Transfer RNA modification status influences retroviral ribosomal frameshifting.
(1/2000)
The possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 3' side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status. In addition, when AAU occurred immediately upstream of UUU as the penultimate frameshift site codon, addition of tRNAAsn without the Q base reduced the stimulatory effect of tRNAPhe without the Y base, whereas addition of tRNAAsn with the Q base did not alter the stimulatory effect. The addition of tRNAAsn without the Q base and tRNAPhe with the Y base inhibited frameshifting. The latter studies suggest an interplay between the tRNAs decoded at the penulimate frameshift and frameshift site codons that is also influenced by tRNA modification status. These data may be intrepreted as indicating that a hypomodified isoacceptor modulates frameshifting in an upward manner when utilized at the frameshift site codon, but modulates frameshifting in a downward manner when utilized at the penultimate frameshift site codon. (+info)
Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy.
(2/2000)
The congenital nemaline myopathies are rare hereditary muscle disorders characterized by the presence in the muscle fibers of nemaline bodies consisting of proteins derived from the Z disc and thin filament. In a single large Australian family with an autosomal dominant form of nemaline myopathy, the disease is caused by a mutation in the alpha-tropomyosin gene TPM3. The typical form of nemaline myopathy is inherited as an autosomal recessive trait, the locus of which we previously assigned to chromosome 2q21.2-q22. We show here that mutations in the nebulin gene located within this region are associated with the disease. The nebulin protein is a giant protein found in the thin filaments of striated muscle. A variety of nebulin isoforms are thought to contribute to the molecular diversity of Z discs. We have studied the 3' end of the 20. 8-kb cDNA encoding the Z disc part of the 800-kDa protein and describe six disease-associated mutations in patients from five families of different ethnic origins. In two families with consanguineous parents, the patients were homozygous for point mutations. In one family with nonconsanguineous parents, the affected siblings were compound heterozygotes for two different mutations, and in two further families with one detected mutation each, haplotypes are compatible with compound heterozygosity. Immunofluorescence studies with antibodies specific to the C-terminal region of nebulin indicate that the mutations may cause protein truncation possibly associated with loss of fiber-type diversity, which may be relevant to disease pathogenesis. (+info)
Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells.
(3/2000)
Epithelial (E)-cadherin and its associated cytoplasmic proteins (alpha-, beta-, and gamma-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin-catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2. (+info)
Mutations in the organic cation/carnitine transporter OCTN2 in primary carnitine deficiency.
(4/2000)
Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease presents early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. The gene for this condition maps to 5q31.2-32 and OCTN2, an organic cation/carnitine transporter, also maps to the same chromosomal region. Here we test the causative role of OCTN2 in primary carnitine deficiency by searching for mutations in this gene in affected patients. Fibroblasts from patients with primary carnitine deficiency lacked mediated carnitine transport. Transfection of patient's fibroblasts with the OCTN2 cDNA partially restored carnitine transport. Sequencing of the OCTN2 gene revealed different mutations in two unrelated patients. The first patient was homozygous (and both parents heterozygous) for a single base pair substitution converting the codon for Arg-282 to a STOP codon (R282X). The second patient was a compound heterozygote for a paternal 1-bp insertion producing a STOP codon (Y401X) and a maternal 1-bp deletion that produced a frameshift creating a subsequent STOP codon (458X). These mutations decreased the levels of mature OCTN2 mRNA and resulted in nonfunctional transporters, confirming that defects in the organic cation/carnitine transporter OCTN2 are responsible for primary carnitine deficiency. (+info)
Infrequent translation of a nonsense codon is sufficient to decrease mRNA level.
(5/2000)
In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin mu heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of mu mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only approximately 4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD. (+info)
p53 mutations in human cutaneous melanoma correlate with sun exposure but are not always involved in melanomagenesis.
(6/2000)
In melanoma, the relationship between sun exposure and the origin of mutations in either the N-ras oncogene or the p53 tumour-suppressor gene is not as clear as in other types of skin cancer. We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced. To investigate whether sun exposure also affects p53 in melanoma, we analysed 81 melanoma specimens for mutations in the p53 gene. The mutation frequency is higher than thus far reported: 17 specimens (21%) harbour one or more p53 mutations. Strikingly, 17 out of 22 mutations in p53 are of the C:G to TA or CC:GG to TT:AA transitional type, strongly suggesting an aetiology involving UV exposure. Interestingly, the p53 mutation frequency in metastases was much lower than in primary tumours. In the case of metastases, a role for sun exposure was indicated by the finding that the mutations are present exclusively in skin metastases and not in internal metastases. Together with a relatively frequent occurrence of silent third-base pair mutations in primary melanomas, this indicates that the p53 mutations, at least in these tumours, have not contributed to melanomagenesis and may have originated after establishment of the primary tumour. (+info)
High frequency of germ-line BRCA2 mutations among Hungarian male breast cancer patients without family history.
(7/2000)
To determine the contribution of BRCA1 and BRCA2 mutations to the pathogenesis of male breast cancer in Hungary, the country with the highest male breast cancer mortality rates in continental Europe, a series of 18 male breast cancer patients and three patients with gynecomastia was analyzed for germ-line mutations in both BRCA1 and BRCA2. Although no germ-line BRCA1 mutation was observed, 6 of the 18 male breast cancer cases (33%) carried truncating mutations in the BRCA2 gene. Unexpectedly, none of them reported a family history for breast/ovarian cancer. Four of six truncating mutations were novel, and two mutations were recurrent. Four patients (22%) had a family history of breast/ovarian cancer in at least one first- or second-degree relative; however, no BRCA2 mutation was identified among them. No mutation was identified in either of the genes in the gynecomastias. These results provide evidence for a strong genetic component of male breast cancer in Hungary. (+info)
How translational accuracy influences reading frame maintenance.
(8/2000)
Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from a second error occurring after recruitment of an errant aminoacyl-tRNA, either spontaneous dissociation causing premature termination or translational frameshifting. This is clearest in programmed translational frameshifting where the mRNA programs errant reading by a near-cognate tRNA; this error promotes a second frameshifting error (a dual-error model of frameshifting). The same mechanism can explain frameshifting by suppressor tRNAs, even those with expanded anticodon loops. The previous model that suppressor tRNAs induce quadruplet translocation now appears incorrect for most, and perhaps for all of them. We suggest that the 'spontaneous' tRNA-induced frameshifting and 'programmed' mRNA-induced frameshifting use the same mechanism, although the frequency of frameshifting is very different. This new model of frameshifting suggests that the tRNA is not acting as the yardstick to measure out the length of the translocation step. Rather, the translocation of 3 nucleotides may be an inherent feature of the ribosome. (+info)