Isolation, cloning and expression of a putative abalone gene relating to egg-laying hormone

A putative abalone egg-laying hormone has been amplified by polymerase chain reaction (PCR) from abalone genomic DNA. The PCR product was found to hybridize to Lymnaea stagnalis egg-laying hormone (CDCH) cDNA probe and the PCR product was then cloned and sequenced. Nucleotide sequences of putative abalone egg-laying hormone were determined.

Isolation of a gene preferentially expressed in mature anthers of rice (Oryza sativa L.)

Using monoclonal antibodies raised against pollen-specific proteins, we have isolated a cDNA clone, designatedOry-Cl from a rice anther cDNA expression library. A transcript corresponding to theOry-Cl gene showed preferential expression in anthers. This transcript was not detected in any vegetative tissues analysed. RNA gel blot analysis of different developmental stages of anthers showed that theOry-Cl gene is expressed at later stages of pollen development. In situ hybridisation showed that theOry-Cl transcript is only present in mature pollen.

Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species : screening of strains and optimization of omega-3 production

An isolation program targeting Thraustochytrids (marine fungoid protists) from 19 different Atlantic Canadian locations was performed. Sixty-eight isolates were screened for biomass, total fatty acid (TFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) content. Analysis of fatty acid methyl ester results discerned four distinctive clusters based on fatty acid profiles, with biomass ranging from 0.1 to 2.3 g L−1, and lipid, EPA, and DHA contents ranging from 27.1 to 321.14, 2.97 to 21.25, and 5.18 to 83.63 mg g−1 biomass, respectively. ONC-T18, was subsequently chosen for further manipulations. Identified using 18S rRNA gene sequencing techniques as a Thraustochytrium sp., most closely related to Thraustochytrium striatum T91-6, ONC-T18 produced up to 28.0 g L−1 biomass, 81.7% TFA, 31.4% (w/w biomass) DHA, and 4.6 g L−1 DHA under optimal fermentation conditions. Furthermore, this strain was found to produce the carotenoids and xanthophylls astaxanthin, zeaxanthin, canthaxanthin, echinenone, and β-carotene. Given this strain’s impressive productivity when compared to commercial strains, such as Schizochytrium sp. SR21 (which has only 50% TFA), coupled with its ability to grow at economical nitrogen and very low salt concentrations (2 g L−1), ONC-T18 is seen as an ideal candidate for both scale-up and commercialization.

Isolation of sperms from the pollen tube of flowering plants during fertilization

Sperm cells have been isolated from pollen tubes growing in style segments of the dicotlyledon Rhododendron macgregoriae and the monocotyledon Gladiolus gandavensis by the in vivo/in vitro method at various stages of fertilization. Pollen tubes emerged from the cut end of the style into agar medium, and more than 95% contained sperm cells. Sperm cells were released from the pollen tubes by osmotic shock or by placing styles in wall-degrading enzymes: 0.5% macerozyme and 1% cellulase. The isolated sperms were ellipsoidal protoplasts of diameter about 2 × 3 micrometers in Gladiolus and about 3 × 4 micrometers in Rhododendron. After isolation, a proportion of the sperm cells occurred in pairs linked at one end by finger-like connections. The pairs of isolated sperms were dimorphic in terms of surface area and volume. By cutting the styles at various positions and times after pollination, the potential exists to detect changes in sperm gene expression associated with fertilization.

Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name “Orientia chuto,” and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Isolation and characterisation via 454 sequencing of microsatellites from the tawny frogmouth, Podargus strigoides (Class Aves, Family Podargidae)

We isolated 24 novel polymorphic microsatellite markers from the tawny frogmouth, a nocturnal bird endemic to Australia, which has successfully adapted to urban environments. Initially, 454 shotgun sequencing was used to identify 733 loci with primers designed. Of these, we trialled 30 in the target species of which all amplified a product of expected size. Subsequently, all 30 of these loci were screened for variation in 25 individuals, from a single population in Melbourne, Victoria, Australia. Twenty-eight loci were polymorphic with observed heterozygosity ranging from 0.03 to 0.96 (mean 0.58) and the number of alleles per locus ranged from 2 to 18 (average of 6.5); we confirmed that 24 loci conformed to Hardy–Weinberg expectations. The 24 loci identified here will be sufficient to unequivocally identify individuals and will be useful in understanding the reproductive ecology, population genetics and the gene flow amongst localities in urban environments where this bird thrives.

