Cytogenetic manifestations associated with the reversion, by gene amplification, at the HGPRT locus in V79 Chinese hamster cells

Some HGPRT spontaneous revertants were isolated from a mutant line (E2) of V79 Chinese hamster cells and phenotypically characterized. Dot-Blot hybridization with a 32P-labelled HGPRT probe revealed an increase in the number of HGPRT sequences in some of these revertants, suggesting the occurrence of gene amplification. Cytogenetic analysis performed in three of these revertants showed a characteristic abnormally banding region (ABR) on the elongated p arm of the X chromosome. In situ hybridization in one revertant (RHE2) showed that the amplified sequences reside on the p+ arm of the X chromosome in two different localizations. Because of the very probably clonal origin of the revertant, these features indicate that the amplified sequences might rearrange after their integration into the chromosome.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

Immunostaining free oligosaccharides directly on thin-layer chromatograms

Oligosaccharides are chromatographed on amino-bonded high-performance thin-layer chromatography silica gel plates and after chromatography the aldehydes on the reducing ends of the oligosaccharides react with the amino groups on the silica gel. Bound oligosaccharides are immunostained directly on the chromatograms by monoclonal antibodies. The binding of antibodies is detected by autoradiography after a second incubation with 125I-labeled goat anti-mouse immunoglobulin. Using this method, 10 pmol of lacto-N-difucopentaose I (Leb hapten) and lacto-N-fucopentaose III (Lex hapten) are detected directly on the thin-layer chromatograms by monoclonal antibodies 10c17 and 534F8, respectively. Previously undescribed larger oligosaccharides containing these epitopes are also detected in human milk. This method may be used to identify and characterize antibody-binding oligosaccharides liberated from glycoconjugates by hydrazinolysis, by trifluoroacetolysis, by ozonolysis, or by treatment with endoglycosidases. This technique may also be used to determine the structural specificity of other carbohydrate-binding proteins such as lectins, toxins, and hormones or of bacteria and viruses that bind to cell surface glycoproteins.     

Comparative studies of galactokinase activity on mammal's red blood cells.

1. ATP: D-galactose-1-phosphotransferase activity was measured in human, pig, cow, rabbit, mouse and rat red blood cells. Mean values of galactokinase activity was markedly lower in the human and pig erythrocyte as compared to those of the other species. 2. The permeability to galactose of the red cells studied was always higher than galactose phosphorylation. 3. The affinity constants of galactokinase for galactose ranged from 119 to 291 microM and from 178 to 406 microM for ATPMg2-. 4. The thermostability values of the galactokinase of the species studied were similar. The pH-optimum is pH 7.5 for the human, mouse and rabbit enzyme and pH 8.0 for cow, pig and rat galactokinase.     

Short direct repeats at the breakpoints of a novel large deletion in the CFTR gene suggest a likely slipped mispairing mechanism

In the cystic fibrosis conductance transmembrane regulator (CFTR) gene a few small deletions and only a large, complex, 50-kb deletion have been described so far. We report a second large deletion, which had been hypothesized in a patient affected by cystic fibrosis on the basis of an abnormal pattern of inheritance of the intragenic microsatellites IVS17b/TA and IVS17b/CA. Southern blot analysis revealed the presence of an anomalous band in the patient and her father, in the region encompassing exons 13 – 19, approximately 0.6 kb shorten than the one present in normal controls, in addition to the band of the correct size. Cloning and sequencing the DNA fragments spanning the region of interest demonstrated the presence of a 703-bp deletion causing complete removal of exon 17b in the paternal cystic fibrosis chromosome. This analysis revealed the presence of two short direct repeats flanking the breakpoints. The 3′ repeat partially overlapped the IVS17b/CA microsatellite and the number of CA repeated units present in the paternal cystic fibrosis allele was the shortest ever found among chromosomes so far analyzed. These data may suggest that the mechanism for the generation of the deletion may have involved a slipped mispairing during DNA replication, which has not previously been described in the CFTR gene. Received: 27 December 1995 / Accepted: 30 January 1996