an individual homozygous for the common Gln368STOP myocilin mutation and to discuss the other family members. DESIGN: Cascade screening was performed for Australian families that had been identified as having the myocilin Gln368STOP mutation. METHODS: Recruited subjects underwent comprehensive clinical examination and mutation analysis for the Gln368STOP myocilin mutation by direct sequencing. RESULTS: One 49-year-old woman was found to be homozygous for the mutation. Her maximal recorded intraocular pressure was 17 mm Hg. Bilateral optic disk examination revealed small, healthy optic discs. Automated perimetry testing was normal. CONCLUSIONS: Neither the individual homozygous for the Gln368STOP myocilin mutation nor her younger heterozygous siblings displayed any signs suggestive of glaucoma. One of the two heterozygous parents did manifest glaucoma. Although there is the possibility of the homozygous individual developing glaucoma in the future, she does not manifest a phenotype that is more severe than usual.

Aged↗

PubMed2004

A tRNA(TRP) gene mediates the suppression of cbs2-223 previously attributed to ABC1/COQ8.

The Saccharomyces cerevisiae gene ABC1 was originally isolated as a multicopy suppressor of a yeast strain harboring a mutation in a cytochrome b translational activator (cbs2-223). Based on this identification, Abc1p was postulated to activate the bc1 complex and function as a chaperone of cytochrome b. ABC1 was subsequently identified as COQ8 and found to be necessary for yeast coenzyme Q synthesis. In this work we show that a segment of yeast genomic DNA containing ABC1/COQ8 and neighboring genes suppresses the respiratory and Q-deficient phenotypes of the coq6 mutant, coq6-1. COQ6 is essential for yeast coenzyme Q biosynthesis. We show that a tRNA(TRP) gene located downstream of ABC1/COQ8 mediates suppression of the cbs2-223 and coq6-1 mutations, and each is identified here as containing UGA nonsense codons. The inability of ABC1/COQ8 to suppress the cbs2-223 allele in multicopy indicates it may not be a chaperone as previously reported.

Gene Expression Regulation, Fungal↗

PubMed2006

The decapping enzyme Dcp1 participates in translation termination through its interaction with the release factor eRF3 in budding yeast.

One of the rate-limiting steps in messenger RNA decay pathway is the 5'-cap cleavage of mRNAs, decapping reaction, which is conducted by the protein complex of Dcp1 and Dcp2. We find here that Dcp1p can interact with the release factor eRF3p (Sup35p) in Saccharomyces cerevisiae. Knockout of DCP1 caused not only the accumulation of nonsense mRNAs possibly due to the impaired decapping activity but also the enhancement of the read-through of nonsense codon. To examine the relationship between the two DCP1-knockout phenotypes, we produced DCP1 point mutants that lack the ability to support the translation termination. Interestingly, decapping activity of Dcp1p was still intact, but its interaction with eRF3p was abolished in the DCP1 mutants, indicating that the two functions originated from different entities of Dcp1p. These results suggest that the decapping enzyme Dcp1p may have an additional role in the translation termination through its interaction with eRF3p.

Codon, Terminator↗

PubMed2006

Natural human isolates of Staphylococcus aureus selected for high production of proteases and alpha-hemolysin are sigmaB deficient.

It has been reported that high production of proteases and alpha-hemolysin in the prototype Staphylococcus aureus strain 8325-4 was associated with its sigmaB deficiency. Here we analyzed one fresh clinical isolate (KS26) and two ancient human isolates (Wood46 and V8) selected for high production of proteases and alpha-hemolysin. All three strains lacked yellow pigment and showed a low level of expression of sigB-dependent promoters, indicating sigmaB deficiency. Nucleotide sequencing of the sigB operon revealed that KS26 and Wood46 had stop codons in rsbU and sigB, respectively, while V8 had an insertion of an IS element in rsbU. Complementation experiments with sigB on a plasmid reduced expression of proteases and alpha-hemolysin dramatically, indicating that the high production of these exoproteins was associated with sigmaB deficiency. Although sigmaB-deficient strains show attenuated virulence in some animal models, our results indicate that such strains can cause infection in humans.

Bacterial Proteins↗

PubMed2006

Compensatory evolution reveals functional interactions between ribosomal proteins S12, L14 and L19.

