Using these Pgt sequences, PtC

Using these Pgt sequences, PtContig18 and PtContig7347 were identified by a BLASTN Pt EST data base search. A PCR product from the cDNA clone, Pt EST PT0061b. D10. TB that aligned to Contig18, was used as a probe to identify Pt BAC PtHSP02. Sequencing of this BAC resulted in four assembled contigs. Gaps could be spanned and thus the contigs could be ordered and oriented. Sizes of the con tigs in bp were 16,991, 30,055, 5,014, and 60,277 for a total of 112,337 bp. Gaps were present in regions of repeated DNA and could not be assembled. GC content was 46. 3% and FGENESH pre dicted 31 ORFs in the contig ranging from 174 bp to 7,167 bp in length. The smaller ORFs were generally within repeated elements. The bean rust effector UfHSP42c Uf011 matched three predicted protein sequences in Pgt, PGTG 17547, PGTG 17548 and PGTG 17549.

UfHSP42c matched five Pt ESTs, including clone PT0131d. B10. Inhibitors,Modulators,Libraries BR from Inhibitors,Modulators,Libraries which probes were derived to identify Pt BAC clone HSP04. Sequencing of HSP04 pro duced two contiguous sequences of 9,276 bp and 157,027 bp for a total of 166,303 bp. GC content was 46. 3% and 61 ORFs were predicted ranging from 120 bp to 5,214 bp in length. BAC annotation The predicted ORFs from each BAC clone were aligned using BLASTN to the Pgt genome, Pgt predicted transcripts and Pt ESTs, and using BLASTX, to the Pgt, Mlp, and U. maydis predicted proteomes. Pt1F16 had nine ORFs with synteny in Pgt. Identity across the protein sequences ranged from 37 87% in these alignments and putative annotations could be assigned to five of the proteins. Pt1F16 4 contained many gaps when compared to PGTG 13013.

Proteins Brefeldin_A Pt1F16 5, 6, 7, 8 and 9 aligned with two proteins each from Pgt. Pt1F16 7 aligned with PgtRAD18, which has one copy in each of the Pgt haplotype Inhibitors,Modulators,Libraries genomes. All but one homolog could also be found in Mlp and four were represented in Um. Nine predicted proteins in PtHSP02 were confirmed through EST sequence alignment and a putative function could be assigned to eight of them. Alignment identity ranged from 30 100% in PtHSP02. Eight homologs could be found in both Mlp and Um in PtHSP02. The most highly conserved protein is PtHSP02 6, a G protein ? subunit containing a conserved WD 40 repeat motif. The first 343 amino acids were 100% identical to PGTG 03727 and 99% to Mlp accession GL883091. Conversely, PtHSP02 3 was only 30% identical to PGTG 3706 and had no homologs in the other two fungi.

PtHSP02 4 and PtHSP02 5 aligned with Mlp HESP 379, the haustorial expressed predicted Inhibitors,Modulators,Libraries secreted protein homolog from M. lini, and a homolog was found for each in Pgt. Two insertions deletions were found in PtHSP02 4 and PGTG 3708. PtHSP02 5 and PGTG 3709 aligned to homologs from M. lini, Mlp, M. medusae deltoidis, and U. maydis. The N terminal half of the protein was conserved between Puccinia and Melampsora. There appeared to be 48 genus specific amino acid changes across the protein.

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