Transposition of mobile genetic elements in interspecific hybrids of Drosophila

In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using ³²P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions (jumping) of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with synthetic karyotoypes characterized by different combinations of D. virilis homologous chromosomes and hybrid chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition (jumping) took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such synthetic stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.     

Ultrastrukturelle Untersuchungen zur Morphologie und Genese der Spermien von Archaeogastropoda

The sperm cells ofPatella coerulea (Patellacea),Monodonta turbinata, andGibbula tumida (Trochacea) were investigated by means of transmission electron microscopy. They belong to the primitive type (sensu Franzén) and have more features in common with primitive Bivalvia sperms than with Neritacea. Their head contains an apical acrosome and a roundish nucleus followed by 4 or 5 mitochondria and a centriolar apparatus which consists of two centrioles, one of which bears a flagellum. The sperm cells ofMonodonta andGibbula are very similar to each other and differ mainly in size;Patella exhibits more differences (very small acrosome, subacrosomal space, variable number of spherical mitochondria (origin of spermic dimorphism ?). The development of the sperm cells shows no peculiarities.     

Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline

The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l-1 x min-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19-kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L-norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10(3) M-1 min-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L-norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.     

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.     

Acidosomes from Dictyostelium. Initial biochemical characterization.

The acidosome, a newly described organelle in Dictyostelium discoideum, is rich in vacuolar proton pumps (V-H(+)-ATPases) and is responsible for the acidification of endocytic vacuoles. Purified acidosomes were not significantly contaminated by lysosomes, endosomes, or plasma membranes but contained a small fraction of contractile vacuole markers. The specific activity of the proton pump in these acidosomes reached 30 mumol/min/mg protein, the highest yet reported for any V-H(+)-ATPase. The V-H(+)-ATPase was the predominant protein in acidosomes. Based on gel electrophoresis and densitometry, its 8 polypeptides had the following apparent molecular mass (in kDa) and stoichiometry: 90(1), 68(3), 53(3), 42(1), 37(3), 25(3), 17(6), and 15(1). These values suggested a Mr congruent to 8 x 10(5), consistent with the hydrodynamic properties and electron microscopic image of the purified pump. The 90- and 17-kDa polypeptides were integral, while the others were peripheral; only the 90-kDa subunit was biosynthetically labeled by [3H]glucosamine and 35SO4. The specific content of phosphatidylcholine and phosphatidylserine in the acidosomes was the highest of any subcellular fraction tested, while sterols and sphingolipids were the lowest. Acidosomes had congruent to 10% of the lipid biosynthetically labeled with [3H]glucosamine. This organelle contributed 5% of cellular protein and 15% of the phospholipid in stationary cultures. We conclude that the acidosome in Dictyostelium is a biochemically discrete organelle, produced by the endoplasmic reticulum/Golgi apparatus but distinct from other endomembranes as well as from the plasma membrane.     

Functional domains of the 67-kDa laminin receptor precursor

We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.     

Design of bioactive peptides based on antibody hypervariable region structures. Development of conformationally constrained and dimeric peptides with enhanced affinity

The variable regions of antibody molecules bind antigens with high affinity and specificity. This binding is imparted largely by the hypervariable portions of the variable region. Hypervariable regions typically fold into reverse turn or loop structures. Peptides derived from antibody hypervariable region sequences can bind antigens with similar specificity, albeit with markedly lower affinity. In this study, cyclic and dimeric peptide analogs of an anti-idiotypic/antireceptor antibody hypervariable region were developed. This antibody (87.92.6) binds to reovirus type 3 receptors on cells as well as to a neutralizing anti-reovirus type 3 monoclonal antibody (9B.G5). The cyclic peptides were utilized to probe the optimal conformation for binding to both the receptor and 9B.G5. By dimerizing or constraining the conformation of these peptides, higher affinity binding was produced. By utilizing several different cyclic peptides, the optimal conformation for binding was established. The conformationally optimized cyclic peptide possessed greater than 40-fold higher affinity for the receptor and the idiotype than the linear analog. This study suggests that conformationally constrained and dimeric peptides derived from antibody hypervariable loop sequences can bind antigens (including receptors) with reasonable affinity. hypervariable loop sequences can bind antigens (including