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PubMed1995

Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus vaccine grown under different cell culture conditions.

Mutations that consistently accumulated in the attenuated Sabin 2 strain of poliovirus during propagation in cell cultures were identified by sequence heterogeneity assay and quantified by mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Eight additional sites previously identified in stool isolates were also examined by MAPREC in the virus passages. The pattern of selectable mutations and the rate of their accumulation depended on the type and confluence of the cell culture and the temperature of virus growth. Five unstable genomic sites were identified in Sabin 2 virus passaged 10 times at 34 degrees in African green monkey kidney (AGMK) cells, with the mutations accumulating in the range 1 to 24%. Accumulation of these mutations did not appear to result in a loss of attenuated phenotype since the virus passaged under these conditions passed the monkey neurovirulence test (MNVT). The content of the 481-G revertant known to be related to neurovirulence in monkeys did not increase. Thus, our results suggest that upon growth of Sabin 2 virus in AGMK cells at 34 degrees, the key determinant(s) of attenuation remained stable, and the mutations that occurred did not affect monkey neurovirulence. In virus passaged 10 times at 37 degrees in AGMK cells, 4 unstable genomic sites were identified, in some of them accumulating up to 12% of the mutants. This virus sample severely failed the MNVT. Virus passaged in Vero cells at 34 and 37 degrees accumulated mutants at 7 and 14 genomic sites, respectively, including 481-G in both cases, with almost complete substitution of the original nucleotides at some of the sites. We tested 44 commercial monopools of Type 2 OPV and found out that all of them contained 481-G revertants in the range 0.4-1.1%. An increase in the 481-G revertants in passaged viruses to the level of 4% and above correlated with failure of these samples by the MNVT. Since the pattern of selectable mutations differed in viruses grown in the two cell cultures used in this study, specific mutation profiles should be determined for each cell substrate used for vaccine production to assess manufacturing consistency.

Amino Acid Sequence↗
PubMed2005

Infrequent mutation of APC, AXIN1, and GSK3B in human pituitary adenomas with abnormal accumulation of CTNNB1.

We analyzed mutation of the APC, AXIN1, and GSK3genes in 14 pituitary adenomas with abnormal nuclear accumulations of CTNNB1. These tumors did not harbor mutation of the CTNNB1 gene. The genes analyzed encode proteins associated with ubiquitin-mediated degradation of CTNNB1. Although the regions encoding functional domains of these protein products were analyzed, no significant genetic alterations were found. Furthermore, the antibody for the C-terminus of APC detected normal expression of the APC protein in these pituitary adenomas. Our present results imply that an unknown mechanism(s) accelerates the accumulation of CTNNB1 that plays an important role in the pathogenesis of human pituitary adenomas. However, the possibility that mutation of regions outside of our survey or epigenetic mechanism play an important role cannot be excluded.

Adenoma↗
PubMed2026

HLA class I escape drives the evolution of SARS-CoV-2 in human populations.

The role of escape from the cytotoxic T cell (CTL) response in Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) evolution remains controversial. Here, we study the origin and spread of SARS-CoV-2 variants whose mutations reduce presentation by the human leukocyte antigen (HLA) class I alleles common in human populations. We find that 35% of mutations that are characteristic of the variants of concern, and 39% of all subsequent viral mutations, facilitate escape of viral epitopes from presentation. Mutations allowing escape from more common HLA alleles reach higher frequencies, particularly in those countries where these HLA alleles are more frequent, indicating that escape is selected by the local genetic composition of the human host population. We also show that viral mutations that accumulated in general-population transmission chains matched population HLA class I allele frequencies as well as, or better than, mutations acquired during persistent infections, suggesting that selection in favor of escape mutations is not limited to immunocompromised individuals. Together, these data reveal CTL escape as a facet of selection, a driver of evolution, and an epidemiological concern for SARS-CoV-2.

Humans↗
PubMed2006

Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase.

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

Anti-HIV Agents↗
PubMed2025

Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6.

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation reduced host lipid droplet content by NSP6. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6's key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications.

SARS-CoV-2↗
PubMed1994

Growth suppression of normal mammary epithelial cells by wild-type p53.

p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.

Alleles↗
PubMed1986

Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function.

Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Heterozygotes of these insertional mutations possess levels of MHC intermediate between those of haploids and diploids. An additional mutation has no gross alteration of the MHC gene or its RNA transcripts. Although leg and larval muscles function normally in each mutant heterozygote, indirect flight muscles are defective and possess disorganized myofibrils. Homozygous mutants die during embryonic or larval development and display abnormal muscle function prior to death. These findings provide direct genetic evidence that the MHC gene at 36B (2L) is essential for both larval and adult muscle development and function. The results are consistent with the previous molecular evidence that Drosophila, unlike other organisms, has only a single muscle MHC gene per haploid genome. Quantitative expression of both copies of the MHC gene is required for function of indirect flight muscle, whereas expression of a single MHC gene is sufficient for function of larval muscles and adult tubular muscles.

