Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.     

Effect of environmental stress on release of norepinephrine in posterior nucleus of the hypothalamus in awake rats: role of sinoaortic nerves

Norepinephrine(NE) release in posterior nucleus(PH) of the hypothalamus was examined before and during acute shaker (oscillation) stress in sinoaortic denervated(SAD) and sham-operated(SO) rats. NE in PH extracellular fluid of freely moving rats was collected by microdialysis and measured by sensitive radioenzymatic assay. Three days after SAD or SO operation, mean arterial pressure(MAP) and heart rate(HR) were significantly higher in SAD rats than SO rats. Baseline levels of NE in PH dialysate were also significantly elevated in SAD rats. Although five minutes of shaker stress elicited pressor and tachycardic responses coupled with increased NE release in PH of both groups, the increases in MAP and dialysate NE were larger in SAD than SO rats. These findings indicate that noradrenergic neurons in the PH respond to stress-induced stimuli and receive tonic input from baroreflex pathways.     

Fractionation of Nitrogen Isotopes during the Uptake and Assimilation of Ammonia by Plants

Two varieties (Nihonbare and Koshihikari) of rice plants (Oryzasativa L.) were grown hydro-ponically with two levels (20 and100 mg N liter ^–1) of ammonia. Variations in levels ofnatural abundance of ¹⁵N (¹⁵N) were analyzed in the ammoniaand organic nitrogen of shoots and roots, as well as in theammonia in the culture solution. There was substantial fractionationof nitrogen isotopes during the uptake of ammonia. When plantsabsorbed a large proportion of ammonia from a solution witha low concentration, less negative ¹⁵N values in plants andhigh positive ¹⁵N values in the ammonia remaining in solutionwere observed. The reverse was found when a smaller fractionof ammonia was absorbed from a solution with a higher concentrationof ammonia. The ^l5N values of ammonia in shoots and roots werehigher than in the respective constituent organic nitrogen,suggesting the fractionation of nitrogen isotopes during theassimilation of ammonia. Wild-type and mutant cells of the cyanobacterium(blue-green alga) Synechococcus PCC 7942 were grown in nitrate-or ammonia-containing medium as the source of nitrogen. Fractionationof nitrogen isotopes during the uptake of nitrate was limited,whereas that during the uptake of ammonia was considerable. ¹ In this report, the term ammonia refers indiscriminately toboth NH₃ or NH₄⁺. (Received June 13, 1991; Accepted September 12, 1991)     

Formation of nodules on non-nodulating soybean T201 after treatment with 2,4-dichlorophenoxyacetate

The formation of effective root nodules on a non-nodulating line (T201) of soybean (Glycine max (L.) Merr.,) was induced by a treatment with 2,4-dichlorophenoxyacetate (2,4-D). The induced nodules, inoculated with mixed Bradyrhizobium japonicum strains A1017 and IRj2101, had a normal internal structure, red in colour and the cells being filled with bacteroids. Externally, the induced nodules were of unusual shape, being paired or gourd-like in form and were attached to thickened roots. The nodules were capable of acetylene reduction (3.1–3.5 moles g^-1 fresh weight nodules h^-1), allowing the growth of plants with dark green leaves.     

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.     

Properties of the Escherichia coli RuvA and RuvB proteins involved in DNA repair, recombination and mutagenesis

The ruvA and ruvB genes constitute an operon, which is regulated by the SOS system and involved in DNA repair, recombination and mutagenesis. RuvA protein binds to both single-stranded and double-stranded DNA. RuvB protein has weak ATPase activity. RuvA bound to DNA greatly enhances ATPase activity of RuvB. UV-irradiation to supercoiled DNA further enhances the stimulatory effect of RuvA on the RuvB ATPase activity. In the presence of ATP the RuvA-RuvB complex has an activity that renatures cruciform structures formed by heating and gradually cooling supercoiled DNA with an inverted repeat. These findings suggest that the RuvA-RuvB complex interacts with an irregular conformation in damaged DNA and induces conformational changes in DNA using energy provided by ATP hydrolysis, so that it facilitates DNA repair, recombination and error prone replication.     

8-Methyladenosine-substituted analogues of 2-5A: synthesis and their biological activities.

8-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me8A), p5'A2'p5'(me8A)2'p5'(me8A), p5'(me8A)2'p5'(me8A)2'p5'(me8A), and p5'(me8A) 2'p5'A2'p5'A, were prepared via a modification of a lead ion-catalyzed ligation reaction. These 2-5A monophosphates were converted into the corresponding 5'-triphosphates. Substitution of an 8-methyladenosine residue at the third position (2'-terminus) of the oligonucleotides increased the stability to snake venom phosphodiesterase digestion. Both binding and activation of mouse liver 2-5A dependent ribonuclease (RNase L) by the various 8-methyladenosine-substituted 2-5A analogues were examined. Among the 8-methyladenosine-substituted trimer analogues, the analogues with 8-methyladenosine residing in the 2'-terminal position showed the strongest binding affinity and were several times more effective than 2-5A itself as an inhibitor of translation.     

Prediction of splice junctions in mRNA sequences.

A general method based on the statistical technique of discriminant analysis is developed to distinguish boundaries of coding and non-coding regions in nucleic acid sequences. In particular, the method is applied to the prediction of splicing sites in messenger RNA precursors. Information used for discrimination includes consensus sequence patterns around splice junctions, free energy of snRNA and mRNA base pairing, and statistical differences between coding and non-coding regions such as periodic appearance of specific bases in coding regions reflecting the non-random usage of degenerate codons. Given the reading frame of an exon (but not the exon/intron boundaries), the method will predict the following exon, namely, the intron to be excised out. When applied to human sequences in the GenBank database, the method correctly identified 80% of true splice junctions.