Cloning and characterization of the immunity region of phage ?80

Summary The immunity region of phage 80 has been localized. It codes for at least three proteins: a protein of 34 kDa which has the biological properties of the phage repressor, and two other proteins of 9 kDa and 18 kDa which are the first proteins on the rightward operon. These two proteins are negatively regulated by the 34 kDa protein at a divergent promoter site. By position analogy with phage , but not by its biological activity, the 9 kDa protein could be the cro roduct. The 18 kDa protein is able to block totally UV induction of phage 80.     

Molecular characterization of the zerknüllt region of the Antennapedia complex of D. subobscura

We have characterized at the molecular level the zerknüllt (zen) region of the Drosophila subobscura Antennapedia complex. The sequence comparison between D. subobscura and D. melanogaster shows an irregular distribution of the conserved and diverged regions, with the homeobox and a putative activating domain completely conserved. Comparisons of the promoter sequence and pattern of expression of the gene during development suggest that the regulation of zen has been conserved during evolution. The conservation of zen expression in a subpopulation of the polar cells indicates the existence of an important role in such cells. We describe a transitory segmented pattern of expression of zen in both species, suggesting the existence of interactions with a pair rule gene. Some indirect clues indicate that the z2 gene might be absent from the D. subobscura genome. A chromosome walk initiated to reach the proboscipedia gene of D. subobscura reveals that the distance between pb and zen is at least four times the one described for D. melanogaster and for D. pseudoobscura. Finally, we present cytological evidence showing that the ANT-C is inverted in D. subobscura as compared to D. melanogaster.     

Antithrombin conformation and the catalytic role of heparin. I. Does cleavage by thrombin induce structural changes in the heparin-binding region of antithrombin?

Heparin has been shown to exhibit lower affinity for the antithrombin-thrombin complex than for antithrombin alone (Carlstrom, A.-S., Lieden, K., and Bjork, I. (1977) Thromb. Res. 11, 785-797), suggesting that structural alterations in antithrombin may accompany its reaction with thrombin. The hydroxy-nitrobenzyl (HNB) group attached to a unique tryptophan has been used in the present study as an extrinsic probe for localization of conformational changes to the heparin-binding region within antithrombin III using immunochemical and spectral techniques. Site-specific modification of tryptophan-49 in antithrombin with the hydroxynitrobenzyl reagent blocks heparin binding to the protein and provides a chemical label in the heparin-binding region of the protein (Blackburn, M. N., Smith, R. L., Carson, J., and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941). Antibodies specific for the hydroxynitrobenzyl hapten, which bind to HNB-tryptophan-49 in antithrombin, were used to detect a change in conformation in the region of tryptophan-49 which occurs upon thrombin binding to antithrombin. This thrombin-induced structural change was also apparent from spectral perturbations which were detected with the environmentally sensitive HNB moiety. Thus, the HNB group was used as an immunochemical probe as well as a spectral reporter group to provide insight into an allosteric mechanism of control in the catalytic role of heparin. The thrombin-promoted alteration of the structure in the heparin-binding region is presumably responsible for recycling of heparin, allowing it to catalyze further reactions between antithrombin and thrombin.     

Physical mapping and evolution of the centromeric class I gene-containing region of the rat MHC

We physically mapped the centromeric part of the BN rat MHC (RT1n haplotype) in a contig of overlapping P1-derived artificial chromosome (PAC) clones encompassing about 300 kb. The following genes were identified and ordered as: (Syngap, Hset, Daxx, Bing1)-Tapbp-Rgl2-Ke2-Bing4-B3galt4- Rps18-Sacm2l-RT1-A1-RT1-A2-RT1-A3-Ring1-Ring2-++ +Ke4-Rxrb-Col11a2-RT1-Hb-Ring3-RT1-DMb. Thus, in contrast to other RT1 haplotypes, RT1n contains three class I genes, RT1-A1, RT1-A2, and RT1-A3, mapping between the Sacm2l and Ring1 genes. Comparisons of the sequences flanking the Sacm2L and Ring1 genes in rat, human, and mouse suggest that the class I gene-containing region was inserted between these genes in rat and mouse at a similar position. Thus, this insertion is likely to have occurred in a common ancestor of these rodents, although the presence of a site particularly permissive for insertions cannot be excluded.     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

Evolution of the primate β-Globin gene region: Nucleotide sequence of the δ-β-globin Intergenic region of gorilla and phylogenetic relationships between African Apes and Man

Summary A 6.0-kb DNA fragment from Gorilla gorilla including the 5 part of the -globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque - intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 ± 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 ± 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the - intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the — intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human. Offprint requests to: P. Perrin-Pecontal     

Sequence simplicity and evolution of the 3′ untranslated region of the histone H1° Gene

The H1° gene has a long 3′ untranslated region (3′UTR) of 1,125 nucleotides in the rat and 1,310 in humans. Analysis of the sequences shows that they have features of simple DNA that suggest involvement of replication slippage in their evolution. These features include the length imbalance between the rat and human sequences; the abundance of single-base repeats, two-base runs and other simple motifs clustered along the sequence; and the presence of single-base repeat length polymorphisms in the rat and mouse sequences. Pairwise comparisons show numerous short insertions/deletions, often flanked by direct repeats. In addition, a proportion of short insertions/deletions results from length differences in conserved single-base repeats. Quantification of the sequence simplicity shows that simple sequences have been more actively incorporated in the human lineage than in the rodent lineage. The combination of insertions/deletions and nucleotide substitutions along the sequence gives rise to three main regions of homology: a highly variable central region flanked by more conserved regions nearest the coding region and the polyA addition site. Correspondence to: P. Suau     

The development of the region between the preexisting and nascent membranes during the first cleavage of Rana amurensis eggs

The region between the preexisting and nascentmembranes of a cleaving Rana egg is a dense protrusionregion of nearly 40μm wide at 180°stage.Later,thisregion differentiate,into an upper part,a strip withlong, branched and randomly distributed protrusionswhich are derived largely from the preexistingmembrane,and a lower part,a band with microvilli.During 4-and 8-cel1 stages,the strip is almost vanishedand microvilli in the band is shortened.The nascentmembrane is smooth at the 180°stage,then a rough areaappears below the smooth region and quickly expands.Wheat germ agglutinin which can bind to preexistingmembrane interrupts the development of the regionbetween the preexisting and nascent membranes.Detergent,Brij,having the property to increase the areaof nascent membrane,does not interrupt the developmentof the region between the preexisting and nascentmembranes.     

Unigenic and multigenicI region control of the immune responses of mice to the GAT10 and GLΦ-GLT terpolymers

Recombinant strains of mice with known alleles in theI region of theH-2 complex were used to map theH-2 linked immune response genes controlling responsiveness to random terpolymers GAT¹⁰ and GL. TheIr-GAT gene was mapped to either theIA orIB subregions. In contrast, data obtained in the GL-GLT system indicated multigenic control. The responsiveness of the B10.A(3R), B10.A(5R), and B10.S(9R) recombinants indicated that one immune response gene,IrGL-GLT _A, mapped to the right ofIB, i.e., in theIC subregion. The nonresponsiveness of the B10.A(1R), B10.A(2R), B10.M(17R), and AQR mice having responderIC ^d alleles butIA ^k-IB ^k nonresponder alleles and the positive response of a (C57BL/6 × A/J)F₁ hybrid derived from two nonresponder parental strains indicated the presence of a second gene inIA-IB subregions,Ir-GL-GLT _B. The interaction between these two genes, each present in a differentI subregion, controls the immune response.