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A Chinese Hamster Mutant Cell Line with a Defect in the Integral Membrane Protein CII-3 of Complex II of the Mitochondrial Electron Transport Chain

In this study, a respiration-deficient Chinese hamster cell line with a defect in succinate dehydrogenase activity is shown to result from a single base change in a codon in the coding sequence for the membrane anchor protein CII-3 (also referred to as QPs-1). A premature translation stop results in the truncation of 33 amino acids from the C terminus. Bovine cDNA encoding this peptide complements the mutation. There is about 82% identity between these two mammalian proteins. The gene for CII-3 was mapped on human chromosome 1, and because it is also found on minichromosomes characterized by our laboratory, we can localize it on the short arm within 1-2 megabases from the centromere.

Identification of a CD40L gene mutation and genetic counselling in a family with immunodeficiency with hyperimmunoglobulinemia M.

A 13-year-old boy with immunodeficiency with hyper-IgM was analyzed for mutations in the CD40L gene. An insertional mutation of an extra T in a run of four T's was found in the second exon of the gene, leading to a premature translation stop. Genetic counselling of the family was performed, based on mutation detection by PCR/oligohybridization.

Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy.

Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively. Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.

Characterization of von Willebrand factor gene defects in two unrelated patients with type IIC von Willebrand disease

Genetic studies were performed in two unrelated patients with the IIC phenotype of von Willebrand disease (vWD) characterized by the increased concentration of the protomeric form of von Willebrand factor (vWF). In patient B, the sequencing of both exons 15 and 16 of the vWF gene showed two sequence alterations: a 3-bp insertion in exon 15 resulting in the insertion of a Glycine at position 625 (625insGly) and a 2-bp deletion in exon 16 leading to a premature translational stop at codon 711 (711 ter), at the heterozygote state. Patient A was found homozygous for a single point mutation also localized in exon 15 and responsible for the substitution Cys623Trp. These candidate mutations were not found in a panel of 96 normal chromosomes, suggesting a causal relationship with IIC vWD phenotypic expression. The composite heterozygote or homozygote state of both patients supports the recessive mode of inheritance already described for this phenotype. Furthermore, the localization of these gene defects in the D2 domain of vWF propeptide, known to play an important role in vWF multimerization, provides another argument in favor of their causative effect regarding the peculiar multimeric pattern of vWF in these patients.

Characterization of von Willebrand Factor Gene Defects in Two Unrelated Patients With Type IIC von Willebrand Disease

Genetic studies were performed in two unrelated patients with the IIC phenotype of von Willebrand disease (vWD) characterized by the increased concentration of the protomeric form of von Willebrand factor (vWF). In patient B, the sequencing of both exons 15 and 16 of the vWF gene showed two sequence alterations: a 3-bp insertion in exon 15 resulting in the insertion of a Glycine at position 625 (625insGly) and a 2-bp deletion in exon 16 leading to a premature translational stop at codon 711 (711 ter), at the heterozygote state. Patient A was found homozygous for a single point mutation also localized in exon 15 and responsible for the substitution Cys623Trp. These candidate mutations were not found in a panel of 96 normal chromosomes, suggesting a causal relationship with IIC vWD phenotypic expression. The composite heterozygote or homozygote state of both patients supports the recessive mode of inheritance already described for this phenotype. Furthermore, the localization of these gene defects in the D2 domain of vWF propeptide, known to play an important role in vWF multimerization, provides another argument in favor of their causative effect regarding the peculiar multimeric pattern of vWF in these patients.

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels

The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

Human lymphocyte-specific pp52 gene is a member of a highly conserved dispersed family.

For a better understanding of genes that potentially function in B lymphocyte cell signaling, we isolated the human genomic counterpart of the murine pp52 or LSP1 gene. We unexpectedly found that the human pp52 gene is one of four closely related loci. Representative cosmids from each of the four family members were isolated and chromosomally localized by fluorescence in situ hybridization. Nucleotide sequence was obtained from an exon common to each locus and demonstrated very close similarity among all four loci. Two of the four loci harbored dysfunctional frameshift mutations or premature translation stop sites. The exon of one locus was flanked by an 80-bp perfect inverted repeat, suggesting that it may have originated through a looped intermediate DNA structure. Through a series of cDNA hybridization studies and nucleotide sequence analyses we were able to unambiguously link the lymphocyte-expressed gene to the locus mapped to chromosome 11p15.5. This same chromosomal band has been involved in tumor-related chromosomal translocations found in chronic lymphocytic leukemia.

Mechanism and Consequences of the Duplication of the Human C4/P450c21/Gene X Locus

The adjacent C4 and P450c21 genes encode the fourth component of serum complement and steroid 21-hydroxylase respectively, and are tandemly duplicated in the human, murine, and bovine genomes. We recently cloned a cDNA for another duplicated gene, operationally termed X, which overlaps the 3' end of human P450c21 and has the opposite transcriptional orientation. Thus, the organization of the locus is 5'-C4A-21A-XA-C4B-21B-XB-3' (Y. Morel, J. Bristow, S. E. Gitelman, and W. L. Miller, Proc. Natl. Acad. Sci. USA 86:6582-6586, 1989). To determine how this locus was duplicated, we sequenced the DNA at the duplication boundaries and the 7 kb between P450c21A and C4B comprising the XA locus. The sequences located the duplication boundaries precisely and indicate that the duplication occurred by nonhomologous recombination. The boundaries are substantially different from those of the corresponding duplication in the mouse genome, suggesting that similar gene duplications may have occurred independently in ancestors of rodents and primates after mammalian speciation. Compared with XB, the XA gene is truncated at its 5' end and bears a 121-bp intragenic deletion causing a frameshift and premature translational stop signal. Nevertheless, XA is transcribed into a stable 2.6-kb polyadenylated RNA that is expressed uniquely in the adrenal gland.

Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus.

The adjacent C4 and P450c21 genes encode the fourth component of serum complement and steroid 21-hydroxylase respectively, and are tandemly duplicated in the human, murine, and bovine genomes. We recently cloned a cDNA for another duplicated gene, operationally termed X, which overlaps the 3' end of human P450c21 and has the opposite transcriptional orientation. Thus, the organization of the locus is 5'-C4A-21A-XA-C4B-21B-XB-3' (Y. Morel, J. Bristow, S. E. Gitelman, and W. L. Miller, Proc. Natl. Acad. Sci. USA 86:6582-6586, 1989). To determine how this locus was duplicated, we sequenced the DNA at the duplication boundaries and the 7 kb between P450c21A and C4B comprising the XA locus. The sequences located the duplication boundaries precisely and indicate that the duplication occurred by nonhomologous recombination. The boundaries are substantially different from those of the corresponding duplication in the mouse genome, suggesting that similar gene duplications may have occurred independently in ancestors of rodents and primates after mammalian speciation. Compared with XB, the XA gene is truncated at its 5' end and bears a 121-bp intragenic deletion causing a frameshift and premature translational stop signal. Nevertheless, XA is transcribed into a stable 2.6-kb polyadenylated RNA that is expressed uniquely in the adrenal gland.

Expression analysis of a pseudogene in transgenic tobacco: a frameshift mutation prevents mRNA accumulation.

Seeds of the Pinto cultivar of the common bean, Phaseolus vulgaris, are deficient in phytohemagglutinin (PHA), a lectin normally composed of two different polypeptides (PHA-E and PHA-L). In Pinto seeds, there is no PHA-E and only small amounts of PHA-L. The gene coding for the Pinto PHA-E, Pdlec1, is a pseudogene as a result of a single base pair deletion in codon 11, causing a frameshift and premature termination of translation. This mutation explains the absence of the PHA-E polypeptide but not the several-hundredfold reduction of the cytoplasmic Pdlec1 mRNA in developing seeds when compared with a normal PHA-E gene. To find the cause for this reduction in mRNA levels, we swapped gene fragments of Pdlec1 with the homologous parts of a normal PHA gene from the cultivar Greensleeves and introduced these fusions into tobacco. Analysis of the transgenic seeds showed that the Pdlec1 promoter is fully functional. We also repaired the Pdlec1 coding frame in vitro and inserted the repaired and unrepaired versions into a PHA gene expression cassette. In transgenic tobacco, both constructs showed Pdlec1 transcript accumulation in the second half of seed maturation. The single-base frame repair boosted the peak transcript levels by a factor of 40 and resulted in the synthesis of PHA-E at normal levels. We propose that the premature translational stop caused by the frameshift leads to a faster breakdown of the Pdlec1 mRNA, thereby preventing this transcript from accumulating to high levels.

Molecular defects in the non-expressed H-2 E alpha genes of the f and q haplotypes.

The expression and sequences of the mouse MHC class II genes, E alpha f and E alpha q, have been studied to determine the molecular basis for their defective expression. Previous work in our laboratory showed that H-2f and H-2q mice produce no detectable E alpha protein. Northern blot analysis confirms previous results showing normal amounts of E alpha f message and a 100-fold reduction in E alpha q mRNA. Despite that observation, the rates of transcription of both defective E alpha genes, measured by nuclear run-on transcription assays, are comparable to that of the normally expressed E alpha k gene. The nucleotide sequences of the E alpha f and E alpha q genes reveal mutations generating premature translation stop codons in both genes. A single base substitution has created the stop codon TGA at amino acid -2 in the E alpha f leader sequence. A nucleotide insertion at codon 64 in the second exon of the E alpha q gene results in a frame shift and a premature stop codon at amino acid residue 69 of a mature E alpha protein. The low steady state level of E alpha q mRNA may be correlated with the unusual size distribution of the RNA, possibly due to altered RNA processing.

Characterization of single base substitutions In edited apolipoprotein B transcripts

Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.

Human apolipoprotein B (apoB) mRNA: identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine.

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides (nucleotides 6523-6552 of apoB mRNA), designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. Analysis of intestinal apoB mRNA by hybridization with apoB-Stop and apoB-Gln probes and sequence analysis of apoB clones in two independent human small intestinal cDNA libraries established that intestinal apoB mRNA contained both the apoB mRNA that codes for apoB-100 and the apoB mRNA containing the premature in-frame stop codon, which codes for apoB-48. Investigation of hepatic apoB mRNA and two hepatic cDNA libraries by hybridization with the apoB-Stop and apoB-Gln synthetic probes as well as by cDNA sequencing revealed that liver apoB mRNA also contains both the apoB-100 mRNA and the apoB-48 mRNA containing the stop codon. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine.