EVIDENCE FOR A MOSAIC HYBRID ZONE IN THE GRASS SHRIMP PALAEMONETES KADIAKENSIS (PALAEMONIDAE) AS REVEALED BY MULTIPLE GENETIC MARKERS

Molecular techniques provide powerful tools for studying the geographic structure of hybrid zones and the dynamics of gene exchange between incipient species. We examined allozyme variation at five loci (PGM, GPI, MDH-1, MDH-2, and LDH) for 27 populations of Palaemonetes kadiakensis from the central, coastal, and eastern regions of Texas. Central Texas populations of P. kadiakensis exhibited highly significant linkage disequilibrium and departures from Hardy-Weinberg genotype proportions. In populations with linkage disequilibrium, allelic differences at GPI defined two types of P. kadiakensis, designated A and B. Both types existed in central Texas with little or no evidence of interbreeding, whereas the populations from all other localities showed complete introgression of type B alleles into the type A gene pool. We also examined ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) variation in a subset of populations, chosen to cover a range of geographic locations and levels of linkage disequilibrium. Two groups of mtDNA haplotypes and two restriction fragment patterns for the rDNA corresponded to allozyme type A and B individuals in populations exhibiting linkage disequilibrium. In populations with ongoing hybridization, all hybrid animals (N= 15) exhibited type A mtDNA. Exhibition of type A mtDNA indicated that type A females had mated successfully with type B males, but type B females had not mated successfully with type A males. Genotype distributions suggest reduced reproduction by hybrid offspring in central Texas populations. These patterns are consistent with a mosaic model of hybrid zone dynamics.     

Genetic and morphological divergence between populations of the flatfish Platichthys flesus (L.) (Pleuronectidae)

Populations of the flounder, Platichthys flesus , were screened for electrophoretically detectable protein variation at up to 37 loci. Atlantic and North Sea populations (subspecies flesus ) were genetically very similar to one another ( I >0.99) but different from Adriatic (subspecies italicus ) and Black Sea (subspecies luscus ) populations. The values for genetic identity between subspecies were around 0.9. Diagnostic loci enabled specimens of flesus, italicus and luscus to be differentiated from one another. Samples of flesus showed two to three times the heterozygosity levels of italicus or luscus , consistent with the greater population size of flesus . Morphological comparisons enabled populations to be categorized to subspecies. Platichthys flesus italicus and luscus are valid subspecies: the terms are not synonyms. The genetic distance data provide an estimate of divergence time of the three subspecies at about 2 My ago, an estimate that seems reasonable in the light of present knowledge concerning the recent geological histories of the Mediterranean and Black Sea basins.     

Detection of a natural point mutation in Potato virus A that overcomes resistance to vascular movement in Nicandra physaloides., and studies on seed-transmissibility of the mutant virus

Isolate M of Potato virus A (PVA‐M; genus Potyvirus) is avirulent in Nicandra physaloides L. (family Solanaceae). The inoculated leaves are infected but no systemic infection is observed. Forty plants of ‘Black Pod’ (BP) and ‘Black Pod Alba’ (BPA), two variants of N. physaloides described in this study, were inoculated with PVA‐M. Two plants of BP and one plant of BPA were systemically infected. Mosaic, blistering and dark green islands developed on the systemically infected leaves, and flowers showed colour‐break symptoms. PVAprogeny were sequence‐characterised for the 6K2 protein and viral genome‐linked protein (VPg) encoding regions known to control the long distance movement of PVA in N. physaloides. All virus progeny (designated as PVA‐Mm) in the systemically infected leaves of the plants inoculated with PVA‐M contained only a single amino acid substitution (Vail 16Met) in the central part of VPg due to a nucleotide substitution G6033A, as compared to PVA‐M. Other PVA isolates that infected N. physaloides systemically also contained Metll6 in VPg. In a previous study using chimeric viruses, Metl16 in VPg was shown to be a major determinant for vascular movement of PVA in N. physaloides, and this study reveals that the mutation for Metl16 can occur in vivo during replication of the avirulent PVA‐M in infected plants. Immunolocalisation studies on BP and BPA plants showed that the pods (berries) and seed coat contained PVA‐Mm in the developing seeds, but no virus was detected in embryons. Up to 27% of the mature seeds contained PVA‐Mm but no transmission to seedlings was observed in a total of 450 seeds tested, and no test plants were infected following mechanical inoculation with extracts prepared from the seeds.     

