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.     

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.     

Determination of the origin of nondisjunction in a 49,XXXXY male using hypervariable dinucleotide repeat sequences

Summary We present a patient with a 49,XXXXY chromosome constitution in whom the origin of the extra X chromosomes was determined by analysis of five polymorphic CA (or GT) dinucleotide repeat sequences. This class of DNA marker has recently been demonstrated to be hypervariable with heterozygosity values up to 80%. By polymerase chain reaction (PCR) analysis of the dinucleotide repeat length polymorphisms, we have shown that all four X chromosomes were of maternal origin.     

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.     

Synthetic magainin analogues with improved antimicrobial activity

Based on modifications to enhance the alpha-helical structure of the broad spectrum antibiotic magainin 2, a series of analogues have been synthesized which display an increase up to two orders of magnitude in antimicrobial activity and, in the most favorable case, no appreciable increase in hemolytic activity over magainin 1 at the concentrations tested.     

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.