Computational protocol: Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping

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Protocol publication

[…] To construct a cDNA library for C. siamensis, poly(A) mRNA was isolated from brain tissue and cloned into the λ uni-ZAP vector (Stratagene) using standard protocols. λ uni-ZAP clones were converted into pBluescript SK(+) clones, and transformed into XL1-Blue bacterial cells (Stratagene). Colonies were picked randomly and transferred into 96-well plates using the ‘Q’ Pix colony picker (GENETIX). The clones were grown overnight and plasmid DNA was prepared using MultiScreen-NA and FB plates (Millipore). Sequencing reactions were performed with dye-labeled dideoxy terminators using the SK primer in accordance with the manufacturer’s protocol (Applied Biosystems), and the nucleotide sequences were determined using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems). Nucleotide sequences were compared against the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) database using the BLASTX program. Individual ESTs were translated in all reading frames and compared against the NCBI “non-redundant” nucleotide and/or peptide sequence database.For chromosome mapping of P. sinensis, a large number of EST clones, which were isolated from cDNA libraries constructed from the brain tissue of a female individual and whole 14-day embryos in our previous study , were subjected to a homology search as described above. EST clones of the African clawed frog (Xenopus laevis) were used for chromosome mapping of X. tropicalis. The genes that were used for chromosome mapping of X. tropicalis were chosen on the basis of the chicken chromosome map using a search with the BLASTN programs of Ensembl (http://www.ensembl.org/) and/or NCBI, and we confirmed that these genes used for mapping were each located in different scaffolds of X. tropicalis using the genome map of the X. tropicalis (the Ensembl Xenopus tropicalis Genome Browser, http://www.ensembl.org/Xenopus_tropicalis). The nucleotide sequences of X. tropicalis homologs of the genes were subjected to a search with the BLASTN program of Ensembl, and homologous clones from X. laevis were selected from a web data catalogue of the NIBB/NIG/NBRP Xenopus laevis EST project (XDB3, http://xenopus.nibb.ac.jp/). All the fragments of X. laevis EST clones that were used for chromosome mapping were more than 1.5 kb in size. […]

Pipeline specifications

Software tools BLASTX, BLASTN
Application Genome data visualization
Organisms Xenopus laevis, Gallus gallus, Pelodiscus sinensis, Crocodylus siamensis, Xenopus tropicalis, Homo sapiens