Detection of squirrel poxvirus by nested and real-time PCR from red (<Emphasis Type="Italic">Sciurus vulgaris</Emphasis>) and grey (<Emphasis Type="Italic">Sciurus carolinensis</Emphasis>) squirrels

Background  

Squirrel poxvirus (SQPV) is highly pathogenic to red squirrels (Sciurus vulgaris), and is a significant contributing factor to the local extinction of the species in most parts of England and Wales, where infection is endemic in Eastern grey squirrel (Sciurus carolinensis) populations. Although a nested PCR assay has been used successfully to study the epidemiology of SQPV, samples have a long processing time and the assay is not quantifiable.     

UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Colonization of the Gastrointestinal Tract of the Farmed South African Abalone Haliotis midae by the Probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1

Viable cell counts and/or in situ hybridization were used to determine whether the probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1 can colonize the gastrointestinal tract of the South African abalone Haliotis midae. The number of culturable probiotic cells reisolated from H. midae fed probiotic-supplemented feed for 3 weeks ranged from 10⁶ to 10⁷ cfu/g gut material. A significant decrease (P < 0.05) in probiont numbers 2 days after feeding the probiotic-supplemented feed had been halted correlated with a significant decrease (P < 0.05) in intestinal protease and amylase activity. There was a positive correlation between Cryptococcus sp. SS1 and amylase activity (r² = 0.681) and V. midae SY9.8 and protease activity (r² = 0.711) in the H. midae intestine. Although culturable probionts were isolated from abalone that had not been fed probiotic-supplemented feed for a 2-week period, the drop in the number of probiotic cells colonizing the abalone digestive tract 2 days after feeding with the probiotic-supplemented feed had been halted indicates that farmed abalone should be fed probiotic-supplemented feed at least every second day for maximum benefit.     

Cloning and characterisation of a novel ompB operon from Vibrio cholerae 569B

The ompB operon of Vibrio cholerae 569B has been cloned and fully sequenced. The operon encodes two proteins, OmpR and EnvZ, which share sequence identity with the OmpR and EnvZ proteins of a variety of other bacteria. Although the order of the ompR and envZ genes of V. cholerae is similar to that of the ompB operon of E. coli, S. typhimurium and X. nematophilus, the Vibrio operon exhibits a number of novel features. The structural organisation and features of the V. cholerae ompB operon are described.     

The interleukin 1 gene cluster contains a major susceptibility locus for ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly heritable inflammatory arthropathy. Although the gene HLA-B27 is almost essential for the inheritance of the condition, it alone is not sufficient to explain the pattern of familial recurrence of the disease. We have previously demonstrated suggestive linkage of AS to chromosome 2q13, a region containing the interleukin 1 (IL-1) family gene cluster, which includes several strong candidates for involvement in the disease. In the current study, we describe strong association and transmission of IL-1 family gene cluster single-nucleotide polymorphisms and haplotypes with AS.     

Quantitative trait loci for partial resistance to Aphanomyces root rot in pea

Aphanomyces root rot, caused by Aphanomyces euteiches Drechs, is the most-important disease of pea ( Pisum sativum L.) worldwide. No efficient chemicals are available to control the pathogen. To facilitate breeding for Aphanomyces root rot resistance and to better understand the inheritance of partial resistance, our goal was to identify QTLs associated with field partial resistance. A population of 127 RILs from the cross Puget (susceptible) x 90-2079 (partially resistant) was used. The lines were assessed for resistance to A. euteiches under field conditions at two locations in the United States (Pullman, Wash. and LeSueur, Minn.) in 1996 and 1998 for three criteria based on symptom intensity and disease effects on the whole plant. The RILs were genotyped using automated AFLPs, RAPDs, SSRs, ISSRs, STSs, isozymes and morphological markers. The resulting genetic map consisted of 324 linked markers distributed over 13 linkage groups covering 1,094 cM (Kosambi). Twenty seven markers were anchored to other published pea genetic maps. A total of seven genomic regions were associated with Aphanomyces root rot resistance. The first one, located on LG IVb and named Aph1, was considered as "major" since it was highly consistent over the years, locations and resistance criteria studied, and it explained up to 47% of the variation in the 1998 Minnesota trial. Two other year-specific QTLs, namely Aph2 and Aph3, were revealed from different scoring criteria on LG V and Ia, respectively. Aph2 and Aph3 mapped near the r (wrinkled/round seeds) and af (normal/afila leaves) genes, and accounted for up to 32% and 11% of the variation, respectively. Four other "minor" QTLs, identified on LG Ib, VII and B, were specific to one environment and one resistance criterion. The resistance alleles of Aph3 and the two "minor" QTLs on LG Ib were derived from the susceptible parent. Flanking markers for the major Aphanomyces resistance QTL, Aph1, have been identified for use in marker-assisted selection to improve breeding efficiency.     

Colonisation pattern of nonpathogenic Fusarium oxysporum, a potential biological control agent, in roots and rhizomes of tissue cultured Musa plantlets

Under laboratory conditions, nonpathogenic, endophytic Fusarium oxysporum inflicts high mortality among banana weevils and nematodes. Following inoculation into banana (Musa spp.) tissue cultured plants, successful colonisation is necessary for efficient biological control of these pests. The pattern of root and rhizome colonisation by two nonpathogenic Ugandan F. oxysporum strains (V2w2 and III4w1) in cv. Nabusa (AAA‐EA) was investigated using light microscopy. Percentage of colonisation in the rhizomes (93%) was higher than in the roots (56%), but hyphal density in the roots (0.30 mm^?2) was higher than in the rhizomes (0.21 mm^?2). The root bases were better colonised (76%) than root midsections (53%) or tips (39%). Both the strains colonised the roots and the rhizomes, with numerous hyphae infecting the hypodermis but fewer infecting the cortex. Colonisation of vascular tissues was not recorded. Despite the presence of hyphae in intercellular and intracellular spaces of the roots and the rhizomes, normal cell structure was observed. Our report provides the first in situ observation and quantification of endophyte colonisation in banana. The study demonstrated the ability of F. oxysporum strains V2w2 and III4w1 to penetrate intact host tissues and recolonise the host internally upon inoculation, an important step for their suitability as biological control agents.