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Xeroderma pigmentosum (XP) is an autosomal recessive disease in which the major clinical manifestation is a 2,000-fold enhanced probability of developing sunlight-induced skin tumors, and the molecular basis for the disease is a defective DNA excision repair system. To clone the gene defective in XP complementation group A (XP-A), cDNA clones were isolated by a competition hybridization strategy in which the corresponding mRNAs were more abundant in cells of the obligately heterozygous parents relative to cells of the homozygous proband affected with the disease. In this report, a human genomic DNA clone that contains this cDNA was transformed into two independent homozygous XP-A cell lines, and these transformants displayed partial restoration of resistance to the killing effects of UV irradiation. The abundance of mRNA corresponding to this cDNA appears to correlate well with the observed UV cell survival. The results of unscheduled DNA synthesis after UV exposure indicate that the transformed cells are repair proficient relative to that of the control XP-A cells. However, using this same genomic DNA, transformation of an XP-F cell line did not confer any enhancement of UV survival or promote unscheduled DNA synthesis after UV exposure.

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.

P-450NF is the major enzyme in human liver involved in the metabolism of the calcium-channel blocker nifedipine. By screening a bacteriophage lambda gt11 expression library, a cDNA clone designated NF 10 with an insert length of 2.8 kilobases (kb) was isolated. This clone was sequenced and found to be highly similar in its overlapping section with sequences of two other cDNA clones previously isolated from the same expression library, NF 25 (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068) and HLp (Molowa, D. T., Schuetz, E. G., Wrighton, S. A., Watkins, P. B., Kremers, P., Mendez-Picon, G., Parker, G. A., and Guzelian, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5311-5315). However, clone NF 10 had an extra 814 or 813 bases of 3'-noncoding sequence relative to NF 25 or HLp, respectively, and this additional sequence contained a second consensus polyadenylation signal. Specific oligonucleotides were synthesized to differentiate between these three clones at the mRNA level. Oligonucleotides specific to the protein coding region of each clone were found to hybridize to mRNAs of 2.2 and 3.0 kb in size at a ratio of approximately 10:1. The major species of hybridizable mRNA was specific to clone NF 25, and levels of this mRNA could be correlated with levels of immunochemically detectable P-450NF and nifedipine oxidase activity in individual human liver samples. In addition, an oligonucleotide specific to the 3'-noncoding region of clone NF 10 hybridized only with the 3.0-kb mRNA. We conclude that alternative use of the second polyadenylation signal present in clone NF 10 results in production of the 3.0-kb mRNA species and that a pretranslational control mechanism is primarily involved in the regulation of nifedipine oxidase activity.

Using indirect immunofluorescence, comparisons were made of patterns of staining of five tissue substrates with 28 RI reticulin antibody-positive coeliac sera, and 23 RI reticulin antibody-positive non-coeliac sera. IgA and IgG fluorescein conjugates were used separately. IgA antibodies were seen in 22 coeliac patients (78%) compared with three non-coeliacs (13%). Concomitant sinusoidal fluorescence (RS pattern) was seen more frequently with the non-coeliac (60.9%) than with the coeliac sera (10.7%). Such differences may help to distinguish those patients who should have a jejunal biopsy when using the reticulin antibody as a screening test for coeliac disease.

Reaction of covalently closed circular PM2 bacteriophage DNA with the anticancer drug bleomycin produces nicked circular (form II) and linear duplex (form III) DNA [Lloyd, R.S., Haidle, C.W. & Robberson, D.L. (1978) Biochemistry 17, 1890-1896]. As the reaction proceeds, the frequencies of both form II and form III DNA increase and, concomitantly, an increasing fraction of the DNA mass is found to be in crosslinked structures. Approximately 16% of the PM2 DNA mass is found to be crosslinked after 30 min of reaction with bleomycin at 0.5 microgram/ml. The proportion of each form found in any given crosslinked structure is directly related to the concentration of uncrosslinked (monomeric) forms. Multiple sites of crosslinking occur, and these frequently extend over a region of approximately 500 nucleotide pairs. The intermolecular crosslinked bonds are dissociated by extensive dialysis or by the addition of salt at high concentration (0.8 M NaCl), as would be expected if the bonds were noncovalent. Because intramolecular covalent crosslinks between complementary strands are not detected, it is suggested that intermolecular crosslinks are formed by noncovalent association of bleomycin molecules bound to each of the forms of DNA.

