Molecular basis and PCR-DNA typing of the Fya/fyb blood group polymorphism

Spondyloepiphyseal dysplasia tarda (SEDL) is an X-linked recessive disorder characterized in affected males by short stature resulting from a growth defect of the vertebral bodies. We have extended our earlier studies by analyzing 15 families with newly identified microsatellite DNA markers; analysis of recombination events with these markers indicates that the gene responsible for SEDL is located in Xp22 between DXS 16 and DXS 987 on an interval spanning approximately 2 Mb.     

A High-Resolution Interval Map of the q21 Region of the Human X Chromosome

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.     

Interaction of Gila monster venom with secretin receptors in rat pancreatic membranes

The stimulatory effect of Gila monster venom on adenylate cyclase activity in rat pancreatic membranes was compared to that of porcine secretin and porcine VIP. The maximal effect exerted by the venom was identical to that of VIP but significantly lower than that of secretin. The effect of Gila monster venom could, however, be attributed to its interaction with secretin receptors rather than with VIP receptors, at variance with its previously described action on guinea pig pancreatic acini. Adenylate cyclase activation by both Gila monster venom and secretin in rat pancreatic membranes was, indeed: (1) dose-dependently inhibited by two secretin fragments secretin-(4-27) and secretin-(7-27), and (2) more severely depressed than VIP stimulation, after pretreating pancreatic membranes with dithiothreitol (DTT).     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

Effect of Bordetella pertussis toxin on ADP-ribosylation of membrane proteins, adenylate cyclase activity and insulin release in rat pancreatic islets

Exposure of rat pancreatic islet membranes to [alpha-32P]-NAD+ in the presence of Bordetella Pertussis toxin (islet-activating protein) reveals the ADP-ribosylation of a peptide with a Mr close to 41 kDa, which corresponds to the alpha-subunit of the guanine nucleotide regulatory protein Ni. Islets removed from rats pretreated with the Bordetella Pertussis toxin display a specific increase in adenylate cyclase responsiveness to GTP and are characterized by a resistance to the inhibitory action of alpha2-adrenergic agonists upon either adenylate cyclase activity or glucose-induced insulin release.     

Isolation and characterization of a fragment of rat thyroglobulin gene

A rat genomic library was screened for thyroglobulin gene clones with recombinant plasmids containing rat thyroglobulin complementary DNA inserts. Two identical recombinant phages were found. A map of the inserted genomic sequence established by restriction and blotting experiments, and electronic microscopy revealed that this fraction of the gene was extensively split. Exons were ≤ 200 base pair long while the introns represented 93% of the insert. A fragment subcloned in plasmid pBR 322 was shown to contain repetitive sequences when used in Southern blot experiments with rat total genomic DNA.     

Evidence for the existence of actomyosin ATPase in the rat pancreas. Isolation and biochemical characterization

In a crude extract of rat pancreas, myosin was associated with a protein having the same electrophoretic mobility as actin. This myosin was purified after dissociation of the actomyosin complex with KI-ATP. On sodium dodecylsulfate/acrylamide gel electrophoresis, the isolated pancreatic myosin showed a major component of approximately 200 kDa, and two smaller components with apparent molecular weight of 22 and 15 kDa, respectively. This purified myosin exhibited high ATPase activity in the presence of K+ + EDTA or Ca2+ and very little activity in the presence of Mg2+. (K+ + EDTA)-ATPase activity showed one pH optimum at 8.0, while Ca2+-ATPase activity showed two pH optima at 6.0 and 9.0, respectively. (K+ + EDTA)-stimulated enzyme activity was specific for ATP whereas Ca2+-stimulated activity showed low specificity for nucleoside triphosphates.     

Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1.

125I-[Tyr2]scyllatoxin allowed to label a single class of high-affinity receptors in membranes from the human neuroblastoma cell line NB-OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the Bmax was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5-fold after a 24-h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.     

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

Summary A characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.     

Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts

Auxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts. Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr1 gene product (ABP1), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10–fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABP1 raised the sensitivity more than 1000–fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr¹ gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface of tobacco protoplasts and for the effects of hetero-logous maize ABP on auxin sensitivity is proposed.