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Biomedical subjects

R A Lewis

Publications and source records attributed to R A Lewis.

At least 181 records · Page 10Linked to original sources
PubMed1990

Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity.

The inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [3H]dGTP in the absence of, and 1 molecule of [3H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 +/- 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 +/- 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Genetic and physical mapping of Xq24-q26 markers flanking the Lowe oculocerebrorenal syndrome.

The Lowe oculocerebrorenal syndrome (OCRL) is characterized by congenital cataract, mental retardation, and renal tubular dysfunction. We are using the approaches of linkage analysis, mapping with somatic cell hybrids, and long-range restriction mapping to determine the order of Xq24-q26 markers with respect to each other and to the OCRL locus. DXS42 and DXS100 are proximal to the translocation breakpoint in a female patient with OCRL and a de novo translocation t(X;3)(q25;q27). DXS10, DXS86, HPRT, and DXS177 are distal to the breakpoint. These flanking markers show tight linkage to the disease locus in 11 families segregating for OCRL. Results from field inversion gel analysis show that DXS86 and DXS10 share a 460-kb BssHII fragment. Multipoint analysis to determine the position of HPRT with respect to (DXS10,DXS86) suggests that HPRT is proximal to (DXS10,DXS86). We propose the following order for markers in Xq24-q26: Xcen-(DXS42,DXS37,DXS100)-OCRL-DXS53 -HPRT-[(DXS10,DXS86),DXS177]-Xqter. The identification of additional tightly linked flanking markers extends the number of markers available for use in genetic counseling and begins to define the physical map of the region containing the gene for OCRL.

Blotting, Southern↗
PubMed1990

DXS165 detects a translocation breakpoint in a woman with choroideremia and a de novo X; 13 translocation.

The search for the gene for choroideremia (MIM 30310), a rare retinal dystrophy, has been of great interest due to the existence of several choroideremia patients with well-defined structural chromosome aberrations, thus providing the basis for a reverse genetics approach to the isolation of this disease gene. This report details our molecular studies of a woman with choroideremia and a de novo X; 13 translocation. Pulsed-field gel electrophoresis using a contour-clamped homogeneous electric field apparatus has allowed detection of the translocation breakpoint with the anonymous DNA marker p1bD5 (DXS165) and the mapping of this probe to within 120 kb of the breakpoint. In addition, we have used this probe to isolate a clone (pCH4) from a 100-kb jumping library which has crossed a rare-cutting restriction site (XhoI) between DXS165 and the choroideremia gene and detects the translocation breakpoint using this enzyme. Although DXS165 lies within 120 kb of the breakpoint and Cremers et al. (1987, Clin. Genet. 32: 421-423; 1989, PNAS 86: 7510-7514) have detected deletions of DXS165 in 3 of 30 choroideremia probands, we have detected no deletions of this marker or of pCH4 in 42 unrelated probands with this retinal disease.

Animals↗
PubMed1990

Mapping recessive ophthalmic diseases: linkage of the locus for Usher syndrome type II to a DNA marker on chromosome 1q.

Usher syndrome is a heterogeneous group of autosomal recessive disorders that combines variably severe congenital neurosensory hearing impairment with progressive night-blindness and visual loss similar to that in retinitis pigmentosa. Usher syndrome type I is distinguished by profound congenital (preverbal) deafness and retinal disease with onset in the first decade of life. Usher syndrome type II is characterized by partial hearing impairment and retinal dystrophy that occurs in late adolescence or early adulthood. The chromosomal assignment and the regional localization of the genetic mutation(s) causing the Usher syndromes are unknown. We analyzed a panel of polymorphic genomic markers for linkage to the disease gene among six families with Usher syndrome type I and 22 families with Usher syndrome type II. Significant linkage was established between Usher syndrome type II and the DNA marker locus THH33 (D1S81), which maps to chromosome 1q. The most likely location of the disease gene is at a map distance of 9 cM from THH33 (lod score 6.5). The same marker failed to show linkage in families segregating an allele for Usher syndrome type I. These data confirm the provisional assignment of the locus for Usher syndrome type II to the distal end of chromosome 1q and demonstrate that the clinical heterogeneity between Usher types I and II is caused by mutational events at different genetic loci. Regional localization has the potential to improve carrier detection and to provide antenatal diagnosis in families at risk for the disease.