A recently described scoring system designed to assess the individual risk of finding serious pathology in patients referred for upper gastrointestinal investigation has been prospectively tested in 1279 patients undergoing first-time endoscopy and 321 patients undergoing radiologic examination. The scoring system has been confirmed to give a reasonable prediction of the likelihood of finding serious pathology in two hospitals with differing endoscopic practice, and also to be applicable to patients attending for radiology. The system works best at defining a low-risk group (score less than 412, 26% of total) in which the incidence of serious pathology was 3%. All cases of malignancy (n = 55) occurred in patients scoring greater than 464 (50% of total). A simple table is described that allows for the easy calculation of score at a glance without the use of a computer. We believe that this scoring system, which can be implemented in seconds, is the simplest yet described and that it could prove to be a useful educational aid.

The autoantibody profile results from 750 randomly selected patients with rheumatoid factor (RF) and/or antinuclear antibody (ANA) positive tests were retrospectively analysed in order to discover if there was any evidence to show that the presence of polyclonal rheumatoid factors affects the incidence and titre of IgG-ANA in these patients. The incidence of IgG-ANA in the RF-positive group (34.4%) was significantly greater than that found in the RF-negative groups (19.6%) (P < 0.001), and there was no significant difference between the mean IgG-ANA titres of the two groups (P = 0.987). The possibility that the presence of IgM-RF in serum might be responsible for the inhibition of IgG-ANA was examined by treating the sera of selected patients who were RF-positive but IgG-ANA-negative with the dissociating agent D-penicillamine (DP). After treatment, IgG-ANA could not be detected in any of the sera. Similar studies were carried out to ascertain if there was a masking effect by RF on two other IgG autoantibodies, anti-keratin antibody (AKA) and gastric parietal cell antibody (GPCA). There was no evidence from these studies that the presence of RF affects the incidence of either AKA or GPCA. We conclude from these results that the presence of RF is not a significant factor controlling the incidence of either IgG-ANA or other IgG autoantibodies and that the routine treatment of RF-containing serum with a dissociating agent before testing for autoantibodies would be unnecessary.

The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)

Campylobacter pyloridis was isolated from 77% of 220 (35%) unselected adults undergoing gastroscopy. Isolation was significantly associated with histological gastritis (p less than 0.0001), duodenal ulcer (p less than 0.0001), and to a much lesser extent, with gastric ulcer (p less than 0.05). The relation between the isolation of C pyloridis and peptic ulcer seemed to be independent of coexisting gastritis. In those with no endoscopic or histological evidence of disease there was no relation between isolation and increasing age. Antibody responses to a whole cell sonicate of a strain of C pyloridis were measured by means of an enzyme linked immunosorbent assay (ELISA). Increased IgA (p less than 0.0001) and IgG (p less than 0.0001) antibody titres were found in patients with C pyloridis. Peptic ulceration or gastritis were present in 78% and 100% of patients with a high concentration of IgG and IgA, respectively, but in only 9% and 18% of those with low titres. These results provide further evidence for a possible pathogenic role of these organisms in gastric disease and suggest that immunological markers of their presence might be useful non-invasive indicators of disease.

In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.

The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS)

A competition hybridization strategy using size-cut cDNA libraries as both probe and competitor was designed for the cloning of genes whose mRNAs are either regulated transcriptionally or vary in abundance as a function of cell line or cell cycle. We used this strategy to construct cDNA libraries from a particular size fraction of mRNA from three members of a xeroderma pigmentosum (XP), complementation group A family. Size-cut cDNA libraries derived from the father's, mother's, and child's fibroblast cell line were used in a competition scheme to screen two lambda gt11 human cDNA libraries. Of the 15 positive lambda gt11 clones which have been characterized, at least 14 clones represent the same gene which is present in greater abundance in both the mother and father XP obligate heterozygotes relative to the homozygous affected child.

