Identification of two amino acids in the hemagglutinin glycoprotein of measles virus (MV) that govern hemadsorption, HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains.

We have used site-directed mutagenesis of the hemagglutinin (H) glycoprotein of measles virus (MV) to investigate the molecular basis for the phenotypic differences observed between MV vaccine strains and recently isolated wild-type MV strains. The former downregulate CD46, the putative cellular receptor of MV, are positive for hemadsorption, and are fusogenic in HeLa cells, whereas the latter are negative for these phenotypic markers. CD46 downregulation in particular, could have profound consequences for the immunopathology of MV infection, as this molecule protects the cell from complement lysis. Mutagenesis of two amino acids, valine and tyrosine at positions 451 and 481, respectively, in the H protein from the vaccine-like Hallé MV strain to their counterparts, glutamate and asparagine, in the H protein from the wild-type Ma93F MV strain (creating the V451E/Y481N double mutation) abrogated CD46 downregulation, HeLa cell fusion, and hemadsorption. The converse double mutagenesis of the Ma93F H protein (E451V/N481Y) transferred the CD46-downregulating, fusogenic, and hemadsorption functions to this protein. The data provide the first mapping study of the functional domains of MV H. The consequences of these results for MV vaccine design and the role of CD46 in MV infection are discussed.     

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.     

Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways

Biodegradation of 2,4,6-trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate-controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate-limiting due to enzyme control. These rate-limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate-limiting steps. Identification of these rate-controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels.This article identifies likely rate-controlling steps (or sets of steps) in reported TNT biodegradation pathways by estimating the Gibbs free energy change for each step and for the overall pathways. The biological standard Gibbs free energy change of reaction was calculated for each pathway step using a group contribution method specifically tailored for biomolecules. The method was also applied to hypothetical "pathways" constructed to mineralize TNT using several different microorganisms. Pathways steps that have large negative Gibbs free energy changes are postulated to be potentially rate-controlling. The microorganisms which utilize degradation pathways with the largest overall (from TNT to citrate) negatiave Gibbs free energy changes were also determined. Such microorganisms can extract more energy from the starting substrate and are thus assumed to have a competitive advantage over other microorganisms. Results from this modeling-based research are consistent with much experimental work available in the literature. (c) 1996 John Wiley & Sons, Inc.     

Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene

Strain TEA, a strictly anaerobic, motile rod with one to four lateral flagella and a crystalline surface layer was isolated from a mixed culture that completely reduces chlorinated ethenes to ethene. The organism coupled reductive dehalogenation of tetrachloroethene or trichloroethene to cis-1,2-dichloroethene to growth, using molecular hydrogen as the electron donor. It was unable to grow fermentatively or in the presence of tri- or tetrachloroethene with glucose, pyruvate, lactate, acetate or formate. The 16S rDNA sequence of strain TEA was 99.7% identical to that of Dehalobacter restrictus. The two organisms thus are representatives of the same species or the same genus within the Bacillus/Clostridium subphylum of the gram-positive bacteria.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.