Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.     

Genotype dependent variation in mycorrhizal colonization and response to inoculation of pearl millet

Summary Genotypes of pearl millet (Pennisetum americanum L. Leeke) were examined for differences in vesicular-arbuscular mycorrhizal (VAM) colonization and response to inoculation. For thirty genotypes tested across three field locations there was a range of mycorrhizal colonization intensity between 25 and 56%. In another experiment with two male-sterile lines, restorer lines and their derived crosses, grown in pots filled with non-sterilized soil there were significant differences between genotypes for colonization by mycorrhiza. This showed hostgenotype dependence for mycorrhizal colonization.Root growth rates, mycorrhizal root length, percentage root colonization and plant growth and P uptake were studied in ten genotypes. A set of 3 genotypes with similar root lengths varied significantly with regard to mycorrhizal root length and the percentage colonization. This supports the suggestion that VAM colonization and spread is dependent on the host genotype. The growth responses differed significantly between the genotypes and they also differed in their responses to P uptake and VAM inoculation. The utility of host-genotype dependent differences in VAM symbiosis in plant breeding is discussed.Journal Article No. 453     

A Mouse β-Globin Mutant That Is an Exact Model of Hemoglobin Rainier in Man

A mutation induced by ethylnitrosourea in a spermatogonial stem cell of a 101/H mouse has resulted in a structurally altered beta-diffuse major globin in one of his offspring. The mutant hemoglobin is associated with polycythemia, rubor, increased oxygen affinity and decreased hem-hem interaction. The mutant haplotype has been designated Hbbd4, polycythemia. Amino acid analysis of the mutant globin has shown that a single substitution beta 145 Tyr----Cys has occurred, and it is proposed that ethylnitrosourea induced an A----G transition in the tyrosine codon (TAC----TGC). This murine polycythemia is homologous with hemoglobin Rainier in man, in which the amino acid substitution is also beta 145 Tyr----Cys and which is associated with similar physiological consequences.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.     

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.     

Membrane properties of isolated winter wheat cells in relation to icing stress

Isolated cell preparations of winter wheat (Triticum aestivum L.) were utilized to examine the effect of ice encasement at −1°C and exposure to ethanol on metabolic and biochemical properties of cells. Following icing and ethanol treatments, passive efflux of amino acids increased gradually with duration of exposure to the stress, and closely paralleled the decline in viability of cells. In contrast, uptake of ⁸⁶Rb declined much more rapidly than viability following exposure to icing or ethanol. Electron spin resonance spectroscopy studies revealed no significant change in molecular ordering within the cell membranes following icing or exposure to ethanol, whereas a small but significant increase in order was detected in the noniced controls. O₂ consumption by isolated cells declined only gradually due to icing and ethanol treatments, and remained relatively high even when cell viability was severely reduced. These results indicate that the plasma membrane is a primary site of injury during ice encasement and that damage to the ion transport system is the earliest manifestation of this injury.     

Structure and function in the excretory systems of some terrestrial prosobranch snails (Cyclophoridae)

The excretory systems of terrestrial prosobranch snails of the family Cyclophoridae, collected in Jamaica, Costa Rica and South Africa, have been examined physiologically and as regards their gross and fine structure. The process of urine formation commences in the heart, where fluid is filtered across the wall of the ventricle. Filtration through the auricular wall is believed to be negligible. The kidney, which contains three types of cell, modifies the composition of the filtrate. One type of resorptive cell, characterized by basal infoldings associated with mitochondria, takes up salts. Another type, with basal subcellular spaces, may be responsible for taking up water. The third type of cell is secretory, producing concretions of uric acid and phospholipid which are liberated into the kidney lumen when the cell degenerates.
The rate and mechanism of urine production have been investigated using injections of inulin. The filtration rate at 25°C is 0.5 μl/g/min, and in 100% R.H. the average rate of urine production is 0–39 μl/g/min.
An accessory excretory organ has been developed from the hypobranchial gland of aquatic forms. It is composed of groups of subepithelial tubular glands opening into the mantle cavity by one or a series of pores, and secreting purines, phospholipids and mucus. There is evidence that this organ becomes progressively more complex in forms occupying drier habitats.
The systems of excretion and osmoregulation in the Cyclophoridae are considered to be very similar to those in their aquatic relatives, the Viviparidae and Ampullariidae. Certainly the cyclophorids are not as well adapted to a terrestrial life as are the Pulmonata, and in many respects they may be considered "aquatic" snails living on land.