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J Lebowitz

Publications and source records attributed to J Lebowitz.

At least 37 records · Page 2Linked to original sources
PubMed1994

Human immunodeficiency virus-1 reverse transcriptase heterodimer stability.

Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus-1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer-dimer-trimer and monomer-dimer-tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 degrees C, with a dimerization Ka value of 5.2 x 10(5) M-1 for both models. Ka values of 2.1 x 10(5) and 3.8 x 10(5) M-1 were obtained for the respective association models at 20 degrees C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 degrees C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 degrees C to 37 degrees C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.

Cold Temperature↗
PubMed1992

Conditional immortalization of bicarbonate-secreting intercalated cells from rabbit.

We have derived an immortalized cell line from primary cultures of bicarbonate-secreting intercalated cells from rabbit. Cells were transfected with a plasmid encoding a temperature-sensitive large T antigen of SV40 plus the neomycin resistance gene under the control of an SV40 promoter. Transfectants were selected for resistance to G418. One stably transfected clone, designated IC250, was subcloned to ensure clonality, and a subclone (clone C) was characterized in detail. The cells divide continuously at permissive temperature. At restrictive temperature, they cease dividing and assume morphological and transport properties of true bicarbonate-secreting intercalated cells. They express appropriate ultrastructural features, bind peanut lectin in an apical pattern, are rich in carbonic anhydrase, stain for proton-adenosinetriphosphatase in a basolateral pattern, and do not stain with antibodies to erythrocyte band 3. Most monolayers of transformed type B intercalated cells do not achieve a significant transepithelial resistance; those monolayers that are sufficiently electrically tight for electrophysiological studies are capable of chloride-dependent bicarbonate transport.

Adenosine Triphosphatases↗
PubMed1991

Differential sequence dynamics of homopolymeric and alternating AT tracts in a small plasmid DNA.

The location of OsO4 bispyridine hyper- and hyporeactivity in a small deletion derivative of plasmid ColE1 (PTC12, 1727 bp) has been determined for approximately 70% of the molecule. Thymine bases in homopolymeric (dA)n.(dT)n tracts (n greater than or equal to 4) were always found to be resistant toward OsO4 modification. DNA supercoiling did not destabilize these tracts. The extent of OsO4 bispyridine reactivity of homopolymeric (dA)n.(dT)n tracts, where n = 3, was found to be dependent on the rate of base unpairing of the sequence immediately 5' and 3' to the tract. Repressed OsO4 reactivity of thymine bases in (dA)3.(dT)3 tracts was observed if immediately both 5' and 3' to the tract were stable DNA sequences composed of GC base pairs and/or a homopolymeric (dA)n.(dT)n tract (n greater than or equal to 4). Homopolymeric tracts of n = 3 not having adjacent sequences with repressed unpairing rates did not show reduced levels of OsO4 bispyridine reactivity. Alternating d(TA)n tracts (n greater than or equal to 2) were found to exhibit hyperreactivity with OsO4. The extent of this hyperreactivity was dependent on the length of the tract and superhelical torsional stress. The distribution and frequency of homopolymeric (dA)n.(dT)n (n greater than or equal to 4) tracts in Escherichia coli promoter sequences were examined, and the possible implications of these tracts on promoter function are discussed.

Base Sequence↗
PubMed1990

Site-directed mutagenesis of the -10 region of the lacUV5 promoter. Introduction of dA4.dT4 tract suppresses open complex formation.

Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.

Base Sequence↗
PubMed1990

Through the looking glass: the discovery of supercoiled DNA.

The discovery of supercoiled DNA was first reported 25 years ago in a paper entitled, 'The Twisted Circular Form of Polyoma Viral DNA' by Vinograd, Lebowitz, Radloff, Watson and Laipis. This personal reflection describes the different experimental and conceptual processes that eventually led to the discovery as they actually occurred.

DNA, Superhelical↗
PubMed1989

Site-directed chemical modification for probing DNA-protein interactions. Osmium tetroxide modification of the -10 site of the lacUV5 promoter enhances open complex formation.

A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function. For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed. Osmium tetroxide was selected as the thymine modifying reagent. Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue. The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts. OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter. DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter. However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes. The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes. A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct. The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites.

Base Sequence↗
PubMed1989

Immunoassays for carbodiimide modified DNA-detection of unpairing transitions in supercoiled ColE1 DNA.

