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PubMed1999

Exponential-phase glycogen recycling is essential for growth of Mycobacterium smegmatis.

Bacterial glycogen is a polyglucose storage compound that is thought to prolong viability during stationary phase. However, a specific role for glycogen has not been determined. We have characterized SMEG53, a temperature-sensitive mutant of Mycobacterium smegmatis that contains a mutation in glgE, encoding a putative glucanase. This mutation causes exponentially growing SMEG53 cells to stop growing at 42 degrees C in response to high levels of glycogen accumulation. The mutation in glgE is also associated with an altered growth rate and colony morphology at permissive temperatures; the severity of these phenotypes correlates with the amount of glycogen accumulated by the mutant. Suppression of the temperature-sensitive phenotype, via a decrease in glycogen accumulation, is mediated by growth in certain media or multicopy expression of garA. The function of GarA is unknown, but the presence of a forkhead-associated domain suggests that this protein is a member of a serine-threonine kinase signal transduction pathway. Our results suggest that in M. smegmatis glycogen is continuously synthesized and then degraded by GlgE throughout exponential growth. In turn, this constant recycling of glycogen controls the downstream availability of carbon and energy. Thus, in addition to its conventional storage role, glycogen may also serve as a carbon capacitor for glycolysis during the exponential growth of M. smegmatis.

Amino Acid Sequence↗
PubMed2000

Molecular genetics of hereditary nonpolyposis colorectal cancer.

The initial paradigm developed for colorectal carcinogenesis was derived from the observation that these tumors suffer a large number of chromosomal losses. This phenomenon results from unbalanced mitoses, and to date there is no clear explanation for this type of genomic instability. In 1993, a second type of genomic instability was detected and linked to 12-15% of sporadic tumors, as well as 90% or more of the colon cancers in hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC tumors are typically diploid and do not have the inactivating mutations at the tumor suppressor genes commonly found in the other cancers. These tumors were found because they have a very large number (perhaps in excess of 10(5)) of insertion or deletion mutations at microsatellite sequences; as a result, this has been termed microsatellite instability (MSI). The majority of HNPCC families can be linked to germline mutations in the DNA mismatch repair (MMR) genes hMSH2 or hMLH1. Germline mutations in hMSH6 and hPMS2 in HNPCC families are much less common. HNPCC tumors develop through the accumulation of mutations at genes that control cellular growth, and these genes are not the same as those recognized in the initial pathway outlined by Vogelstein et al. The genetic targets of MSI all contain repetitive sequences in coding regions that are unstable when the DNA MMR system is inoperative. Certain pathological features have been identified that suggest that colon cancers have developed in the setting of defective DNA MMR.

Adaptor Proteins, Signal Transducing↗
PubMed1997

[Study on the molecular mechanism of multi-drug resistance in clinical isolates of Mycobacterium tuberculosis].

OBJECTIVE: To study the molecular mechanism of multi-drug resistance in M. Tuberculosis, and to develop a new method for detecting genes related with multi-drug resistance. METHOD: The ropB, rpsL, katG genes and inhA regulatory sequence in clinical isolates of M. tuberculosis were analyzed with PCR and PCR-SSCP techniques. RESULT: The sensitivity of amplifing the drug-resistant genes with PCR was 1-10 pg DNA. Of the 20 multiple resistant strains with reduced sensitivity to streptomycin, rifampin and isoniazid, 90% showed mutations in more than two genetic markers associated with resistance to each of these three drugs, 10% revealed only mutations in rpoB gene. CONCLUSION: Multi-drug resistance in M. tuberculosis could be caused by an accumulation of mutations in chromosomal genes encoding drug targets or an alteration at a single multiple resistance locus. PCR and PCR-SSCP techniques might become simple, rapid and reliable diagnostic tests for multi-drug resistance.

Antitubercular Agents↗
PubMedDate not supplied

[A possible role of genetic translation ambiguity in evolution ].

A hypothesis has been suggested that the variability of translation machinery is one of the key factors of evolutionary transformations of genetic material. It considers a module principle of the evolution theory based on the concepts of duplication and divergence of genetic material, which is required for origination of new genes and proteins with new functions. The duplication results in the appearance of pseudogenes, functionally inactive, but serving a material for creating new functions. The possible mechanisms changing the translation machinery have been considered, which may lead to the sporadic pseudogene activation "supplying" natural selection with mutational changes accumulated by pseudogenes to assess their adaptive value. This takes into account not only potential possibilities of mutational variability of the translation machinery, but also the possibility of protein prioritization: a prion mechanism of inheritance is also considered which is intensively studied nowadays.

