Construction and characterization of a gridded cattle BAC library

A bovine genomic large-insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein-Friesian male. Size fractionated DpnII-digested genomic DNA was ligated to the dephosphorylated BamH1 ends of a pBACe3.6 vector. Approximately 8.3 x 10(4) individual BAC clones were picked into 384-well plates. Two-hundred and sixty-seven randomly chosen clones were characterized by pulsed-field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5.5%. Thirty-four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http:// www.rzpd.de).     

Cytogenetics of the Anopheles gambiae complex in Sudan, with special reference to An. arabiensis: relationships with East and West African populations

The species composition of malaria vector mosquitoes belonging to the Anopheles gambiae complex (Diptera: Culicidae) from >40 localities in Sudan, representing most ecological situations, was determined by analysis of ovarian polytene chromosomes. Of 2162 females, 93% were identified as An. arabiensis Patton and 7% were An. gambiae Giles sensu stricto. No hybrids were found between the two species. Anopheles arabiensis occurred in all but two sites, whereas An. gambiae s.s. was effectively limited to the southernmost, more humid localities. For chromosomal paracentric inversions, the degree of polymorphism was low in An. gambiae s.s. (inversions 2La, 2Rb and 2Rd), higher in An. arabiensis (inversions Xe, 2Ra, b, bc, d1, s; 3Ra, d). Anopheles gambiae samples from Sudan were all apparently panmictic, i.e. they did not show restricted gene flow such as observed among West African populations (interpreted as incipient speciation). Chromosomal inversion patterns of An. gambiae in southern Sudan showed characteristics of intergrading Savanna/Forest populations similar to those observed in comparable eco-climatic situations of West Africa. Anopheles arabiensis was polymorphic for inversion systems recorded in West Africa (2Ra, 2Rb, 2Rdl, 3Ra) and for a novel 2Rs polymorphism, overlapping with inversion systems 2Rb and 2Rd1. Samples carrying the 2Rs inversion were mostly from Khashm-el-Girba area in central-eastern Sudan. In the great majority of the samples all polymorphic inversions were found to be in Hardy-Weinberg equilibrium. Sudan populations of An. arabiensis should therefore be considered as generally panmictic. Anopheles arabiensis shows more inversion polymorphism in west than in east African populations. Sudan populations have more evident similarities with those from westwards than those from eastwards of the Great Rift Valley. The possible influence of the Rift on evolution of An. arabiensis is discussed.     

Geographic and breed distribution of an Msp I PCR-RFLP in the bovine growth hormone (bGH) gene

Information is presented on the frequency of the Msp I (-) allele in the third intron of the bovine growth hormone gene in a large number of cattle breeds. Consideration of the breed frequencies in relation to their geographic origin shows a low frequency for breeds originating in Northern Europe, moderate frequencies for breeds originating in Eastern Europe or the countries surrounding the Mediterranean basin, and very high frequencies for breeds originating in the Indian subcontinent. Consideration of breed frequencies in relation to breed type, shows low to moderate frequencies for the humpless breeds, high frequencies for the humped breeds. Various explanations for this distribution are discussed, among them the possibility that the Msp I (-) allele originated in the Bos indicus breeds of the Indian subcontinent, from which it diffused through the humpless Bos taurus breeds of Eastern Europe, the Mediterranean basin, eventually reaching Western, Northern Europe, Western Africa in low frequencies.     

GnRH-Bik/Bax/Bak chimeric proteins target and kill adenocarcinoma cells; the general use of pro-apoptotic proteins of the Bcl-2 family as novel killing components of targeting chimeric proteins