Blotting, Southern↗
PubMed1990

Mapping X-linked ophthalmic diseases. IV. Provisional assignment of the locus for X-linked congenital cataracts and microcornea (the Nance-Horan syndrome) to Xp22.2-p22.3.

The Nance-Horan syndrome (NHS) is an infrequent X-linked disorder typified by dense congenital central cataracts, microcornea, anteverted and simplex pinnae, brachymetacarpalia, and numerous dental anomalies. The regional location of the genetic mutation causing NHS is unknown. The authors applied the modern molecular techniques of analysis of restriction fragment length polymorphisms to five multigenerational kindreds in which NHS segregated. Provisional linkage is established to two DNA markers--DXS143 at Xp22.3-p22.2 and DXS43 at Xp22.2. Regional localization of NHS will provide potential antenatal diagnosis in families at risk for the disease and will enhance understanding of the multifaceted genetic defects.

Abnormalities, Multiple↗
PubMed1990

Nance-Horan syndrome: localization within the region Xp21.1-Xp22.3 by linkage analysis.

Nance-Horan Syndrome (NHS) or X-linked cataract-dental syndrome (MIM 302350) is a disease of unknown pathogenesis characterized by congenital cataracts and dental anomalies. We performed linkage analysis in three kindreds with NHS by using six RFLP markers between Xp11.3 and Xp22.3. Close linkage was found between NHS and polymorphic loci DXS43 (theta = 0 with lod score 2.89), DXS41 (theta = 0 with lod score 3.44), and DXS67 (theta = 0 with lod score 2.74), defined by probes pD2, p99-6, and pB24, respectively. Recombinations were found with the marker loci DXS84 (theta = .04 with lod score 4.13), DXS143 (theta = .06 with lod score 3.11) and DXS7 (theta = .09 with lod score 1.68). Multipoint linkage analysis determined the NHS locus to be linked completely to DXS41 (lod score = 7.07). Our linkage results, combined with analysis of Xp interstitial deletions, suggest that the NHS locus is located within or close to the Xp22.1-Xp22.2 region.

Abnormalities, Multiple↗
PubMed1989

Molecular genetics of human blue cone monochromacy.

Blue cone monochromacy is a rare X-linked disorder of color vision characterized by the absence of both red and green cone sensitivities. In 12 of 12 families carrying this trait, alterations are observed in the red and green visual pigment gene cluster. The alterations fall into two classes. One class arose from the wild type by a two-step pathway consisting of unequal homologous recombination and point mutation. The second class arose by nonhomologous deletion of genomic DNA adjacent to the red and green pigment gene cluster. These deletions define a 579-base pair region that is located 4 kilobases upstream of the red pigment gene and 43 kilobases upstream of the nearest green pigment gene; this 579-base pair region is essential for the activity of both pigment genes.

Adolescent↗
PubMed1989

Phosphorylation of arabinosyl guanine by a mitochondrial enzyme of bovine liver.