The water soluble reagent N-cyclohexyl-N'-beta-(4-methylmorpholinium) ethyl carbodiimide-p-toluene sulphonate (CMC) can be used to probe for unpaired and mismatched sites in DNA. Polyclonal antibodies for CMC modified DNA were produced in order to develop immunological assays for the localization and quantitation of CMC adducts. Immunoslot blot analysis of modified DNA exhibited antibody binding proportional to the extent of CMC modification with adduct detection in the femtamole range. Unmodified DNA did not cross react under the conditions of the assay. The distribution of CMC reactivity for supercoiled ColE1 DNA modified at 100, 200 and 300 mM NaCl was determined by immunoanalysis of EcoRI-Hae2-NruI restriction fragments Southern transferred to nylon membranes. Reactivity above random expectation occurred in the A2-II fragment which can be accounted for by its high A-T content of 71.3%. Reactivity below random expectation occurred in the C fragment which can be accounted for by its low AT content of 43%. CMC modification for the other restriction fragments appeared random.

Antibodies↗
PubMed1989

Raman spectroscopy of supercoiled and nicked ColE1 plasmid.

Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy. ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils. An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart. This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide. The intensities of several Raman bands were altered between the spectra of the two topological forms. Notably absent were any changes in bands arising from cytosine and guanine vibrations. The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies. The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure. Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption. Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species. No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.

Bacteriocin Plasmids↗
PubMed1989

Acid-induced dimerization of skeletal troponin C.

We have investigated pH-dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low-speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20 degrees C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20 degrees C, and was completely prevented at a rotor speed of 40,000 rpm and 4 degrees C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met-25 and Cys-98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH-dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The proton-induced increase in the energy transfer distance is related to the acid-induced dimerization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Estimation of the effect of coumermycin A1 on Salmonella typhimurium promoters by using random operon fusions.

We have estimated the extent to which relaxation of supercoiling by the DNA gyrase inhibitor coumermycin A1 affects gene expression in vivo in Salmonella typhimurium. We isolated a set of Mu d1-8 Lac+ operon fusions to random promoters and measured the effect of coumermycin A1 on the expression of 67 fusions. The differential rate of synthesis was increased for 70% of the fusions and decreased for 16%, and 13% of the fusions had less than a 25% change in expression. The coumermycin A1 response was found to correlate well (P = 0.067) with the basal level of expression such that coumermycin A1 tended to stimulate fusions with low expression and inhibit those with high expression. Since the vast majority of the fusions were sensitive to coumermycin A1 addition and, therefore, to the level of supercoiling, these results indicate that if the level of supercoiling were to vary under physiological conditions, then major readjustments in the cellular economy would occur.

Aminocoumarins↗
PubMed1985

Nucleotide sequence and gene organization of ColE1 DNA.

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).

Base Sequence↗
PubMed1984

Effect of supercoiling on the abortive initiation kinetics of the RNA-I promoter of ColE1 plasmid DNA.

An analysis of the effect of DNA supercoiling on transcription of the RNA-I promoter of ColE1 has been undertaken using the abortive initiation assay developed by McClure (McClure, W. R., Cech, C. L., and Johnston, D. E. (1978) J. Biol. Chem. 253, 8941-8948). A tau plot was constructed, enabling the equilibrium constant for closed complex formation (KI) and the isomerization rate constant (k2) to be determined. The effect of supercoiling on RNA-I is an apparent 100-fold increase in the isomerization rate, k2, with little or no effect on KI. This is the first reported abortive initiation analysis of RNA-I transcription which serves in the regulation of plasmid replication.

DNA, Bacterial↗
PubMed1984

Evaluation of an automated image analysis system for counting human tumor colonies.

The Omnicon FAS II image analysis system was applied to counting tumor colonies grown in a soft agar human tumor clonogenic assay with a detailed protocol designed to assess the instrument's sensitivity, specificity, precision, and accuracy. Comparisons of technician and instrument counts were done on a blinded basis. Sensitivity studies (which used metal microspheres) yielded a correlation coefficient (r) of 0.999 between technicians and the counter. A field-by-field analysis of the instrument's specificity for identifying individual objects correctly as tumor colonies rather than artifacts (as identified by the technician) was excellent (r = 0.95). In the precision studies (determined with repeated automated counting of the same samples for five days), the median coefficient of variation was less than 7%. Accuracy was evaluated on cultures of fresh biopsies from 30 human cancers obtained for drug sensitivity testing as well as on a series of tumor cell lines. The correlation between the mean number of colonies counted by the technicians and by the colony counter was greater than 0.91. Similar comparisons of mean percent survival of tumor colony-forming cells after drug exposure between technician and machine were also quite acceptable (r = 0.85). We conclude that the colony counter provided sufficient reliability to be applied to counting human tumor colonies grown in vitro. In addition, the colony counter performed the Petri dish counts ten times faster than experienced technicians and without associated operator fatigue.

Animals↗
PubMed1983

Carbodiimide inactivation of Escherichia coli RNA polymerase promoters on supercoiled simian virus 40 and ColE1 DNAs occurs by a one-hit process at salt concentrations in the physiological range.