Certain mutations in S12, a ribosomal protein involved in translation elongation rate and translation accuracy, confer resistance to the aminoglycoside streptomycin. Previously we showed in Salmonella typhimurium that the fitness cost, i.e. reduced growth rate, due to the amino acid substitution K42N in S12 could be compensated by at least 35 different mutations located in the ribosomal proteins S4, S5 and L19. Here, we have characterized in vivo the fitness, translation speed and translation accuracy of four different L19 mutants. When separated from the resistance mutation located in S12, the three different compensatory amino acid substitutions in L19 at position 40 (Q40H, Q40L and Q40R) caused a decrease in fitness while the G104A change had no effect on bacterial growth. The rate of protein synthesis was unaffected or increased by the mutations at position 40 and the level of read-through of a UGA nonsense codon was increased in vivo, indicating a loss of translational accuracy. The mutations in L19 increased sensitivity to aminoglycosides active at the A-site, further indicating a perturbation of the decoding step. These phenotypes are similar to those of the classical S4 and S5 ram (ribosomal ambiguity) mutants. By evolving low-fitness L19 mutants by serial passage, we showed that the fitness cost conferred by the L19 mutations could be compensated by additional mutations in the ribosomal protein L19 itself, in S12 and in L14, a protein located close to L19. Our results reveal a novel functional role for the 50 S ribosomal protein L19 during protein synthesis, supporting published structural data suggesting that the interaction of L14 and L19 with 16 S rRNA could influence function of the 30 S subunit. Moreover, our study demonstrates how compensatory fitness-evolution can be used to discover new molecular functions of ribosomal proteins.

Amino Acid Substitution↗

PubMed2006

Stop-codon read-through for patients affected by a lysosomal storage disorder.

Lysosomal storage disorders are a group of inherited diseases that can result in severe and progressive pathology due to a specific lysosomal dysfunction. Current treatment strategies include bone-marrow transplantation, substrate reduction, chemical-chaperone and enzyme-replacement therapy. However, each of these treatments has its limitations. Enhanced stop-codon read-through is a potential alternative or adjunct therapeutic strategy for treating lysosomal-storage-disorder patients. Premature stop-codon mutations have been identified in a large cohort of patients with a lysosomal storage disorder, making stop-codon read-through a possible treatment for this disease. In lysosomal-storage-disorder cells (mucopolysaccharidosis type I, alpha-L-iduronidase deficient), preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage.

Codon, Nonsense↗

PubMed2005

The importance of mutation, then and now: studies with yeast cytochrome c.

The development of a genetic system based on the CYC1 gene was initiated over 40 years ago, primarily because of the anticipated ease of sequencing of the corresponding encoded protein, iso-1-cytochrome c from Saccharomyces cerevisiae. The success of the iso-cytochrome c system was dependent on the early development of methods for detecting and selecting cyc1 defective mutants and CYC1 functional revertants, and of methods for fine-structure genetic mapping using deletions and single-site mutations. The nonsense codons TAA and TAG, and the initiation codon ATG, were determined from the amino acid alterations of iso-1-cytochromes c from intragenic revertants; this represented the first assignments of such codons in a eukaryotic organism. The types of desired sequences were expanded by selecting recombinants from cyc1 x cyc1 nonfunctional mutants or CYC1 x CYC1 functional mutants, permitting the early determination of the rules of translation, which differed from those of prokaryotes by use of the most 5' AUG codon for initiation of translation. The sequence of 44 base pairs of CYC1 was determined with altered iso-1-cytochromes c from revertants of frameshift and initiation mutants, allowing the early cloning of the gene. A method was developed for transforming yeast directly with synthetic oligonucleotides, resulting in the convenient production of CYC1 mutants with defined sequences. At this point in time, Sherman and colleagues have published approximately 240 papers on or using the iso-cytochrome c system, dealing with such diverse topics as translation, informational suppressors, transcription and transcription termination, recombination, ectopic recombination, mutagen specificity, regulation by Ty1 elements, evolution of duplicated chromosomal segments, structure-function relationships of cytochrome c, protein stability and degradation, biosynthesis and mitochondrial import of cytochrome c, mitochondrial proteases, co- and post-translational modifications, and mRNA degradation. Current work on degradation of proteins in mitochondria, on degradation of mRNA in the nucleus, and on N-terminal acetylation stems from properties of CYC1 mutants isolated in early screens more than a decade ago.

Amino Acid Sequence↗

PubMed1983

Analysis of two purified mutants of Escherichia coli aspartate transcarbamylase with single amino acid substitutions.

Two mutant versions of Escherichia coli aspartate transcarbamylase have been purified and analyzed kinetically. Each of these mutant enzymes contains a single amino acid different from the wild-type enzyme, which was introduced by suppression of a nonsense codon within the E. coli pyrB gene. These enzymes exhibited alterations in both homotropic and heterotropic interactions with little change in specific activity. Depending upon the site of the substitution, the allosteric interactions have been either enhanced or diminished over the wild-type enzyme. Carbamyl phosphate saturation curves indicate that aspartate and carbamyl phosphate homotropic co-operativity are separable. Experiments employing the allosteric effectors indicate that the transmission of the regulatory effect is dependent upon the structure of the catalytic subunit, and that CTP inhibition can be partially decoupled from ATP activation. The kinetics of one of the mutants is unusually sensitive to dissociation at elevated temperatures. This sensitivity may be due to weakened interactions between the subunits of the enzyme.

