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Biomedical subjects

M Levine

Publications and source records attributed to M Levine.

At least 379 records · Page 21Linked to original sources
PubMed1988

Inversion events in the HSV-1 genome are directly mediated by the viral DNA replication machinery and lack sequence specificity.

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.

Animals↗
PubMed1988

Divergent homeo box proteins recognize similar DNA sequences in Drosophila.

A member of a small group of genes in Drosophila that define the segmentation pattern of the early embryo even-skipped (eve), which plays a key role in a network of interactions among segmentation genes. It appears to control morphogenesis by regulating the expression of the segmentation gene engrailed (en), and by autoregulating its own expression (M. Frasch and M.L., in preparation). Here we show that these regulatory interactions could occur at the level of transcription as a full-length eve protein binds with high affinity to specific sequences located near the 5' ends of the eve and en genes. The en binding sites contain at least one copy of a 10-base pair consensus sequence: T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve binding sites are relatively G-C rich and do not share obvious similarities with the 10-base pair consensus sequence associated with en. Other homeo box proteins can recognize both classes of eve binding sites, lending support to the proposal that regulatory interactions among homeo box genes involve a competition of different homeo box proteins for similar cis regulatory sequences.

Animals↗
PubMed1988

Gap genes define the limits of antennapedia and bithorax gene expression during early development in Drosophila.

The maintenance of selective patterns of homeotic gene expression within the Drosophila CNS involves cross-regulatory interactions among the genes of the antennapedia and bithorax complexes (ANT-C and BX-C). Such a mechanism does not appear to be responsible for the establishment of these selective expression patterns during early development. Here we show that mutations in several of the gap genes strongly alter the early patterns of Antp and Abd-B expression. The altered patterns that are observed do not always correlate with simple expectations based on cuticular pattern defects observed in advanced-stage mutants. It appears that the initial patterns of Antp and Abd-B expression involve their differential regulation by a common set of gap genes. We propose that the gap genes are largely responsible for integrating the processes of segmentation and homeosis.

Animals↗
PubMed1988

Combinatorial expression of a ftz-zen fusion promoter suggests the occurrence of cis interactions between genes of the ANT-C.

The nine homeobox genes contained within the Antennapedia gene complex (ANT-C) are precisely regulated during embryonic development. It is not known to what extent the physical linkage of these genes contributes to their normal patterns of expression. Here we show that cis regulatory elements associated with one homeobox gene can act over a long distance (approximately 20 kb) to influence the expression of another homeobox gene. Specifically, fushi tarazu (ftz) promoter elements can direct the periodic expression of the z2 gene, which normally shows a simple 'dorsal on/ventral off' pattern of expression. An 80 kb deletion within the ANT-C [Df(3R)LIN] juxtaposes the z2 and ftz promoters, resulting in a hybrid expression pattern whereby z2 transcripts are distributed within periodic stripes that are confined to dorsal and lateral tissues and not observed in the ventral mesoderm. This observation suggests that separate promoter elements of different genes can function in a combinatorial manner, and that the patterns of ANT-C gene expression might depend on cis regulatory interactions.

Animals↗
PubMed1988

Pathogenicity of glycoprotein C negative mutants of herpes simplex virus type 1 for the mouse central nervous system.

A previous study from our laboratory showed that a mutant of herpes simplex virus type 1 (HSV-1), strain KOS-321, carrying a deletion in the structural gene for glycoprotein C (gC) had reduced pathogenicity for the mouse central nervous system when compared to the wild-type virus (Kümel et al., 1985). In this study, eight additional gC negative (gC-) mutants derived from KOS-321 were shown to vary widely in their ability to induce lethal encephalitis in female DBA/2 mice following intracerebral inoculation. This variation in virulence showed no correlation with thymidine kinase activity. One less virulent gC- strain, gC-39, was further studied to determine whether the neurovirulent phenotype could be restored by rescue of the gC gene using standard marker rescue cotransfection procedures. The resulting progeny contained 2% gC+ recombinant virions and was tested for its ability to cause encephalitis. Although this progeny had increased virulence, it was not attributable to the acquisition of the gC gene since passive immunization of mice with a pool of anti-gC monoclonal antibodies had no effect on the development of encephalitis and only gC- viruses were isolated from diseased brain tissues. In agreement with these findings, individual plaque-purified gC positive (gC+) virus recombinants were shown not to have been restored to the wild-type virus level of neurovirulence. It is concluded that gC is not a virulence determinant in this mouse model of HSV-induced encephalitis and that cotransfection procedures can induce additional mutations that affect viral pathogenesis.