Animals↗
PubMed1984

A nuclear mutation that post-transcriptionally blocks accumulation of a yeast mitochondrial gene product can be suppressed by a mitochondrial gene rearrangement.

The nuclear amber mutation, pet494-1, specifically blocks the accumulation of the product of the mitochondrial gene oxi2, cytochrome oxidase subunit III. The pet494-1 mutation does not prevent transcription of the mitochondrial gene since RNA--gel blot hybridizations showed that mutant cells contain normal amounts of an oxi2 transcript, indistinguishable in size from wild-type. A mitochondrial mutation that partially suppresses the nuclear mutation was isolated. The "mitochondrial revertant" behaved as though it contained two different mitochondrial DNAs: one rho+, the other rho-. The suppressor mutation is carried on the rho- mitochondrial DNA and is apparently the result of a gene fusion between oxi2 and another mitochondrial gene, oxi3. This gene rearrangement replaced the normal 5'-non-translated sequence of oxi2 with a portion of the open reading frame of the second intron of oxi3. Novel transcripts of the rearranged gene, containing oxi3 sequences upstream from oxi2 were detected in the mitochondrial revertant. The strain accumulated an electrophoretically variant form of cytochrome oxidase subunit III, probably translated from a new initiation codon. The data are consistent with models in which the PET494 protein acts within the mitochondria to specifically promote the translation of the oxi2 messenger RNA.

DNA, Mitochondrial↗
PubMed1998

Mutation analysis using the restriction site mutation (RSM) assay.

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109-121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1, 2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117-123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.

4-Nitroquinoline-1-oxide↗
PubMed1998

Cancer cells exhibit a mutator phenotype.

This review analyzes the concept and evidence in support of a mutator phenotype in human cancer. The large number of mutations reported in tumor cells cannot be accounted for by the low mutation rates observed in normal somatic cells; rather, it must be a manifestation of a mutator phenotype present early during the tumorigenic process. The interaction between increased mutagenesis and clonal selection provides a mechanism for the selection of cells with increased proliferative advantage. The concept of a mutator phenotype in cancer has gained considerable support from the findings of enormous numbers of somatic mutations in repetitive sequences in human tumors. Moreover, cell lines exhibiting microsatellite instability demonstrate an increased mutation frequency in expressed genes. A knowledge of mechanisms that generate multiple mutations in cancer cells has important implications for prevention. For many tumors, a delay in the rate of accumulation of mutations by a factor of two could drastically reduce the death rates from these tumors.

Animals↗
PubMed2025

Early germline sequestration in a basidiomycete fungus.

In sexual organisms, inheritance of new mutations is highly dependent on the timing of germline definition. Here, we used the fairy ring-forming fungus Marasmius oreades to challenge the general assumption of a late germline separation in the Fungi. We collected mushrooms from different parts of rings over a 7-year period and identified new mutations in different tissues by whole-genome sequencing. We found evidence that fertile and sterile tissues had accumulated different mutations, suggesting that the germ line, destined for spore production, is already defined in the mycelium in this species. Moreover, the germ line carried fewer mutations than sterile tissues, indicating a lower mutation rate. Our findings suggest that early germline sequestration is more widespread than previously considered across multicellular life.

Genome, Fungal↗
PubMed1994

A cluster of cystic fibrosis mutations in exon 17b of the CFTR gene: a site for rare mutations.

Intensive screening has improved our understanding of the profile of mutations in the CFTR gene in which more than 400 mutations have been detected to date. In collaboration with several European laboratories we are involved in such analysis. We have identified 14 new mutations in exon 17b of CFTR, having analysed 780 CF chromosomes, and have compared the frequency of mutations in this exon with that of other regions of the CFTR gene. The results obtained indicate an accumulation of mutations, not only in regions encoding the two nucleotide binding folds, but also in those encoding transmembrane domains of the CFTR gene, in particular exon 17b.

Cystic Fibrosis↗
PubMed1989

Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii.

The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.

Amino Acid Sequence↗
PubMed1994

DNA damage induced p53 mediated transcription is inhibited by human papillomavirus type 18 E6.

Cervical cancer is similar to other human cancers in that it develops through a multistep process. However, infection with oncogenic human papillomaviruses (HPVs) is believed to be essential for the initiation of this disease. Although HPV may play a central role in the early stages of neoplasia, the accumulation of mutations in an assortment of genes precedes the development of malignant cervical carcinoma. The mechanisms by which abnormalities accumulate are various, but it is possible that viral proteins are involved. In particular, the viral E6 oncoprotein has been shown to interact with the cellular tumour suppressor protein p53, which is involved in DNA damage repair pathways. Hence, E6 may contribute to the genomic instability through this interaction with p53. We have tested this hypothesis by monitoring the effects of E6 upon DNA damage induced p53 transcriptional activity. This study shows that HPV-18 E6 inhibits p53 transcriptional activity following genotoxic stress with UV radiation. No effect was observed when a mutant E6 unable to direct the degradation of p53 was included in this assay. These results suggest that continued E6 expression may contribute to the accumulation of DNA damage associated with the progression of cervical cancer.

