Preparation of an active recombinant peptide of crustacean androgenic gland hormone

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.     

Developmental expression of ascidian neurotransmitter synthesis genes

To identify cholinergic neurons, we isolated a choline acetyltransferase (Ci-ChAT) gene from Ciona intestinalis by PCR methods. In the cloning process, we also obtained the gene encoding the vesicular acetylcholine transporter (Ci-vAChTP). These two genes shared the same 5'-UTR sequence as well as similar expression patterns. In both cases, the gene expression was first detected by whole-mount in situ hybridization in the anterior-dorsal region of the caudal nerve cord at the early tailbud stage. In the larva, the expression was seen in several cells of the visceral ganglion. These results suggest that ascidian larval motor neurons exist in the visceral ganglion.     

Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme

To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.     

QTL Analysis of Al Tolerance in Recombinant Inbred Lines of Arabidopsis thaliana

In the December issue of Plant and Cell Physiology     

Reaction of CO with cytochrome c oxidase. Titration of the reaction site with chemical oxidant and reductant

The number of reducing equivalents required to form the reduced cytochrome a₃-CO compound has been determined for suspensions of submitochondrial particles and for isolated cytochrome c oxidase. Anaerobic preparations were titrated reductively with NADH and oxidatively with O₂ in the presence of high concentrations of CO (0.4 to 0.8 mM) while monitoring reduction of cytochrome a and the formation of the reduced cytochrome a₃-CO compound by their characteristic absorbance changes. Analysis of the titration data show that 2.0±0.3 and 2.1±0.2 reducing equivalents per mol of cytochrome oxidase (per cytochrome a) are required for formation of the reduced cytochrome a₃-CO compound in submitochondrial particles and isolated cytochrome c oxidase, respectively. In each case, the formation of the CO compound is proportional to the number of equivalents accepted by the preparation, indicating that the two equivalents are equal and the effective n value for the reaction is 2.0. Potentiometric titrations of cytochrome c oxidase using the cobalt orthophenanthrolene complex (E_{m, 7.0} = 0.37 V) as mediator give the same half-reduction potential values for cytochrome a and a₃ as those obtained using the ferro-ferricyanide couple. The formation of the reduced cytochrome a₃-CO compound at pH 7.0, in the presence of 0.6 mM CO and with CO-orthophenanthrolene as mediator occurs with a half-reduction potential of 0.45 V and requires two electrons. These data confirm and extend the observation of Lindsay et al. (Arch. Biochim. Biophys. (1975) 169, 492–505) that both the “invisible” copper and cytochrome a₃ must be reduced in order for CO to bind with high affinity.     

Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)     

Nucleotide sequences of the mouse globin beta gene cDNAs in a wild derived new haplotype Hbb w1

Haplotypes of the beta-globin gene complex (Hbb) in laboratory mice have been defined as d, p, and s. We previously found a new haplotype w1 in wild mice collected from northwestern China. This study analyzed the nucleotide sequences of b1 and b2-globin gene cDNAs of both the p and w1 haplotypes, in comparison with those of the d haplotype. In Hbb-b1 cDNA, six base substitutions were found between the d and w1 haplotypes and also between p and w1, but none existed between d and p. In Hbb-b2 cDNA, three base substitutions were found between the d and w1 haplotypes and also between d and p, but none between p and w1. This result indicated that the Hbb gene complex of the p haplotype carries b1 ^d and b2 ^w1 genes and is probably a recombinant between d and w1 haplotypes. The hemoglobin containing the W1 phenotype showed oxygen-binding properties identical with those of the hemoglobins containing D and P phenotypes. Received: 5 January 1999 / Accepted: 5 April 1999