Suppression of src transformation by overexpression of full-length GTPase-activating protein (GAP) or of the GAP C terminus.

Overexpression of the full-length GTPase-activating protein (GAP) has recently been shown to suppress c-ras transformation of NIH 3T3 cells but not v-ras transformation (36). Here, we show that focus formation induced by c-src was inhibited by approximately 80% when cotransfected with a plasmid encoding full-length GAP. In a similar assay, focus formation by the activated c-src (Tyr-527 to Phe) gene was inhibited by 33%. Cotransfection of the GAP C terminus coding sequences (which encode the GTPase-accelerating domain) with c-src or c-src527F inhibited transformation more efficiently than did the full-length GAP, while the GAP N terminus coding sequences had no effect on src transformation. When cells transformed by c-ras, c-src, c-src527F, or v-src were transfected with GAP or the GAP C terminus sequence in the presence of a selectable marker, 40 to 85% of the resistant colonies were found to be morphologically revertant. The GAP C terminus induced reversion of each src-transformed cell line more efficiently than the full-length GAP, but this was not the case for reversion of c-ras transformation. Biochemical analysis of v-src revertant subclones showed that the reversion correlated with overexpression of full-length GAP or the GAP C terminus. There was no decrease in the level of pp60src expression or the level of protein-tyrosine phosphorylation in vivo. We conclude that GAP can suppress transformation by src via inhibition of endogenous ras activity, without inhibiting in vivo tyrosine phosphorylation of cellular proteins induced by pp60src, and that src may negatively regulate GAP's inhibitory action on endogenous ras.     

Secondary pulsed field gel electrophoresis: a new method for faster separation of larger DNA molecules.

A novel technique, which we call secondary pulsed field gel electrophoresis (SPFG) has been developed. In SPFG, short pulses are applied in the direction of net migration of the DNA in addition to the reorienting pulses used in conventional pulsed field electrophoresis (PFG). Experimental results show that SPFG extends and improves the electrophoretic resolution of DNA for molecules from 0.5 megabase pairs to over 10 megabase pairs in size. This improved resolution is obtained with dramatically shorter run times. Thus SPFG appears to circumvent a number of the key limitations in previous PFG protocols.     

Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives.

A procedure is described for linking nucleosides covalently to controlled pore glass or cross-linked polystyrene supports by means of an oxalyl anchor. Though stable to triethylamine and diisopropylamine, the nucleoside-oxalyl link can be cleaved within a few minutes at room temperature with ammonium hydroxide in methanol. This new anchor can be used in automated synthesis of conventional oligonucleotides. The primary value, however, is that it enables one to employ solid support methodology to synthesize a variety of base-sensitive oligonucleotide derivatives, as illustrated here by synthesis of oligomers with base protecting groups intact and with methyl phosphotriester groups at the internucleoside links.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Anoxic block of GABAergic IPSPs

In rat hippocampal slices GABAergic IPSPs are very rapidly suppressed by anoxia (in<2 min). Both early (GABA_A) and late (GABA_B) components are affected. After reoxygenation, the IPSPs recover, but only slowly and not always completely. Iontophoretic applications of GABA or baclofen indicated no major depression of responses during anoxia. It is therefore unlikely that the anoxic suppression of IPSPs is caused by desensitizations of GABA receptors. A more probable explanation is a failure of GABAergic neurons to release GABA from inhibitory nerve terminals.Special issue dedicated to Dr. Eugene Roberts.     

Chloroplast DNA Diversity in Populations of Wild and Cultivated Barley

Chloroplast DNA (cpDNA) diversity was found within and among populations (245 accessions total) of wild barley, Hordeum vulgare L. ssp. spontaneum Koch from Israel and Iran. Three polymorphic restriction sites (HindIII, EcoRI, BclI) which define three distinct cpDNA lineages were detected. One lineage is common to populations in the Hule Valley and Kinneret of northern Israel, and in Iran. The second lineage is found predominantly in the Lower Jordan Valley and Negev. The distribution of the third lineage is scattered but widespread throughout Israel. Sixty two accessions of cultivated barleys, H. vulgare L., were found, with two exceptions, to belong to just one cpDNA lineage of wild barley, indicating that the cpDNA of cultivated barley is less variable than its wild ancestor. These results demonstrate the need for assessing intraspecific cpDNA variability prior to choosing single accessions for phylogenetic constructions at the species level and higher.