Distribution of polymorphic forms at the porcine GH locus in a population of day-10 pig embryos

The present study describes an analysis of genotype and allele distribution at the porcine GH locus among day-10 pig embryos. Embryos were collected post mortem from 6 crossbred (Danish Landrace x Yorkshire) sows inseminated with mixed Duroc semen and individually frozen for later analysis. After extraction, DNA was subjected to PCR amplification and restriction analysis with Msp I and Hae II enzymes. The genotype frequencies were: Msp I CD 0.17, DD 0.83; and Hae II AA 0.33, AB 0.58; and BB 0.09. The Msp I CC genotype was not found among analysed embryos. To our knowledge, this is the first report on the genotype and allele distribution at the GH locus among early pig embryos.     

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain

The porcine COL10A1 gene, encoding the alpha1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.     

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.     

Functional and structural characterization of anti-β1-adrenoceptor autoantibodies of spontaneously hypertensive rats

Eighteen month old spontaneously hypertensive rats (SHR-rats) showed myocardial dysfunction and autoantibodies directed against the ₁-adrenoceptor similarly as known in human dilated cardiomyopathy or Chagas' disease. The agonist-like antibodies were able to activate the ₁-adrenoceptor mediated signal transduction cascade in cultured rat cardiomyocytes and induced a long-lasting stimulatory effect resulting in a harmful adrenergic overdrive. The antibodies recognized an epitope of the second extracellular loop of the ₁-adrenoceptor identical to that epitope identified in Chagas' disease. In conclusion, our assumption is supported that old SHR-rat are an useful animal model for investigating the role of anti-₁-adrenoceptor antibodies in the induction of human cardiomyopathy.     

Timing of insemination relative to ovulation in pigs: effects on sex ratio of offspring

The objective of the present study was to identify effects of the interval between insemination and ovulation in pigs on the sex ratio and sex ratio dispersion of offspring. Crossbred sows that had farrowed 2 to 9 litters were weaned (Day 0) and came into estrus between Days 3 and 7 after weaning. Ultrasonography was performed every 6 h, from 12 h after the onset of estrus until ovulation had been observed. The sows were inseminated once at various intervals from the onset of estrus. At farrowing, the numbers of viable piglets and dead piglets were recorded per sow. In four 12-h intervals between insemination and ovulation (36 to 24 h before ovulation, 24 to 12 h before ovulation, 12 to 0 h before ovulation and 0 to 12 h after ovulation), the total number of piglets was (mean+/-SEM) 10.8+/-1.2 (n=15); 13.4+/-0.7 (n=23); 13.2+/-0.9 (n=21); and 12.1+/-1.0 (n=16), respectively (P>0.05). The percentage of male piglets per litter in the four 12-h intervals was 52.1+/-3.6, 50.5+/-2.7, 54.9+/-2.8 and 47.8+/-4.5, respectively (P>0.05). Sex ratio was not influenced by litter size (P>0.05), and its distribution was normally dispersed (i.e., as expected under a binomial distribution) in all 4 intervals between insemination and ovulation (P>0.05).     

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.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.