Amino Acid Sequence↗
PubMed1994

p53 gene mutations and steroid receptor status in breast cancer. Clinicopathologic correlations and prognostic assessment.

BACKGROUND: There is increasing evidence linking development and progression of cancer to an accumulation of mutations at the genomic level. The most frequently mutated gene known to date in sporadic breast cancer appears to be the tumor suppressor gene p53. This study was designed to determine the frequency of p53 gene mutations in primary breast cancer, to correlate the presence of p53 mutations with established clinicopathologic parameters, including the estrogen receptor (ER) and progesterone receptor (PR) status, and to assess the prognostic significance of p53 mutations regarding patient survival. METHODS: We examined the p53 gene in genomic DNA samples from 192 primary breast cancers. Using denaturant gradient gel electrophoresis, the authors analyzed exons 5-9 in all tumors for mutations and performed DNA sequencing in 20 tumors to identify the exact nature of the p53 mutations. RESULTS: p53 gene alterations were identified in 43 of the 192 tumors (22%), the majority localized in exons 5 and 6. DNA sequencing showed mostly missense mutations resulting from G or C substitutions. p53 mutations were found more often in tumors of younger women (P = 0.002), Afro-American women (P = 0.05), and in tumors lacking ER (P = 0.03), PR (P = 0.04), or both (P = 0.06). There were no significant correlations with family history, tumor size, histologic grade or type, nodal status, or disease stage. The overall survival rates showed no significant difference between patients with mutant and wild-type p53 tumors. The same was true when the comparison was limited to node-negative patients or patients with ER-positive or ER-negative tumors. Finally, there was no significant difference in survival between patients with tumors harboring mutations in exons 5 and 6 versus exons 7-9. CONCLUSIONS: The results of this and other studies demonstrate a consistent relationship between ER-positive tumors and wild-type p53 on one hand and ER-negative cancers and p53 mutations on the other. Our data do not support a significant prognostic role for p53 mutations in predicting survival.

Base Sequence↗
PubMed1991

Evolution of the master Alu gene(s).

A comparison of Alu sequences that comprise more recently amplified Alu subfamilies was made. There are 18 individual diagnostic mutations associated with the different subfamilies. This analysis confirmed that the formation of each subfamily can be explained by the sequential accumulation of mutations relative to the previous subfamily. Polymerase chain reaction amplification of orthologous loci in several primate species allowed us to determine the time of insertion of Alu sequences in individual loci. These data suggest that the vast majority of Alu elements amplified at any given time comprised a single Alu subfamily. We find that, although the individual divergence relative to a consensus sequence correlate reasonably well with sequence age, the diagnostic mutations are a more accurate measure of the age of any individual Alu family member. Our data are consistent with a model in which all Alu family members have been made from a single master gene or from a series of sequential master genes. This master gene(s) accumulated diagnostic base changes, resulting in the amplification of different subfamilies from the master gene at different times in primate evolution. The changes in the master gene(s) probably occurred individually, but their appearance is clearly punctuated. Ten of them have occurred within an approximately 15-million-year time span, 40-25 million years ago, and 8 changes have occurred within the last 5 million years. Surprisingly, no changes appeared in the 20 million years separating these periods.

Animals↗
PubMed2005

APC, K-ras, and p53 gene mutations in colorectal cancer patients: correlation to clinicopathologic features and postoperative surveillance.

