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At least 415 records · Page 23Linked to original sources
PubMed2005

Reversal of the ATM/ATR-mediated DNA damage response by the oncogenic phosphatase PPM1D.

The eukaryotic cell has evolved a sophisticated set of cell signaling pathways that respond to DNA damage and efficiently repair that damage, protecting the cell from deleterious mutations, genomic instability, and transformation into a cancerous state. The ATM and ATR serine/threonine kinases are key sensors and transducers of DNA damage signals through phosphorylation of an array of signaling molecules that mediate all aspects of the DNA damage response, including enforcement of cell cycle checkpoints and direct repair of damaged DNA. We have shown that a type 2C serine/threonine phosphatase, PPM1D (or Wip1), can reverse the phosphorylation status of ATM/ATR-phosphorylated proteins p53 and Chk1. This dephosphorylation of p53 and Chk1 by PPM1D may result in reduced functional activities and is accompanied by suppression of DNA damage-induced cell cycle checkpoints and some aspects of DNA repair. Because PPM1D is transcriptionally activated by p53 in response to DNA damage, PPM1D may serve as a critical component of a p53 negative feedback regulatory loop since it now appears that PPM1D can inhibit p53 activity by at least four different molecular mechanisms. This may explain why PPM1D is amplified and overexpressed in a subset of human breast cancers that invariably retain wild type p53 alleles. We hypothesize that PPM1D is a homeostatic regulator of the DNA damage response that returns the cell to a more normal unstressed state following repair of the damage.

Alleles↗
PubMed1995

Arabidopsis phosphoribosylanthranilate isomerase: molecular genetic analysis of triplicate tryptophan pathway genes.

Phosphoribosylanthranilate isomerase (PAI) catalyzes the third step of the tryptophan biosynthetic pathway. Arabidopsis PAI cDNAs were cloned from a cDNA expression library by complementation of an Escherichia coli trpC- PAI deficiency mutation. Genomic DNA blot hybridization analysis detected three nonallelic genes encoding PAI in the Arabidopsis genome. DNA sequence analysis of cDNA and genomic clones indicated that the PAI1 and PAI2. All three PAI polypeptides possess an N-terminal putative plastid target sequence, suggesting that these enzymes all function in plastids. The PAI1 gene is flanked by nearly identical direct repeats of approximately 350 nucleotides. Our results indicate that, in contrast to most microorganisms, the Arabidopsis PAI protein is not fused with indole-3-glycerolphosphate synthase, which catalyzes the next step in the pathway. Yeast artificial chromosome hybridization studies indicated that the PAI2 gene is tightly linked to the anthranilate synthase alpha subunit 1 (ASA1) gene on chromosome 5. PAI1 was mapped to the top of chromosome 1 using recombinant inbred lines, and PAI3 is loosely linked to PAI1. cDNA restriction mapping and sequencing and RNA gel blot hybridization analysis indicated that all three genes are transcribed in wild-type plants. The expression of antisense PAI1 RNA significantly reduced the immunologically observable PAI protein and enzyme activity in transgenic plants. The plants expressing antisense RNA also showed two phenotypes consistent with a block early in the pathway: blue fluorescence under UV light and resistance to the anthranilate analog 6-methylanthranilate. The extreme nucleotide conservation between the unlinked PAI1 and PAI2 loci suggests that this gene family is actively evolving.

Aldose-Ketose Isomerases↗
PubMed1999

Uniparental isodisomy resulting from 46,XX,i(1p),i(1q) in a woman with short stature, ptosis, micro/retrognathia, myopathy, deafness, and sterility.

We report on a 43-year-old woman who was referred for evaluation because of minor facial anomalies, myopathy, sterility, short stature, hearing loss, downward slant of palpebral fissures, bilateral ptosis, severe micro/retrognathia, high arched palate, and scoliosis. Cytogenetic analyses utilizing GTG/CBG bandings showed presence of one i(1p) and one i(1q) without normal chromosome 1 homologues. Fluorescence in situ hybridization analysis showed hybridization to only two chromosomes, consistent with the G-banded interpretation of i(1p) and i(1q). To the best of our knowledge, this is the first case of isochromosomes 1p and 1q replacing the two normal chromosome 1s. Molecular investigations using markers for chromosome 1 showed inheritance of only one set of paternal alleles and absence of any maternal alleles in the patient. The adverse phenotype of the patient may be due to one or more recessive mutations, genomic imprinting, or a combination of both.

