Kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the hprt gene of Chinese hamster ovary cells

The kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the protein coding region of the hprt gene of Chinese hamster ovary (CHO) cells were determined by direct sequencing of polymerase chain reaction (PCR)-amplified cDNA. Primary mutations were found in 15 of 19 of the mutants: 11 were G:C-->T:A transversions, two were A:T-->T:A transversions and two were deletions of single G:C base pairs (-1 frameshifts). The remaining four mutants had large alterations in the cDNA that were explained by mRNA splicing errors. A group of control mutants had more diverse hprt cDNA alterations than MX-induced mutants. Transversions yielding an A:T base pair were the predominant type of MX-induced mutations, in agreement with previous findings in bacteria. This specificity may be explained by the 'A rule', that DNA polymerases preferentially insert adenine nucleotides opposite non-instructional lesions.     

Multimodal cerebral monitoring in comatose head-injured patients

We isolated and characterized the ERCC1 coding sequence from three Chinese hamster ovary (CHO) parental (CHO-AA8, CHO-AT3-2 and CHO-9) and 10 ERCC1 mutant cell lines. Two general classes of mutations were observed: two mutant cell lines exhibited nucleotide additions or deletions to produce frameshift mutations and seven mutant cell lines exhibited point mutations that resulted in transitions or transversions, including nonsense mutations and mutations that generated intron/exon splicing errors. One mutant (UV201) which had been provisionally assigned to ERCC1 complementation group 1 (CG1) had no detectable mutation in its coding sequence. Of the nine ERCC1 mutant alleles characterized two mutations were identified in the XpA binding region of the Ercc1 protein; no mutations were found in the N-terminal portion of the Ercc1 protein. Results of Northern hybridization analysis showed that the relative levels of ERCC1 mRNA differed significantly both among the parental cell lines and among the mutant cell lines derived from each parental cell line. Western analysis with a CHO Ercc1-specific antibody detected Ercc1 protein in each of the parental cell lines and also in UV201. The marked reduction in Ercc1 protein levels observed in all the other mutants examined supports the hypothesis that ERCC1 mutations may destabilize this polypeptide.     

Comparison of hypercholesterolemic men and women at high risk for atherosclerosis. Study of risk factors and response to pravastatin treatment

BACKGROUND: Ornithine decarboxylase (ODC; EC 4.1.1.17) is the first rate-limiting enzyme in the biosynthesis of polyamines. ODC protein has a characteristic amino acid sequence, the PEST sequence, which is related to the enzyme's rapid degradation. ODC cDNA prepared from human hepatoma tissues has been reported to show nonsense or missense mutations. METHODS: We examined somatic mutations of ODC cDNA by RT-PCR-SSCP analysis and mRNA expressions by RT-PCR in 50 colorectal cancer tissues to investigate the involvement of ODC gene alterations in colorectal cancers. RESULTS: Increased expression of the ODC gene was observed in 36 cases (86%) out of the 42 examined by RT-PCR. In one case, a missense mutation was found in the cancer tissue but not in normal mucosa. The missense mutation from Asp to Asn at codon 424, in the PEST region, possibly stabilizes the ODC protein. In colorectal cancer, replication error and a germline mutation in hMSH2 gene were observed. CONCLUSIONS: The missense mutation at codon 424 is speculated to be a cause of stabilization and a passenger mutation owing to the mutator phenotype. Since only one of 50 colorectal cancers exhibited a missense mutation of the ODC gene, mutations in ODC gene are not frequent in colorectal cancer. The increased expression of the ODC gene was noted in 86% of colorectal cancer tissues by RT-PCR, however, it was not due to point mutations in ODC coding exons.     

Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow-derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-gamma, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3' portion of WASP interfere with crucial functions of the protein and cause classic WAS.     

mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(-) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(-) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(-) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(-), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(-) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frame-shift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD.     