Current researches have proposed a genetic model for colorectal cancer (CRC), in which the sequential accumulation of mutations in specific cancer-related genes, including adenomatous polyposis coli (APC), K-ras, and p53, drives the transition from normal epithelium through increasing adenomatous dysplasia to colorectal cancer. To identify patients with an increased risk of tumor recurrence or metastasis and evaluate the prognostic values of APC, K-ras, and p53 gene mutations, we investigated the frequency of these three mutated genes in tumors and sera of CRC patients. APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired preoperative serum samples of 118 CRC patients were detected by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, followed by direct DNA sequencing of the PCR-amplified genomic DNA. Subsequently, serum molecular markers were analyzed for their correlation with patients' clinicopathologic features and presence of postoperative recurrence/metastasis. We did not observe any significant difference in the association of APC or K-ras or p53 gene mutations in primary tumors with patients' demographic data (all were P > 0.05). In contrast, both serum APC and p53 molecular markers were closely correlated with lymph node metastasis and TNM stage (both P < 0.05). Moreover, the serum overall molecular markers (at least one of the three markers) were prominently associated with depth of tumor invasion (P = 0.033), lymph node metastasis (P < 0.001), and TNM stage (P < 0.001). In addition, a significantly higher postoperative metastasis/recurrence rate in patients positive for overall molecular markers compared to those negative for these molecular markers were also demonstrated (P < 0.001). APC and K-ras molecular markers were more frequently observed in patients with locoregional metastasis (both P < 0.05), while p53 molecular marker was usually detected in the cases of peritoneal metastasis (P = 0.004). Our findings suggest that serum molecular markers are potentially useful in the determination of colorectal cancer patients harboring gene mutations at high risk of metastasis. Serial analysis is warranted in order to assess their long-term prognostic significance and the therapeutic implications.

Biomarkers, Tumor↗
PubMed2001

DNA mismatch repair and cancer.

Five human DNA mismatch repair genes have been identified that, when mutated, cause susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Mutational inactivation of both copies of a DNA mismatch repair gene results in a profound repair defect and progressive accumulation of mutations throughout the genome. Some of the mutations confer selective advantage on the cells, giving rise to cancer. Recent discoveries suggest that apart from postreplication repair, DNA mismatch repair proteins have several other functions that are highly relevant to carcinogenesis. These include DNA damage surveillance, prevention of recombination between nonidentical sequences and participation in meiotic processes (chromosome pairing). A brief overview of these different features of the human DNA mismatch repair system will be provided, with the emphasis in their implications in cancer development.

Animals↗
PubMed2001

The ORFO product of Potato leafroll virus is indispensable for virus accumulation.

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.

Genes, Essential↗
PubMed1993

Genetic variability and evolution of the satellite RNA of cucumber mosaic virus during natural epidemics.

The genetic structure of populations of cucumber mosaic virus (CMV) satellite RNA (satRNA) and its evolution were analyzed during the course of a CMV epidemic in tomatoes in eastern Spain. A total of 62 variants of CMV-satRNA from epidemic episodes in 1989, 1990, and 1991 were characterized by RNase protection assay (RPA); RPA patterns defined 60 haplotypes in the CMV-satRNA population. RPA of nine CMV-satRNAs of known sequences showed that numbers of nucleotide substitutions per site (dij) between different satRNAs can be estimated from RPA data. Thus, dij were estimated for any possible pair of field CMV-satRNA types, and nucleotide diversities within and between yearly subpopulations were calculated. Also, phylogenetic relationships among CMV-satRNAs were derived from RPA data (by parsimony) or from dij (by neighbor joining). From these analyses, a model for the evolution of CMV-satRNAs in field epidemics can be built. High genetic variability of CMV-satRNA results in very heterogeneous populations, even compared with those of other RNA genomes. The high diversity of the population is maintained through time by the continuous generation of variants by mutation, counterbalanced by negative selection; this results in a certain replacement of haplotypes from year to year. The sequential accumulation of mutations in CMV-satRNA leads to fast genetic divergence to reach what appears to be an upper permitted threshold.

Biological Evolution↗
PubMed1980

Senescence of cultured human diploid fibroblasts. Are mutations responsible?

Two predictions of the error/mutation hypothesis of cellular senescence (Orgel, '73) namely,a) exponential accumulation of somatic mutations during the replicative lifespan and b) shortening of culture lifespan upon treatment with mutagens have been examined experimentally in a strain of cultured human diploid fibroblasts. Our studies show that as cells traverse the replicative lifespan (from 10 to 75 mean population doublings (MPD); total lifespan congruent to 95 MPD), no rapid and exponential increase occurs in the accumulation of mutations measured by the frequencies of Thgr(thioguanine resistance) and Dipr (diphtheria toxin resistance) mutants. Furthermore, repeated cycles of treatment (from 1- to 14-times) of human fibroblasts with two mutagens, ethyl methane sulfonate (EMS) and N-methyl-N' nitro-nitrosoguanidine, which led to a marked increase in the mutation frequency for the Dipr marker (congruent to 100-fold), failed to shorten the lifespan of cultured fibroblasts. On the contrary, repeated mutagen treatment (12 times with EMS) prolonged the lifespan of one replicative culture (110 MPD versus 94--98 MPD). These results strongly indicate that mutations are unlikely to be the primary event in cellular senescence and suggest instead that senescence is probably controlled by one or more (specific) gene(s) whose expression can be modified by mutations.

Cell Line↗