Animals↗
PubMed1998

Excess of hMLH1 germline mutations in Swiss families with hereditary non-polyposis colorectal cancer.

Lynch syndrome, or hereditary non-polyposis colorectal cancer (HNPCC), is a consequence of a dominantly inherited susceptibility to accumulate somatic mutations. The disorder is manifested as a familial aggregation of colorectal cancers diagnosed at an early age and, to a lesser degree, of cancers of the endometrium, ovary, urinary tract, and organs of the gastrointestinal tract other than the colon. In more than half of the HNPCC families investigated, the cancer predisposition has been linked to germline mutations in one of the 2 genes hMLHI or hMSH2, involved in post-replicative DNA-mismatch repair. Twenty-four Swiss families affected with colorectal cancer were screened for germline mutations in these 2 genes, and pathogenic mutations were identified in over 70% of the families fulfilling the Amsterdam criteria (AC), but in only 10% of the families not completely fulfilling these criteria. One of the reported mutations, discovered in an extended HNPCC kindred from the Swiss Alps, is shown to be a founding mutation. Unexpectedly, all the mutations identified are in the hMLHI gene, where all but one are novel sequence alterations. Our data suggest that an unusually high proportion of Swiss HNPCC patients may harbour a germline mutation in the hMLHI gene.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Metal ions as regulators of the conformation and function of the tumour suppressor protein p53: implications for carcinogenesis.

The p53 protein is a multi-function nuclear factor that is activated in response to multiple forms of stress and controls the proliferation, survival, DNA repair and differentiation of cells exposed to potentially genotoxic DNA damage. Loss of p53 function by mutation is a frequent event in human cancer, and is thought to result in the capacity of cells to acquire and accumulate oncogenic mutations during the progression of neoplasia. The p53 protein is a metal-binding transcription factor that is inactivated by metal chelation and by oxidation in vitro. In intact cells, p53 protein activity is crucially dependent on the availability of Zn ions and is impaired by exposure to Cd, a metal which readily substitutes for Zn in a number of transcription factors. Inactivation by Cd suppresses the p53-dependent responses to DNA damage. Overall, these findings indicate that regulation by metals plays an important role in the control of p53, and that perturbation of this control may explain the carcinogenic potential of several metal compounds.

Cadmium↗
PubMed1998

Inherited breast cancer: an emerging picture.

A role for BRCA1 and BRCA2 in the control of genome integrity easily fits a tumor suppressor model. It is well established that mutations in DNA repair genes lead to genomic instability (138). Genomic instability may directly lead to tumorigenesis by allowing for the accumulation of mutations in key cell cycle regulators (139). The studies summarized here suggest that BRCA1, BRCA2, RAD51. and BARD1 function as a biochemical complex. This complex apparently plays a role in one or more of the DNA damage response pathways. Experimental data suggest that BRCA1 and BRCA2 function as regulators of transcription. These observations highlight some of the fundamental questions that remain to be addressed in the study of the biology of these genes. Are the DNA repair and transcriptional regulatory functions of BRCA1 and BRCA2 related? BRCA1 and BRCA2 may maintain the integrity of the genome by regulating expression of genes directly involved in this process. Alternatively, if the functions are not related, which is required for suppression of tumorigenesis? Researchers also are grappling with another paradox. If BRCA1 and BRCA2 are ubiquitously expressed, why do mutations in BRCA1 and BRCA2 lead specifically to tumors primarily of the breast and ovary, as well as a limited number of other tissues to a lesser degree? Nothing to date has been revealed that would explain how alteration of the transcriptional regulatory function and or the DNA repair function ascribed to BRCA1 and BRCA2 would result in tumor specificity as both of these functions are essential to a broad spectrum of tissues. It is possible that BRCAI and BRCA2 may regulate genes expressed only in the breast and ovary. Similarly, there may be unidentified BRCA1 and BRCA2 co-factors that are active only in the breast and ovary and, therefore, are critical to tumorigenesis. All breast cancer is genetic, although only a small fraction of cases are attributable to inherited genetic predisposition. Most breast cancer is due to genetic alterations that are specific to breast epithelial cells, many of which remain unknown. Integration of genetic approaches into research designed to elucidate biological pathways of breast cancer tumorigenesis will ultimately lead to new information critical to the development of new tools for the diagnosis and treatment of disease.

BRCA1 Protein↗
PubMed1993

Accumulation of the F plasmid TraJ protein in cpx mutants of Escherichia coli.

