A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

Alleviation of water and osmotic stress-induced changes in nitrogen metabolizing enzymes in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. cultivars by potassium

Present communication reports laboratory and pot experiments conducted to study the influence of water and osmotic stress on nitrogen uptake and metabolism in two wheat (Triticum aestivum L) cultivars with and without potassium supplementation. Polyethylene glycol 6000-induced osmotic stress/restricted irrigation caused a considerable decline in the activity of nitrate reductase, glutamate synthase, alanine and aspartate aminotransferases, and glutamate dehydrogenase. Potassium considerably improved nitrogen metabolism under normal water supply conditions and also resulted in amelioration of the negative impact of water and osmotic stresses indicating that potassium supplementation can be used as a potential tool for enhancing the nitrogen use efficiency in wheat for exploiting its genetic potential.     

Identification of Repetitive Elements in the Genome of Oreochromis niloticus: Tilapia Repeat Masker

The large-scale bacterial artificial chromosome-end sequencing project of Nile tilapia (Oreochromis niloticus) has generated extensive sequence data that allowed the examination of the repeat content in this fish genome and building of a repeat library specific for this species. This library was established based on Tilapiini repeat sequences from GenBank, sequences orthologous to the repeat library of zebrafish in Repbase, and novel repeats detected by genome analysis using MIRA assembler. We estimate that repeats constitute about 14% of the tilapia genome and also give estimates for the occurrence of the different repeats based on the Basic Local Alignment Search Tool searches within the database of known tilapia sequences. The frequent occurrence of novel repeats in the tilapia genome indicates the importance of using the species-specific repeat masker prior to sequence analyses. A web tool based on the RepeatMasker software was designed to assist tilapia genomics.     

Genetic structure of the vairone Telestes souffia in the eastern part of Lake Constance, central Europe

Examination of the genetic structure of the vairone Telestes souffia based on 10 nuclear markers (microsatellites) revealed little-to-moderate genetic differentiation between geographically adjacent populations in the eastern part of Lake Constance in central Europe. Results emphasize the critically endangered status of this freshwater fish in the upper Rhine River system.     

Entomopathogenic Nematodes Are Not an Alternative to Fenamiphos for Management of Plant-Parasitic Nematodes on Golf Courses in Florida

With the cancellation of fenamiphos in the near future, alternative nematode management tactics for plant-parasitic nematodes (PPN) on golf courses need to be identified. The use of entomopathogenic nematodes (EPN) has been suggested as one possible alternative. This paper presents the results of 10 experiments evaluating the efficacy of EPN at managing PPN on turfgrasses and improving turf performance. These experiments were conducted at various locations throughout Florida over the course of a decade. In different experiments, different EPN species were tested against different species of PPN. Separate experiments evaluated multiple rates and applications of EPN, compared different EPN species, and compared single EPN species against multiple species of PPN. In a few trials, EPN were associated with reductions in certain plant-parasite species, but in other trials were associated with increases. In most trials, EPN had no effect on plant parasites. Because EPN were so inconsistent in their results, we conclude that EPN are not acceptable alternatives to fenamiphos by most turf managers in Florida at this time.     

Improving baculovirus transduction of mammalian cells by surface display of a RGD-motif

An RGD-containing peptide, comprising 23 amino acids from the foot-and-mouth disease virus (FMDV) VP1 protein was engineered into the envelope of Autographa californica nuclear polyhedrosis virus surface (AcNPV) using two different display strategies. The RGD-motif is a well-described tripeptide, that by binding to cell surface integrins facilitates virus entry into cells. This epitope was displayed, either by directly modifying the native major envelope protein gp64 of AcNPV, or by incorporating a second, modified version of gp64 onto the virus surface. Transduction efficiencies of four mammalian cell lines were compared by detecting the expression of the reporter gene green fluorescent protein (gfp), delivered by the baculovirus genome. Our results showed that insertion of the RGD-peptide into the envelope protein gp64 leads to enhanced specific uptake of baculoviral particles in mammalian cells, only when a combination of wild-type and mutant gp64 was present on the viral surface. Whenever the RGD-peptide was directly inserted into the native gp64, the overall amount of gp64 envelope protein was diminished, leading to decreased viral uptake.     

Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies.