Reversal of the posttranslational modification on Chlamydomonas flagellar alpha-tubulin occurs during flagellar resorption

We previously have shown that a posttranslational modification of alpha-tubulin takes place in the flagellum during Chlamydomonas flagellar assembly (L'Hernault, S. W., and J. L. Rosenbaum, 1983, J. Cell Biol., 97:258-263). In this report, we show that the posttranslationally modified alpha-3 tubulin is changed back to its unmodified alpha-1 precursor form during the microtubular disassembly that takes place during flagellar resorption. These data indicate that the addition and removal of a posttranslational modification on alpha-tubulin might be a control step in the assembly and disassembly of flagella.     

Dopamine- and octopamine-sensitive adenylate cyclase in the brain of adultCulex pipiens mosquitoes

The effects of dopamine and octopamine on adenylate cyclase activity were studied on the head homogenate of adult Culex pipiens mosquitoes in vitro. Both dopamine and octopamine were shown to increase the cyclic AMP content in the homogenate. The antagonist haloperidol blocked the production of cyclic AMP induced from dopamine but had no effect on the production of cyclic AMP induced by octopamine at the concentrations tested. The opiate agonist etorphine was ineffective at reducing cyclic AMP levels induced by either dopamine or octopamine at the concentrations tested.     

Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms

When outer-row dynein arms are extracted from Chlamydomonas flagellar axonemes, they dissociate into two ATPase complexes with sedimentation coefficients of 12S and 18S. We immunized mice with 18S dynein and generated a library of monoclonal antibodies against the polypeptides in this complex. Antibodies were selected which specifically recognize the 18S alpha- and beta-heavy chains and the 83,000-dalton and 70,000-dalton intermediate chains. These antibodies were isolated and characterized for their ability to recognize determinants on both denatured antigens and native 18S dynein; 18S dynein was dissociated in stepwise fashion into smaller aggregates with ionic and nonionic detergents and the resulting subcomplexes were isolated by precipitation with specific monoclonal antibodies. The smallest aggregates isolated were heterodimers between the alpha-chain and a 16,000-dalton light chain and between the two intermediate chains. Additional close associations of the beta-heavy chain with an 18,000-dalton light chain and 70,000-dalton intermediate chain, and a weaker interaction between the intermediate chain heterodimer and light chains of 21,000 daltons and 12,500 daltons, were also observed. We present a model of 18S dynein substructure based upon this information.     

The effect of endotoxin and endotoxin tolerance on inflammation induced by mycobacterial adjuvant

Peptidoglycan, the substance in mycobacteria thought to be responsible for inducing adjuvant arthritis, and endotoxin (lipopolysaccharide or LPS) share many inflammatory properties. Since repeated administration of LPS produces tolerance, i.e., resistance to the toxic and inflammatory effects of LPS, we tested whether LPS and/or LPS tolerance might influence inflammation due to mycobacterial adjuvant. Male Sprague-Dawley rats were injected with Escherichia coli LPS or saline intraperitoneally and then challenged with 100 micrograms killed Mycobacteria butyricum (adjuvant) in the footpad. A single dose of 100 micrograms LPS three or 24 hours before adjuvant markedly, but transiently, reduced the local footpad swelling that begins within hours of the adjuvant injection and histologically resembles a sterile abscess. Animals that received multiple doses of LPS and were therefore tolerant or animals that received LPS 72 hours before adjuvant demonstrated adjuvant-induced footpad swelling nearly equal to controls. The anti-inflammatory effect of LPS was transient since footpad swelling in all groups was nearly comparable six days after the adjuvant injection and LPS failed to inhibit consistently the arthritis that develops two or more weeks after adjuvant injection. These studies establish that LPS can markedly inhibit the prodrome of adjuvant arthritis (footpad swelling due to M. butyricum), that inhibition of this prodrome does not prevent the subsequent development of arthritis, and that LPS tolerance diminishes this anti-inflammatory effect of LPS.     