Abnormalities, Multiple↗
PubMed1996

A simplified method for the repeated replacement of yeast chromosomal sequences with in vitro mutations.

A strategy for gene replacement in Saccharomyces cerevisiae has been modified to facilitate the repeated substitution of a chromosomal locus with in vitro generated variant sequences, so that the resulting locus contains only the desired mutation and is free of extraneous vector DNA. The construction of an internally deleted chromosomal target locus carrying the counterselectable CYH2S marker and a second positively selectable marker has been simplified; the design of the locus has been altered to increase the frequency of authentic gene replacements obtained upon the subsequent integration of in vitro mutated DNA. The modified chromosomal target locus is amenable to replacement using either of two transformation protocols: (i) integration of a second positively selectable plasmid carrying mutant sequences to form a tandem intermediate structure at the locus; upon counterselection on cycloheximide, all vector sequence is excised to give the desired replacement at high frequency (> 70%); (ii) single-step integration of a linear segment of mutated genomic DNA by selection for cycloheximide resistance. A subsequent screen for the loss of the positively selectable target locus marker detects the desired replacement at modest frequency (> 2%). Polymerase chain reaction using multiple primers in a single amplification reaction is useful for monitoring these variously modified chromosomal loci.

Chromosomes, Fungal↗
PubMed1999

Clinical impact of molecular genetic diagnosis, genetic counseling, and management of hereditary cancer. Part I: Studies of cancer in families.

Hereditary cancer represents approximately 5-10% of the total cancer burden and may account for 60,000 to 120,000 new cancer occurrences this year in the United States. New developments in molecular genetics and the cloning of cancer-prone genes have intensely fueled interest in dealing with hereditary forms of cancer. The authors provide an algorithm that depicts the process for the identification, study, and DNA-based genetic counseling of families being investigated under a research proposal at the Hereditary Cancer Institute of Creighton University School of Medicine. They have studied 56 hereditary nonpolyposis colorectal carcinoma families; in 18 of them, associated genomic mutations have been identified in affected members. DNA-based genetic counseling has been provided for seven of these families. The authors have also evaluated 131 hereditary breast-ovarian carcinoma families. BRCA1 and BRCA2 mutation searches have been performed for 76 of these families; BRCA1 mutations were found in 38 families and BRCA2 mutations in 9. The study of cancer-prone families is a powerful approach to cancer control, particularly when the germ-line mutation is identified in the family and individuals at high risk can be tested, once they provide informed consent, and receive DNA-based genetic counseling. Discovery of the germ-line mutation for cancer proneness provides an unparalleled opportunity to predict patients' life-time risk for cancer of specific anatomic sites, inclusive of a pattern of multiple primaries. Surveillance and management protocols, when melded to the particular syndrome's natural history, can be life-saving.

Clinical Protocols↗
PubMed1999

The use of arbitrarily primed polymerase chain reaction in cancer research.

There is a great need for techniques that detect the genome alterations present in cancer cells. Here, we present a review of the arbitrarily primed polymerase chain reaction (AP-PCR), a genomic mutation detection method with some unique advantages: (i) It can detect most types of mutations that usually occur in tumors (except point mutations). (ii) It is especially useful to detect moderate gains in DNA, which most methods currently in use cannot detect. (iii) It allows detection and cloning of alterations in a single experiment. (iv) It is inexpensive and does not require special equipment. We discuss some characteristics of this method and review some of its achievements in cancer research.

Acrylic Resins↗
PubMed2007

Expression of activation-induced cytidine deaminase in human hepatocytes during hepatocarcinogenesis.

