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Gene Function Analysis Using the Chicken B-Cell Line 195sources as a query sequence. Select the “BLAST search” link from the chickentranscript website. The result lists any homologous ESTs or full-length cDNAsamong the sequences from two bursal cDNA libraries. The link may be followedbehind the sequence name to obtain the sequences and other information suchas whether there are other overlapping sequences or SAGE tags derived fromBursal and DT40 SAGE tag libraries. The sequences in The Bursal TranscriptDatabase are from bursal lymphocytes, but DT40 is derived from these cellsand is similar in its transcription profile. Thus, sequences found in The BursalTranscript Database may be expected to be expressed in DT40 as well. On theother hand, tags from the DT40 SAGE library are direct evidence for expressionand their relative frequencies indicate the steady-state level of correspondingtranscripts.Although, the Bursal Transcript Database includes close to 30,000 ESTsfrom different cDNA clones and more than 2250 unique full-length cDNAs, theabsence of sequences matching a candidate gene does not necessarily indicatelack of expression in DT40. The failure may be because of cDNA cloningdifficulties or low transcript abundance. Because there are other large-scalechicken EST and cDNA databases in the public domain, they too can be probedfor chicken transcript sequences (i.e., http://www.chick.umist.ac.uk/ or http://www.chickest.udel.edu/). If this is successful, the expression of the candidategene in DT40 can be confirmed by reverse polymerase chain reaction (PCR) ofDT40 mRNA.1.1.3. Finding the Target LocusThe best ways to define the exon–intron structure of the target locus is byrunning a BLAST search of the full-length chicken cDNA sequence againstthe chicken genome assembly (http://www.ncbi.nlm.nih.gov/projects/genome/guide/chicken/ or http://www.ensembl.org/Gallus_gallus/index.html)or perform a BLAT search (http://genome.ucsc.edu/). However, if the chickencDNA is not available, a search of the cDNA or protein sequence of thehomolog from another species can be tried. The successful identification ofthe coding regions depends on the degree of interspecies transcript conservation.If the chicken full-length cDNA is available, a BLAST or BLAT searchagainst the chicken genome should reveal the precise exon–intron structure ofthe locus. The only possible problem can be errors in the cDNA sequence orgaps or errors in the genome sequence. The chicken genome assembly is estimatedto be 90–95% complete.If only the cDNA sequence of a nonchicken homolog is available, aBLAST/BLAT search against the chicken genome may reveal the conservedcoding regions of the chicken locus. Although both the nucleotide and the aminoacid sequence can be tried, the amino acid sequence may be the most suitable