Isolation and characterisation of the ylmE homologue of Thermus thermophilus

Screening of a Thermus thermophilus genomic library led to the identification of a homologue of the ylmE gene. ylmE is highly conserved in widely divergent organisms from prokaryotes to mammals, suggesting an important, albeit currently unknown, cellular function. The 633 bp gene has a GC content of 69.2% overall and 90% in the third nucleotide position, while the gene product is predicted to be a soluble cytoplasmic protein of 23441 Da. It belongs to a family of conserved proteins of unknown function and exhibits amino acid identities ranging from 45% to 28% to the Aquifex aeolicus and Saccharomyces cerevisiae family members, respectively. We speculate that the gene product may be involved in a cellular stress response in T. thermophilus.

Population genetic structuring in Acanthopagrus butcheri (Pisces: Sparidae): does low gene flow among estuaries apply to both sexes?

Acanthopagrus butcheri completes its entire life history within estuaries and coastal lakes of southern Australia, although adults occasionally move between estuaries via the sea. Consequently, it is expected that populations of A. butcheri in different estuaries will be genetically distinct, with the magnitude of genetic divergence increasing with geographic isolation. However, previous genetic studies of A. butcheri from southeast Australia yielded conflicting results; allozyme variation exhibited minimal spatial structuring (θ = 0.012), whereas mitochondrial DNA distinguished the majority of populations analyzed (θ = 0.263) and genetic divergence was positively correlated with geographic isolation. This discrepancy could reflect high male gene flow, which impacts nuclear but not mitochondrial markers. Here we estimated allele frequencies at five nuclear microsatellite loci across 11 southeast Australian populations (595 individuals). Overall structuring of microsatellite variation was weaker (θ = 0.088) than that observed for mitochondrial DNA, but was able to distinguish a greater number of populations and was positively correlated with geographic distance. Therefore, we reject high male gene flow and invoke a stepping-stone model of infrequent gene flow among estuaries for both sexes. Likewise, management of A. butcheri within the study range should be conducted at the scale of individual or geographically proximate estuaries for both sexes. The lack of allozyme structuring in southeast Australia reflects either the large variance in structuring expected among loci under neutral conditions and the low number of allozymes surveyed or a recent colonization of estuaries such that some but not all nuclear loci have approached migration-drift equilibrium.

Isolation and phylogenetic identification of halotolerant/halophilic bacteria from the salt mines of Karak, Pakistan

Extreme environments like salt mines are inhabited by a variety of bacteria that are well-adapted to such environments. The bacterial populations provide economic benefits in terms of enzymes synthesis. The salt mines of Karak region in Pakistan are extremely saline and the microbial communities found here have not yet been explored. In the present study, 57 halotolerant/halophilic bacterial strains were isolated from the salt mines of Karak. These strains were grown in media with 0-35% NaCl concentration. The morphological and physiological characteristics of the isolated strains were studied to optimize the growth conditions and to classify the isolated bacterial strains into slightly halotolerant/halophilic, moderately halophilic and extreme halophilic. The phylogenetic analyses inferred from 16S rRNA gene sequence of the isolated strains demonstrated that the major population were closely related to species belonging to Planococcus, Jeotgalicoccus, Staphylococcus, Halobacillus, Halomonas, Brevibacterium, Gracilibacillus, Kocuria, Salinivibrio, Salinicoccus, Oceanobacillus and Bacillus genera. Results showed that the salt mines of Karak region are rich in halotolerant/halophilic bacterial population with diverse bacterial communities, which may be utilized in various industrial applications after proper screening and identification.