Adenosine Triphosphate↗

PubMed2000

The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames.

The nonsense-mediated mRNA decay (NMD) pathway functions to degrade transcripts containing nonsense codons. Transcripts containing mutations that insert an upstream open reading frame (uORF) in the 5'-UTR are degraded through NMD. However, several naturally occurring uORF-containing transcripts are resistant to NMD. Here we demonstrate that the GCN4 and YAP1 mRNAs, which contain uORFs, harbor a stabilizer element (STE) that prevents rapid NMD by interacting with the RNA binding protein Pub1. Conversely, a uORF-containing mRNA that lacks an STE, such as CPA1, is degraded by the NMD pathway. These results indicate that uORFs can play a pivotal role regulating both translation and turnover and that the Pub1p is a critical factor that modulates the stability of uORF-containing transcripts.

5' Untranslated Regions↗

PubMed1998

Molecular genetics of C1 inhibitor.

More than 100 different C1 inhibitor gene mutations have been described in hereditary angioedema (HAE) patients. Sixty-nine mutations have been reported in patients with the quantitative C1 inhibitor defect (type 1 HAE) in two recent large-scale studies. These changes were found distributed over all exons and exon/intron boundaries. The molecular defects can be divided as follows: Alu-repeat-mediated deletions or duplications (accounting for 21% of all cases), missense mutations (> 36%), frameshifts (14%), Stop codon mutations (10%), promoter variants (4%), splice site mutations (7-10%), deletions of a few amino acids (less than 3%). Several recent studies indicate that up to 25% of these changes are found in patients without a family history of angioedema and represent de novo mutations. Pathogenic amino acid substitutions were found distributed over the entire length of the coding sequence, except for the 100 amino-acid-long glycosylated amino-terminal extension, whose sequence tolerates extensive variation, as indicated by comparisons across species. Functional studies have been carried out only on a fraction of these amino acid substitutions and indicate that defects affecting intracellular transport are often at the basis of type 1 hereditary angioedema. An interesting promoter variant (a C to T transition at position -103) was found in an exceptional family with recessive transmission of the disease. Regulatory elements in the promoter region and in intron 1 were revealed by their sequence conservation in mouse and man and by functional studies. C1 inhibitor "minigene" constructs directing correct mRNA and protein synthesis in transgenic mice have provided valuable information on hormonal control and cell-type specificity of gene expression.

Amino Acid Sequence↗

PubMed2000

Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination.

Early studies provided evidence that peptide-chain release factors (RFs) bind to both ribosomal subunits and trigger translation termination. Although many ribosomal proteins have been implicated in termination, very few data present direct biochemical evidence for the involvement of rRNA. Particularly absent is direct evidence for a role of a large subunit rRNA in RF binding. Previously we demonstrated in vitro that mutations in Escherichia coli rRNAs, known to cause nonsense codon readthrough in vivo, reduce the efficiency of RF2-driven catalysis of peptidyl-tRNA hydrolysis. This reduction was consistent with the idea that in vivo defective termination at the mutant ribosomes contributes to the readthrough. Nevertheless, other explanations were also possible, because still missing was essential biochemical evidence for that idea, namely, decrease in productive association of RFs with the mutant ribosomes. Here we present such evidence using a new realistic in vitro termination assay. This study directly supports in vivo involvement in termination of conserved rRNA regions that also participate in other translational events. Furthermore, this study provides the first strong evidence for involvement of large subunit rRNA in RF binding, indicating that the same rRNA region interacts with factors that determine both elongation and termination of translation.

Base Sequence↗

PubMed2000

Gene mutation in hereditary progressive dystonia with marked diurnal fluctuation (HPD), strictly defined dopa-responsive dystonia.

Mutations of the guanosine triphosphate (GTP)-cyclohydrolase I (GCH-I) gene, which catalyzes the first step in the tetrahydrobiopterin (the natural cofactor for tyrosine hydroxylase) biosynthesis, are demonstrated to cause HPD, i.e. strictly defined dopa-responsive dystonia. We analyzed the GCH-I gene of patients who fulfilled clinical criteria for typical hereditary progressive dystonia (HPD) to finalize the diagnosis. Two novel point mutations in two independent families and one novel de novo point mutation in one sporadic patient were identified. In a Japanese family, a T-to-C transition was found at exon 2, which resulted in a substitution of Cys 141 to Arg. In another Japanese family, a C-to-T mutation in exon 4 caused a nonsense codon Gln180Stop. In a clinically sporadic Japanese patient, T-to-G transition in exon 1 brought Met 102 Arg missense mutation, which was not observed in its biological parents. These three mutations were not observed in previously reported 57 pedigrees/patients and no polymorphisms in the coding region of the GCH-I gene were identified. None of the mutations of GCH-I gene in HPD reported to date or in this study have been detected more than once in any ethnicity suggesting a relatively high spontaneous mutation rate in this gene.

