The use of λplac-Mu hybrid phages in Klebsiella pneumoniae and the isolation of stable Hfr strains

Klebsiella pneumoniae 1033-5P14 and its P1-sensitive derivative KAY2026 were found to be resistant to lambda although they contained a LamB protein, active as a maltoporin. Sensitive derivatives could only be obtained after introduction of the pTROY9 plasmid which expresses lamB and the corresponding lambda receptor from Escherichia coli K12 at high levels. Lysogenic derivatives from such strains were shown to carry the phage at secondary att sites and to give high titer lysates when induced. The use of lambda plac-Mu hybrid phages allowed the isolation from several operons of lacZ fusions orientated in, or against, the direction of transcription. Such insertions could subsequently be used to isolate stable Hfr strains by allowing homologous recombination to take place between the lac genes in the inserted hybrid phages and those of plasmid F' ts114 lac+ zzf20::Tn10. The Hfr strains were able to transfer K. pneumoniae chromosomal genes and allowed the mapping of such genes. Characteristic differences between this conjugation system and that of Escherichia coli K12 are discussed. The insertions also allowed determination of the direction of transcription of the gut gene, the newly mapped scr gene and of the sor gene cluster encoding enzymes for the metabolism of D-glucitol, sucrose and L-sorbose.     

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G. austeni Newstead and G. p. palpalis R-D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A 'refractory' line of G. m. morsitans Westwood took 170 h to kill 50% infections while a 'susceptible' line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo. Flies fed before an infective feed were able to remove trypanosomes from their midguts more quickly than flies infected as tenerals. Increasing the period of starvation before infection increased the susceptibility to trypanosome infection of non-teneral flies. Teneral flies showed little agglutinating activity in vitro, suggesting that lectin is produced in response to the bloodmeal. Feeding flies before infection also abolished the differences in rate of trypanosome killing found between teneral 'susceptible' and 'refractory' G. m. morsitans, suggesting that maternally inherited susceptibility to trypanosome infection is a phenomenon limited to teneral flies. Electron micrographs of midguts of G. m. morsitans suggest that procyclic trypanosomes are killed by cell lysis, presumably the result of membrane damage caused by lectin action.     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-subunit of human acetylcholine receptor were studied for their binding activity ofp>125p>I-labeled p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">122–138. In addition, low-binding activities were obtained with peptides p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">34–49 and p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">194–210. It is concluded that the region within residues p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

A re-examination of the Na+-independent binding of [3H]β-alanine to rat brain stem-spinal cord

The Nap>+p>-independent binding of [p>3p>H]p3lm5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-alanine to rat brain stem plus spinal cord was reinvestigated, in order to study in more detail the characteristics of previously described p3lm5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-alanine binding processes. Binding was absent when amino acid-free postnuclear supernatants or crude synaptic membranes were used. Experiments performed with several other Nap>+p>-free preparations showed a sole binding component, irrespective of the preparation used. Biochemical characterization of this Nap>+p>-independent binding, using frozen/thawed/washed synaptosomal-mitochodrial fractions, showed that binding reached a plateau between 7 min and 13 min, increasing thereafter. Binding was linear with fraction protein over a range of 200–415 p3lm5/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">g/ml incubation medium. Binding was completely inhibited by glycine, alanine, p3lm5/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-aminobutyric acid, p3lm5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-aminoisobutyric acid, hypotaurine and strychnine, and to a lesser extent by 2,2-dimethyl-p3lm5/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-alanine, brucine and gelsemine. It was insensitive to taurine, p3lm5/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-aminobutyric acid (GABA), 2-guanidinoethanesulfonic acid (GES), carnosine, and bicuculline methiodide. Binding was reversible, saturable (K _D 20 p3lm5/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M), and heat sensitive.     

Genes coding for the major tuberous root protein of sweet potato: Identification of putative regulatory sequence in the 5′ upstream region

The isolation and characterization is described of sporamin A and B genes, gSPO-A1 and gSPO-B1, respectively, which are members of two distinct subfamilies of the multigene family encoding the major soluble protein of sweet potato tuberous root. The nucleotide sequences of the two genes along with the 5′ and 3′ flanking regions were determined. The transcriptional start sites were determined by S1 nuclease mapping. Comparison of the sequences with those of the full-length cDNAs for sporamin A and B reveals that these genes contain no introns. Sequence comparison by dot matrix plot analysis shows that homology of the 5′ flanking regions between gSPO-A1 and gSPO-B1 extends only to 45 bp upstream of the transcription start site, which includes the consensus TATA box sequence. The sequence of this region is also conserved in the sporamin-related gene, gSPO-X1, which dose not seem to be expressed in the tuberous root. The sequences further upstream diverge extensively, but two short sequence blocks of 28 to 29 bp and 19 to 22 bp are found in the 5′ flanking sequence of gSPO-A1 and gSPO-B1 but not in gSPO-X1. The 19–22 bp sequence block is repeated twice in gSPO-A1 and four times in gSPO-B1. These conserved sequence blocks may play regulatory roles in their expression.     

A model for gramicidin A′-phospholipid interactions in bilayers

A model is proposed for the effect of gramicidin Ap723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin Ap723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin Ap723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin Ap723u0004/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine.Abbreviations NMR nuclear magnetic resonance - DMPC dimyristoylphosphatidylcholine - S molecular order parameter - CSA chemical shift anisotropy - DPPC dipalmitoylphosphati-dylcholine - LPC lysophosphatidylcholine     

The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The p92164g02123k284/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the p92164g02123k284/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat p92164g02123k284/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5p3/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.