Distribution of antigens in established cell lines of Drosophila melanogaster

Antibodies prepared against two Drosophila cell lines together with antibodies prepared against other Drosophila extracts were used to study the distribution of antigens in 10 cell lines. The results suggest: (1) that cultured cells express differentiated functions; (2) that each cell line, including sub-clones of a clone, is unique; and (3) that the cell lines are derived from the lateral ectoderm.     

Molecular characterization of choline acetyltransferase from Drosophila melanogaster

Purified acetyl-CoA: choline O-acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster has been shown to contain two major polypeptides of 67 and 54 K Daltons. However, all enzyme activity is found in a single molecular weight form of approx 67 K Daltons as determined by sucrose gradient sedimentation and molecular exclusion chromatography. The latter showed both the 67 and 54 K Dalton polypeptides on polyacrylamide gel electrophoresis in sodium lauryl sulfate (10% acrylamide). Analysis of purified choline acetyltransferase on polyacrylamide gel electrophoresis in sodium lauryl sulfate (15% acrylamide) revealed the presence of an additional polypeptide at 13 K Daltons. Tryptic-peptide maps of the 67, 54 and 13 K Dalton components showed all three to be structurally related. In addition to several common tryptic peptides, the 13 K Dalton polypeptide contained three tryptic-peptides that were also found in the 67 K Dalton polypeptide, but were absent from the 54 K Dalton polypeptide. This evidence suggests that native Drosophila choline acetyltransferase may exist in two forms, one a single polypeptide chain with a molecular weight of 67 K Daltons and the other consisting of two noncovalently bound polypeptide chains with molecular weights of 54 and 13 K Daltons. The latter form is the major one isolated and may be generated by limited proteolysis of the single chain 67 K Dalton form.     

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K_m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.     

Convenient procedure for the isolation of highly enriched,cytochrome P-450-containing microsomal fraction from Candida tropicalis

The suitability of Ca²⁺ ions for the precipitation of the microsomal fraction from the hydrocarbon-grown yeast Candida tropicalis was evaluated. In the final procedure the microsomes were precipitated by the addition of 16 mm CaCl₂. Crude extracts obtained from cells via spheroplast lysis were centrifuged at 12,000g for 15 min and at 25,000g for 15 min prior to precipitation. The cytochrome P-450 content of the fraction was between 0.22 and 0.35 nmol mg^?1 protein. The isolated microsomes exhibited both hexadecane hydroxylation activity and NADPH-cytochrome c reductase activity.     

An effect of mate fertility on the attractiveness and oviposition rates of mated Drosophila melanogaster females

Mate fertility has a strong influence on the sexual receptivity of mated Drosophila melanogaster females, but an effect of mate fertility on the attractiveness of mated females has not previously been demonstrated. We compared the declines in attractiveness over the first 10 h after mating for females mated to fertile (XY) males and those mated to sterile (XO) males, and found a significant effect of mate fertility. A large and significant decrease in attractiveness, that is the same for both XY- and XO-mated females, is evident during the first 4 h after mating. However, a further decline in attractiveness occurs between 4 and 6 h after mating for XY-mated females, but not until between 6 and 10 h after mating for females with sterile mates. Thus, XY-mated females are significantly less attractive than XO-mated females at 6–8 h post-mating, but not at any other time. A sharp increase in oviposition rates for both types of mated females is associated with the decline in attractiveness that occurs between 4 and 10 h after mating.     

Primary structure of the Klebsiella serotype 16 capsular polysaccharide

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α-D-Glcp-(1/ar4)β-D-GlcAp-(1/ar4)-α-L-Fucp-(1/ar chain with a β-D-Galp-(1→ branch at position 4 of the D-glucosyl residue.     

The defensive secretion of the opisthobranch mollusc Onchidella binneyi

Opisthobranch molluscs of the family Onchidiacea have been reported to employ a chemical deterrent as a protection against predators. A single lipid-soluble compound, onchidal, has been isolated from the defensive secretion of Onchidella binneyi. The structure of onchidal was determined from spectral data and from chemical degradation studies.     

