Analyzing the fidelity of reverse transcription and transcription.
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Avian Myeloblastosis Virus↗
Biomedical subjects
J C Boyer
Publications and source records attributed to J C Boyer.
The movement proteins of cowpea mosaic virus and cauliflower mosaic virus induce tubular structures in plant and insect cells.
The movement proteins (MP) of cowpea mosaic virus and cauliflower mosaic virus (CaMV) are associated with tubular structures in vivo which participate in the transmission of virus particles from cell to cell. Both proteins have been expressed in plant protoplasts and insect cells. In all cases, immunofluorescent histochemistry showed that the MPs accumulate intracellularly as tubular extensions projecting from the cell surface. Additionally, electron microscopy revealed intracellular MP aggregates in CaMV MP-expressing cells. The data presented establish common features for the tubule-forming MPs: no other virus gene products are required for tubule formation and unique plant components (e.g. plasmodesmata) are not essential for tubule synthesis.
Animals↗
Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines.
The instability of short repetitive sequences in tumor DNA can result from defective repair of replication errors due to mutations in any of several genes required for mismatch repair. Understanding this repair pathway and how defects lead to cancer is being facilitated by genetic and biochemical studies of tumor cell lines. In the present study, we describe the mismatch repair status of extracts of 22 tumor cell lines derived from several tissue types. Ten were found to be defective in strand-specific mismatch repair, including cell lines from tumors of the colon, ovary, endometrium, and prostate. The repair defects were independent of whether the signal for strand specificity, a nick, was 5' or 3' to the mismatch. All 10 defective cell lines exhibited microsatellite instability. Repair activity was restored to 9 of these 10 extracts by adding a second defective extract made from cell lines having known mutations in either the hMSH2 or hMLH1 genes. Subsequent analyses revealed mutations in hMSH2 (4 lines) and hMLH1 (5 lines) that could explain the observed microsatellite instability and repair defects. Overall, this study strengthens the correlation between microsatellite instability and defective mismatch repair and the suggestion that diminuition in mismatch repair activity is a step in carcinogenesis common to several types of cancer. It also provides an extensive panel of repair-proficient and repair-deficient cell lines for future studies of mismatch repair.
Adaptor Proteins, Signal Transducing↗
Microsatellite instability in gynecological sarcomas and in hMSH2 mutant uterine sarcoma cell lines defective in mismatch repair activity.
We have examined a panel of gynecological sarcomas for microsatellite instability. The genomic DNA from 11 of 44 sarcomas contained somatic alterations in the lengths of one or more di-, tri-, tetra-, or pentanucleotide microsatellite sequence markers, and 6 of these cases had alterations in two or more markers. In addition, di-, tri-, and tetranucleotide microsatellites were found to be highly unstable in single cell clones of two cell lines derived from a uterine mixed mesodermal tumor. Since such instability is characteristic of cells defective in postreplication mismatch repair, we examined mismatch repair activity in extracts made from these lines. Both extracts were repair deficient, while an extract of another gynecological sarcoma cell line not exhibiting microsatellite instability was repair proficient. The repair deficiency was complemented by a colon tumor cell extract that was defective in the hMLH1 protein but not by an extract defective in hMSH2 protein. This suggested that the defect in the uterine sarcoma line could be in hMSH2. Subsequent analysis of the gene revealed a 2-bp deletion in exon 14, leading to premature truncation of the hMSH2 protein at codon 796 and no detectable wild-type gene present. These data suggest that the microsatellite instability observed in these cell lines, and possibly in a significant number of gynecological sarcomas, is due to defective postreplication mismatch repair. There was no apparent correlation with microsatellite instability and clinical outcome.
DNA Repair↗
[Hemoperitoneum caused by spontaneous rupture of hepatocellular carcinoma. Value and prognostic factors of hepatic artery embolization].
To assess efficiency and to determine prognostic factors in emergency embolization for intraperitoneal hemorrhage from hepatocellular carcinoma we retrospectively analyzed 12 consecutive procedures. In order to evaluate the extension of tumor thrombus in the intrahepatic portal system before embolization, a portal perfusion rate was defined as the relative patency of the segmental portal veins demonstrated at arteriography. Serum bilirubin level and portal perfusion rate were correlated with length of survival. Successful hemostasis was achieved in all patients. Mean length of survival was 149 days. Two patients are still alive 545 and 195 days, respectively, after the procedure. A significant correlation (p = 0.005) between portal perfusion rate and length of survival was obtained. There was no correlation between serum bilirubin level and prognosis. Emergency embolization is an effective treatment in patients with intraperitoneal hemorrhage from hepatocellular carcinoma. Prognosis depends of portal perfusion rate.
Aged↗
DNA loop repair by human cell extracts.
An activity in human cell extracts is described that repairs DNA with loops of five or more unpaired bases. Repair is strand-specific and is directed by a nick located 5' or 3' to the loop. This repair is observed in a colorectal cancer cell line that is devoid of a wild-type hMLH1 gene and is deficient in repair of mismatches. However, a cell line with deletions in both hMSH2 alleles is deficient in repair of both loops and mismatches. Defects in loop repair may be relevant to the repetitive-sequence instability observed in cancers and other hereditary diseases.