Procedures have been developed which simplify and expedite the screening of recombinant DNA constructions for those which only exhibit the desired DNA-DNA junctions. A synthetic DNA oligonucleotide designed to span (or "bridge") sequences around correct restriction enzyme junctions was used as a hybridization probe for the rapid identification of those sequences in several molecular cloning methodologies. It facilitated analyses of the products of random ligation reactions, as well as constructions harbored in bacteria and bacteriophage. "Bridge" probes, [32P]-end-labeled to very high specific activity, remained useful after several hybridizations and often circumvented lengthy restriction analyses.

Human liver cytochrome P-450NF is the form of cytochrome P-450 responsible for the oxidation of the calcium-channel blocker nifedipine, which has been reported to show polymorphism in clinical studies. By screening a bacteriophage lambda gt11 expression cDNA library, we isolated two clones: NF95 with an insert length of 0.8 kilobases which gave a stable fusion protein and NF25 with an insert length of 2.2 kilobases. The two clones were both sequenced and shown to be identical in their overlapping section. The sequence of NF25 is 77% similar to that reported for a rat cytochrome "P-450PCN" cDNA (PCN = pregnenolone-16 alpha-carbonitrile). The similarity decreases to 45-53% when the sequence is compared to human cytochromes P-450 belonging to other families [i.e., "pH P-450(1)," "P1-450," "P3-450," and "P-450MP." The deduced amino acid sequence is 73% similar to that of rat cytochrome P-450PCN, and the first 21 amino acids are identical to those reported for human liver cytochrome "P-450p." Sections of these clones were nick-translated and used as probes for analyses of human mRNA and genomic DNA. The number and size of bands indicate that P-450NF belongs to a multigene family, the so-called pregnenolone-16 alpha-carbonitrile-inducible family.

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.

T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.

A sensitive technique was used to estimate two acid hydrolases--N-acetyl-beta-glucosaminidase (N.A.G.) and beta-glucuronidase (B.G.)--produced by peripheral-blood monocytes. Enzyme levels were measured after in-vitro incubation of monocytes with or without stimulation by zymosan and endotoxin. Compared with controls, enzyme production and release in inflammatory bowel disease, chronic liver disease, and rheumatoid arthritis were markedly raised. It is suggested that various stimuli, including immunological ones, may be responsible for the release of such enzymes from monocytes and that such release may be a factor in the production of the chronic inflammation seen in these disorders.

The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme "scans" DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule.

Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.

Non-covalent intermolecular DNA cross-links are created by reaction with the antitumor antibiotic, bleomycin. The cross-links are observed only when the reactant covalently closed circular duplex DNA contains either positive or negative superhelical turns. Multiple sites of cross-linking often extend over 500 base-pairs or more in length.

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.

Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E. coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted. Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI. The IFN-gamma expressed in E. coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter. When examined microscopically, IPTG induced E. coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well in E. coli and not only significantly enhanced the UV survival of repair-deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129----Trp and Tyr-129----Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129----Phe and Tyr-129----Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129----Phe mutant and to 20% that of wild type for the Tyr-129----Trp mutant.

Published data on the structure and mechanism of endonuclease V from bacteriophage T4 are reviewed with the objective of developing a working mechanistic model of this enzyme. Endonuclease V is an interesting and important candidate to be the first DNA-repair enzyme to have its structure determined by crystallography, and a more detailed model of the reaction process is needed to mechanistically interpret such a structure. Such a model should be sufficiently detailed to support future investigations of structure/function relationships between the enzyme and the DNA damage repair pathway it initiates, as probed by site-directed mutagenesis techniques and other methods. The early literature is presented in an historical perspective, followed by a description of prior models and biochemical investigations. The biochemical phenotypes of mutants in the enzyme structural gene are discussed. The results of computer analyses aimed at structural interpretations of the protein sequence are given, together with a brief discussion of the strengths and weaknesses of such experiments.

