The ras oncogene product p21 is not a regulatory component of adenylate cyclase

Harvey (Ha-MSV) and Kirsten (Ki-MSV) murine sarcoma viruses induce tumours in animals and transform various cells in culture because of the expression of the ras oncogene product, p21 (ref. 1). Proto-oncogenes homologous with these genes are highly conserved evolutionarily and activated ras oncogenes have been detected in many human cancers. Whether c-ras oncogenes are directly responsible for human carcinogenesis is uncertain; however, it is clear that p21 mediates virus-induced transformation, although by an unknown mechanism. Epithelial and fibroblast cell lines transformed with Ha-MSV and Ki-MSV express p21 (ref. 8) and exhibit reduced adenylate cyclase activity. Like the guanine nucleotide regulatory proteins, Ns and Ni, which mediate stimulation and inhibition, respectively, of adenylate cyclase, p21 is a membrane-associated GTP binding protein, which exhibits GTPase activity. These similarities suggest that p21 and the adenylate cyclase regulatory proteins are related in cellular function, and that p21 depresses adenylate cyclase by inhibiting the activity of Ns or acting as Ni. We have therefore now examined the structural and functional similarities between p21 and Ns and Ni and find no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.     

Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells

K1 killer toxin secreted by the K1 strain of Saccharomyces cerevisiae, has been well characterized. It is a simple protein of molecular weight (MW) 11,470 (ref. 3), encoded by a double-stranded, linear RNA plasmid, called M RNA, of MW 1.1-1.7 x 10(6) (refs 4-6). It is lethal to sensitive Saccharomyces cerevisiae which does not carry M RNA. Leakage of K+ and ATP is the first distinct response in sensitive cells, and the toxic action is thought to be due to its action as a protonophore or K+ ionophore. Recently, a further killer toxin has been found in Kluyveromyces lactis IFO 1267, and it is associated with the presence of the double-stranded linear DNA plasmids, pGK1-1 (MW 5.4 x 10(6)) and pGK1-2 (MW 8.4 x 10(6)). It has been shown, by curing pGK1-1 or deletion mapping, that the structural gene for the killer toxin and immunity-determining gene reside on the smaller plasmid. Moreover, the plasmids could be transferred from K. lactis to S. cerevisiae by protoplast fusion and protoplast transformation. As the K. lactis toxin is encoded by a DNA plasmid and has a relatively wider action spectrum than K1 killer toxin, the mode of action of the toxin is highly interesting. Here we report that K. lactis toxin inhibits adenylate cyclase in sensitive yeast cells and brings about arrest of the cells at the G1 stage.     

Structural determinants for GoLoco-induced inhibition of nucleotide release by Galpha subunits

Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Galpha subunits and maintain G-protein subunit dissociation in the absence of Galpha activation. Here we describe the structural determinants of GoLoco activity as revealed by the crystal structure of Galpha(i1) GDP bound to the GoLoco region of the 'regulator of G-protein signalling' protein RGS14. Key contacts are described between the GoLoco motif and Galpha protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Galpha to make direct contact with the GDP alpha- and beta-phosphates. The structural organization of the GoLoco Galpha(i1) complex, when combined with supporting data from domain-swapping experiments, suggests that the Galpha all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco Galpha interactions.     

Altered nucleotide sequences of a translocated c-myc gene in Burkitt lymphoma

The nucleotide sequence of a translocated c-myc gene in a Burkitt lymphoma reveals multiple base changes in the coding region. Twenty-five base changes, generating 16 codon alterations, were found in the first coding exon; no changes occur in the second coding exon. These changes are probably the result of somatic mutations that occurred during and after translocation, and may contribute to oncogenesis by allowing synthesis of an altered c-myc gene product.     

A pair of two－component regulatory genes ecrA1／A2 in S. coelicolor

Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed that ecrA1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway, ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecrA 1/A2 was positive regulatory element for red gene cluster.     

A new method for splice site prediction based on the sequence patterns of splicing signals and regulatory elements

It is of significance for splice site prediction to develop novel algorithms that combine the sequence patterns of regulatory elements such as enhancers and silencers with the patterns of splicing signals. In this paper, a statistical model of splicing signals was built based on the entropy density profile （EDP） method, weight array method （WAM） and K test; moreover, the model of splicing regulatory elements was developed by an unsupervised self-learning method to detect motifs associated with regulatory elements. With two models incorporated, a multi-level support vector machine （SVM） system was devised to perform ab initio prediction for splice sites originating from DNA sequence in eukaryotic genome. Results of large scale tests on human genomic splice sites show that the new method achieves a comparative high performance in splice site prediction. The method is demonstrated to be with at least the same level of performance and usually better performance than the existing SpliceScan method based on modeling regulatory elements, and shown to have higher accuracies than the traditional methods with modeling splicing signals such as the GeneSplicer. In particular, the method has evident advantage over splice site prediction for the genes with lower GC content.     

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity and appear to interact with G-proteins, perhaps by causing phosphorylation of them. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predisposition to infection. We show here that experimentally-induced type I diabetes leads to the loss of expression of Gi in rat liver. As it has been suggested that Gi may couple receptors to K+-channels as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.