Animals↗
PubMed1988

Freestanding emergency centers and the health care of adolescents.

We studied the nature of the visits of patients aged 11-20 years to three free-standing emergency centers (FECs) and compared the mean to the national studies of visits by this age group to pediatricians and family/general physicians. Differences in sex distribution, diagnostic procedures, and diagnostic categories of the FECs emerged. Our results suggest that adolescents seek FEC care for reasons different than those prompting visits to the traditional physician's office.

Adolescent↗
PubMed1988

A specific 15-bp TATA box promoter element is required for expression of a herpes simplex virus type 1 late gene.

The herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene is a true late or gamma 2 gene in that its expression shows a strict requirement for viral DNA replication. Elements required for regulated expression of this gene were previously shown to consist of the gC TATA box, transcription start site and a large portion of the leader sequence of the gC gene. In this paper we show that transcription of the gC gene requires a 15-bp sequence, GGGTATAAATTCCGG, which contains the gC TATA box. This sequence contains specific promoter elements because replacement of this sequence with either the TATA box of the HSV-1 early thymidine kinase (tk) gene or two random TATA-like elements results in a transcriptionally inactive gC gene. In addition, we show that temporal expression of HSV beta and gamma genes at early and late times during infection are controlled by separate and distinct regulatory elements; regulatory signals distal to the TATA box are needed for early expression, whereas a gC-like TATA box is needed for late expression. These signals were identified by construction of a chimeric HSV gene that contained the distal control signals of the beta tk gene fused upstream of the TATA sequence of the gamma 2 gC gene. When RNA was isolated at various times postinfection from cells infected with a virus whose genome contained this chimeric tk-gC gene, synthesis of gC mRNA showed both early and late kinetics.

Base Sequence↗
PubMed1988

Molecular analysis of even-skipped mutants in Drosophila development.

The homeo box gene even-skipped (eve) plays a key role in the regulation of the Drosophila segmentation pattern. eve- embryos lack segment borders and show altered activities of several segmentation genes, including fushi tarazu (ftz), engrailed (en), and wingless (wg). Here, we present evidence that eve influences its own expression in a tissue-specific manner. Each of four different eve mutations disrupts the normal eve expression pattern, and null mutations cause a premature loss of eve products in ectodermal, but not mesodermal, tissues. Molecular characterization of eve mutations indicates that disruptions of the eve pattern are not due to alterations in the eve promoter but, instead, involve abnormal eve proteins. Two different eve mutations cause single amino acid substitutions within the homeo box, and we discuss the implications of these changes with regard to homeo box gene function. We also present evidence that eve+ gene activity is not only required for the activation of the odd-numbered en stripes but also for the correct positioning of each ftz stripe. We present a model for the loss of en expression in eve- embryos, based on the concentration-dependent regulation of the ftz pattern by eve+ products.

Alleles↗
PubMed1988

Simple assay for quantitation of Tn5 inversion events in Escherichia coli and use of the assay in determination of plasmid copy number.

In this report, we describe a simple method for measuring the frequency of sequence inversion in the transposable element Tn5 as a result of recombination across its duplicated IS50 elements. The structure of Tn5 was manipulated so that the neomycin phosphotransferase gene of the transposon would be expressed only if a sequence inversion event occurred. This highly sensitive assay also served as the basis for a novel means of estimating plasmid copy number.

Chromosome Inversion↗
PubMed1988

The carboxy-terminal 41 amino acids of herpes simplex virus type 1 glycoprotein B are not essential for production of infectious virus particles.