Activation-induced cytidine deaminase (AID) plays a role as a genome mutator in activated B cells, and inappropriate expression of AID has been implicated in the immunopathological phenotype of human B-cell malignancies. Notably, we found that the transgenic mice overexpressing AID developed lung adenocarcinoma and hepatocellular carcinoma (HCC), suggesting that ectopic expression of AID can lead to tumorigenesis in epithelial tissues as well. To examine the involvement of AID in the development of human HCC, we analyzed the AID expression and its correlation with mutation frequencies of the p53 gene in liver tissues from 51 patients who underwent resection of primary HCCs. The specific expression, inducibility by cytokine stimulation and mutagenic activity of AID were investigated in cultured human hepatocytes. Only trace amounts of AID transcripts were detected in the normal liver; however, endogenous AID was significantly upregulated in both HCC and surrounding noncancerous liver tissues with underlying chronic hepatitis or liver cirrhosis (p < 0.05). Most liver tissues with underlying chronic inflammation with endogenous AID upregulation already contained multiple genetic changes in the p53 gene. In both hepatoma cell lines and cultured human primary hepatocytes, the expression of AID was substantially induced by TGF-beta stimulation. Aberrant activation of AID in hepatocytes resulted in accumulation of multiple genetic alterations in the p53 gene. Our findings suggest that the aberrant expression of AID is observed in human hepatocytes with several pathological settings, including chronic liver disease and HCC, which might enhance the genetic susceptibility to mutagenesis leading to hepatocarcinogenesis.

Adult↗
PubMed1994

Yap1p, a yeast transcriptional activator that mediates multidrug resistance, regulates the metabolic stress response.

Overexpression of the YAP1 transcriptional activator renders yeast cells resistant to multiple metabolic inhibitors. In an effort to identify other gene products required for this phenotype we have isolated genomic mutations which neutralize this effect. One such mutation was further characterized and the affected gene was shown to be identical to TPS2 which encodes trehalose phosphate phosphatase, an enzyme catalysing the second step in trehalose biosynthesis. We have analysed the transcriptional regulation of the TPS2 gene and have shown that its transcription is induced by a variety of stressful conditions caused by metabolic inhibitors, osmotic shock and heat shock. This transcriptional activation is mediated by multiple stress promoter elements (C4T) and requires the function of Yap1p as well as reduced activity of the cAMP-regulated protein kinase. Using an appropriate reporter gene we have shown that Yap1p is generally required for transcriptional regulation through the C4T stress element. These results show that the YAP1 protein has a pivotal role in the metabolic stress response and the acquisition of stress tolerance.

Adaptation, Biological↗
PubMed1983

Recombinant DNA strategies in genetic neurological diseases.

The application of recombinant DNA techniques applied to the study of genetic neurological diseases will play a major role in the practice of neurology in upcoming years. Strategies are now available to develop useful and relatively simple biochemical diagnostic tests for heterozygous individuals with diseases inherited as autosomal dominant traits. In addition, molecular genetic methods will lead to the delineation of the genomic mutations responsible for these diseases. This review will update the current status of research in several neurological genetic diseases including myotonic muscular dystrophy, Huntington's disease, Charcot-Marie-Tooth disease and Duchenne muscular dystrophy (X-linked). An introduction and overview of the methodology is provided. Specific research strategies including random screening of libraries, chromosome walking, messenger RNA selection, and messenger RNA translation are described. These strategies are designed to provide heterozygote identification, prenatal diagnosis and gestational management, the development of rational therapies, and the understanding of the molecular basis of disease expression.

Charcot-Marie-Tooth Disease↗
PubMed1994

An NF1 motif plays a central role in hepatitis B virus enhancer.

The hepatitis B virus enhancer plays an important role in transcription regulation of the viral genes in a liver-specific manner. In animal models a homologous element seems to be involved in activation of cellular oncogenes and tumorigenesis. Previously, the enhancer was divided into several functional domains, whereby each one seemed to be required for optimal transcription activity. To gain more information on the mode of action of these elements and their role in viral genome, we mutagenized the individual enhancer elements and analyzed their functions in three different experimental systems. All show that the NF1b motif of the enhancer plays a central role, with the most dramatic results obtained from the cell-free in vitro transcription assay. Furthermore, an intact viral genome mutated at the NF1b site is a poor template for the synthesis of the 3.5-kb pregenomic RNA. These data are rather unexpected, given the ubiquitous appearance of this factor. On the other hand, our findings are in agreement with a large number of recently reported cases in which NF1 seems to determine tissue-specific expression of a wide range of cellular and viral promoters.