Juvenile hormone-induced biosynthesis of vitellogenin in Leucophaea maderae

Isolated abdomens of virgin female Leucophaea maderae, a South American cockroach, could be induced by a single injection of juvenile hormone I to synthesize vitellogenin. Synthetic capacity was assayed by removal of the fat body at various times after induction and incubation in organ culture with radioactive leucine for 5 h. Under these conditions, active tissues secrete radioactive vitellogenin into the medium after a lag of 2 h and continue secretion at a fixed rate for up to 8 h. Vitellogenin in the tissue reaches a steady-state level after about 3 h. By 5 h, approximately 80% of the newly synthesized vitellogenin can be found in the medium and constitutes 75% of the secreted protein.Vitellogenin does not appear in the medium until 18 h after juvenile hormone induction, rises to a maximum between 72 and 96 h, and has declined to half by 120 h postinduction. At the maximum, about 75% of the total protein secreted is vitellogenin, a more than 50-fold stimulation over injected controls. Since virtually the same quantitative effect of juvenile hormone was found in abdomens with and without ovaries, secondary stimulation by ovarian hormones does not appear to be involved in vitellogenin synthesis in this insect.Oocyte vitellogenin exists as a 560,000 molecular weight monomer complex and a 1,590,000 molecular weight trimer. The smaller complex is only found in young oocytes. By sodium dodecyl sulfate-gel electrophoresis, we find that the 560,000 molecular weight 14S monomer consists of four discrete peptides in the ratio A₁B₁C₂D₂ with molecular weights 118,000, 87,000, 57,500, and 96,000, respectively. The 28S trimer contains only the three peptides A, B, and C in the ratio A₁B₃C₂. These stoichiometries satisfactorily account for the molecular weights of both complexes when corrected for the lipid content. Maturation of 14S to 28S probably involves specific proteolytic conversion of D to B^D, a different peptide of the same size as B, suggesting that the true composition of 28S is A₁B₁B₂^DC₂.The 14S vitellogenin synthesized and secreted by fat body in tissue culture consists mostly of larger polypeptides. There is a major fraction of 179,000 molecular weight, a minor fraction of >260,000 molecular weight, and other smaller polypeptides. Although the exact relationship between these polypeptides and the mature subunits of vitellogenin are not yet clear, it is evident that the multiple protein subunits of this complex protein are synthesized as one or more precursor proteins, followed by proteolytic processing to the mature size.     

Diacylglycerol-transporting lipoproteins and flight in Locusta

Evidence from chromatographic and heparin precipitation studies shows that the ‘heparin-soluble’ lipoprotein, A⁺, forms in the haemolymph during flight. In locusts flown continuously for 60 min, lipoprotein A⁺ occurs in the haemolymph at low concentrations but accumulates during a short rest period following flight. After injections of tissue extracts containing adipokinetic hormone (AKH), A⁺ accumulates in the haemolymph but disappears more rapidly in flying locusts than in resting locusts. This difference in the rate of disappearance of diacylglycerol from the lipoprotein A⁺ can be used to estimate its rate of utilization during sustained flight (approx. 100μg. min^?1 from 45–90 min of flight). It is suggested that lipoprotein A⁺ is the major carrier of diacylglycerol from the fat body to the flight muscles during prolonged flight. The steady state concentrations of total diacylglycerol and ‘heparin-soluble’ diacylglycerol during continuous flight are unaffected when tissue extracts containing AKH are injected before flight. This suggests that there is a close homeostatic control over the steady state concentration of haemolymph lipid during flight.     

α-Amanitin-sensitive DNA-dependent RNA polymerase I from cherry salmon (Oncorhynchus masou) liver nuclei

DNA-dependent RNA polymerases I and II were purified approximately 3900- and 13300 fold, respectively, from a sonicated nuclear extract of the cherry salmon liver by column chromatographies on DEAE-Sephadex, heparin-Sepharose and DNA-cellulose. The RNA polymerases were examined with respect to template-specificity, the effects of Mn²⁺, Mg²⁺ and ammonium sulfate, α-amanitin sensitivity. Results showed that the RNA polymerase I differed from other eukaryotic RNA polymerase I in α-amanitin sensitivity.