Adaptor Proteins, Signal Transducing↗
Defective mismatch repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability.
A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.
Cell Line↗
Infectious transcripts and cDNA clones of RNA viruses.
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Cloning, Molecular↗
Radioanatomic study of the gastrocolic venous trunk.
The mode of formation, measurements and frequency of occurrence of the gastrocolic venous trunk were studied by the injection-corrosion technique in a series of 54 anatomic specimens and by the analysis of 50 CT studies in patients without hepatic or pancreatic disease. The gastrocolic trunk was found in 51 of the 54 anatomic specimens and in 27 of the 50 CT studies. The great variability in its formation, whether bipodal, tripodal or quadripodal, was noted. With a mean diameter of 4.9 mm, it opened into the anterior, right or antero-left aspects of the superior mesenteric v. at a mean distance of 15 mm below the inferior border of the spleno-mesenteric confluence. The value of preliminary CT assessment before an operation for portal hypertension or a pancreatic tumor or in the diagnosis of a splenic thrombosis is emphasised.
Aged↗
[Results of endoprostheses in the iliac arteries; an experience apropos of 47 implantations].
Obstruction or stenosis of the iliac artery was treated by placement of an endoprothesis in 41 patients. A total of 47 lesions was treated. 26 patients had poor results of balloon angioplasty including residual stenosis (7 cases), restenosis post angioplasty (7 cases), and dissection (12 cases). 15 patients had iliac occlusion. The endoprothesis used was a self expandable flexible endoprothesis (Wallstent) in 26 cases and a balloon expandable flexible endoprothesis (Strecker) in 21 cases. Complications included one thrombosis of the endoprothesis occurred immediately after placement, one hematoma. No distal embolization occurred. Patency at the end of the study was 70% at 9 months and 50% at 21 months. Patency was better with the Wallstent endoprothesis (60% at 21 months) than with the Strecker endoprothesis (38% at 21 months). Results was better with lesions of the common iliac artery, and when the indication was dissection or failure of angioplasty. During the 21 months of follow-up 18 occlusions or restenosis occurred. At the end of the study 20 patients (49%) were asymptomatic and improvement was achieved in 9 (22%), the clinical success rate was 71%. One amputation was required.
Aortic Dissection↗
[Contribution of x-ray computed tomography in the diagnosis of cervical suppuration].
The authors report their experience with computed tomography (CT) in 27 cases of cervical suppurations. The diagnosis was confirmed surgically in 24 cases and by the improvement of clinical signs and CT after antibiotics in 3. Two types of CT features were identified: collected abscesses (n = 22) visualized as encapsulated heterogeneous hypodense formations and extensive suppurative cellulitis (n = 5) formed by large diffuse hypodense collections without a clearly visible peripheral wall. The extension of the suppurative process could be evaluated precisely with CT, especially in certain anatomic areas where physical examination is difficult including mediastinal suppurations (n = 2), extension of parapharyngeal suppurations to the retrostyleal area (n = 1), and pterygo-maxillary and epidural collections (1 each) the latter in combination with a tuberculous abscess. Computed tomography is also useful in the etiological diagnosis (search for foreign body, cystic malformations, tuberculosis) and in post-treatment follow-up to visualize the absence of collections after cure in cases of favourable outcome and to determine the extent of the suppurative process in case of complications.
Adult↗
Transgenic plants that express genes including the 3' untranslated region of the turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection.
In order to evaluate new possibilities for protecting plants against virus infection by interference with viral replication, two chimeric genes were constructed in which the (+) strand 3'-terminal 100 nucleotides (nt) of the noncoding region of the turnip yellow mosaic virus (TYMV) genome were placed downstream from the sense or antisense cat coding region. The two chimeric genes were then introduced into the genome of rapeseed (Brassica napus) using an Agrobacterium rhizogenes vector system. Plants expressing high levels of either chimeric gene showed partial protection against infection by TYMV RNA or virions. One interesting feature of the protection is that a proportion of the inoculated transgenic plants does not become infected. Protection was overcome when the inoculum concentration was increased. RNA complementary to the initial transcript was detected after infection.
Base Sequence↗
Fidelity of DNA replication by extracts of normal and malignantly transformed human cells.
To test the hypothesis that a mutator phenotype may be associated with carcinogenesis (L. A. Loeb, Cancer Res., 51: 3074-3079, 1991), we have compared the fidelity of double-stranded DNA replication and the efficiency of mismatch repair in extracts from normal diploid and malignantly transformed human cells. Included was a diploid fibroblast strain and its transformed derivative, as well as a second diploid fibroblast strain and HeLa cells. The fidelity of DNA replication by cytoplasmic extracts in the presence of simian virus 40 large tumor antigen (SV40 T-antigen) was measured using a forward mutagenesis assay. The replicated DNA consisted of double-stranded M13 mp2 DNA containing the SV40 origin of replication and the lacZ alpha complementation gene as a target sequence for scoring mutations. T-antigen-dependent replication was detected in all cell extracts, with those from transformed cells having the greatest activity. No differences in replication fidelity were detected between normal and transformed cell extracts. Using a heteroduplex containing a G.G mispair, we also detected mismatch repair activity in the cell extracts, including efficient repair in extracts from malignantly transformed cells. While these data do not eliminate the possibility that a mutator phenotype may be associated with carcinogenesis, they do suggest that genetic instability associated with transformation does not involve reduced fidelity of replication of undamaged DNA or reduced mismatch repair efficiency.
