Genetic and morphometric differentiation among island populations of two Norops lizards (Reptilia: Sauria: Polychrotidae) on independently colonized islands of the Islas de Bahia (Honduras)

Aim Anole lizards (Reptilia: Sauria: Polychrotidae) display remarkable morphological and genetic differentiation between island populations. Morphological differences between islands are probably due to both adaptive (e.g. differential resource exploitation and intra‐ or interspecific competition) and non‐adaptive differentiation in allopatry. Anoles are well known for their extreme diversity and rapid adaptive speciation on islands. The main aim of this study was to use tests of morphological and genetic differentiation to investigate the population structure and colonization history of islands of the Islas de Bahia, off the coast of Honduras. Location Five populations of Norops bicaorum and Norops lemurinus were sampled, four from islands of the Islas de Bahia and one from the mainland of Honduras. Methods Body size and weight differentiation were measured in order to test for significant differences between sexes and populations. In addition, individuals were genotyped using the amplified fragment length polymorphism technique. Bayesian model‐based and assignment/exclusion methods were used to study genetic differentiation between island and mainland populations and to test colonization hypotheses. Results Assignment tests suggested migration from the mainland to the Cayos Cochinos, and from there independently to both Utila and Roatán, whereas migration between Utila and Roatán was lacking. Migration from the mainland to Utila was inferred, but was much less frequent. Morphologically, individuals from Utila appeared to be significantly different in comparison with all other localities. Significant differentiation between males of Roatán and the mainland was found in body size, whereas no significant difference was detected between the mainland and the Cayos Cochinos. Main conclusions Significant genetic and morphological differentiation was found among populations. A stepping‐stone model for colonization, in combination with an independent migration to Utila and Roatán, was suggested by assignment tests and was compatible with the observed morphological differentiation.     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Metabolism and excretion of acetylchromenes by the migratory grasshopper

The extent of metabolism and excretion of three acetylchromenes (two toxic, one relatively nontoxic) were examined in adult migratory grasshoppers (Melanoplus sanguinipes) following topical administration. Both the total amount excreted (parent plus metabolites) and the proportion of parent compound in the excreta were inversely correlated with contact toxicity. Both toxic and nontoxic acetylchromenes are rapidly absorbed from the cuticle, with maximum excretion of parent and metabolite chromenes from 4 to 8 h posttreatment in each case. Much of the applied compounds (60–80%) apparently remains within the insect, and cannot be recovered by extraction of the insect. Metabolites formed result from simple oxidative and reductive transformations. For all of the compounds tested (including the allatocidin precocene II), the major mode of metabolism results from aliphatic hydroxylation of one of the geminal methyl groups on the chromene. No conjugated metabolites were found in the excreta.     

Immunoaffinity-purified DNA polymerase alpha displays novel properties

The purification and characterization of a novel and more intact form of the DNA polymerase alpha-primase complex from calf thymus are described. The polymerase-primase was enriched 10,000-fold to apparent homogeneity by chromatography on phosphocellulose, heparin-Sepharose, and an immobilized anti-human DNA polymerase alpha monoclonal antibody [SJK287-38; Tanaka, S., Hu, S., Wang, T. S.-F., & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390]. A quantitative elution from the antibody column was achieved by shifting the pH from neutrality to between 12.5 and 13. From 1 kg of calf thymus, the procedure yields 1-2 mg of polymerase-primase with a specific activity of 30,000-40,000 units/mg for the polymerase and 15,000-20,000 units/mg for the primase. The complex sediments at 9 S through a sucrose gradient and exhibits a Stokes radius of 6.0 nm, yielding a native molecular mass of 335,000. Denaturing gel electrophoresis of the complex gives bands of Mrs 180,000, 155,000, 148,000, 73,000, 59,000, and 48,000 with a relative abundance of the two smallest subunits. Primase activity was partially resolved from the complex by centrifugation through sucrose gradients. The primer-forming activity was found to be associated with the Mr 59,000 and 48,000 polypeptides. In contrast to conventional preparations, the immunopurified polymerase displays several features which show it is the most intact form of the enzyme known to date. The deoxynucleoside triphosphate Km values are all within the range of 0.6-0.9 microM. The Km for binding to a single RNA primer on M13 DNA is 3.5 nM; the Ki for nonspecific binding to unprimed DNA is 70 microM (nucleotide).(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.     

Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI)

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.25 nm and 2.01 +/- 0.13 nm respectively. This is in good agreement with the structurally related bovine peripheral myelin P2 protein (about 70% amino acid sequence homology). CD measurements have revealed MDGI to be a protein with about 50% beta structure and less than 20% alpha helix similarly as in fatty acid-binding proteins. Removal of endogenous long-chain fatty acid by lipidex or storage in the frozen state lead to a destabilization of the active MDGI conformation which is accompanied by a loss of its activity with regard to growth inhibition of Ehrlich Ascites cells.     

The Cellular Mechanism of Orcadian Rhythms-A View on Evidence, Hypotheses and Problems

A stable period length is a characteristic property of circadian oscillations. The question about whether higher frequency oscillators (0.5-8 hr) contribute to or establish the stable circadian periodicity cannot be answered at present. A sequential coupling of quantal subcycles appears possible on the basis of known “ultradian” oscillations. There is, however, no supporting evidence for such a concept. Phase response curves of the circadian clock derived from various perturbing pulses allow qualitative conclusions concerning the perturbed clock process. Deductions from computer simulations also allow conclusions about the phase of this oscillatory process.

The distinction between processes (a) essential to the clock mechanism, (b) maintaining and controlling the clock (inputs) and (c) depending on the clock (outputs) on the basis of “oscillatory” and “change of φ or τ after perturbation” seems to be useful but not stringent. Protein synthesis may be an essential or input process. Oscillatory changes of this process may be due to periodic translational control or RNA-supply. Circadian changes in protein concentration and/or activity may depend on periodic synthesis, proteolysis, covalent modifications or aggregations. Specific essential proteins have not been identified conclusively. The large overlap between the group of agents and treatments that phase shift the clock and the group that induces stress proteins suggest that the latter may play a role in the controlling (input) or essential domain.

The role of membranes in the clock mechanism is not clear: concepts assuming an essential function are based on circumstantial evidence. The membrane potential as well as Ca²⁺ may be involved in either input or essential function. Ca²⁺ -calmodulin may also be important as concluded from inhibitor experiments. It is tempting to assume that a calmodulin-dependent kinase is part of a periodic protein phosphorylation process, yet it is not clear whether the periodic protein phosphorylation that has been observed is essential or is just another output process.     

Comparison of salt- and heat-induced alterations of protein synthesis in the cyanobacterium Synechocystis sp. PCC 6803

Protein synthesis of the cyanobacterium Synechocystis spec. PCC 6803 decreases after a 684 mM NaCl salt shock. Qualitative changes were observed during the shock and the subsequent adaptation process using one-dimensional polyacrylamide electrophoresis. Proteins of apparent molecular masses of 13.0, 14.2, 16.6, 20.0, 21.0, 23.0, 33.0, 47.0, 52.0, 65.0 and 72.0 kDa are synthesized at enhanced rates after salt stress. The proteins of 14.2, 21.1 and 52.0 kDa are transiently induced during the first hours of the adaptation phase, while the other proteins are also synthesized at enhanced rates in salt-adapted cells. The proteins of 14.2, 23.0, 33.0 and 65.0 kDa are also induced by heat shock (43°C). Heat shock proteins of about 88.0, 75.0, 58.0, 17.5 and 13.8 kDa, in contrast, are induced by heat shock but not by salt. Two-dimensional polyacrylamide electrophoresis showed that the induced salt and heat shock proteins in some cases consisted of isoforms of different isoelectric points.Abbreviations IP isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

DNA polymerase alpha-DNA primase from human lymphoblasts

The DNA polymerase alpha-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458-8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195,000 and 180,000 for the DNA synthesizing alpha-subunit, of 68,000 for the beta-subunit, and of 55,000 and 48,000 for the primase-carrying gamma- and delta-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the alpha-subunit, at 5.5 for the beta-subunit, and at 7.5 and 8.0 for the gamma- and delta-subunit, respectively. The purified polymerase-primase converted single-stranded phi X174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3-10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.