Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation.

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.     

Intervening sequences in the ribosomal RNA genes of Ascaris lumbricoides: DNA sequences at junctions and genomic organization

An rDNA size class in the genome of the nematode Ascaris lumbricoides is described which is interrupted by a 4.5-kb long intervening sequence located in the 26S coding region. This molecular form occurs in approximately 15 copies per haploid genome and amounts to approximately 5% of the total nuclear rDNA. Intervening sequences are present only in the 8.8-kb rDNA, but not in the 8.4-kb rDNA repeating units of A. lumbricoides. Cloning of the interrupted rDNA units revealed, in addition to the main 4.5-kb insertion, shorter intervening sequences of 4-kb and 119-bp length. Both shorter rDNA forms are present in the single copy range of the haploid genome. Sequence analyses of the intervening sequence/rDNA junctions show an identical right-hand junction for all of the three different rDNA forms. The two shorter intervening sequences are a coterminal subset of the right-hand end of the main 4.5-kb insertion, whereas all three insertions have a different left-hand junction with the coding region of rDNA. Each intervening sequence is flanked by a short direct repeat of variable length, being only once present in the uninterrupted rDNA. The intervening sequences of A. lumbricoides show striking similarity to the organization of type I insertion family in dipteran flies, even though they are inserted at different positions in the 26S coding region. Additional rDNA intervening sequences may be present outside of the rDNA cluster, but in not more than 15-20 homologous copies per haploid genome.     

Nucleotide sequence and genome organization of foot-and-mouth disease virus.

A continuous 7802 nucleotide sequence spanning the 94% of foot and mouth disease virus RNA between the 5'-proximal poly(C) tract and the 3'-terminal poly(A) was obtained from cloned cDNA, and the total size of the RNA genome was corrected to 8450 nucleotides. A long open reading frame was identified within this sequence starting about 1300 bases from the 5' end of the RNA genome and extending to a termination codon 92 bases from its polyadenylated 3' end. The protein sequence of 2332 amino acids deduced from this coding sequence was correlated with the 260 K FMDV polyprotein. Its processing sites and twelve mature viral proteins were inferred from protein data, available for some proteins, a predicted cleavage specificity of an FMDV encoded protease for Glu/Gly(Thr, Ser) linkages, and homologies to related proteins from poliovirus. In addition, a short unlinked reading frame of 92 codons has been identified by sequence homology to the polyprotein initiation signal and by in vitro translation studies.     

Antennal sensory system of Periplaneta americana L.

The morphology of the antennal hair-sensilla of Periplaneta americana, their distribution and frequency on the antennal flagellum have been examined by transmission- and scanning-electron microscopy. The types of sensilla were distinguished with respect to physiologically relevant criteria such as wall structure and number of sensory cells. Among the sensilla of the antenna of the adult male, long, single-walled sensilla with four sensory cells (types sw B), Probably responsible for reception of sexual pheromones, are most frequent, representing about 54% of the antennal sensilla. About half of these sensilla are newly-formed at the imaginal ecdysis; the other half are derived from the shorter type sw B sensilla of the nymphal antenna. Short type sw B sensilla are present in all larval stages of both sexes and in adult females as well. During the imaginal ecdysis of males, however, the length of these sensilla increases to double that found in nymphs. Dendritic branches also increase in number. During postembryonic development, the number of sensory fibers in the antennal flagellum increases nearly 20-fold, from 14,000 in the first larval instar to about 270,000 in the adult male. The greatest increase, approximately 90%, occurs during the last developmental stage.     

Penicillinase from Bacillus licheniformis: nucleotide sequence of the gene and implications for the biosynthesis of a secretory protein in a Gram-positive bacterium.

The gene for the penicillinase from B. licheniformis has been cloned in a functional stat on a 1.5 kb DNA fragment and its nucleotide sequence has been determined. A sequence of 307 amino acid residues is infered for the penicillinase precursor. Of these 34 amino acids precede the sequence of the secreted form of the enzyme. This peptide extension shows the features of a signal for secretion and also provides the hydrophobic anchor for the membrane-bound form of the enzyme.     

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.     

Presence of nadph-cytochrome P-450 reductase in rat liver golgi membranes: Evidence obtained by immunoadsorption method

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 μm) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.     

The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase.

Genomic and cDNA clones that code for a protein with structural and biochemical properties similar to the receptor protein kinases from animals were obtained from Arabidopsis. Structural features of the predicted polypeptide include an amino-terminal membrane targeting signal sequence, a region containing blocks of leucine-rich repeat elements, a single putative membrane spanning domain, and a characteristic serine/threonine-specific protein kinase domain. The gene coding for this receptor-like transmembrane kinase was designated TMK1. Portions of the TMK1 gene were expressed in Escherichia coli, and antibodies were raised against the recombinant polypeptides. These antibodies immunodecorated a 120-kD polypeptide present in crude extracts and membrane preparations. The immunodetectable band was present in extracts from leaf, stem, root, and floral tissues. The kinase domain of TMK1 was expressed as a fusion protein in E. coli, and the purified fusion protein was found capable of autophosphorylation on serine and threonine residues. The possible role of the TMK1 gene product in transmembrane signaling is discussed.     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Identification of the disulfide bonds of human complement C1s

C1s, one of the three subcomponents of C1, the first component of the complement system, is a complex serine protease. To determine the disulfide-bonding pattern, fragments of C1s were generated by cleavage with pepsin, thermolysin, or subtilisin. Disulfide bonds have been identified by several methods, for example, direct observation of the phenylthiohydantoin derivative of cystine during Edman degradation of isolated peptides and placement in the known cDNA sequence. All of the 26 half-cystines are linked in disulfide bonds occurring at positions 50-68, 120-132, 128-141, 143-156, 160-187, 219-236, 279-326, 306-339, 344-388, 371-406, 410-534, 580-603, and 613-644. All of the disulfide bonds of the earlier described substructures of C1s, the EGF-homologous part, the two SCR units, and the two domains typical for C1s and C1r are localized within these domains.