Extracts of mitochondria isolated from bovine liver were shown to phosphorylate araG, forming araGMP as the sole product. When other nucleosides were used as competitors with araG as the substrate for phosphorylation, deoxycytidine, deoxythymidine and guanosine were not significantly inhibitory. However, the phosphorylation of araG was blocked by deoxyguanosine, deoxyadenosine and deoxyinosine. Deoxyguanosine was shown to be a competitive inhibitor of araG phosphorylation (apparent Ki for deoxyguanosine = 9 microM; apparent Km for araG = 66 microM). Likewise, araG was determined to be a competitive inhibitor of mitochondrial deoxyguanosine kinase activity (apparent Km for deoxyguanosine = 16 microM; apparent Ki for araG = 55 microM). These data suggest that the two nucleosides were phosphorylated by the same enzyme. Disc gel electrophoresis showed that the phosphorylating activity for araG migrated with deoxyguanosine kinase activity. The pH profiles of the araG and deoxyguanosine kinase activities were dissimilar. The optimum pH for deoxyguanosine kinase was 5.5; for araG kinase, it was 8.0. Collectively, these data suggest that araG is phosphorylated by mitochondrial deoxyguanosine kinase; however, separate forms of the enzyme or different reaction conditions may be involved in the optimal activities of the two catalytic events.

Animals↗
PubMed1989

Binding of captan to DNA polymerase I from Escherichia coli and the concomitant effect on 5'----3' exonuclease activity.

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Captan↗
PubMed1989

Automated site-directed drug design: the concept of spacer skeletons for primary structure generation.

This paper examines the problem of automated structure generation at specified binding sites. The objective is to obtain molecular graphs that span the binding site and incorporate predicted ligand points at their vertices. Three approaches are considered: brute-force techniques, subgraph addition and spacer skeletons. Spacer skeletons are assemblies of molecular subgraphs and are used to reduce the combinatorial problems of structure generation to a practicable level for future analysis. This description is restricted to structure generation in two dimensions. Assemblies of rings are examined for planarity by searching the Cambridge Structural Database. Appropriate spacer skeletons may then be fitted to arrays of site points.

Computer Simulation↗
PubMed1989

Automated site-directed drug design: the formation of molecular templates in primary structure generation.

In this paper the spacer skeleton concept is used to produce molecular graphs of putative ligands for binding sites. The skeletons are transformed into molecular templates within the constraints of the accessible surface of the ligand-binding site. A distance-matrix method is used to compare ligand points with vertices of the spacer skeleton through a permutation of all possible correspondences. A tolerance parameter is used to screen for poor matches. As a result, a small number of matched vertices and ligand points are produced. These are fitted into the site by a constrained optimization routine using an analytical function. Ligand points fall within the site and are optimally positioned adjacent to the corresponding site points; other vertices of the spacer skeleton lying beneath the accessible surface of the site are clipped off. A molecular template is thereby formed with its vertices linked to the ligand points. The final step is to verify that the bonding integrity of the skeleton remains. The computational methods outlined in this paper have been tested at two binding sites: the pteridine binding site in dihydrofolate reductase and the amidinophenylpyruvate site of trypsin. Molecular graphs for both sites were generated automatically; they showed strong similarity to those of the natural ligands.

Drug Design↗
PubMed1989

Intraocular pressure after cardiopulmonary bypass surgery.

The ocular effects of cardiopulmonary bypass surgery were prospectively studied in 46 patients. We examined preoperative and post-operative visual acuity, intraocular pressure, body weight, fluids infused during surgery, hematocrit, and cardiopulmonary bypass time. On the first postoperative day mean intraocular pressure increased 2.1 mm Hg (P = .003) from baseline preoperative levels. Over the first postoperative day intraocular pressure increased in 29 (63%) of the patients. Three patients (7%) had a greater than 10 mm Hg rise. The mean intraocular pressure returned to baseline by the third postoperative day. Patient weight increased from preoperative levels an average of 9.3 lbs (P less than .0001) on day 1 and 6.5 lbs on day 3. Mean hematocrit decreased 11.3% (P less than .0001) on day 1 from baseline and remained at that level through day 3. None of the patients complained of visual dysfunction during the course of this study and none showed more than a two-line decrease in near visual acuity. The increase in intraocular pressure did not correlate with the postoperative weight gain or hemodilution. However, the medications necessary after cardiac surgery may be a significant confounding variable. This study demonstrates that one cause of ocular problems from cardiopulmonary bypass surgery may be related to the dynamics of intraocular pressure.

Adult↗