Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.     

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences

Summary A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the thylakoid-transfer domain of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Biotransformation of mercury by bacteria isolated from a river collecting cinnabar mine waters

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10g/ml Hg (as HgCl₂) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10g/ml HgCl₂ was diagnostic for Hg detoxification based on reduction.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Three species of Trebius Krøyer, 1838 (Copepoda: Siphonostomatoida) parasitic on Pacific elasmobranchs

The paratypes of Trebius akajeii Shiino, 1954, originally collected from Dasyatis akajei (Müller & Henle) at Owase, Japan, and the paratypes and newly collected specimens of T. latifurcatus Wilson, 1921, previously collected from Urolophus and Myliobatis at Venice, California, are redescribed. New host records for T. latifurcatus are: Torpedo californica Ayres, Squatina californica Ayres, Rhinobatos productus (Ayres), Platyrhinoides triseriata (Jordan & Gilbert), Raja inornata (Jordan & Gilbert) and Gymnura marmorata (Cooper). A new species, Trebius heterodonti, is described from the horn shark Heterodontus francisci (Girard) collected from southern California waters. These three species of Trebius are apparently closely related to each other. Diagnostic characters of the species are provided along with a discussion on the taxonomic value of selected features.     

The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum

Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms.     

Class II MHC molecules and the HIV gp 120 envelope protein interact with functionally distinct regions of the CD4 molecule.

A murine T cell hybridoma with a receptor specific for the class I molecule H-2 Dd was transfected with an expressible cDNA for human CD4. Expression of the human class II MHC molecule HLA-DP on Dd-positive murine fibroblasts resulted in a greatly enhanced response of the CD4-positive T cell hybridoma, measured either by lymphokine production or by rosette formation. Inhibition of these functional assays with anti-CD4 monoclonal antibodies implicated the two amino-terminal domains of CD4 in an interaction with the HLA-DP molecule. This interaction was blocked by incubation with recombinant gp120 envelope protein of HIV. In contrast, recombinant soluble CD4 did not inhibit and was able to prevent the inhibition by gp120. Anti-CD4 antibody blocking experiments clearly indicated that distinct regions of CD4 interact respectively with gp120 and with class II MHC molecules.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

Effects of the wasp venom peptide, mastoparan, on a phosphoinositide-specific phospholipase C purified from rabbit brain membranes

The wasp venom peptide, mastoparan (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2), activated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis as catalyzed by a phosphoinositide-specific phospholipase C (PLC-Im) purified from rabbit brain membranes. This activation was found when the molar ratio of mastoparan to PIP2 was less than 1 and when the concentration of PIP2 exceeded 10 microM. PIP2 breakdown was inhibited at both high and low substrate concentrations if the molar ratio of mastoparan to PIP2 was greater than 1. The stimulatory effect of mastoparan correlated with its ability to restrict aggregation of PIP2 into higher order structures (liposomes or mixed deoxycholate/phospholipid micelles) as the concentration of PIP2 was increased to 10 microM or greater. Mastoparan stimulation of PIP2 breakdown required the presence of a higher calcium concentration than was necessary for detection of enzyme activity. Both the stimulatory and inhibitory effects of mastoparan on PIP2 hydrolysis were lost if 2.5 mM deoxycholate was present in the assays. Hydrolysis of phosphatidylinositol (PI) by PLC-Im was inhibited at all concentrations of mastoparan tested. These results show that both PIP2 and PI are suitable substrates for PLC-Im, depending on the physical characteristics of their aggregates in aqueous suspension. An amphiphilic alpha-helix-forming peptide such as mastoparan may modulate phospholipase C activity due to the peptide's interaction with phospholipid substrates.