A bacterial artificial chromosome based physical map of the Ustilago maydis genome.

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.

Pyruvate dehydrogenase specific T cells in primary biliary cirrhosis show restricted antigen recognition sites.

BACKGROUND/AIMS: The aim was to characterise the antigen recognition sites of the variable T cell receptor alpha-chain (TCRAV) and beta-chain (TCRBV) of T cells specific to the pyruvate dehydrogenase (PDC) in primary biliary cirrhosis. METHODS: In 21 PDC-specific T cell clones isolated from five patients we analysed TCRAV and TCRBV usage by RT-PCR and sequenced the CDR3 regions. RESULTS: Preferential expression of the TCR elements BV6 (6 clones), BV12 (4 clones) and BV1 (3 clones), and frequent usage of the joining elements JB2.3 and JB2.1 were seen. Analysis of the alpha chain revealed rearrangement of AV2 in 7 clones (35%) and AV7 in 3 clones, however, distribution of the joining elements was heterogenous and no common sequence motifs were detected. Evaluation of the physicochemical properties of the beta-chain demonstrated a positive charge at position P4 in several clones of two patients and a hydrophobic residue at position P5 in two different patients. Further, a conserved glycine at position P7 and neutral residues at positions P6 and P8 were frequently detected. CONCLUSIONS: Our data define TCR variable region restriction and preferred CDR3 features of PDC-specific T cells and support the notion that few relevant epitopes on the PDC complex are recognised by selected T cells.