C1 proteins: a class of high-mobility-group non-histone chromosomal proteins from the fruit fly Ceratitis capitata

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodríguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2.     

Compositional analysis of Helicobacter pylori rough-form lipopolysaccharides.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the macromolecular heterogeneity of lipopolysaccharides (LPS) from seven fresh clinical isolates and three culture collection strains of the human pathogen Helicobacter pylori. All the clinical isolates produced smooth-form LPS with O side chains of relatively homogeneous chain length, whereas the culture collection strains yielded rough-form LPS. A better yield of the latter LPS was obtained when combined protease pretreatment and hot phenol-water extraction were used than when the conventional phenol-water technique alone was used for extraction. The LPS of the three culture collection strains (S-24, C-5437, and NCTC 11637) were chemically characterized. Constituents common to all the LPS were fucose, D-mannose, D-glucose, D-galactose, D-glycero-D-manno-heptose, L-glycero-D-manno-heptose, and 3-deoxy-D-manno-2-octulosonic acid. The molar ratios of the hexoses differed between different strains, thereby reflecting structural differences. Phosphate, phosphorylethanolamine, and pyrophosphorylethanolamine were present also. Free lipid A contained D-glucosamine and fatty acids, with phosphate and a minor amount of ethanolamine. The major fatty acids were ester- and amide-bound 3-hydroxyoctadecanoic acid and ester-bound octadecanioc and 3-hydroxyhexadecanoic acids, with minor amounts of ester-bound tetradecanoic and hexadecanoic acids. In addition to the uncommonly long 3-hydroxy fatty acids, an unusual phosphorylation pattern was deduced to be present in the lipid A.     

Proteins containing an uncleaved signal for glycophosphatidylinositol membrane anchor attachment are retained in a post-ER compartment

Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleavable COOH-terminal extension that signals anchor attachment. Overexpression in COS cells of hGH-DAF fusion proteins containing the GPI signal of decay accelerating factor (DAF) fused to the COOH-terminus of human growth hormone (hGH), produces both GPI-anchored hGH-DAF and uncleaved precursors that retain the GPI signal. Using hGH-DAF fusion proteins containing a mutated, noncleavable GPI signal, we show that uncleaved polypeptides are retained inside the cell and accumulate in a brefeldin A-sensitive, Golgi-like juxtanuclear structure. Retention requires the presence of either a functional or a noncleavable GPI signal; hGH-DAF fusion proteins containing only the COOH-terminal hydrophobic domain (a component of the GPI signal) are secreted. Immunofluorescence analysis shows colocalization of the retained, uncleaved fusion proteins with both a Golgi marker and with p53, a marker of the ER-Golgi intermediate compartment. Since N-linked glycosylation is postulated to facilitate the transport of proteins to the cell surface, we engineered a glycosylation site into hGH-DAF. Glycosylation failed to completely override the transport block, but allowed some uncleaved hGH-DAF to pass through the secretory pathway and acquire endoglycosidase H resistance. The retained molecules remained endoglycosidase H sensitive. We suggest that the uncleaved fusion protein is retained in a sorting compartment between the ER and the medial Golgi complex. We speculate that a mechanism exists to retain proteins containing an uncleaved GPI signal as part of a system for quality control.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl2.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Branched-chain amino acid transport in Streptococcus agalactiae.

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.     

A comparison of chromosomal and allozymal variation across a narrow hybrid zone in the grasshopper Caledia captiva

A hybrid zone between the Moreton and Torresian taxa of the grasshopper Caledia captiva in S.E. Queensland has been characterised in terms of allozyme and chromosome variation within the same individuals. — On chromosomal criteria (pericentric rearrangements), the zone is asymmetrical with evidence of high levels of introgression of Torresian chromosomes into the Moreton taxon. This is apparent from the analysis of two independent transects across the hybrid zone. Major changes in chromosomal frequency occur over distances of less than 0.5 km. and the level of introgression differs between the two transects, with much higher levels in the northern Moreton populations, characterised by an acrocentric X-chromosome, when compared with the southern metacentric-X Moreton populations. Chromosome analysis of samples taken from the same transect over two years has revealed no major changes in the structure of the zone. Moreover, a Moreton population located only 0.5 km. from the null point was found to be stable over 6 generations with evidence for a new balanced genome having originated following the differential incorportation of Torresian chromosomes. — Contrary to the chromosomal situation, the same hybrid zone was found to be symmetrical with respect to allozyme variation with evidence of movement of diagnostic alleles in both directions across the zone. The alleles are independent and not tightly linked to any of the pericentric rearrangements. Thus these 5 alleles are acting as markers of the background genome and reveal the relatively free movement of genes which are located outside the pericentric rearrangements. — It is proposed that the hybrid zone in Caledia captiva is unstable and is moving slowly in a westerly direction into the Torresian territory. This is due to the ability of the Moreton taxon to incorporate more readily into its genome those Torresian chromosomes or chromosome segments which increase the fitness of the Moreton taxon. On chromosomal criteria, the Torresian taxon does not share the same capacity. — It is suggested that, so long as the two taxa retain their ability to hybridise with subsequent asymmetrical introgression, the zone will continue to move westwards and eventually lead to the selective incorporation of the Torresian genome into the Moreton taxon. This will result in a polymorphic situation with clinal variation in chromosomal frequencies. The structure of the zone is dependent upon a fine balance between genomic reorganisation in recombinant genotypes and the relative dispersal capacities of the two hybridising taxa.     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.