Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Characterization of serological properties of polyclonal antibodies produced against enzymes involved in the L-selective cleavage of hydantoin derivatives

Summary Polyclonal antibodies were produced against the highly purified enzymes L-hydantoinase, hydantoin-racemase and L-N-carbamoylamino acid amidohydrolase of Arthrobacter aurescens DSM 3747. In order to exploit these antibodies for basic research (molecular biology) or bioengineering (process development), the serological properties had to be characterized. Both, the hydantoinase- and carbamoylase-antibodies were observed to be monofunctional, whereas the hydantoin-racemase-antibody was found to be additionally specific against the L-hydantoinase. Monospecificity was realized after affinity chromatography. Investigations on serological crossreactions with several linear- and cyclic amidases (e.g. hydantoinases) as well as hydantoin-racemases are demonstrated in this paper.Deticated to Prof. Dr. Klaus Mosbach on the occation of his 60th birthday.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

The microaerophily and photosensitivity of Propionibacterium acnes

E. MARTIN GRIBBON, J.G. SHOESMITH, W.J. CUNLIFFE AND K.T. HOLLAND. 1994. The effect of oxygen on the in vitro propagation of Propionibacterium acnes was investigated under defined culture conditions. This micro-organism is the predominant bacterial resident within the pilosebaceous follicles of sebum-rich areas of human skin. The organism was grown in continuous culture in defined synthetic medium with glucose as the main carbon-energy source at various air saturation concentrations and in the presence and absence of light. Steady state continuous cultures were achieved at very low oxygen tensions in the presence of light, and at higher levels of oxygen when non-illuminated. Culture biomass yields were higher than those of anaerobic cultures. Bacterial cells were inactivated in the presence of light at high oxygen concentrations because of photosensitization reactions involving excess oxygen and microbial porphyrin species.     

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α₁-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc.     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

Constructs carrying the entire or part of the tobacco nitrate reductase cDNA (NIA) cloned between the promoter and terminator sequences of the 35S RNA of the cauliflower mosaic virus were introduced into tobacco, in an attempt to improve nitrate assimilation. Several transgenic plants that had elevated NIA mRNA and nitrate reductase (NR) activity were obtained. In addition, a few plants that exhibited a chlorotic phenotype characteristic of NR-deficient mutants were also obtained. One of these plants contained no NIA mRNA, no NR activity and accumulated nitrate. This phenotype was therefore assumed to result from co-suppression of 35S-NIA transgenes and host NIA genes. NR-deficient plants were also found among the progeny of transformants overexpressing NIA mRNA. Genetic analyses indicated that these NR-deficient plants were homozygous for the 35S-NIA transgene, although not all homozygous plants were deficient for NR. The ratio of normal to NR-deficient plants in the progeny of homozygous plants remained constant at each generation, irrespective of the state of expression of the NIA genes (active or inactive) in the previous generation. This ratio also remained unchanged when field trials were performed in two areas of France: Versailles and Bergerac. The analysis of homozygous plants revealed that co-suppression was reversible at some stage of sexual reproduction. Indeed, host genes and transgenes reactivated at each generation, and co-suppression always appeared after a lag period of normal growth, suggesting that the phenomenon is developmentaly regulated. We observed that the triggering of cosuppression was delayed when plants were initially grown under limited light and/or watered with limited nitrate supply (light and nitrate both being required for the expression of the host NIA genes). However, this delay did not affect the final ratio between normal and NR-deficient plants after transfer to nitrate-fertilized fields. Independent transformants exhibited either different co-suppression ratios or no co-suppression at all, irrespective of the transgene copy number, suggesting that genomic sequences surrounding the transgene might play a role in determining co-suppression.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.