FLAGELLAR ELONGATION AND SHORTENING IN CHLAMYDOMONAS : The Use of Cycloheximide and Colchicine to Study the Synthesis and Assembly of Flagellar Proteins

Flagella can be removed from the biflagellate Chlamydomonas and the cells begin to regenerate flagella almost immediately by deceleratory kinetics. Under usual conditions of deflagellation, more than 98% of all flagella are removed. Under less drastic conditions, cells can be selected in which one flagellum is removed and the other left intact. When only one of the two flagella is amputated, the intact flagellum shortens by linear kinetics while the amputated one regenerates. The two flagella attain an equal intermediate length and then approach their initial length at the same rate. A concentration of cycloheximide which inhibits protein synthesis permits less than one-third of each flagellum to form when both flagella are amputated. When only one is amputated in cycloheximide, shortening proceeds normally and the degree of elongation in the amputated flagellum is greater than if both were amputated in the presence of cycloheximide. The shortening process is therefore independent of protein synthesis, and the protein from the shortening flagellum probably enters the pool of precursors available for flagellar formation. Partial regeneration of flagella occurs in concentrations of cycloheximide inhibitory to protein synthesis suggesting that some flagellar precursors are present. Cycloheximide and flagellar pulse-labeling studies indicate that precursor is used during the first part of elongation, is resynthesized at mid-elongation, and approaches its original level as the flagella reach their initial length. Colchicine completely blocks regeneration without affecting protein synthesis, and extended exposure of deflagellated cells to colchicine increases the amount of flagellar growth upon transfer to cycloheximide. When colchicine is applied to cells with only one flagellum removed, shortening continues normally but regeneration is blocked. Therefore, colchicine can be used to separate the processes of shortening and elongation. Radioautographic studies of the growth zone of Chlamydomonas flagella corroborate previous findings that assembly is occurring at the distal end (tip growth) of the organelle.     

Derepression of Phosphomannose Isomerase by Regulator Gene Mutations Involved in Capsular Polysaccharide Synthesis in Escherichia coli K-12

A regulator gene mutation (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation (capS, which maps separately from capR) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR(+) to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR(+)man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.     

An effective method for isolating DNA from historical specimens of baleen

DNA was isolated from an early twentieth century museum specimen of northern right whale baleen. A system of stringent controls and a novel set of cetacean specific primers eliminated contamination from external sources and ensured the authenticity of the results. Sequence analysis revealed that there were informative nucleotide positions between the museum specimen and extant members of the population and closely related species. The results indicate that museum specimens of baleen can be used to assess historical genetic population structure of the great whales.     

Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Sexual Selection in the Three-Spined Stickleback

The purpose of this study was to seek evidence for the influence of sexual selection on both male and female three-spined sticklebacks, a species in which the ♂ alone has parental responsibilities. We studied the intrasexual relationships of ♂♂ and ♀♀ prior to courtship, and the intrasexual and intersexual relationships of both sexes during courtship. Results demonstrated that both ♂♂ and ♀♀ compete intrasexually, and suggested that both sexes are discriminative in mate selection. These results are compatible with Trivers' (1972) model of the role of parental investment in sexual selection.     

Chromosomal deletion 4p15.32----p14 in a Treacher Collins syndrome patient: exclusion of the disease locus from and mapping of anonymous DNA sequences to this region.

Treacher Collins syndrome is an autosomal dominant condition of bilateral craniofacial abnormalities of structures derived from the first and second branchial arches. A patient with severe manifestations of Treacher Collins syndrome and a de novo chromosomal deletion in region 4p15.32----p14 was identified. Anonymous DNA sequences of loci D4S18, D4S19, D4S20, D4S22, and D4S23 were mapped to the deleted region. DNA probes previously mapped to loci on chromosome 4p (D4S10, D4S15, D4S16, D4S26, D4S35, D4S95, D4S144, RAF1P1, QDPR, and HOX7) were not deleted in this patient. Linkage analysis between the D4S18, D4S23, and QDPR loci and Treacher Collins syndrome in eight families excluded the Treacher Collins syndrome locus from the region of the deletion.