We report here studies of the cellular control of F plasmid TraJ protein levels, focusing on the effects of chromosomal cpx mutations. The principal conclusion from our results is that the cpx mutations impair accumulation of the TraJ protein, thereby reducing tra gene expression. We measured TraJ activity in vivo by expression of a traY'-'lacZ fusion gene and TraJ protein by immuno-overlay blot. In strains with normal TraJ levels, traY expression and donor-related functions were reduced in cells carrying any of four cpxA mutations. In the strain background used to isolate cpx mutants, these reductions were especially evident in cells grown to high density, when traY expression and donor activity both increased in cpx+ cells. In each of the four cpxA mutants tested, TraJ levels were lower than in the otherwise isogenic cpxA+ strain. In cells grown to high density, the differences ranged from 4-fold in the cpxA6 strain to > 10-fold in the cpxA2, cpxA5, and cpxA9 strains. The cpxA2 mutation had little or no effect on traY expression or on donor-related functions when TraJ was present in excess of its limiting level in F' or Hfr cells or on a mutant traY promoter whose expression in vivo was independent of TraJ.

Bacterial Outer Membrane Proteins↗
PubMed2000

DNA polymerase zeta introduces multiple mutations when bypassing spontaneous DNA damage in Saccharomyces cerevisiae.

Spontaneous DNA damage can be dealt with by multiple repair/bypass pathways that have overlapping specificities. We have used a frameshift reversion assay to examine spontaneous mutations that accumulate in yeast strains defective for the high-fidelity nucleotide excision repair or recombination pathways. In contrast to the simple frameshift mutations that occur in wild-type strains, the reversion events in mutant strains are often complex in nature, with the selected frameshift mutation being accompanied by one or more base substitutions. Genetic analyses demonstrate that the complex events are dependent on the Pol zeta translesion polymerase, thus implicating the DNA damage bypass activity of low-fidelity translesion polymerases in hypermutation phenomena.

Base Sequence↗
PubMed2003

HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus.

HLA-B*0702 transgenic mice (expressing a chimeric heavy chain with a murine alpha 3 domain: HLA-B7(m alpha 3)) in which the H-2K(b) and H-2D(b) class I-a (Cl I-a(-/-)) genes have been inactivated were compared with H-2K(b)D(b) Cl I-a(+/+) positive controls. Expression of the HLA-B7(m alpha 3) molecules resulted in a 3- to 4-fold increase in peripheral CD8(+) T lymphocyte numbers compared to H-2 Cl I-a(-/-) knockout mice. These cells show a diversified TCR repertoire. Following influenza infection, a significant improvement in HLA-B0702-restricted cytotoxic T lymphocyte (CTL) responses was observed in HLA-B7(m alpha 3), H-2 Cl I-a(-/-) compared to HLA-B7(m alpha 3), H-2 Cl I-a(+/+) mice. The CTL response of infected HLA-B7(m alpha 3), H-2 Cl I-a(-/-) mice was directed against the nucleoprotein (NP) 418-426 epitope in which mutations have accumulated. Whereas all NP 418-426 variant peptides induced a CTL response, cross-reactivity to the variants was affected. These NP mutations could have been selected over time in humans for the virus to escape HLA-B0702-restricted CTL responses since a similar response was seen in humans with, as in mice, altered cross-recognition of the NP 418-426 variants. These animals may prove a suitable model to study HLA-B0702-restricted CTL responses.

Animals↗
PubMed1989

Genetic and biochemical characterization of a mutation (fatA) that allows trans unsaturated fatty acids to replace the essential cis unsaturated fatty acids of Escherichia coli.

Unsaturated fatty acid auxotrophs of Escherichia coli are able to use only unsaturated fatty acids of the cis configuration as the required growth supplement. A mutation in the fatA gene allows such auxotrophs to utilize unsaturated fatty acids with a trans double bond as well as fatty acids having a cis double bond. The fatA gene was mapped to min 69 near argG, and the allele studied (fatA1) was found to be dominant over the wild-type gene. fatA1 mutant strains grew at similar rates when supplemented with elaidate (trans-9-octadecenoate) or oleate (cis-9-octadecenoate). The fat+ strain, however, lysed when supplemented with the trans fatty acid. Physiological characterization of the fatA mutant strain was undertaken. The mutation appeared not to be involved with long-chain fatty acid transport. Introduction of lesions in known fatty acid transport genes abolished trans fatty acid utilization in the fatA mutant strain. Also, growth characteristics of the fat+ and the fatA1 mutant strains on elaidate as the sole carbon source were identical, which indicated comparables rate of fatty acid accumulation. The mutation appeared to be involved with recognition of the trans configuration after uptake into the cell. The levels of trans fatty acid incorporation into the phospholipids of the fat+ and the fatA strains differed considerably, with the mutant incorporating much higher levels. No significant accumulation of elaidate into nonphospholipid cellular components was observed. The fatA mutation did not appear to be involved with the cellular metabolic state, as cyclic AMP had no effect on the ability of the strains to utilize trans fatty acids.

