Microdissection of the Prader-Willi syndrome chromosome region and identification of potential gene sequences

The Prader-Willi syndrome chromosome region on the long arm of human chromosome 15 was microdissected and microcloned from 20 GTG-banded metaphase chromosomes, and 5000 recombinant clones were obtained. Of these clones, 39% identify single-copy human DNA sequences, most of which map to the dissected chromosome region and are evolutionarily conserved in other species. Three of eleven clones studied in detail are deleted in several patients with Prader-Willi syndrome. The microclones will be useful for the physical characterization of the Prader-Willi syndrome chromosome region and the identification of the affected genes in this disease.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Neutrophil migration into the bovine uterine lumen following intrauterine inoculation with killed Haemophilus somnus.

Polymorphonuclear neutrophils (PMN) in bovine uterine flushings following intrauterine deposition of killed bacteria were measured and the effect of immune status on the influx of PMN into the uterine lumen during oestrus was determined. Holstein heifers were immunized with a 270-kDa outer-membrane protein (omp-270) from Haemophilus somnus. During oestrus, immunized heifers (n = 21) received an intrauterine inoculum of either a heat-killed suspension of a homologous strain of H. somnus containing omp-270 (n = 7), a heterologous strain of H. somnus lacking omp-270 (n = 7), or phosphate-buffered saline (n = 7). Five additional heifers were inseminated with extended bovine semen. Uterine contents were collected in saline lavage immediately before inoculation (t0) and at 6, 24, 48, 72, 96, and 120 h after inoculation. The semen-inoculated heifers were lavaged only at t120. All groups experienced PMN infiltration which peaked 6 h after inoculation and tended to decline thereafter. Differences were not observed between treatment groups, indicating that neither bacterial inoculation nor immune status was as important in eliciting PMN effusion as the flushing procedure itself.     

Binding of 125I-succinylated concanavalin A to bovine spermatozoa

This study characterizes interactions of 125I-succinylated concanavalin A (125I-sucConA) with plasma membranes of intact bovine spermatozoa. Maximum binding was achieved by 30 min at 24 degrees C. Reversibility of binding was established by displacement of bound ligand with nonradioactive sucConA. Seventy-five percent of the bound sucConA was removed as a single kinetics class. When alpha-methylmannoside was used as a competitive ligand, 90% of the sucConA was removed. Saturability of binding sites, however, was not achieved over the concentration range of 125I-sucConA examined (0.13 micrograms/ml to 77 micrograms/ml). Binding kinetics of this system was complex and linear Scatchard analysis was not appropriate. The degree of 125I-sucConA binding to spermatozoa was influenced (P less than 0.01) by different iodination batches of 125I-sucConA. Complications due to iodination of ligand and the complex nature of its interaction with the membrane preclude the use of 125I-sucConA for a quantitative study of sperm membrane features.     

Adaptation, Readaptation and Synthesis of Hydrogenases in Scenedesmus obliquus

Scenedesmus cells reach full hydrogen activity after 2.5 h ofanaerobic adaptation. Exposure to oxygen inactivates the hydrogenaseimmediately. Readaptation occurs with the same kinetics as primaryadaptation. The fast activation and reactivation as well asthe insensibility to cycloheximide suggest that hydrogenase,inactive or activated, is present in the chloroplast all thetime. The activation and even more the readaptation are sensitiveto chloramphenicol. Thus, we propose that hydrogenase itselfor an activating protein in the chloroplast have a rather fastturnover. ¹ Present address: Pharathiar University, Dept. of Botany, Coimbatore,India.     

Evidence for the Presence of Chlorophyllide b in the Green Alga Scenedesmus obliquus in vivo

Chlorophyllide b could be extracted from the wild type of Scenedesmus obliquus and its pigment mutant C-2A'. Its identity was proved by absorption and fluorescence spectroscopy and by a positive hydroxylamine test. Chlorophyllide b could be transformed into pheophorbide b and methylpheophorbide b. The formation of chlorophyllide b from chlorophyll b by dephytylation with chlorophyllase could be ruled out. The stimulation of chlorophyllide b biosynthesis with o-phenanthroline, as described in the literature, could not be confirmed under physiological conditions.     

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green algaScenedesmus obliquus,mutant C-2A′

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA^Glu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K _m value of 2.3 M ± 0.3 for glutamate.Abbreviations ALA 5-aminolevulinic acid - FPLC fast protein liquid chromatography - Glu glutamate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNA^Glufrom E. coli.     

Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.     

Solubilization and hydrophobicity test by Triton X-114-partitioning of NADPH-protochlorophyllide oxidoreductase from the unicellular alga Scenedesmus obliquus, mutant C-2A'

NADPH-protochlorophyllide oxidoreductase (PChilde reductase, EC 1.3.1.33), a key enzyme in light-dependent greening and the conversion of etioplasts into chloroplasts was investigated in the the greening mutant C-2A' of the unicellular green alga Scenedesmus obliquus. In the absence of detergent, the solubilization of the enzyme increased with high glycerol concentrations in the buffer. Solubilization capacities of 4 non-ionic or zwitterionic detergents, Triton X-100, CHAPS, octylglucoside and decyl-maltopyranoside, were compared. Due to the addition of these detergents, the enzyme activity in the soluble fraction was increased severalfold. Hydrophobicity of the enzyme was analyzed by Triton X-114 phase partitioning. The protein had a preference for the aqueous phase, but its distribution was strongly influenced by the glycerol concentration of the buffer. These results indicate that the PChlide reductase of the green alga Scenedesmus obliquus is a hydrophobic, membrane-associated enzyme, but not an integral membrane protein.     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.