Construction of transposons encoding genes for β-glucosidase,amylase and polygalacturonatetrans-eliminase fromKlebsiella oxytoca and their expression in a range of gram-negative bacteria

DNA fragments containing theKlebsiella oxytoca genes encoding -glucosidase and amylase were cloned into the kanamycin resistance transposon Tn5. Another DNA fragment containing two genes for polygalacturonatetrans-eliminase was cloned into Tn1721. These newly constructed transposons were then each transposed in vivo onto the broad-host-range plasmid pR751 and conjugally transferred to a variety of Gram-negative bacteria. These were then screened for the newly acquired phenotypes.     

Enhanced recognition of a modified peptide antigen by cytotoxic T cells specific for influenza nucleoprotein

A previously identified Kd restricted epitope of influenza A virus nucleoprotein (147-161) was modified, resulting in recognition by Kd restricted cytotoxic T cells at significantly lower concentrations than the natural peptide sequence. This was achieved by first refining the epitope to the minimum determinant 147-158. Deletion of arginine 156 resulted in a peptide that was shown to be greatly superior in both dose response titrations and in its rate of association with cells to form targets. Analog peptides were tested to determine the important amino acid changes. These data suggest that T cell epitopes can be modified to result in improved immunological recognition.     

Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4.

Plasmid pJP4 is an 80-kilobase, IncP1, broad-host-range conjugative plasmid of Alcaligenes eutrophus encoding resistance to mercuric chloride and phenyl mercury acetate and degradation of 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoate. By the use of cloning, transposon mutagenesis, and restriction endonuclease analysis, a biophysical and genetic map of pJP4 was generated.     

Use of plasmid pULB113 (RP4::Mini-Mu) to construct a genomic map ofAeromonas hydrophila

Plasmid pULB113 (RP4::Mini-Mu) promoted homologous gene transfer inAeromonas hydrophila; transfer of chromosomal markers occurred at frequencies of between 10^–3 and 10^–4 per donor cell regardless of the marker selected; this indicated chromosome transfer from multiple origins. With a variety of amino acid biosynthetic markers, a single circular map of this bacterium was constructed.     

Cloning of a haemolysin from Citrobacter freundii and its expression in phylogenetically related bacteria

Abstract The determinants for a haemolysin from an extraintestinal isolate of Citrobacter freundii have been cloned and expressed in both Escherichia coli K12 and phylogenetically related bacteria. Compared with E. coli , where the haemolytic determinants are encoded in 7.5 kb, the hemolysin determinants of C. freundii are located on a 2.5-kb Hin dIII fragment in the recombinant plasmid PJP71. Chicken embryo tests indicate that this haemolysin does contribute to the pathogenicity of C. freundii .     

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system

Albicidin blocked DNA synthesis in intact cells of a PolA- EndA- Escherichia coli strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication. Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidin-resistant (Albr) E. coli host-cells. All stable spontaneous Albr mutants of E. coli simultaneously became resistant to phage T6. The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations. Albicidin does not closely resemble a nucleoside in structure. However, Albs E. coli strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations. An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance. Albicidin supplied at approximately 0.1 microM blocked DNA replication within seconds in intact Albs E. coli cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells. We conclude that albicidin is effective at very low concentrations against E. coli because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake. Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

GENETIC VARIATION AND JUVENILE SURVIVAL IN RED DEER

The survival of red deer (Cervus elaphus L.) calves to two years of age was examined in relation to electrophoretic variation in a population on the Scottish island of Rhum. Survival was analyzed using logistic analysis in which the “phenotypic” factors birth weight, birth date, subdivision of the study area, cohort, and sex, which affect the probability of a calf's survival, were taken into account. All three polymorphic loci examined, Mpi, Idh-2, and Trf (each with two detected alleles) are significantly associated with juvenile survival. At Mpi, there is selection against one allele, f (or an allele at a linked locus), and there are indications that this effect is stronger in females than males. For Idh-2, overall, the heterozygote class survives better than the two homozygotes, which survive equally well. However, again there is a difference between the sexes; female heterozygotes survive much better than homozygotes, whereas male homozygotes survive better than heterozygotes, and the difference in survival is smaller. Furthermore, there is an interaction involving Mpi, Idh-2, and survival in which Mpi^f carriers that are also Idh-2 homozygotes survive very badly compared with other Mpi-Idh-2 combinations, which all survive equally well. For Trf, the heterozygote class survives best, and there is also a difference in survival between the two homozygote classes. Genotype frequencies in the adult population are consistent with the results for calf survival, in that the Mpi^f frequency is lower in succeeding cohorts of surviving adults, whereas no significant gene frequency change is apparent for Idh-2 or Trf.     

Interaction of subtilisins with serpins.

Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that alpha 1 proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and alpha 1 antichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 degrees C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.