Chromosome Mapping↗
PubMed1994

Hereditary non-polyposis colorectal cancer--morphologies, genes and mutations.

Mutations in a human homologue of the yeast DNA mismatch repair gene MSH2 (equivalent to bacterial MutS) cause the condition hereditary non-polyposis colorectal cancer (HNPCC). Cancers in HNPCC originate within benign neoplastic polyps termed adenomas. Adenomas are clonal and each may serve as a marker of a single initiating mutation. The progression of adenomas is marked by increasing size, dysplasia and villosity. These characteristics can be taken as the morphological counterparts of the stepwise accumulation of mutations implicating oncogenes and tumour suppressor genes. The aim of this study was to link the morphogenesis of hereditary colorectal cancer with recent insights into the role of DNA mismatch repair genes. The frequency and anatomical distribution of adenomas in at-risk members of HNPCC families was the same as in an autopsy population. This suggests that the HNPCC gene does not initiate the process of neoplastic transformation. On the other hand, adenomas in at-risk members of HNPCC families were more likely to show villosity (p < 0.001), high grade dysplasia (p = 0.002) and probably increased size (p = 0.15). These findings are consistent with the observation that the HNPCC gene causes DNA replication errors to develop and accumulate within neoplastic but not normal tissues. The effect of the HNPCC gene is to accelerate the progression of adenoma to carcinoma, but not to initiate adenoma development.

Adenoma↗
PubMed2001

The homeobox gene CDX2 in colorectal carcinoma: a genetic analysis.

Accumulation of mutations in tumour suppressor genes and oncogenes has been proposed to underlie the initiation and progression of sporadic colorectal cancer (CRC). Evidence is accumulating to suggest that the caudal homeobox gene CDX2 is implicated in the pathogenesis of CRC. The CDX2 transcription factor is expressed in intestinal epithelium and is markedly down-regulated in colon tumours. Furthermore, Cdx2 heterozygous null mice develop multiple intestinal tumours. In this present study, we have investigated CDX2 as a potential candidate gene for sporadic CRC by a thorough search of all exons and exon/intron boundaries for DNA polymorphisms and rare variants in a panel of CRC tumours. 6 polymorphisms were identified and the haplotypes determined. In addition two rare variants were found, one of which was only identified in DNA from a CRC case. Loss of heterozygosity was observed in 3 out of 28 informative CRC cases. A possible association between particular haplotypes and tumour progression was also suggested by the data. In addition a preliminary analysis of the relative expression of CDX2 alleles in tumour/normal tissue suggested some variation in the levels, however further analysis is required before any conclusions can be drawn. While CDX2 mutations predisposing to sporadic CRC have not been identified, this study has established that loss of CDX2 contributes towards the progression of some sporadic CRC tumours.

Adult↗
PubMed1999

Hprt mutant frequency and molecular analysis of Hprt mutations in rats treated with mutagenic carcinogens.

Much of the progress in the field of cancer research has come from the increased understanding of the molecular events associated with the initiation and accumulation of mutational events associated with carcinogenesis. Genetic toxicologists have developed a number of in vitro and in vivo non-mammalian and mammalian systems to predict those genetic events required to induce the cancer process. Several model rodent systems have been proposed that have the ability to detect and quantify in vivo somatic mutation in endogenous genes and transgenes and relate the nature of the mutation to the specific type of chemical damage. One such system, the rat lymphocyte hypoxanthine guanine phosphoribosyl transferase (Hprt) assay is described in this review. Data are presented that describe mutant induction and mutational spectra in N-ethyl-N-nitrosourea (ENU), 7,12-dimethylbenzo[a]anthracene (DMBA) and thiotepa (TEPA) treated rats.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Spontaneous mutations affecting glycerol-3-phosphate dehydrogenase enzyme activity in Drosophila melanogaster.

Significant genetic variance in glycerol-3-phosphate dehydrogenase (GPDH) activity was observed between chromosome lines of Drosophila melanogaster that had each accumulated spontaneous mutations for approximately 300 generations. No restriction map variation was found in a 26-kb region surrounding the entire Gpdh gene. The restriction analysis used is capable of detecting insertions/deletions larger than 0.05 kb. The survey would also detect chromosomal recombinations that include the entire Gpdh coding region. Therefore, if the spontaneous mutations that affected the enzyme activity are located inside the Gpdh gene region, then they are base pair substitutions or structural changes that are smaller than the limit in resolution described above.

Alcohol Dehydrogenase↗