The UvrABC endonuclease system of Escherichia coli--a view from Baltimore

Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP. Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP. Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn. The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase. The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex. The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions. The final integrity of the DNA strands is restored with polynucleotide ligase.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Summary Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations. This investigation was supported by grants from the John S. Dunn Foundation, Houston, TX, the Esther Knispel Fund administered by The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Department of Health and Human Services PHS grant DE 07007.     

Interaction of poly(ADP-ribose)polymerase with DNA polymerase α

Homogeneously purified poly(ADP-ribose) polymerase (PARP) specifically stimulated the activity of immunoaffinity-purified calf or human DNA polymerase by about 6 to 60-fold. Apparently, poly(ADP-ribosyl)ation of DNA polymerase was not necessary for the stimulation. The effects of PARP on DNA polymerase were biphasic: at very low concentrations of DNA, it rather inhibited its activity, whereas, at higher DNA concentrations, PARP greatly stimulated it. The autopoly(ADP-ribosyl)ation of PARP suppressed both its stimulatory and inhibitory effects. By immunoprecipitation with an anti-DNA polymerase antibody, it was clearly shown that PARP may be physically associated with DNA polymerase . Stimulation of DNA polymerase may be attributed to the physical association between the two, rather than to the DNA-binding capacity of PARP, since the PARP fragment containing only the DNA binding domain showed little stimulatory activity. The existence of PARP-DNA polymerase complexes were also detected in crude extracts of calf thymus.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.     

Transport and transformation of hexavalent chromium through soils and into ground water

A detailed characterization of the underlying and adjacent soils of a chrome‐plating shop was performed to provide information on the extent of soil and aquifer contamination at the site and on the potential for off‐site migration and environmental impact. Intact, moist cores were obtained from more than 40 different locations, resulting in more than 200 discrete samples for total metal analysis, selective extraction tests, and adsorption‐reduction experiments, to assess the chemical speciation and distribution of chromium on the contaminated soils and its leaching potential. Surface analytical techniques were also used to determine chemical speciation and to further elucidate mineral fractions responsible for retention of the chromium on the soils and sediments. Adsorption and reduction capacities of the saturated aquifer sediments were variable and low, while the unsaturated soils’ reduction capacities were much greater and were correlated with depth (decreasing capacity with increasing depth). The soils’ adsorption and reduction capacities were eventually overwhelmed, however, and permitted the passage of Cr(VI) into the underlying ground water. Adsorption capacity differences were primarily related to clay content and pH, and less so to the presence of amorphous iron oxide coatings on matrix minerals as operationally defined by the selective extraction methods used in the study. Reduction of Cr(VI) to Cr(III) and subsequent precipitation as (Fe, Cr)(OH)₃ is proposed as the primary attenuation mechanism in the unsaturated soils immediately beneath the shop, based on extraction and surface analyses results.