Localization of the D5 dopamine receptor gene to human chromosome 4p15.1-p15.3, centromeric to the Huntington's disease locus.

Genes encoding G-protein-coupled receptors, including dopamine, serotonin, muscarinic cholinergic, and adrenergic receptors, play an important role in neurotransmission and may be involved in the pathophysiology of diseases such as Alzheimer's disease, Parkinson's disease, or Huntington's disease (HD). We mapped the gene encoding the D5 dopamine receptor (DRD5) to human chromosome 4p, an area implicated in HD and the Wolf-Hirschhorn syndrome, using gene-specific amplification with the polymerase chain reaction on a panel of somatic cell hybrids carrying different human chromosomes. Further localization of the DRD5 gene was carried out through the isolation and analysis of yeast artificial chromosomes, fluorescence in situ suppression hybridization to human metaphase chromosomes, and analysis of a panel of somatic cell hybrids subdividing human chromosome 4 into nine regions. The human DRD5 gene is located at 4p15.1-p15.33, centromeric to the location of the Huntington's disease locus although not in the obligate area containing the HD gene. The localization of the DRD5 gene to 4p15.1-p15.33 suggests the possibility that cis-position effects could be responsible for the altered D1-type dopamine receptor number observed in HD tissues or that the DRD5 gene could be a candidate for some of the abnormalities associated with the Wolf-Hirschhorn syndrome.     

Isolation and expression of linked zinc finger gene clusters on human chromosome 11q

Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.     

Several CD4 domains can play a role in human immunodeficiency virus infection in cells.

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.     

Mutagenicity of 2-amino-N6-hydroxyadenine at the tk locus in L5178Y strains differing in repair capabilities and karyotype

The cytotoxicity and mutagenicity of 2-amino-N6-hydroxyadenine (AHA) were measured in strains of L5178Y differing in repair capabilities and karyotype. Strain LY-R83 is monosomic for chromosome 11 and is therefore hemizygous for the tk gene, while strains LY-R16 and LY-S1 are TK+/- heterozygotes. Both strain LY-R83 and LY-R16 are sensitive to UV light and are presumed to be deficient in the excision of pyrimidine dimers as shown for the parental strain, LY-R (Hagen et al., 1988; Szumiel et al., 1988). Strain LY-S1 is sensitive to the cytotoxic effects of ionizing radiation and is presumed to be defective in the repair of radiation-induced DNA double-strand breaks, as shown for the parental strain, LY-S (Evans et al., 1987a; Wlodek and Hittelman, 1987). The sensitivities of the three strains to the cytotoxic effects of AHA were similar. After a 4-hour treatment with AHA at 37 degrees C, the D37 for all three strains was approximately 35 ng/ml. The AHA-induced mutant frequency was similar for the hemizygous TK+ strain LY-R83 and the heterozygous TK +/- strain LY-R16, but was slightly higher for strain LY-S1 than for either LY-R strain at an AHA concentration of 100 ng/ml. The proportion of AHA-induced LY-S1 TK -/- mutants forming colonies with diameters less than 0.3 mm was much lower than following treatment with X radiation (24% vs. 61% for AHA and X radiation, respectively). These results indicate that the vast majority of AHA-induced TK -/- mutants harbor single gene mutations. AHA did not result in cyanide-insensitive oxygen uptake, and treatment with this compound did not induce a significant number of DNA single-strand breaks, DNA alkali labile lesions, or DNA degradation in either strain. However, two hours after AHA removal, DNA single-strand breaks and/or alkali-labile lesions, possibly due to the occurrence of DNA repair, were apparent in the DNA of both strain LY-R16 and strain LY-S1.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Glucose-stimulated insulin release by individual pancreatic beta cells: potentiation by glyburide.

To investigate the effect of glyburide on insulin secretion by individual beta cells from normal rats, we employed a reverse hemolytic plaque assay. Pancreata were harvested from female Wistar-Furth rats, the pancreatic islets isolated, and the latter dispersed into single cells. These cells were mixed with protein A-coated ox erythrocytes, the mixture was placed in a Cunningham chamber in the presence of insulin antiserum, and the cells were exposed to the various test substances. Having developed hemolytic plaques around the insulin-secreting cells with complement, the percentage of plaque-forming cells was determined and the plaque areas (reflecting the amount of insulin secreted) were quantitated. For the purpose of validation, we demonstrated that (i) plaque-forming (but not nonplaque-forming) cells could be identified as insulin secreting by an independent immunofluorescent technique, (ii), plaques did not form if insulin antiserum was deleted from the preparation, (iii) plaques failed to develop if insulin antiserum was preabsorbed with insulin, and (iv) incubation with non-protein A-coated RBC or omission of complement resulted in no plaque formation. In addition, both the percentage of plaque-forming cells and the mean plaque are increased upon exposure to glucose (0.75-20 mM) in a concentration-dependent manner at 5- and 60-min incubation times. Moreover, somatostatin suppressed the percentage of plaque-forming cells and diminished the mean plaque area of cells which continued to secrete insulin in response to glucose. Exposure of cells to 100 nM glyburide in the presence of 5 mM or 20 mM glucose had no effect on the percentage of plaque-forming cells present at 5 min or 60 min. Similarly, glyburide did not alter mean plaque area at 5 or 60 min when cells were co-incubated with 5 mM glucose. However, mean plaque area was markedly enhanced at 5 and 60 min in response to glyburide and 20 mM glucose. These results demonstrate that glyburide (i) does appear to enhance insulin secretion by an effect directly on the pancreatic beta cell; (ii) does not act by recruiting previously noninsulin-secreting cells into a secretory pool; (iii) does not potentiate the effect of glucose, at fed concentrations, on insulin secretion by individual cells; but (iv) does augment insulin secretion by beta cells stimulated with supraphysiologic concentrations of glucose.     

Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures

Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Poliovirus antigen chimeras.

Poliovirus, the aetiological agent of paralytic poliomyelitis, is arguably the best characterized of all animal viruses. Using recombinant-DNA technology, this information, together with the availability of infectious cDNA clones of the notably safe and efficacious live attenuated Sabin 1 vaccine strains of poliovirus, has enabled the creation of hybrid viruses (chimeras) possessing novel antigenicity. The potential applications of these 'epitope-presentation systems' include their use as immunogens, as antigens for serodiagnosis, and as vaccines.