DNA Mutational Analysis↗

PubMed1998

ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines.

In mammalian nucleotide excision repair (NER), the ERCC1 protein is known to act as a complex with ERCC4 (XPF) protein, which is necessary for stability of ERCC1, and this complex introduces an incision on the 5' side of a damaged site in DNA. ERCC1 also binds to XPA protein to make a large protein complex at the site of DNA damage. Since no human disease associated with ERCC1 has been identified, Chinese hamster ovary (CHO) cell lines defective in ERCC1 are a unique source for characterization of ERCC1 deficiency in mammalian cells. We have isolated the full length ERCC1 cDNA from a wild-type CHO cell line and analyzed mutations in two CHO cell lines which fall into complementation group 1 of UV-sensitive rodent cell lines. One cell line, 43-3B, has a missense mutation at the 98th residue (V98E). The in vitro translated mutant protein of 43-3B is unable to bind to XPA protein. Although the mutant protein is able to bind to XPF protein in vitro, the mutant protein is highly unstable in vivo. These defects presumably cause the NER deficiency of this cell line. Another mutant, UV-4, has an insertion mutation in the middle of the coding sequence, resulting in a truncated protein due to a nonsense codon arising from the frameshift. Thus, these two mutant cell lines are deficient in the function of the ERCC1 gene for NER.

Amino Acid Sequence↗

PubMed2000

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

Adenomatous Polyposis Coli Protein↗

PubMed2001

A general approach for the generation of orthogonal tRNAs.

BACKGROUND: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli. RESULTS: A library of amber suppressor tRNAs derived from M. jannaschii tRNA(Tyr) was generated. tRNA(Tyr)(CUA)s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E. coli synthetases than M. jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M. jannaschii TyrRS. CONCLUSIONS: The mutant suppressor tRNA(Tyr)(CUA) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.

Amino Acyl-tRNA Synthetases↗

PubMed2003

Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats.

Mice and rats are naturally deficient in cholesteryl ester transfer protein (CETP) activity, although the reason behind the deficiency in activity is unknown. A search of mouse genome databases revealed sequences resembling 7 of the 16 human exons. However, these sequences could not code for a functional CETP. Analysis of the rat genome using Southern blotting revealed sequences complementary to human CETP cDNA, but RNase protection assays were unable to detect any Cetp gene expression in liver, adipose, or muscle. A search of rat whole-genome shotgun databases revealed exon-like sequences that would be unable to code for a functional CETP. An Ap3s1 pseudogene lay immediately upstream of the CETP-like sequences in mouse, but was nearly identical to the functional gene and unlikely to have been inserted prior to mouse-rat divergence. In contrast, a deletion leading to a nonsense codon was found in the exon 11-like sequences of both rat and mouse and not in any other species. Thus, the lack of CETP activity in both the mouse and the rat is most likely due to an evolutionary event that occurred before these species diverged and not to altered regulation of the gene or function of the gene product.

Adaptor Protein Complex 3↗

PubMed1998

Mutagenesis of a single AT basepair in mice transgenic for PhiX174 am3 cs70 I. Spleen and testis.

Mutations induced in a single AT base pair were studied in spleen and testis by using mice transgenic for PhiX174 am3, cs70 and ethylnitrosourea (ENU) as the mutagen. The transgenic mice were produced on the C57BL6/J background. The line (am54), which carries 50 copies of PhiX per haploid genome integrated in a tandem array, was selected for experimental use and was maintained by random breeding. The animals for mutagenesis studies were produced by mating homozygous am54 males to wildtype C57BL6/J females. Hemizygous male offspring (8 to 10 weeks old) from this cross were injected i.p. with 150 mg ENU per kg and were euthanized 3, 10 or 110 days after treatment. The spontaneous revertant frequency in the spleen was 1.42 x 10(-6) per plaque forming unit (pfu) and in the testis it was 1.41 x 10(-6) per pfu. There was no significant difference between the two tissues. In spleen, it was not until 110 days after ENU treatment that the average revertant frequency among treated animals was significantly higher than the revertant frequency among the control animals. In spleen, the induced frequency of basepair substitutions in the center AT basepair in the am3 nonsense codon was 2 x 10(-6). Also at this post-injection interval the variance of revertant frequencies in the spleen was not different from control variance. In testis, the average revertant frequency 110 days post ENU injection was not significantly different from the control. However, two important observations were made regarding the testis data. First, one animal had a significantly increased revertant frequency 110 days after ENU treatment in comparison to the other four animals in the group that had revertant frequencies equal to or lower than the average control frequency. Second, the variance of revertant frequencies in the testis among the treated animals increased as the post injection period increased. Taken together, these observations may indicate that the revertants formed large clusters in one testis sample.

Adenine↗