Identification of diverse molecular forms of GnRH in reptile brain

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His⁵,Trp⁷,Tyr⁸-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp⁷,Leu⁸-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln⁸-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.     

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis

AIMS: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping. METHODS AND RESULTS: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively. CONCLUSION: recA gene amplification and HhaI and Sau3AI restriction digestion was demonstrated to increase the differentiating power between isolates of Salmonella enteritidis phage type 4 by combining the patterns of the random amplified polymorphic DNA analysis procedure using a primer ERIC2. Significance and Impact of the Study: A novel restriction fragment length polymorphism assay for isolates of Salmonella enteritidis phage type 4, based on the amplification of the recA gene was attained and its comparison and its combination with random amplified polymorphic DNA analysis was provided.     

Mitochondrial acyl carrier proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> are predominantly soluble matrix proteins and none can be confirmed as subunits of respiratory Complex I

Arabidopsis mitochondria are predicted to contain three acyl carrier proteins (ACPs). These small proteins are involved in fatty acid and lipoic acid synthesis in other organisms and have been previously reported to be subunits of respiratory Complex I in mitochondria in mammals, fungi and plants. Recently, the mammalian mitochondrial ACP (mtACP) has been shown to be largely a soluble matrix protein but also to be minimally associated with Complex I (Cronan et al. 2005), consistent with its involvement in synthesis of lipoic acid for TCA cycle decarboxylating dehydrogenases in the matrix but contrary to earlier claims it was primarily a Complex I subunit. We have investigated the localization of the ACPs in Arabidopsis mitochondria. Evidence is presented that mtACP1 and mtACP2 dominate the ACP composition in Arabidopsis mitochondria, and both are present in the mitochondrial matrix rather than in the membrane. No significant amounts of mtACPs were detected in Complex I isolated by blue native gel electrophoresis, rather mtACPs were detected at low molecular mass in the soluble fraction, showing that in A. thaliana mtACPs are predominately free soluble matrix proteins.     

Detection and resolution of genetic loci affecting circadian period in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Circadian rhythms regulate many aspects of plant growth, fitness and vigour. The components and detailed mechanism of circadian regulation to date have been dissected in the reference species Arabidopsis thaliana. To determine the genetic basis and range of natural allelic variation for intrinsic circadian period in the closest crop relatives, we used an accurate and high throughput data capture system to record rhythmic cotyledon movement in two immortal segregating populations of Brassica oleracea, derived from parent lines representing different crop types. Periods varied between 24.4 and 26.1 h between the parent lines, with transgressive segregation between extreme recombinant lines in both populations of ∼3.5 h. The additive effect of individual QTL identified in each population varied from 0.17 to 0.36 h. QTL detected in one doubled haploid population were verified and the mapping intervals further resolved by determining circadian period in genomic substitution lines derived from the parental lines. Comparative genomic analysis based on collinearity between Brassica and Arabidopsis also allowed identification of candidate orthologous genes known to regulate period in Arabidopsis, that may account for the additive circadian effects of specific QTL. The distinct QTL positions detected in the two populations, and the extent of transgressive segregation suggest that there is likely to be considerable scope for modulating the range of available circadian periods in natural populations and crop species of Brassica. This may provide adaptive advantage for optimising growth and development in different latitudes, seasons or climate conditions.     

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.     

Behavioral phenotypes of Disc1 missense mutations in mice

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.     

Sex pheromone of the cloaked pug moth,Eupithecia abietaria (Lepidoptera: Geometridae), a pest of spruce cones

The sex pheromone of the cloaked pug moth, Eupithecia abietaria Götze, an important cone‐feeding pest in spruce seed orchards in Europe, was investigated. Chemical and electrophysiological analyses of pheromone gland extracts of female moths and analogous analyses of synthetic hydrocarbons and epoxides of chain length C₁₉ and C₂₁ revealed (3Z,6Z,9Z)‐3,6,9‐nonadecatriene (3Z,6Z,9Z‐19:H) and 3Z,6Z‐cis‐9,10‐epoxynonadecadiene (3Z,6Z‐cis‐9,10‐epoxy‐19:H) as candidate pheromone components, which were found in a gland extract in a ratio of 95 : 5. In field trapping experiments, conspecific males were only attracted to a combination of 3Z,6Z,9Z‐19:H and the (9S,10R)‐enantiomer of 3Z,6Z‐cis‐9,10‐epoxy‐19:H. The (9R,10S)‐enantiomer was not attractive, which is in agreement with studies on other Eupithecia species, for which males have only been attracted by the (9S,10R)‐enantiomer of epoxides. Subsequent experiments showed that E. abietaria males were attracted to a wide range of ratios of the two active compounds and that trap catches increased with increasing dose of the binary blend. A two‐component bait containing 300 μg 3Z,6Z,9Z‐19:H and 33 μg of the (9S,10R)‐enantiomer of 3Z,6Z‐cis‐9,10‐epoxy‐19:H was efficient for monitoring E. abietaria in spruce seed orchards in southern Sweden, where this species has probably been overlooked as an important pest in the past. With sex pheromones recently identified for two other moths that are major pests on spruce cones, the spruce seed moth, Cydia strobilella L., and the spruce coneworm, Dioryctria abietella Denis & Schiffermüller, pheromone‐based monitoring can now be achieved for the whole guild of cone‐feeding moths in European spruce seed orchards.     

Ancient population genomics and the study of evolution

Recently, the study of ancient DNA (aDNA) has been greatly enhanced by the development of second-generation DNA sequencing technologies and targeted enrichment strategies. These developments have allowed the recovery of several complete ancient genomes, a result that would have been considered virtually impossible only a decade ago. Prior to these developments, aDNA research was largely focused on the recovery of short DNA sequences and their use in the study of phylogenetic relationships, molecular rates, species identification and population structure. However, it is now possible to sequence a large number of modern and ancient complete genomes from a single species and thereby study the genomic patterns of evolutionary change over time. Such a study would herald the beginnings of ancient population genomics and its use in the study of evolution. Species that are amenable to such large-scale studies warrant increased research effort. We report here progress on a population genomic study of the Adélie penguin (Pygoscelis adeliae). This species is ideally suited to ancient population genomic research because both modern and ancient samples are abundant in the permafrost conditions of Antarctica. This species will enable us to directly address many of the fundamental questions in ecology and evolution.