One class of growth hormone (GH) receptor and binding protein messenger ribonucleic acid in rat liver, GHR1, is sexually dimorphic and regulated by GH

In the rat, alternatively spliced messenger RNA (mRNA) species encode GH receptor (GHR) and GH-binding protein (GHBP). Additionally, these mRNAs are alternatively spliced in the 5'-untranslated region, resulting in at least two classes of GHR and GHBP mRNA with distinct first exons and identical coding regions. These alternative first exons define two unique classes of GHR and GHBP mRNA (called GHR1 and GHR2). The GHR1 class of RNA is expressed only in the liver, is far more abundant in females than males, and is particularly abundant during pregnancy. GHR1 RNA is induced later in development than is GHR2. Additional classes of GHR and GHBP RNA may also exist. The genomic structure of the GHR1 first exon reveals a putative promotor region with no TATA box, CAAT box, or other sequence elements suggesting specific responses. An in vivo approach was used to investigate the regulation of GHR1 expression. In female rats, gonadectomy was found to reduce the percentage of steady state GHR1 RNA levels in the liver, whereas male castration resulted in an induction of GHR1 RNA. However, short-term treatment with estrogen or testosterone had little effect, suggesting that direct regulation of GHR1 expression may occur through effector(s) other than gonadal steroids. Hypophysectomy abolished GHR1 RNA in females. Treatment of hypophysectomized females and castrated males with GH by single injection did not significantly induce GHR1 RNA, but treatment by continuous infusion of GH did. Little change in non-GHR1 RNA levels was observed for each of these treatments. The results suggest that: 1) the sexual dimorphism observed in total GHR and GHBP RNA in rat liver is attributable to the sexually dimorphic expression of the GHR1 class of RNA; 2) the sexually dimorphic pattern of GH release in rats regulates the GHR1 class of RNA; 3) changes in GHR and GHBP expression observed on gonadectomy, hypophysectomy, GH treatment, and pregnancy are best attributed to GHR1 regulation; and 4) since GHR1 is liver specific, the observed increases in serum GHBP concentration in response to sex steroids, GH pattern, and pregnancy are likely to originate from the liver.     

FTDP-17 mutations N279K and S305N in tau produce increased splicing of exon 10

Missense mutations and intronic mutations in the tau gene cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Known missense mutations reduce the ability of tau to promote microtubule assembly. Intronic mutations lead to increased mRNA splicing of the alternatively spliced exon 10, resulting in an overproduction of tau isoforms with four microtubule-binding repeats. We show here that the recently identified FTDP-17 missense mutations N279K and S305N do not reduce the ability of tau to promote microtubule assembly. Instead they lead to increased splicing of exon 10, like the intronic mutations. The N279K and S305N mutations define a class of missense mutations in tau whose primary effects are at the RNA level.     

Gene amplification in Chinese hamster DNA repair deficient mutants

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2-5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.     

Mutation analysis of the RSK2 gene in Coffin-Lowry patients: extensive allelic heterogeneity and a high rate of de novo mutations

Coffin-Lowry syndrome (CLS) is an X-linked disorder characterized by severe psychomotor retardation, facial and digital dysmorphisms, and progressive skeletal deformations. By using a positional cloning approach, we have recently shown that mutations in the gene coding for the RSK2 serine-threonine protein kinase are responsible for this syndrome. To facilitate mutational analysis, we have now determined the genomic structure of the human RSK2 gene. The open reading frame of the RSK2 coding region is split into 22 exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single-strand conformation polymorphism analysis. We screened 37 patients with clinical features suggestive of CLS. Twenty-five nucleotide changes predicted to be disease-causing mutations were identified, including eight splice-site alterations, seven nonsense mutations, five frameshift mutations, and five missense mutations. Twenty-three of them were novel mutations. Coupled with previously reported mutations, these findings bring the total of different RSK2 mutations to 34. These are distributed throughout the RSK2 gene, with no clustering, and all but two, which have been found in two independent patients, are unique. A very high (68%) rate of de novo mutations was observed. It is noteworthy also that three mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms. No obvious correlation was observed between the position or type of the RSK2 mutations and the severity or particular clinical features of CLS.     

Effect of folate deficiency on mutations at the hprt locus in Chinese hamster ovary cells exposed to monofunctional alkylating agents

Multiplex PCR amplification of hprt exons from 113 Chinese hamster ovary cell clones selected for resistance to 6-thioguanine was performed to investigate the molecular basis for the synergistic mutagenic effects of nutritional folic acid deficiency and alkylating agents. In cells treated with ethyl methanesulfonate, intragenic deletions were detected in 9 of 46 (19.6%) clones derived from folate-deficient cells, but in none of 16 mutants grown in folate-replete medium. The number of deletions found in mutants generated by N-nitroso-N-ethylurea was low in both folate-deficient (1 of 25; 4%) and folate-replete (1 of 26; 3.8%) cells. Correction of folate deficiency may decrease the frequency of intragenic deletions caused by some alkylating agents.