ABSTRACT
Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Subject(s)
Chromosomes, Artificial , Codon, Terminator , Metabolism , Euplotes , Chemistry , Peptide Termination Factors , Genetics , Metabolism , Peptides , Metabolism , Protein Biosynthesis , Genetics , Protozoan Proteins , Genetics , Ribosomal Proteins , GeneticsABSTRACT
Hv-S/TPK gene, a resistance related gene to powdery mildew, was cloned by using genechip, and its expression was upregulated after the inoculation of Blumeria graminis to Haynaldia villosa. Using the specific primers of Hv-S/TPK to screen a genomic TAC (Transformation-competent artificial chromosome) library of translocation line 6VS/6AL, a positive TAC was screened. A 5-kb fragment containing Hv-S/TPK was subcloned and identified. This 5160-bp fragment (GenBank Accession No. EU153366) was determined by specific primer walking. The analysis of Hv-S/TPK genomic sequence showed three introns and four extrons between start code and stop code. In the promoter region of Hv-S/TPK, there were W-box and OCS-like elements which were the elements related to disease resistance. In this study, the positive TAC clone was used to as probe in situ hybridized to mitotic metaphase chromosomes of translocation line. The result of fluorescence in situ hybridization (FISH) indicated that the TAC clone containing Hv-S/TPK was from Haynaldia villosa chromosome.
Subject(s)
Base Sequence , Chromosomes, Artificial , Cloning, Molecular , Genomic Library , Molecular Sequence Data , Plant Diseases , Genetics , Poaceae , Genetics , Protein Serine-Threonine Kinases , Genetics , Serine , Genetics , Translocation, Genetic , Triticum , Genetics , VirologyABSTRACT
A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.