In recent years chimeric proteins carrying bacterial toxins as their killing moiety, have been developed to selectively recognize and kill cell populations expressing speciific receptors. The involvement of Gonadotropin releasing hormone (GnRH) has been demonstrated in several adenocarcinomas and a GnRH-bacterial toxin chimeric protein (GnRH-PE66) was thus developed and found to specifically target and kill adenocarcinoma cells both in vitro and in vivo. Because of the immunogenicity and the non-specific toxicity of the bacterial toxins, we have developed new chimeric proteins, introducing apoptosis inducing proteins of the Bcl-2 family as novel killing components. Sequences encoding the human Bik, Bak or Bax proteins were fused to the GnRH coding sequence at the DNA level and were expressed in E. coli. GnRH-Bik, GnRH-Bak and GnRH-Bax new chimeric proteins efficiently and specifically inhibited the cell growth of adenocarcinoma cell lines and eventually led to cell death. All three Bcl2-proteins-based chimeric proteins seem to induce apoptosis within the target cells, without any additional cell death stimulus. Apoptosis-inducing-proteins of the Bcl-2 family targeted by the GnRH are novel potential therapeutic reagents for adenocarcinoma treatment in humans. This novel approach could be widely applied, using any molecule that binds a specific cell type, fused to an apoptosis-inducing protein.     

Differential responses to chronic hypoxia and dietary restriction of aerobic capacity and enzyme levels in the rat myocardium

Chronic exposure of mammals to hypoxia induces a state of anorexia. We aimed to determine the role played by diet restriction in the alterations of myocardial energy metabolism occurring under chronic hypoxia in order to detect the specific effects of hypoxia per se.Adult female rats were exposed to normobaric hypoxia (Fi O2 = 0.10) for three weeks; pair-fed rats, kept under normoxic conditions, received the same amount of food as hypoxic rats. The oxidative capacity of myocardial ventricles and some skeletal muscles was evaluated using permeabilized fibers. Several metabolic enzyme activities were measured on extracts from myocardium and soleus.Diet restriction increased the activity of lactate dehydrogenase in both ventricles while it augmented phosphofructokinase and pyruvate kinase activities only in the left ventricle and depressed the respiratory rate in the right ventricle only.Hypoxia per se induced a rise in hexokinase activity in all studied oxidative muscles and a fall of hydroxy-acyl CoA-dehydrogenase activity in both myocardial ventricles. The respiratory rate and the citrate synthase activities were unaffected by hypoxia.We conclude that chronic hypoxia per se leads to specific alterations in myocardial metabolism that could favor the use of exogenous glucose at the expense of free fatty acids without any change in the oxidative capacity.     

Protected polymorphism in the two-locus haploid model with unpredictable fitnesses

 In an unpredictable environment, the distributions of alleles from which polymorphism can be maintained forever belong to a certain set, the C-viability kernel. Such a set is calculated in the two-locus haploid model, as well as the corresponding fitnesses at any time which make this maintenance possible. The dependence of the C-viability kernel on the set U of admissible fitnesses and on the recombination rate r is studied. Notably, the C-viability kernel varies rapidly in the neighborhood of equal fitness of AB and ab; it becomes empty when ab has a fitness below a certain function, which is delineated, of the recombination rate. The properties of the two-locus model under constraints, out of equilibrium and with unpredictable selection are thus presented. Received: 20 May 1999     

Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.     

Dimer formation by a "monomeric" protein

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 10(5)-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 degrees C results in a 10(3)-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 degrees C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer-dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 degrees C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of "monomeric" proteins could be more prevalent than is usually appreciated.     

Chloroplast DNA polymorphism reveals little geographical structure in Castanea sativa Mill. (Fagaceae) throughout southern European countries

The distribution of haplotypic diversity of 38 European chestnut (Castanea sativa Mill.) populations was investigated by PCR/RFLP analysis of regions of the chloroplast and mitochondrial genomes in order to shed light on the history of this heavily managed species. The rapid expansion of chestnut starting from 3000 years ago is strongly related to human activities such as agricultural practice. This demonstrates the importance of human impact, which lasted some thousands of years, on the present-day distribution of the species. No polymorphism was detected for the single mitochondrial analysed region, while a total of 11 different chloroplast (cp) haplotypes were scored. The distribution of the cpDNA haplotypes revealed low geographical structure of the genetic diversity. The value of population subdivision, as measured by GSTc, is strikingly lower than in the other species of the family Fagaceae investigated. The actual distribution of haplotypic diversity may be explained by the strong human impact on this species, particularly during the Roman civilization of the continent, and to the long period of cultivation experienced during the last thousand years.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.