Computational protocol: Hecate/Grip2a Acts to Reorganize the Cytoskeleton in the Symmetry-Breaking Event of Embryonic Axis Induction

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Protocol publication

[…] Initial linkage was identified by bulk segregant analysis with SSLP markers. Once initial linkage of the mutation was obtained, genotypically identified homozygous mutant males were crossed to heterozygous females to generate large numbers of fish for fine mapping .Chromosome walking was conducted by screening the CHORI-211 BAC library using marker z67047 and zC150E8z (0 recombination/1762 genomes). PCR-based screening of the primary pool and secondary pools identified 2 positive BAC clones. Individual BAC clones were ordered from BACPAC resources center (http://bacpac.chori.org).To find the mutation in hec/grip2a, 5 fragments of hec/grip2a cDNA from mutant and wild-type 1-cell embryo cDNA were amplified by RT-PCR with 5 primer pairs, which cover the entire hec/grip2a coding region:1. 5′-ATGTCCTGCATCTTGCTTCCAGAG-3′ and 5′-CCTCAGTGGGAATCCCATTAATGG-3′ 2. 5′-TGGAGTGTTACAAGTTGGCGACAG-3′ and 5′-TGAATGGCTTCGCTCAGAGGTTTG-3′ 3. 5′-TTCATATCGGTGACCGAGTTTTGG-3′ and 5′-GACATTATTGTAGCCTCAAGCTCG-3′ 4. 5′-GAGACCTGCGGTCAGTCAGAAATC-3′ and 5′-GTGCTCTGTGTTTCTCATTTGTGG-3′ 5. 5′-AGGACACTTCCCAACAGTCTGCAC-3′ and 5′-ACCTGATCACTTCTAACCCAACAG-3′ All PCR products were cloned into pGEM-T easy vector and sequenced.For Phylogenetic analysis, homologous grip1 and grip2 genes were found using BLAST searches on Ensembl and NCBI genome databases. A phylogenetic tree was constructed and drawn using ClustalW in the MegAlign program from Lasergen. PDZ domains were identified using CD-search in the Conserved Domain Database (CDD) in NCBI . Schematic diagram of the protein domain structures for each gene were drawn using DomainDraw . […]

Pipeline specifications

Software tools Clustal W, DomainDraw
Applications Phylogenetics, Proteome data visualization
Diseases Movement Disorders
Chemicals Glutamic Acid