Cell Line, Transformed↗
Electrotransfection of turnip yellow mosaic virus RNA into Brassica leaf protoplasts and detection of viral RNA products with a non-radioactive probe.
We describe here a convenient and efficient system for studying turnip yellow mosaic virus (TYMV) replication in leaf protoplasts. Inoculation of rapeseed (Brassica napus) or Chinese cabbage (B. sinensis) protoplasts was achieved via electroporation, and sensitive detection of viral RNA products was performed by Northern blot analyses using a non-radioactive digoxigenin-labelled cDNA probe. Virus replication was detected when 1.5 x 10(6) rapeseed protoplasts were inoculated with 20 ng of TYMV RNA. Electrotransfection of TYMV RNA was more efficient in rapeseed than in Chinese cabbage protoplasts, and gave somewhat higher signals than those of TYMV virions. TYMV RNA appeared to replicate equally well whether the protoplasts were incubated in the dark or under constant light.
Blotting, Northern↗
Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates.
Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was greater than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.
Avian Myeloblastosis Virus↗
Role of postreplication repair in transformation of human fibroblasts to anchorage independence.
Cellular capacity for postreplication repair (PRR) and sensitivity to transformation to anchorage independence (AI) were quantified in normal foreskin and xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo(a)pyrene-diol-epoxide I (BPDE-I). PRR is defined here as a collection of pathways that facilitate the replication of DNA damaged by genotoxic agents. It is recognized biochemically as the process by which nascent DNA grows longer than the average distance between two lesions in the DNA template. PRR refers more directly to the elimination of gaps in the daughter-strand DNA by mechanisms which remain to be determined for human cells, but which may include translesion replication and recombination. PRR was measured in diploid human fibroblasts by analysis of the dose kinetics for inhibition of DNA strand growth in carcinogen-treated cells. Logarithmically growing foreskin fibroblasts (NHF1) displayed D0 values of 4.3 J/m2 and 0.14 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. XP variant cells (CRL1162) exhibited corresponding D0 values of 1.5 J/m2 and 0.16 microM. The increased sensitivity to inhibition of DNA replication by UV in these XP variant fibroblasts (2.9-fold greater than normal) was mirrored by an enhanced frequency of transformation to AI. XP variant fibroblasts (CRL1162) were 3.2 times more sensitive to transformation to AI by UV than were the normal foreskin fibroblasts. As predicted by the PRR studies, both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m2; XP variant fibroblasts, 0.3 J/m2) and BPDE-I (both, 0.05 microM). Doses of UV and BPDE-I above these thresholds produced proportional increases in the inhibition of DNA replication in operating replicons and in the induced frequency of anchorage-independent colonies. At doses of UV and BPDE-I that produced the same degree of inhibition of DNA strand growth, BPDE-I induced a greater number of cells capable of anchorage-independent growth than did UV in both normal and XP variant fibroblasts.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.
Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.
Amsacrine↗
Defective postreplication repair in xeroderma pigmentosum variant fibroblasts.
Postreplication repair (PRR) was quantified in normal human fibroblasts and in xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo[a]pyrene diol epoxide-I (BPDE-I). PRR may be defined as the elimination of discontinuities in the daughter-strand DNA and the replicative bypass of lesions in the DNA template. Pathways of PRR reduce the number of DNA growing points that are blocked at template lesions and increase the rate of growth of nascent DNA on damaged templates. Rates of DNA synthesis and strand growth were measured in solvent- and carcinogen-treated cells by velocity sedimentation analyses of radiolabeled nascent DNA in alkaline sucrose gradients. Logarithmically growing normal fibroblasts displayed D0 values of 6.3 J/m2 and 0.37 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. Under identical conditions, the XP variant cells exhibited D0 values of 1.5-2.0 J/m2 and 0.27-0.31 microM. Pulse-chase experiments were performed in cells synchronized at the beginning of the S phase. Normal and XP variant cells displayed inhibition of DNA strand growth by UV, with D0 values of 21.6 and 7.0 J/m2, respectively. The D0 values for the inhibition of DNA strand growth by BPDE-I were 0.85 microM for the normal cells and 0.62-0.79 microM for the XP variant cells. The inhibitions of DNA replication by UV and BPDE-I were also analyzed in terms of DNA lesion frequencies. Based on the D0 values for inhibition of DNA replication, we concluded that the XP variant cells express maximally 25-33% of the total PRR activity observed in normal fibroblasts after UV treatment. Conceivably, this deficiency in PRR activity results in the XP variant's increased risk of cancers induced by sunlight, because XP variant cells and normal fibroblasts are equally proficient in excision repair. Both normal and XP variant fibroblasts, however, displayed similar PRR activities in response to BPDE-I treatment.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