The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream of the bacteriophage lambda hybrid promoter-operator oLpR, was analyzed under a variety of growth parameters. Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli (uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation.

To assess its value as a screen for avian antibodies, indirect immunofluorescence against avian intestinal tissue has been to test sera from thirty-nine patients with documented bird-fancier's lung disease, thirty-eight asymptomatic bird-fanciers and 257 controls without known avian contact. Immunofluorescent antibodies occurred more frequently than precipitins among patients with BFL and asymptomatic bird-fanicers. Globular fluorescence within the mucus occurred only in patients with avian contact, although other fluorescent antibodies were seen with control patients. No particular pattern was confined to patients with the lung disease. When included in an autoantibody profile, indirect immunofluorescence provides a sensitive and convenient alternative to precipitin methods in screening for avian antibodies.

A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.

A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.

Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases. The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate DNA backbone. Arginine-3 was chosen as a potential candidate for involvement in this protein-nontarget DNA interaction and was extensively mutated to assess its role. The mutations include changes to Asp, Glu, Leu, and Lys and deleting it from the enzyme. Deletion of Arg-3 resulted in an enzyme that retained marginal levels of AP specificity, but no other detectable activity. Charge reversal to Glu-3 and Asp-3 results in proteins that exhibit AP-specific nicking and low levels of dimer-specific nicking. These enzymes are incapable of affecting cellular survival of repair-deficient Escherichia coli after irradiation. Mutations of Arg-3 to Lys-3 or Leu-3 also are unable to complement repair-deficient E. coli. However, these two proteins do exhibit a substantial level of in vitro dimer- and AP-specific nicking. The mechanism by which the Leu-3 and Lys-3 mutant enzymes locate pyrimidine dimers within a population of heavily irradiated plasmid DNA molecules appears to be significantly different from that for the wild-type enzyme. The wild-type endonuclease V processively incises all dimers on an individual plasmid prior to dissociation from that plasmid and subsequent reassociation with other plasmids, yet neither of these mutants exhibits any of the characteristics of this processive nicking activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of thyroid microsomal and/or thyroglobulin antibodies has been recorded over a 2 year period of 15 000 consecutive autoimmune profile request. Where there had been no initial clinical suspicion of thyroid diseases, 332 requests showed positive thyroid antibodies, and of these 63 (19%) had abnormal in vitro thyroid function tests (TFT). No differences were observed between the abnormal and normal groups with respect to the presence of different autoantibodies or to the age and sex distributions. Of these subjects with clinically unsuspected hypothyroidism but with abnormal TFTs, 29% were commenced on thyroxine therapy and experienced a symptomatic improvement, 25% remain well on no therapy and 9% continue on no treatment but with symptoms possibly attributable to hypothyroidism. 3% became clinically hypothyroid during a follow-up period of 2 years. 5% died of unrelated causes and there was inadequate follow-up information on the remainder. This study provides further confirmation that when thyroid antibodies, and in particular thyroid microsomal antibody, are found unexpectedly, a significant proportion of patients will have biochemical evidence of hypothyroidism and may benefit from appropriate treatment.

Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated. We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo. An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA. In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred. These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.

An autoantibody screen is often requested in patients with non-specific symptoms such as pyrexia of unknown origin. However, in a survey of 100 consecutive requests for this investigation in such patients only one patient was encountered in whom the investigation led to a positive diagnosis at an estimated cost of pound 2500. These requests accounted for 5% of the total workload. These observations reaffirm that this should remain a second line investigation and should be deferred until more likely diagnoses have been excluded.

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A small proportion of sera submitted for routine immunofluorescent antibody screening contains heterophile antibodies. The reaction patterns produced by the heterophile antibody may be confused with or mask specific autoantibody patterns, leading to false positive or false negative autoantibody results. In this study we report the development of a method in which fresh sheep red cells are used to absorb heterophile antibody from sera without affecting specific autoantibody which may be present. This technique was used on a panel of 142 sera known to contain heterophile antibody but not initially reported as containing specific autoantibodies by immunofluorescence. After absorption with sheep red cells the heterophile antibody was completely removed from the sera under test and 27 (19%) specimens were shown to contain previously undetected autoantibodies. Only 6 (4%) of the sera, however, contained autoantibodies at a titre which would be reported as a positive result on routine screening. These results suggest that there may be a significant number of sera submitted for routine autoantibody screening which contain autoantibodies that are masked by the presence of heterophile antibody. Selective use of the absorption technique offers a simple solution to this problem.

