Bacterial artificial chromosome–based physical map of Gibberella zeae (Fusarium graminearum)

Fusarium graminearum is the primary causal pathogen of Fusarium head blight of wheat and barley. To accelerate genomic analysis of F. graminearum, we developed a bacterial artificial chromosome (BAC)–based physical map and integrated it with the genome sequence and genetic map. One BAC library, developed in the HindIII restriction enzyme site, consists of 4608 clones with an insert size of approximately 107 kb and covers about 13.5 genome equivalents. The other library, developed in the BamHI restriction enzyme site, consists of 3072 clones with an insert size of approximately 95 kb and covers about 8.0 genome equivalents. We fingerprinted 2688 clones from the HindIII library and 1536 clones from the BamHI library and developed a physical map of F. graminearum consisting of 26 contigs covering 39.2 Mb. Comparison of our map with the F. graminearum genome sequence showed that the size of our physical map is equivalent to the 36.1 Mb of the genome sequence. We used 31 sequence-based genetic markers, randomly spaced throughout the genome, to integrate the physical map with the genetic map. We also end-sequenced 17 BamHI BAC clones and identified 4 clones that spanned gaps in the genome sequence. Our new integrated map is highly reliable and useful for a variety of genomics studies.

Characterization of pathogenic and non-pathogenic African swine fever virus isolates from Ornithodoros erraticus inhabiting pig premises in Portugal

Ten African swine fever virus isolates from the soft tick Ornithodoros erraticus collected on three farms in the province of Alentejo in Portugal were characterized by their ability to cause haemadsorption (HAD) of red blood cells to infected pig macrophages, using restriction enzyme site mapping of the virus genomes and by experimental infection of pigs. Six virus isolates induced haemadsorption and four were non-haemadsorbing (non-HAD) in pig macrophage cell cultures. The restriction enzyme site maps of two non-HAD viruses, when compared with a virulent HAD isolate, showed a deletion of 9·6 kbp in the fragment adjacent to the left terminal fragment and of 1·6 kbp in the right terminal fragment and an insertion of 0·2 kbp in the central region. The six HAD viruses isolated were pathogenic and produced typical acute African swine fever in pigs and the four non-HAD isolates were non-pathogenic. Pigs that were infected with non-HAD viruses were fully resistant or had a delay of up to 14 days in the onset of disease, after challenge with pathogenic Portuguese viruses. Non-HAD viruses could be transmitted by contact but with a lower efficiency (42–50 %) compared with HAD viruses (100 %). The clinical differences found between the virus isolates from the ticks could have implications for the long-term persistence of virus in the field because of the cross-protection produced by the non-pathogenic isolates. This may also explain the presence of seropositive pigs in herds in Alentejo where no clinical disease had been reported.

Mechanisms of Double-Strand-Break Repair During Gene Targeting in Mammalian Cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin μ-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal μ-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal μ-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process: The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an “end” bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence.Formation of hDNA was frequently associated with gene targeting and, in most cases, began ∼645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.