DNA Sequencing Of Rag1 Gene In Channel Catfish And The Value Of Rag1 Gene Expression On Detection Of MRD In Patients With Neoplasm Originated From Lymphocyte

Objective: The recombination activating genes (RAG, rag1 and rag2),encoding components of the recombinase which initiate V(D)J recombination of the immunoglobulin and T-cell receptor genes and play an important role in the formation of Ig and TCR diversity, usually expressed together in immature lymphocytes. Meanwhile, because the expression of RAG expression in the different phases of lymphocyte development changes regulatively, it is useful marker for tracing the development procedure of lymphoid tissues. Channel catfish (Ictalurus punctatus) has the feature that is easy for the identication of specific mutant phenotypes and genetic screens. Furthermore, bony fish is the earliest vertebrate to evolve structurally distinct DH gene segments. The channel catfish has been demonstrated as a good model organism for the study of early bony fish development and immune system evolution. The sequence of rag1 gene was necessary for detection of rag1 expression in the channel catfish. The degenerate primers, designed based on the highly conservative character of the rag1 gene, were used to amplify the fragments which would be cloned and sequenced of rag1 gene from the channel catfish genomic DNA. The sequence would be analyzed by using several kinds of bioinformatic methods and submitted to GenBank. It would be conduced to provide new insights into the immune system evolution, early evolutionary patterns and mechanisms of genetic diversity of the rag1 gene and the action of Rag1 protein. Besides, it was investigated that the expression of rag1 gene in peripheral blood cell was applied to detect the minimal residual disease (MRD) in patients with neoplasm originated from lymphocyte. Methods:1. Genomic DNA of the channel catfish was isolated from the anterior kidney of the adult (4-to 6-mo-old ) channel catfish. 2. Fragments of the rag1 gene in the channel catfish were obtained by PCR amplification of genomic DNA using degenerate primers. The fragments were purified and cloned into a T/A plasmid. Clones were randomly chosen for sequencing and all sequences were obtained on both strands.3. The programs of "ORF Finder"(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)and "NetGene" (http://www.cbs.dtu.dk/services/NetGene2/) were applied to analyze open reading frames and introns. Database comparison between the base sequences and the derived nucleic acid sequences was conducted with BLAST-N and BLAST-X respectively. Selected sequences were also analyzed by using the program of "CLUSTAL W"(http://www. ebi. ac. uk/ clustalw/) for the multiple alignment and unrooted phyligenetic tree. The secondary structure of the predicted AA sequence was analyzed by using the "discovery studio gene V1.1" program. The tertiary structure of Rag1 protein was modeled by using "SWISS-MODEL"(http://www.expasy.org/swissmod/SWISS-MODEL.html). 4. Sequences were submitted to GenBank by using the program "Bankit" in NCBI (http://www.ncbi.nlm.nih.gov/BankIt/) to apply for the sequence numbers in the GenBank.5. Expressions of the rag1 gene and IgH, TCRγ gene rearrangements of 57 peripheral blood cells samples from 35 patients with neoplasm originated from lymphocyte were detected by reverse transcription polymerase chain reaction (RT-PCR) and consensus primers polymerase chain reaction, respectively.Results:1. Three fragments of rag1 gene in channel catfish were obtained by PCR amplification of genomic DNA using degenerate primers of which sequences were verified with BLAST algorithm.2. An ORF of 2235bp was found (The initiation codon was ATG from the 624th base and the termination codon was TGA from the 2856th base). 3. An intron of 293bp was identified (from the 228th to the 520th) whose identity percents with that of the zebrafish and the rainbow trout were 49.1% and 44.3%, respectively.4. Sequences comparison using BLAST-N and BLAST-X indicated that identity percents of the rag1 gene in the channel catfish with that of other species were mostly more than 70% and the identity percents of the core regions in the predicted...