The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular DNa. Form I ColE1 DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule were treated with T4 endonuclease V and analysed by agarose gel electrophoresis. At all dimer frequencies examined, the production of form III DNA was linear with time and the double-strand scissions were made randomly on the ColE1 DNA genome. Since the observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of the DNA population while some DNA molecules contained no scissions. When DNA containing an average of 25 dimers per circle was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimmer sites in some molecules before additional strand scissions were produced in other DNA molecules. The results support a processive model for the interaction of T4 endonuclease V with UV-irradiated DNA.

In a study of selenium status in 391 apparently healthy subjects resident in the south of England statistical examination of the data showed a significant effect with regard to age, smoking, alcohol, and oral contraceptives. The most important of these factors seems to be a combination of alcohol and smoking habits in men over 30. Reference ranges have been established for glutathione peroxidase activities and the concentrations of selenium in whole blood plasma and erythrocytes.

T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.

Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms. The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing. The complexity and frequency of these structures increased with time of reaction with endonuclease V. Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation. Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecules as did preparations estimated to be only 5-10% pure. In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded 'loops' 200-300 base pairs in length. DNA 'bubble structures' were also observed and may represent folding of the 'loops' onto adjacent segments of DNA. These results suggest that at least one active form of T4 endonuclease V may be a multimeric complex of enzyme molecules in association with DNA.

Fasting blood samples taken from 116 apparently healthy men aged 30-50 years were assayed for selenium, glutathione peroxidase activity, vitamin E, cadmium, lead, glucose, lipids, and albumin. Blood pressure was measured in each subject, and details of height, weight, smoking habits, and alcohol consumption were recorded. Multivariate analysis of the data showed that the decrease in blood and serum concentrations of selenium and the increase in whole blood cadmium concentrations in the cigarette smokers was independent of alcohol consumption. There was no correlation between blood selenium concentrations or glutathione peroxidase activities and the risk factors for cardiovascular disease. Neither alcohol consumption nor smoking had an effect on the vitamin E concentrations. There was a strong association, however, between vitamin E and serum lipid concentrations, although the increase in triglyceride concentrations in the smokers was not matched by a comparable increase in vitamin E. The possible role of selenium in the aetiology of heart disease remains unresolved.

To test the hypothesis of an association between schizophrenia and coeliac disease, the sera of 380 chronic schizophrenic in-patients in two mental hospitals in the West of Ireland have been screened for the presence of reticulin antibodies. Antibodies were found in 26 patients. Twenty-one of these patients were further studied by proximal duodenal mucosal biopsy. None of the biopsies showed the morphological and histological features found in untreated coeliac disease. The incidence of reticulin antibodies in schizophrenic patients and controls is similar. The findings of this study lead to the rejection of the hypothesis of a positive genetic relationship between schizophrenia and coeliac disease.

Ambulatory oesophageal pH, oesophageal manometry and fasting serum gastrin concentrations were carried out on 28 patients with reflux oesophagitis, before and during treatment with ranitidine 300 mg bd. Fourteen patients healed endoscopically at six weeks (group A) and 14 had residual oesophagitis (group B). Group A were characterised by a lower serum gastrin concentration before treatment (4.52 pmol/l; 2.4-10: mean and range) than group B (11.1 pmol/l; 3.5-21: p less than 0.05) and showed a marked reduction in acid reflux on treatment to near normal values. Mean per cent time below pH4 fell from 14.9 to 4.2 in group A (p less than 0.05) but was not affected in group B (14.2-15.6, not significant). Abnormal oesophageal motility was found in 13 patients from each group. This did not inhibit the response to ranitidine, and was not improved by healing of oesophagitis.