Animals↗
PubMed1996

Neutralizing epitope on penetration protein of vaccinia virus.

The monoclonal antibody 2D5 neutralized vaccinia virus by preventing penetration of the virus and reacting with VP23-29K. The conformation of the VP23-29K was maintained by a disulfide bond(s), and the 2D5mAb reacted stronger with the nonreduced 23-kDa form than with the reduced 29-kDa form. We selected several escape mutants. Sequences of the A17L genes, which were thought to encode the VP23-29K, did not show cognate mutation. Genomic DNA of a 2D5mAb-resistant mutant (M4) was cleaved with HindIII, and all the fragments were introduced into parental IHD-J strain vaccinia virus by transfection. Only the L fragment produced a 2D5mAb-resistant virus. Dissection of the L fragment and subsequent transfection revealed that the L1R gene induced the 2D5mAb-resistant virus. The 2D5mAb-resistant mutants showed a consensus G to A conversion at nucleotide 101 of their LIRs which would replace asparatic acid 35 with asparagine. Ishibashi-111 strain mousepox virus spontaneously resistant to 2D5mAb also had the same sequence at this region. Moreover, the VP23-29K was myristoylated as predicted by the L1R gene. The coding gene of the VP23-29K was L1R.

Amino Acid Sequence↗
PubMed1985

Homologous recombination of polyoma virus DNA in mouse cells.

We have produced nonviable deletion mutants of polyoma virus in order to study homologous recombination after DNA transfection into mouse cells. The frequency of recombination was determined by the formation of infectious virus. It was dependent on the amount of DNA transfected and the size of the region of homology between the mutations. Recombination frequencies were highest when both mutated genomes were transfected in closed circular form rather than after linearization of one or both of the recombination partners. The system described may be useful for a more detailed analysis of physiological and genetic conditions influencing the frequency of homologous recombination in mouse cells as well as to study enzymes involved and intermediates produced in this process.

Animals↗
PubMed1997

Association of diabetic neuropathy with Na/K ATPase gene polymorphism.

Diabetes mellitus induces a decrease in Na/K ATPase activity in man and animals, and this decrease plays a role in the development of diabetic neuropathy. Na/K ATPase is encoded by various genes, of which the ATP1 A1 gene is expressed predominantly in peripheral nerves and in erythrocytes. To investigate whether a polymorphism in the Na/K ATPase genes could explain the predisposition of some patients with insulin-dependent diabetes mellitus (IDDM) to develop polyneuropathy, a restriction fragment length polymorphism (RFLP) of the ATP1 A1 gene was studied together with erythrocyte Na/K ATPase activity in 81 Caucasian patients with more than 10 years' duration of IDDM. Associations with diabetic neuropathy, retinopathy and nephropathy were sought. Digestion of the first intron of the ATP1 A1 gene by the Bgl II restriction enzyme revealed a dimorphic allelism. Frequency of the restricted allele was 0.18 in this selected series (however, it was 0.10 in representative samples of IDDM patients and of normal subjects in our area). Mean erythrocyte Na/K ATPase activity was lower in diabetic patients than in 42 control subjects (292 +/- 10, vs 402 +/- 13 nmol Pi.mg protein-1.h-1, p < 0.0001) and was not related to HbA1c value or to diabetes duration. It was lower in the group of the 28 patients bearing the restricted allele (241 +/- 10 vs 319 +/- 11 nmol Pi.mg protein-1.h-1, p < 0.0001). Neuropathy was absent in 50 patients, mild in 15 and severe in 16. When classified accordingly the three groups of patients did not differ with respect to sex, age and duration of diabetes. The respective frequency of the restricted allele among the groups was 10, 73 and 81%, (p < 0.0001) and mean erythrocyte Na/K ATPase activity was respectively: 322 +/- 10.7 nmol Pi.mg protein-1.h-1, 268 +/- 15 and 229 +/- 17, (p < 0.001). A borderline association between renal status or retinal status and repartition of polymorphism and a borderline correlation between renal status and Na/K ATPase activity were found, but significance disappeared after checking for the presence or absence of neuropathy. IDDM patients bearing the ATP1 A1 variant detected by Bgl II RFLP are much more frequently affected by neuropathy (relative risk 6.5, with 95% CI 3.3-13). Identification of this risk factor may help to prevent this complication. It is suggested that the restricted allele is in linkage disequilibrium with a genomic mutation allowing diabetes to induce a greater impairment of Na/K ATPase activity which could in turn favour the development of neuropathy.

Adult↗
PubMed1998

Structural organization of the human short-chain L-3-hydroxyacyl-CoA dehydrogenase gene.

The third step in the mitochondrial beta-oxidation spiral of short-chain fatty acids is catalyzed by short-chain L-3-hydroxyacyl-CoA dehydrogenase (HADHSC; EC 1.1.1.35). We have determined the structural organization of the human HADHSC gene by sequencing of cloned genomic amplification products, obtained using HADHSC-specific cDNA-based primers, as well as by direct sequencing of an isolated PAC clone containing the HADHSC gene. Upon comparison with the HADHSC cDNA sequence, HADHSC was shown to encompass at least eight exons, ranging in size from 73 to 158 bp, and 7 introns. The total HADHSC gene spans approximately 49 kb. The HADHSC 5'-flanking region was characterized with an AluI plasmid library constructed from a partially AluI-digested PAC clone containing the human HADHSC gene. Several typical promoter elements such as a CAAT-box, Sp1, AP1, and AP2 sites were found, while a TATA-box was apparently absent. Among other putative regulatory elements, a NRRE-1 site was identified. By radiation hybrid panel, assisted fine-mapping HADHSC was linked to marker AFM070TH5, corresponding to Chromosome (Chr) 4q22-26, and a putative HADHSC pseudogene was linked to marker D15S1324, located at Chr 15q17-21. Knowledge of the genomic organization and 5'-flanking region of HADHSC will enable genomic mutation analysis of patients suspected of HADHSC deficiency, as well as facilitate the investigation into the transcriptional regulation of short-chain fatty acid oxidizing gene products in general and HADHSC expression in particular.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2004

The peptide nucleic acids (PNAs): introduction to a new class of probes for chromosomal investigation.

Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed. Peptide nucleic acids hybridize with complementary nucleic acids with remarkably high affinity and specificity, essentially because of their uncharged and flexible polyamide backbone. The unique physicochemical properties of PNAs have led to the development of a large variety of biological research assays, and, over the last few years, PNAs have proved their powerful usefulness in genetic and cytogenetic diagnostic procedures. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic mutation or capturing nucleic acids. The more recent applications of PNA involve their use as molecular hybridization probes. Thus, the in situ detection of several human chromosomes has been reported in various types of tissues.

Chromosomes, Human↗
PubMed1997

Interstrain variation of immediate early DNA sequences and glycoprotein B genotypes in cytomegalovirus clinical isolates.

Cytomegalovirus (CMV) disease is associated with a high mortality in recipients of an allogeneic stem cell transplant. Apparent differences in biological behaviour have been noted among clinical CMV isolates. By amplifying specific functionally relevant regions of the CMV genome [immediate early (IE) exon 3, glycoprotein B (gB)], a possible association of strain variation and clinical symptoms of infection was analysed in 24 patients. A high number of genome mutations of the IE exon 3 region could be documented translating into amino acid changes of viral isolated of 8 out of 15 patients with symptomatic and 2 out of 9 patients with asymptomatic CMV infection. Identical IE mutations and gB types were observed in isolates from two different sites in 6 patients. gB strain 2 was found to be associated with symptomatic CMV infection (P = 0.03). Thus, apart from host factors viral factors might influence the virus-host interaction in severely immunosuppressed patients.

Amino Acid Sequence↗
PubMed1989

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif.

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.

Base Composition↗
PubMed1992

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.

Adenosine Triphosphatases↗