Effect of a selectiveβ 2-adrenergic agonist (Cenbuterol) on energy balance and body composition in normal and protein deficient rats

In rats fed a normal (22% protein) diet, injection of clenbuterol (1 mg/kg/d for 21 d) did not affect energy intake, energy expenditure or weight gain, but reduced energetic efficiency, and fat and energy gains and increased body protein content. Presenting a low-protein (8%) diet reduced energy intake, gain and efficiency, body protein content and the mass of the gastrocnemius muscle when compared to rats fed the control diet. Injection of the protein-deficient rats with clenbuterol (1 mg/kg/d for 21 d) caused hypophagia and reduced body weight and energy gains, energy expenditure and total body fat. However, the total body content of protein was not significantly reduced and the percentage of body protein in this protein deficient, clenbuterol-treated group was greater than that of untreated rats on both the high- and low-protein diets. The ratio of body protein to fat following clenbuterol treatment was increased by over 50% in both normal and protein-deficient rats. The results show that in protein deficient animals, clenbuterol treatment may help conserve body protein at the expense of fat, resulting in a smaller, but leaner body mass.     

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

Gramicidin A-phospholipid model systems

Gramicidin A forms ion-conducting channels which can traverse the hydrocarbon core of lipid bilayer membranes. The structures formed by gramicidin A are among the best characterized of all membrane-bound polypeptides or proteins. In this review a brief summary is given of the occurrence, conformation, and synthesis of gramicidin A, and of its use as a model for ion transport and the interaction of proteins and lipids in biological membranes.     

Insertion of the precursor of the light-harvesting chlorophylla/b-protein into the thylakoids requires the presence of a developmentally regulated stromal factor

The precursor of the major light-harvesting chlorophylla/b-proteins of photosystem II was synthesizedin vitro from a gene fromLemna gibba. When the labelled precursor was incubated with developing barley plastids, the precursor and the processed polypeptide were incorporated in the thylakoids in proportions that varied depending on the developmental stage of plastids. At early stages of development most of the precursor associated with the thylakoids could be removed by washing with 0.1 M NaOH, while in more mature plastids most of its was resistant to a NaOH wash. Insertion of the precursor into thylakoids required the presence of a stromal factor and Mg-ATP. The stromal factor is probably a protein. The insertion reaction has an optimal temperature of 25°C and a pH of 8. The appearance of the stromal factor and the thylakoid membrane's receptivity for the insertion of the precursor depended on the stage of plastid development. These observations are consistent with the hypothesis that the insertion of the precursor into the thylakoid prior to its proteolytic processing, is one of the steps involved in the assembly of the light-harvesting complex of photosystem II.     

Developmental biochemistry of cottonseed embryogenesis and germination. XIX. Sequences and genomic organization of the α globulin (vicilin) genes of cottonseed

The globulin storage protein genes of cotton are found to exist as gene tandems that contain a gene from each of the 2 globulin subfamilies separated by a spacer region of about 2700 or 3400 base pairs. Three different tandems have been identified by restriction endonuclease mapping of genomic DNA. A cDNA that is different from the genes of the tandems in map sites and/or in nucleotide sequence indicates that a fourth tandem probably exists in the cotton genome. Since the species of cotton used here (Gossypium hirsutum) is an amphidiploid, it is likely that two of the tandems are contributed from each genome.Considerable divergence in nucleotide sequence (18%) and in derived amino acid sequence (28%) is found when the 2 genes of a sequenced tandem are compared. The sequence of the cDNA closely resembles one of the genes in the tandem showing only a 4% divergence in nucleotides and a 4.2% divergence in amino acids. Thus the 2 genes of each tandem represent a relatively ancient gene duplication that has given rise to the two globulin subfamilies of cotton. Only one subfamily has a glycosylation site and the glycosylation of its derived proteins gives rise to the 2 molecular weight sets of globulins seen on gel electrophoresis.Other basic features of these genes and their derived proteins are presented.     

The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.Abbreviations Chl Chlorophyll - CL Continuous light - CPa the reaction center complex of PSII - CPI the reaction center complex of PSI - CPIa Chlorophyll protein complex containing the CPI and the light harvesting complex of PSI - fr w fresh weight - LDC Light dark cycles - LHC-I Light-harvesting complex of PSI - LHC-II Light harvesting complex of PSII - PS photosystem - PSI photosystem I - PSII photosystem II