Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.     

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.     

Assessment of a rigorous transitive profile based search method to detect remotely similar proteins

Profile-based sequence search procedures are commonly employed to detect remote relationships between proteins. We provide an assessment of a Cascade PSI-BLAST protocol that rigorously employs intermediate sequences in detecting remote relationships between proteins. In this approach we detect using PSI-BLAST, which involves multiple rounds of iteration, an initial set of homologues for a protein in a 'first generation' search by querying a database. We propagate a 'second generation' search in the database, involving multiple runs of PSI-BLAST using each of the homologues identified in the previous generation as queries to recognize homologues not detected earlier. This non-directed search process can be viewed as an iteration of iterations that is continued to detect further homologues until no new hits are detectable. We present an assessment of the coverage of this 'cascaded' intermediate sequence search on diverse folds and find that searches for up to three generations detect most known homologues of a query. Our assessments show that this approach appears to perform better than the traditional use of PSI-BLAST by detecting 15% more relationships within a family and 35% more relationships within a superfamily. We show that such searches can be performed on generalized sequence databases and non-trivial relationships between proteins can be detected effectively. Such a propagation of searches maximizes the chances of detecting distant homologies by effectively scanning protein "fold space".     

Low dose insemination in cattle with the Ghent device

A new artificial insemination device for semen deposition near the uterotubal junction (UTJ) in cattle (Ghent device) was developed at Ghent University (Belgium). In this study, UTJ insemination of dairy cows with the Ghent device was compared with the conventional insemination technique to evaluate the effect on pregnancy rates after insemination with different doses of semen. In each of three field trials, the cows (n=795, 659, 360) and heifers (n=253, 182, 231) were randomly assigned to receive 12 million sperm deposited in the uterine body using conventional techniques (control) or a reduced sperm dose (RSD) deposited in the same manner as the control or bilateral deposition near the uterotubal junction using the Ghent device (Ghent). Sperm dosages for RSD and Ghent inseminations were 8, 4, and 2 million sperm for field trials 1-3, respectively. In the multivariable analysis, the pregnancy rates were significantly affected by the parity of the cow (p相似文献   

Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures

Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was ompA.     

Differential distribution of leaf chemistry in eucalypt seedlings due to variation in whole-plant nutrient availability

We investigated the effects of whole-plant nutrient-availability on the degree of distribution of some plant primary and secondary (nitrogen, fibre, flavonols, gallotannins and cineole) chemicals across young, mature and old leaves of seedlings of Eucalyptus nitens. Four treatments that ranged from low to high nutrient-application rates resulted in mean whole-plant foliar concentrations of 0.63%, 0.85%, 1.11% and 1.82% nitrogen dry matter (N%DM) for treatments A, B, C and D, respectively. Within-plant distribution (across the leaf age profile of young, mature and old leaves within a eucalypt seedling) of N%DM ranged from zero in treatment A to a wide range of distribution in treatment D (low N%DM concentrations in old leaves to high N%DM concentrations in young leaves). Similarly, the distribution of fibre ranged from zero in treatment A to a wide range of distribution in treatment D, but with high concentrations in old leaves and low concentrations in young leaves. In contrast, flavonols (weakly) and gallotannins had a wide range of distribution in treatment A (low concentrations in old leaves to high concentrations in young leaves) but were little or not distributed in the other treatments. Again in contrast, cineole was strongly distributed between old and young leaves (low concentrations in old leaves to high concentrations in young leaves) across all treatments while concentrations in mature leaves reflected one or other leaf age depending on treatment. Protein precipitable phenols in treatments A, B and C were high in young, and low in old leaves; whereas in treatment D they were low and similar between leaves of different ages.