Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Attachment as a factor in the protection of Enterobacter cloacae from chlorination

Enterobacter cloacae attached to drinking water distribution particles was subjected to chlorination. Attachment resulted in the protection of these organisms from disinfection. This effect was found to be dependent upon both the level of chlorine in the system and attachment time. The results obtained in this study indicate that attached organisms may play an important role in coliform outbreaks.     

Comparisons of tests for aneuploidy

The fundamental problems that face us in the development of suitable assay systems for the detection of potentially aneugenic (aneuploidy-inducing) chemicals include: (a) the diversity of cellular targets and mechanisms where perturbations of structure and function may give rise to changes in chromosome number, and (b) the phylogenetic differences that exist between species in their mechanism and kinetics of cell division and their metabolic profiles. A diverse range of assay systems have been developed, which have been shown to have potential for use in the detection of either changes in chromosome number or of perturbations of the events which may be causal in the induction of aneuploidy.

Chromosome number changes may be detected cytologically by karyotypic analysis, or by the use of specialised strains in which aneuploid progeny may be observed due to phenotypic differences with aneuploid parental cells or whole organisms. Techniques for the detection of cellular target modifications range from in vitro studies of tubulin polymerisation to observations of the behaviour of various cellular organelles and their fidelity of action during the division cycle.

The diversity of mechanisms which may give rise to aneuploidy and the qualitative relevance of events observed in experimental organisms compared to man make it unlikely that the detection and risk assessment of the aneugenic activity of chemicals will be possible using a single assay system. Optimal screening and assessment procedures will thus be dependent upon the selection of an appropriate battery of predictive tests for the measurement of the potentially damaging effects of aneuploidy induction.     

Antigenic structure and variation in an influenza virus N9 neuraminidase.

We previously determined, by X-ray crystallography, the three-dimensional structure of a complex between influenza virus N9 neuraminidase (NA) and the Fab fragments of monoclonal antibody NC-41 [P. M. Colman, W. G. Laver, J. N. Varghese, A. T. Baker, P. A. Tulloch, G. M. Air, and R. G. Webster, Nature (London) 326:358-363, 1987]. This antibody binds to an epitope on the upper surface of the NA which is made up of four polypeptide loops over an area of approximately 600 A2 (60 nm2). We now describe properties of NC-41 and other monoclonal antibodies to N9 NA and the properties of variants selected with these antibodies (escape mutants). All except one of the escape mutants had single amino acid sequence changes which affected the binding of NC-41 and which therefore are located within the NC-41 epitope. The other one had a change outside the epitope which did not affect the binding of any of the other antibodies. All the antibodies which selected variants inhibited enzyme activity with fetuin (molecular weight, 50,000) as the substrate, but only five, including NC-41, also inhibited enzyme activity with the small substrate N-acetylneuramin-lactose (molecular weight, 600). These five probably inhibited enzyme activity by distorting the catalytic site of the NA. Isolated, intact N9 NA molecules form rosettes in the absence of detergent, and these possess high levels of hemagglutinin activity (W.G. Laver, P.M. Colman, R.G. Webster, V.S. Hinshaw, and G.M. Air, Virology 137:314-323, 1984). The enzyme activity of N9 NA was inhibited efficiently by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, whereas hemagglutinin activity was unaffected. The NAs of several variants with sequence changes in the NC-41 epitope lost hemagglutinin activity without any loss of enzyme activity, suggesting that the two activities are associated with separate sites on the N9 NA head.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

Relationships among pure cultured strains of Frankia based on host specificity

Fifty strains of Frankia were tested for their ability to nodulate six species of actinorhizal plants. Pure cultured strains were used to inoculate seedlings of Alnus glutinosa (L.) Gaertn., Alnus rubra Bong., Casuarina equisetifolia L., Elaeagnus angustifolia L., Hippophaë rhamnoides L. and Myrica cerifera L. in nutrient solution culture. From the results of this study, host inoculation groups among the actinorhizal plants were defined. Although overlap between host inoculation groups appears to be common, the results from this study did not support the view that Frankia strains are promiscuous. All Frankia strains tested in this study could easily be classified into four major host-specificity groups.     

Transport and Compartmentation of 1-Aminocyclopropane-1-Carboxylic Acid and Its Structural Analog, alpha-Aminoisobutyric Acid, in Tomato Pericarp Slices

The uptakes of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene, and its structural analog, α-aminoisobutyric acid (αAIB) by tomato pericarp slices were investigated. Both uptakes show a biphasic (saturable-linear) dependence on external concentration of the transported amino acid. At low concentrations, ACC uptake is competitively inhibited by αAIB and vice versa. Both uptakes also are inhibited by other neutral amino acids but not by acidic or basic amino acids. ACC and αAIB uptakes are metabolically dependent and are increased with time of tissue incubation. αAIB efflux patterns from pericarp slices indicated three distinct αAIB compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole. The bulk of the αAIB taken up by pericarp tissue is sequestered into the vacuole. The ability of pericarp tissue to accumulate αAIB in the vacuole declines with fruit development.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin.

Recently, we have identified a novel topogenic sequence at the C terminus of Escherichia coli haemolysin (HlyA) which is essential for its efficient secretion into the medium. This discovery has introduced the possibility of using this secretion system for the release of chimeric proteins from E. coli directly into the medium. We have now successfully fused this C-terminal signal to a hybrid protein containing a few residues of beta-galactosidase and the majority of the E. coli outer membrane porin OmpF lacking its own N-terminal signal sequence. We find that this chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. In addition, we have further localized the HlyA secretion signal to the final 113 amino acids of the C terminus. In fact, a specific secretion signal appears to reside at least in part within the last 27 amino acids of HlyA.