Glycoprotein B (gB) is a virally encoded protein that is found in the envelope of herpes simplex virus type 1 and membranes of cells infected with herpes simplex virus type 1. It is essential for the production of infectious virus particles. An amber mutation was introduced into the gB gene by oligonucleotide-directed mutagenesis at the codon for amino acid 863 of the protein. Virus carrying this mutation should synthesize gB molecules lacking the last 41 amino acids of the cytoplasmic domain. Immunoprecipitation of infected cell extracts demonstrated the synthesis of appropriately truncated gB molecules. Characterization of the mutant virus indicated that the loss of the carboxy-terminal 41 amino acids has little effect on gB function.

Amino Acids↗
PubMed1988

Monoclonal antibodies define a domain on herpes simplex virus glycoprotein B involved in virus penetration.

In an earlier report (S.D. Marlin, S.L. Highlander, T.C. Holland, M. Levine, and J.C. Glorioso, J. Virol. 59: 142-153), we described the production and use of complement-dependent virus-neutralizing monoclonal antibodies (MAbs) and MAb-resistant (mar) mutants to identify five antigenic sites (I to V) on herpes simplex virus type 1 glycoprotein B (gB). In the present study, the mechanism of virus neutralization was determined for a MAb specific for site III (B4), the only site recognized by MAbs which exhibited complement-independent virus-neutralizing ability. This antibody had no detectable effect on virus attachment but neutralized viruses after adsorption to cell monolayers. These findings implied that the mechanism of B4 neutralization involved blocking of virus penetration. The remaining antibodies, which recognized sites I, II, and IV, required active complement for effective neutralization. These were further studied for their ability to impede virus infectivity in the absence of complement. Antibodies to sites I (B1 and B3) and IV (B6) slowed the rate at which viruses penetrated cell surfaces, supporting the conclusion that antibody binding to gB can inhibit penetration by a virus. The data suggest that MAbs can interfere with penetration by a virus by binding to a domain within gB which is involved in this process. In another assay of virus infection, MAb B6 significantly reduced plaque development, indicating that antibody binding to gB expressed on infected-cell surfaces can also interfere with the ability of a virus to spread from cell to cell. In contrast to these results, antibodies to site II (B2 and B5) had no effect on virus infectivity; this suggests that they recognized structures which do not play a direct role in the infectious process. To localize regions of gB involved in these phenomena, antibody-binding sites were operationally mapped by radioimmunoprecipitation of a panel of truncated gB molecules produced in transient-expression assays. Residues critical to recognition by antibodies which affect penetration by a virus (sites I, III, and IV) mapped to a region of the molecule (amino acid residues 241 to 441) which is centrally located within the external domain. Antibodies which had no effect on penetration (site II) recognized sequences distal to this region (residues 596 to 737) near the transmembrane domain. The data suggest that these gB-specific MAbs recognize two major antigenic sites which reside in physically distinct components of the external domain of gB.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

Cloning, Molecular↗
PubMed1988

DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context.

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.

Amino Acid Sequence↗
PubMed1988

Possible interaction of ranitidine with phenytoin.

Clinical and animal studies have shown that cimetidine and ranitidine can inhibit hepatic cytochrome P-450-mediated metabolism of a variety of other drugs. This occurs to a lesser extent with ranitidine than with cimetidine at doses commonly used to treat gastric acid-related disorders. We recently observed a 66-year-old man whose steady-state serum phenytoin concentration increased 40 percent during one month after the addition to his regimen of ranitidine 150 mg bid. Because the ranitidine had been prescribed postsurgically for prophylaxis, it was discontinued and the patient's serum phenytoin concentration declined to the previous steady-state level with no change in dose or other drug therapy. This case indicates that the serum phenytoin concentration should be monitored for the first month after the addition of ranitidine to the regimens of patients on chronic phenytoin therapy. As well, further clinical investigation of factors affecting the interindividual inhibitory action of ranitidine is warranted.

Aged↗