Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster

An ovary transplanted from a Drosophila melanogaster female into a male will mature and form morphologically normal yolk-filled oocytes. Since it has been supposed that the yolk polypeptides come only from the female fat body, it was hypothesized that the implanted ovary induces the fat body of the male host to synthesize and secrete yolk polypeptides (YPs). To test this hypothesis, fat body preparations from females, untreated males, and males containing transplanted ovaries were cultured in vitro with ³⁵S-methionine and the medium was examined for the presence of newly labeled YPs. Female fat body secreted newly labeled YPs, but no freshly synthesized YPs were secreted by fat bodies from untreated males or from males containing transplanted ovaries. In vitro cultured ovaries, however, both from females and from male hosts did secrete newly synthesized YPs. Therefore, the YPs in an ovary that matured in a male come mainly from endogenous synthesis by the implanted ovary. To find whether males were responsive to the hormones that stimulate YP production in isolated female abdomens, we treated males with the juvenile hormone analogue ZR-515 and with 20-hydroxyecdysone. The latter, but not the former, was able to cause synthesis and secretion of three bands migrating precisely as YPs in SDS gels. Partial peptide digests of the 20-hydroxyecdysone-stimulated polypeptides in males showed them to be identical with those stimulated by 20-hydroxyecdysone or ZR-515 in isolated female abdomens and with the three YPs found in normal female hemolymph. Finally, YP synthesis was assayed in mutants that affect the phenotypic sex of a fly. It was found that flies bearing two X chromosomes and the mutations dsx, dsx^D, ix or three sets of autosomes continued to make YPs, but tra-3-pseudomales did not. These results suggest that the process of sex determination involves steps leading to synthesis of an ecdysteroid in females, which then activates synthesis of the YPs by the fat body. A hypothesis is suggested to explain the fact that two different hormones can stimulate YP synthesis and two different organs can synthesize YPs.     

The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km² region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.     

The pattern of segment formation,as revealed by engrailed expression,in a centipede with a variable number of segments

Arthropods vary enormously in segment number, from less than 20 to more than 200. This between-species variation must have originated, in evolution, through divergent selection operating in ancestral arthropod species with variable segment numbers. Although most present-day arthropod species are invariant in this respect, some are variable and so can serve as model systems. Here, we describe a study based on one such species, the coastal geophilomorph centipede Strigamia maritima. We investigate the way in which segments are formed using in situ hybridization to demonstrate the expression pattern of the engrailed gene during embryogenesis. We also analyze segment number data in mother-offspring broods and thereby demonstrate a significant heritable component of the variation. We consider how natural selection might act on this intraspecific developmental variation, and we discuss the similarities and differences in segment formation between the geophilomorphs and their phylogenetic sister-group.     

DNA integrity and transgene expression after passage through the NOGA needle catheter used for therapeutic myocardial angiogenesis

BACKGROUND: The NOGA (Biosense Webster, Markham, ON, Canada) injection catheter is an innovative navigational device that provides an ideal platform for intra-myocardial injection material. However, injection through a long (1.91 m), narrow (27G) nitinol needle could result in deterioration in the integrity and functionality of DNA. METHODS: To test this possibility, DNA in plasmid form (pcDNA3.1) containing the Lac Z transgene (250 micro l) was passed through the NOGA needle using a hand-held 1 cc syringe at a gentle hand injection pressure (43 +/- 3 PSI, 3.0 +/- 0.2 kg/cm(2)) or at maximal manual pressure (90 +/- 6 PSI, 6.3 +/- 0.4 kg/cm(2)), either once or 20 times. This DNA, compared to DNA not passed through the NOGA needle (control), was then used to transfect primary cultures of rat skin fibroblasts (FB) from Fisher 344 rats and the cells were subsequently stained for beta galactosidase (betagal). RESULTS: Transfection efficiency was significantly reduced by passing the DNA through the needle at both 43 +/- 3 PSI (78 +/- 4% of control, n = 10, P < 0.05 versus control) and 90 +/- 6 PSI (66 +/- 4 % of control, n = 10, P < 0.01 versus control, P < 0.02 versus 43 +/- 3 PSI). Passage of the DNA through the NOGA needle 20 times resulted in a transfection efficiency of only 5 +/- 1% of control (n = 20, P < 0.1 x 10(-11) versus control). Capillary Electrophoresis revealed that the reduction in transfection efficiency was due to a conformational change in the DNA from predominantly supercoiled to nicked and linearized DNA. Transfection efficiency as compared with control decreased as the concentration of the DNA solution which was passed through the needle was increased from 0.3 micro g/ micro l to 2.4 micro g/ micro l. Recovery experiments confirmed that the reduction in transfection efficiency was not due to loss of DNA by binding to the NOGA needle. CONCLUSION: These results suggest that DNA is susceptible to shear forces when injected through the NOGA needle even at nominal clinical injection pressures, suggesting that careful and controlled injections will be required to achieve optimal gene integrity and expression.