A previous study (Hale, P., Woodward, R. W., and Lebowitz, J. (1980) Nature 284, 640-644) showed that Escherichia coli RNA polymerase promoters on superhelical SV40 DNA are highly selective targets for chemical modification by the water-soluble carbodiimide, N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). To extend the inactivation analysis of supercoiled DNAs, we determined the number and location of RNA polymerase binding sites on the supercoiled and linear forms of ColE1 DNA. We also determined the site distribution of [3H] CMC on the superhelical form. This information, coupled with per cent inhibition of transcription versus CMC-bound curves, allowed a test of the specificity of the CMC inactivation by the Poisson equation. Curves were obtained for supercoiled SV40 DNA modified at 0 and 100 mM NaCl (2 mM NaPi, pH 7.0) and for supercoiled ColE1 DNA modified at 0, 100, and 320 mM NaCl. For supercoiled SV40 DNA, these data, coupled to our knowledge of the number of RNA polymerase binding sites from the study cited above, revealed an excellent fit to a one-hit inactivation by the Poisson equation for DNA modified at 100 mM NaCl. For ColE1 DNA, we obtained an excellent fit to a Poisson distribution when supercoiled DNA was modified at 320 mM NaCl. The Poisson distribution can be applied to [3H] CMC restriction fragment data with equivalent results. These results suggest that promoter sites can be forced into different structural conformations with variable degrees of unpairing.

Affinity Labels↗
PubMed1983

The coupled use of 'footprinting' and exonuclease III methodology for RNA polymerase binding and initiation. Application for the analysis of three tandem promoters at the control region of colicin El.

In order to determine the initiation site for three promoters P1, P2 and P3 (5' to 3') in close proximity in the colicin E1 control region we developed a new methodology that couples ternary complex formation and the analysis of the 3' border protected from exonuclease III digestion. The initiation of transcription could be detected by measuring the shift in the position of the 3' protected border when RNA polymerase moved from its binary complex position to its ternary complex position. The latter stops at a specific nucleotide because transcription is initiated with one or more NTPs missing. This approach, coupled with "footprinting", can also be used to decide whether the formation of an RNA polymerase binary or ternary complex at one site excludes or weakens binding at neighboring sites. The location of 3' protected borders reveals the formation of respective binary and ternary complexes at non-saturating RNA polymerase conditions, whereas at saturating conditions only the distal 3' boundary is seen and exonuclease cannot penetrate further. However, if "footprinting" reveals proximal 5' patterns this establishes that simultaneous binding has occurred on the same DNA fragment. The data showed that this was true for P1 and P3 which are only 8 nucleotides apart. P2 could only be detected at non-saturating conditions since it overlaps both P1 and P3. The evidence from the literature and this study establishes P1 as the true colicin E1 promoter with the possibility that supercoiling may eliminate any role for P2 and P3.

Amino Acid Sequence↗
PubMed1982

Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1.

Interactions between Escherichia coli RNA polymerase holoenzyme and three small plasmid DNAs (pSM1, pSM2, and pSM15) derived from the drug resistant factor R12 have been studied. These plasmids carry the copy number and incompatibility determinants, the origin of DNA replication and the rep gene(s) necessary for plasmid replication. They also contain the insertion element IS1 and the putative finO cistron. Thirteen DNA segments within the largest of the three plasmids (pSM2) were able to form either a binary and/or ternary complex with RNA polymerase. A unique strong binding site was mapped within the left end of IS1. Five binding sites were found within the rep-cop-inc region. Four of these are weak binding sites whereas the fifth does not form a stable binary complex and was detected by ternary complex formation. A strong binding site was located in the putative finO region whereas the remaining six binding sites are located in regions with unidentified genetic functions.

Base Sequence↗
PubMed1980

Decay of hormone responsiveness in mouse melanoma cells in culture as a function of cell density.

Cloudman S91 mouse melanoma cells lose their ability to demonstrate an MSH-induced increase in tyrosinase activity as cell density increases. This loss in hormone responsiveness occurs before confluency is reached and cannot be reversed by exposure of cells to increasing concentrations of MSH. The failure of high-density cultures to respond to MSH is apparently not the result of an inability of MSH to stimulate cAMP production, since either low- or high-density cultures exposed to MSH demonstrate equivalent increases in intracellular levels of cAMP. Further, neither theophylline (1mM), dibutyryl cyclic AMP (10(-4)M), or prostaglandin E1 (10(-6)M) is effective in stimulating tyrosinase activity in melanoma cells cultured at densities exceeding 6 X 10(4) cells/cm2. This finding suggests that the decay of hormone responsiveness occurs at a cellular site distal to cAMP production. The decrease in tyrosinase stimulation by MSH as cell density increases is also apparently not the result of an increase in activity of any soluble inhibitor of the enzyme, for cytosol preparations from high-density cultures (10(5) cells/cm2) fail to inhibit tyrosinase activity in cell homogenates from low-density cultures treated with MSH.

Animals↗