Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.     

SH2-B is required for growth hormone-induced actin reorganization

The Src homology-2 (SH2) domain-containing protein SH2-Bbeta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-Bbeta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-Bbeta present at the plasma membrane and in the cytosol. SH2-Bbeta colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-Bbeta is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-Bbeta in 3T3-F442A cells and assayed for GH- and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-Bbeta enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-Bbeta (R555E) and truncation mutant DeltaC555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant DeltaN504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. DeltaN504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-Bbeta is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-Bbeta as a stimulator of JAK2 kinase activity.     

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.     

A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments

Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus “Scalindua” group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem.     

Disabled is a bona fide component of the Abl signaling network

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.     

The demise of Leptotheca Thélohan, 1895 (Myxozoa: Myxosporea: Ceratomyxidae) and assignment of its species to Ceratomyxa Thélohan, 1892 (Myxosporea: Ceratomyxidae), Ellipsomyxa K?ie, 2003 (Myxosporea: Ceratomyxidae), Myxobolus Bütschli, 1882 and Sphaerospora Thélohan, 1892 (Myxosporea: Sphaerosporidae)

A revision of Leptotheca Thélohan, 1895 is presented. The boundaries that separate Leptotheca from Ceratomyxa Thélohan, 1892 and Sphaerospora Thélohan, 1892 are vague and have been highlighted as an area of concern within myxosporean classification. A survey of the literature revealed 63 species that are currently assigned to Leptotheca and a further 11 species that have been relegated as synonyms in Ceratomyxa, Sphaerospora or Myxobolus Bütschli, 1882. The placement of some species in the genus is unclear and demonstrates the need for a revision. The type-species of Leptotheca (L. agilis Thélohan 1892) has many Ceratomyxa-like characters, such that a minor amendation of the diagnosis of Ceratomyxa will then accept the type-species of Leptotheca, rendering the latter genus its synonym. We propose the suppression of Leptotheca, with all species currently assigned to that genus reassigned to Ceratomyxa, Ellipsomyxa K?ie, 2003, Myxobolus or Sphaerospora on the basis of appropriate morphological and biological traits. The diagnoses of Ceratomyxa and Ellipsomyxa are amended appropriately. Molecular analysis may change the placement of some species in the future; however, the aim of this review was to eliminate the ambiguity of assignment of species in the genera Leptotheca, Ceratomyxa and Sphaerospora by suppressing Leptotheca.