Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli.

Incubation of purified Escherichia coli biodegradative threonine dehydratase with glyoxylate resulted in covalent binding of 1 mol of glyoxylate per mol of protein with concomitant loss of enzyme activity. The glyoxylate-binding site was identified as a heptapeptide representing amino acid residues Ser-33-Asn-Tyr-Phe-Ser-Glu-Arg-39 in the protein primary structure. Addition of glyoxylate to a culture of E. coli cells led to time-dependent enzyme inactivation. Immunoprecipitation with anti-dehydratase antibody of extract from [14C]glyoxylate-treated cells revealed labeled dehydratase polypeptide. These results are interpreted to mean that enzyme inactivation by glyoxylate in E. coli cells is associated with covalent protein modification.     

EPR studies with 77Se-enriched (NiFeSe) hydrogenase of Desulfovibrio baculatus. Evidence for a selenium ligand to the active site nickel

The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.     

Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice

Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Cam^r Amp^r Tet^s), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10. TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Mechanisms of beta-adrenergic stimulation of cardiac Ca2+ channels revealed by discrete-time Markov analysis of slow gating.

Individual cardiac Ca2+ channels cycle slowly between a mode of gating in which the channel is available to open, and one in which the channel remains silent. The regulation of this multisecond cycling process by isoproterenol was investigated by single-channel recording and the development of a discrete-time Markov model that describes the slow switching among modes in terms of (de) phosphorylation reactions. The results provide evidence that isoproterenol increases Ca2+ channel activity by a reciprocal regulatory mechanism: not only is the phosphorylation rate of the channel increased, but also the dephosphorylation rate decreased. The discrete-time Markov formalism should prove useful as a general tool for understanding the mode switching demonstrated by a number of ionic channels.     

Life histories ofPsyllaephagus yaseeni (Hym., Encyrtidae) andTamarixia leucaenae (Hym., Eulophidae), parasitoids of the leucaena psyllidHeteropsylla cubana

Age specific fecundity of two parasitoids,P. yaseeni andT. leucaenae, of the leucaena psyllidH. cubana, were studied under laboratory conditions. At 25 °C,P. yaseeni had a greater fecundity (R₀=192.9)_thanT. leucaenae (R₀=71.2);T. leucaenae however had a lower sex ratio (about 99 % females) thanP. yaseeni (about 50 % females). Innate capacity for increase (r_m=0.236) ofT. leucaenae was higher thanP. yaseeni (r_m=0.188). Developmental rates of the parasitoids were examined at constant and fluctuating temperatures and equations of the rate of development against temperature were calculated. At 25 °C, mean generation times were 28.0 and 18.1 days forP. yaeseeni andT. leucaenae respectively. At temperatures of 21.5, 25, and 30 °C total development times (egg to adult) were 28.5, 21.9, and 14.7 days inP. yaseeni and 19.2, 12.6, and 9.5 days inT. leucaenae respectively. The level of parasitism was low and pupal mortality was high at the lower temperature of 21.5 °C for both parasitoids. Both parasitoids showed poor survivorship at 100 % RH,P. yaseeni survived particularly well (32 days) at a temperature of 21.5 °C and 44 or 76 % RH. P. yaseeni allocated about 58 % females to first instar psyllid nymphs but only 12 % females to second instars. About 99 % of allT. leucaenae births were females. Significantly largerT. leucaenae females emerged from fifth instar parasitized nymphs than third or fourth instars.     

Minute supernumerary ring chromosome 22 associated with cat eye syndrome: further delineation of the critical region.

Cat eye syndrome (CES) is typically associated with a supernumerary bisatellited marker chromosome (inv dup 22pter-22q11.2) resulting in four copies of this region. We describe an individual showing the inheritance of a minute supernumerary double ring chromosome 22, which resulted in expression of all cardinal features of CES. The size of the ring was determined by DNA dosage analysis and FISH analysis for five loci mapping to 22q11.2. The probes to the loci D22S9, D22S43, and ATP6E were present in four copies, whereas D22S57 and D22S181 were present in two copies. This finding further delineates the distal boundary of the critical region of CES, with ATP6E being the most distal duplicated locus identified. The phenotypically normal father and grandfather of the patient each had a small supernumerary ring chromosome and demonstrated three copies for the loci D22S9, D22S43, and ATP6E. Although three copies of this region have been reported in other cases with CES features, it is possible that the presence of four copies leads to greater susceptibility.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Chemotaxis ofRhizobium sp. Towards root exudate ofCicer Arietinum L.

Summary Root exudate from seedlings ofCicer arietinum L. was collected in a chamber under aseptic conditions. The exudate was fractionated into anion, cation and neutral fractions. The anionic fraction was made up of galacturonic acid, gluconic acid, mannuronic acid and two unidentified compounds withR _f values 0.56 and 0.62. The cationic fraction contained alanine, arginine, aspartic acid, cystine, glycine, histidine, isoleucine, leucine, lysine and serine. The neutral fraction was made up of arabinose, galactose, glucose, ribose and xylose. The amino acids contributed to the bulk of the root exudate. The ratio of anionic, cationic and neutral fraction was 1∶7∶2. The crude root exudate was tested for its chemotactic ability using the capillary tube method. It was highly chemotactic for theRhizobium sp. The individual fractions and their various combinations were tested for chemotaxis. The chemotactic response of the Cicer strain of Rhizobium was least with anionic fraction most with cationic fraction and intermediate with neutral fraction. Maximum chemotactic response among the fractional combinations was obtained with all the three fractions and least with cationic plus neutral factions. Individual compounds constituting the various fractions were also tried for their ability to elicit chemotactic response. The organism exhibited maximum positive chemotactic response to histidine and negative response to alanine among the amino acids and to glucose and gluconic acid among the sugars and sugar acids.