Mapping a putative pyruvoyl decarboxylase active site to human cytomegalovirus open reading frame UL77.

Human cytomegalovirus is present as an apparently innocuous infection among a large proportion of the adult population that causes serious disease when congenitally transferred (1). Human cytomegalovirus disease may become life threatening when found as an infection of immunocompromised transplant and AIDS patients (1,2). We now map a putative pyruvoyl decarboxylase enzyme prosthetic group, known to be essential to the active site of this class of enzymes, to Human cytomegalovirus open reading frame UL77.

Direct modulation of HBV surface antigen in a human, HBsAg-producing hepatocellular carcinoma cell line by alpha, beta, or gamma interferons.

A transient depression of HBV serologic markers has been reported for some chronically infected patients treated with human interferons. To determine a molecular basis for these observations, a human, HBV-carrying, hepatocellular carcinoma cell line (PLC/PRF/5) was treated with human alpha, beta, or gamma interferons. Administration of these interferons resulted in a marked depression of HBV surface antigen (HG-sAg) levels in the culture medium.

Preneoplastic and neoplastic growth of xenotransplanted lung-derived human cell lines using deepithelialized rat tracheas.

The human lung tumor-derived cell lines A549, Calu-1, Calu-3, HuT292, and SW900 and the transformed human bronchial epithelial cell line TBE-1, that was transfected with the v-Harvey-ras oncogene, were inoculated into deepithelialized Fisher 344 rat tracheas (5 X 10(5) cells/trachea). After the ends of the tracheas were sealed, the tracheas were transplanted into s.c.

The purification of the Escherichia coli UvrABC incision system.

The UvrA, UvrB and UvrC proteins of Escherichia coli have been purified in good yields to homogeneity with rapid three- or four-step purification procedures. The cloned uvrA and uvrB genes were placed under control of the E. coli bacteriophage lambda PL promoter for amplification of expression.

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen.

Induction of hepatitis B virus core gene in human cells by cytosine demethylation in the promoter.

A recombinant human cell line constructed by transfection of epithelial cells with a plasmid containing the hepatitis B virus core gene (HBc) was used to study the regulation of HBc gene expression. Methylation of a single Hpa II site 280 base pairs upstream from the structural gene was found to regulate the expression of the core gene. Expression increased in cells treated with 5'-azacytidine as a result of cytosine demethylation at this site, and there was a fivefold increase in the number of HBc gene transcripts in total cellular messenger RNA.

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene.

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells.

In vitro studies of human lung carcinogenesis.

Advances in the methodology to culture normal human lung cells have provided opportunities to investigate fundamental problems in biomedical research, including the mechanism(s) of carcinogenesis. Using the strategy schematically shown in Figure 1, we have initiated studies of the effects of carcinogens on the normal progenitor cells of the human cancers caused by these carcinogens. Extended lifespans and aneuploidy were found after exposure of mesothelial cells to asbestos and bronchial epithelial cells to nickel sulfate.

High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells.

Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the pL promoter of pKC30.

We have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, we developed a genetic selection using the basal level of expression (30 degrees C) from pL in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-UV-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under pL control rapidly.

Isolation of plasmids carrying either the uvrC or uvrC uvrA and ssb genes of Escherichia coli K-12.

A 3.4 kb PstI fragment containing the uvrC gene of Escherichia coli K-12 has been cloned into pBR322. Plasmids carrying this PstI fragment, in either orientation (pGY3233, pGY4211) relative to the cloning vehicle, complement uvrC mutants.

Cross-linking and relaxation of supercoiled DNA by psoralen and light.

Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA.

Role of ATP in removal of psoralen cross-links from DNA of Escherichia coli permeabilized by treatment with toluene.

Removal of interstrand cross-linked from DNA was examined in Escherichia coli permeabilized by treatment with toluene. Under these conditions, the reaction requires ATP and Mg2+, and the mechanism appears to be similar to that occurring in whole cells. Under optimum conditions, the rate constant was 0.

Tryptophan photoproduct(s): sensitized induction of strand breaks (or alkali-labile bonds) in bacterial deoxyribonucleic acid during near-ultraviolet irradiation.

Long-wavelength ultraviolet light (300 to 400 nm) converts L-tryptophan to a photoproduct that is toxic for bacterial cells in dark conditions. We now report that similar photoproducts of l-tryptophan sensitize bacterial deoxyribonucleic acid to 365-nm radiation, increasing the yield of deoxyribonucleic acid strand breaks (or alkali-labile bonds) by approximately 11.5-fold.

Near-UV photoproduct(s) of L-typtophan: an inhibitor of medium-dependent repair of X-ray-induced single-strand breaks in DNA which also inhibits replication-gap closure in Escherichia coli DNA.

Near-UV photoproducts of L-tryptophan (TP), which are especially toxic for recombination-deficient (rec) mutants, were found to inhibit medium-dependent repair of X-ray-induced single-strand breaks. This inhibitor also slows the rate of closure of replication gaps, suggesting that these two processes may have a common pathway (or share a required step which TP can inhibit).

Inhibition of replication gap closure in Escherichia coli by near-ultraviolet light photoproducts of L-tryptophan.

Near-ultraviolet photoproducts of l-tryptophan (TP) differentially inhibited deoxyribonucleic acid (DNA) replication in wild-type cells and uvrA, polA1, and recA recB double mutants of Escherichia coli. Wild-type cells labeled in their DNA with [(3)H]thymidine in the presence of TP produced small pieces of DNA (7 x 10(6) daltons), which corresponded in size to late replicative intermediates of discontinuous DNA synthesis. Moreover, when TP was present, it took five times longer to chase the low-molecular-weight DNA pieces into high-molecular-weight DNA.

Toxicity of L-Tryptophan photoproduct on recombinationless (rec) mutants of Salmonella typhimurium.

l-Tryptophan after exposure to black light becomes toxic for recombinationless (rec) mutants of Salmonella typhimurium. Fifty-six radiation-sensitive mutants were screened for sensitivity to the tryptophan photoproduct; the rec and exr (X-ray sensitive) mutants are sensitive, whereas the uvr, hcr, and wild-type strains are resistant. A number of catabolic products of tryptophan and compounds related to tryptophan were screened for toxicity to rec strain; these are nontoxic or far less toxic for rec strains than irradiated l-tryptophan.