A rapid method has been developed for the detection of the solitary long terminal repeat ev15, a member of the avian leukosis virus (ALV) family of endogenous viral elements (ev genes) in chickens. Detection is accomplished by a polymerase chain reaction (PCR) assay that can be performed on purified genomic DNA samples or crude preparations of partially purified whole blood lysates. The test discriminates unambiguously between birds that are homozygous ev15-, homozygous ev15+, or heterozygous ev15-/ev15+. The incorporation of a modified touchdown amplification profile significantly improved the specificity of the PCR assay. Small-scale screening of birds from a variety of chicken breeds has revealed that ev15 is present in populations of both egg-strain birds and broilers.
Experiments were carried out to transform laboratory mice and domestic chickens by use of sperm incubated with bacterial plasmid DNA. Following demonstration of "uptake" of such DNA by sperm of both species, attempts were made to replicate a previously published procedure (Lavitrano et al. 1989, Cell 57: 717–723) for producing transgenic mice through in vitro fertilization (IVF). Also, female mice and hens were inseminated (AI) with sperm which had been incubated in a DNA solution. Such incubation did not influence the fertility or hatchability of the hens' eggs. However, no transformed progeny were detected among 45 mice produced by IVF or among 69 mice and 470 chickens produced by AI. Key words: Sperm-mediated DNA transfer, mice, chickens
Plants possess an exceedingly complex innate immune system to defend against most pathogens. However, a relative proportion of the pathogens overcome host's innate immunity and impair plant growth and productivity. We previously showed that mutation in purple acid phosphatase (PAP5) lead to enhanced susceptibility of Arabidopsis to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Here, we report that an optimal level of PAP5 is crucial for mounting complete basal resistance. Overexpression of PAP5 impaired ICS1, PR1 expression and salicylic acid (SA) accumulation similar to pap5 knockout mutant plants. Moreover, plant overexpressing PAP5 was impaired in H2O2 accumulation in response to Pst DC3000. PAP5 is localized in to peroxisomes, a known site of generation of reactive oxygen species for activation of defense responses. Taken together, our results demonstrate that optimal levels of PAP5 is required for mounting resistance against Pst DC3000 as both knockout and overexpression of PAP5 lead to compromised basal resistance.
A short fragment of chicken genomic DNA encompassing the insertion site of the endogenous avian leucosis viral element ev3 was isolated using the inverse polymerase chain reaction (inverse PCR) technique. The nucleotide sequence of the unoccupied site was used to design PCR primers that can be used to unambiguously determine the genetic status of any chicken, with respect to ev3. Screening of a small number of individuals from exotic breeds of chickens suggested that the frequency of ev3 is highly variable. The ev3 integration site shows a high degree of sequence homology with the macrophage-specific tyrosine kinase gene, bmk, in mice.
A quick and simple method has been developed to detect the presence or absence of the endogenous Rous-associated virus (RAV) element ev1 in chickens. The procedure consists of a one-tube multiplex polymerase chain reaction (PCR) involving three oligonucleotide primers that are specific for the upstream flanking region, the long terminal repeat (LTR), and the downstream flanking region of the proviral insert, respectively. The multiplex reaction allows for the unambiguous discrimination between ev1+/ev1+ homozygote, ev1-/ev1- homozygote, and ev1+/ev1- heterozygote birds. The method works best with purified genomic DNA as substrate, but can also be used with rapidly prepared, "crude" DNA samples. The combination of speed with the safety of a nonradioactive procedure, and the ability to perform large numbers of assays by a semi-automated procedure, make this method attractive for large-scale screening projects. The ev1 locus has been used as a model system to demonstrate the feasibility of the PCR diagnostic approach. However the same principle should be applicable to the analysis of other RAV-type ev loci, as well as endogenous elements belonging to other families of viruses as sequence information for the flanking regions of these inserts becomes available.
Abstract To assess the value of DNA fingerprints for the prediction of heterosis in chickens, retrospective analyses of data from three crossbreeding experiments and DNA fingerprints (DFP) of parental strains were conducted using two minisatellite and one middle-repetitive DNA probes. DFP bands were assessed on pooled DNA samples of 10-15 individuals per parental genetic group. The number of DFP bands evaluated in the experiments ranged from 81 to 139. The probes varied in their predictive value, but predictability of heterosis generally increased with multiple probes. Highly significant correlations (0.68-0.87) between band sharing ratios (SH) and heterosis were found in 25 crosses of White Leghorns in the first egg production cycle for age at sexual maturity, egg production, and mature body weight: traits with heterosis of 10% or more of the means. Regressions on SH explained 78.4% of the variation in heterosis in age at sexual maturity, 60.2% in egg production and 46.4% in mature body weight. For “broiler” traits with heterosis of <1%, none of the correlations, based on 13 crosses, were significant. It was concluded that multilocus probe DFP of pooled DNA samples show promise as predictors of heterosis.
Previous work has shown that the alpha-amylase gene of Drosophila melanogaster is subject to repression by dietary glucose. Moreover, glucose repression of this gene is mediated by promoter elements that lie upstream of the transcriptional start site. In this study, we examined the activity of the glucose-repressible Drosophila promoter in transformed yeast cells. We show that the amylase promoter region can mediate glucose repression of a heterologous reporter gene in yeast. The implication of this result is that the yeast regulatory machinery can recognize the Drosophila promoter signals. This, in turn, implies an unexpectedly high degree of evolutionary conservation in the mechanism of glucose repression among eukaryotes. It also shows that genes that have acquired complex patterns of developmental regulation-e.g., the Drosophila amylase gene, can still retain, intact, more primitive forms of regulation, such as glucose repression.
