New CSNP Loci In Rattus Hrh1 Gene And Its Relevant Study

Histamine is derived from the decarboxylation of the amino acid histidine, a reaction catalyzed by the enzyme L-histidine decarboxylase. It is one of monamines which have the most extensively influences on the functions of the body. Histamine is the major response factor in hypersensitivity type I. Mediated by Hrh1 ( histamine receptor H1 ) , histamine regulates comprehensive physiological function in eucaryotic organism. Therefore, Hrh1 is an important agency to study the nerve-incretion-immunity-effector axis.As a monocopy gene, Hrh1 is localized at Rattus norvegicus chromosome 4, 4q42 region. Homo Sapiens Hrh1 gene was thought to be intronless until recently. Up to April 2009, the numbers of human's intronless gene reported at NCBI site were about 180, far less than the number of human's intron gene. But eukaryotic intronless genes are important datasets for comparative genomics and evolutionary studies, and have evoked interesting of researchers. Rattus Hrh1 is also intronless gene and its full length is entire exon.There are few studies about the Hrh1 gene polymorphism and base mutation so far, and there is no report about the mutation of Rattus Hrh1 gene in domestic research. Rattus Hrh1 gene is highly consensus with human genomic Hrh1 gene. Their protein structures are very similar: both have two 7-transmembraneαhelix regions, in which homologous amino acid sequence is about 80%. According to this fact, we chose Rattus Hrh1 gene to study its base mutation and nucleotide polymorphism in genomic DNA and DNA transcript (mRNA), to lay the experimental foundation of polymorphism, relevant mutation, pharmacogenetics, and pharmacogenomics about human Hrh1 gene and its express product (histamine H1 receptor) in the future.The subject study is mainly in four parts for the purpose of our investigation.In experiment part I, we studied the sequence variation of rattus Hrh1 gene from different origins respectively, i.e. genomic DNA extracted from rat whole blood, transcript mRNA in rat brain tissue and transcript mRNA in rat hepatic tissue.We extracted a male SD rat genomic DNA from whole blood using KI method and extracted total RNA from rat brain tissue and hepatic tissue with Trizole. Ultraviolet spectrophotometry and agarose gel electrophoresis were used to detect purity and concentration of obtained nucleic acid. RevertAid? First Strand cDNA Synthesis Kits was used to synthesize first strand cDNA chain with brain total RNA and hepatic total RNA as reaction template. We apply Vector NTI Suite 10.0 to design the primers in compliance with coding sequence of rattus genomic Hrh1 DNA sequences (gi: 62750804) and mRNA sequence (gi: 220770) reported at NCBI site. According to the restriction enzymes digest site at 5'terminus in sense and antisense primer, we can conveniently construct the recombinant express vector in the future. PrimeSTAR? HS DNA Polymerase has very high fidelity, so we used it to clone the rat Hrh1 gene entire coding sequence by polymerase chain reaction with rat genomic DNA, brain cDNA, and hepatic cDNA as template. PCR purification kit was used to purify and recover the product of PCR. Double enzyme restriction was used to digest PCR product of genomic DNA, brain cDNA, and hepatic cDNA, and cloning vector pUC19 Gel extraction kit was used to purify and recover the product of double digestion. T4 DNA Ligase jointed the digestion product of genomic DNA, brain cDNA, hepatic cDNA and vector pUC19 respectively with their cohesive termini. By this, we construct pUC19-Hrh1 recombinant vector.The Inoue Method was used to prepare super competent of Ecoli DH5α. pUC19-Hrh1 recombinant vector was transformed into DH5αsuper competent by heat shock, and IPTG, X-gal was used to do a blue-white screen. Colony PCR was used to quickly identify the positive colony with pUC19-Hrh1. The selective positive colony was cultured overnight. Plasmid was extracted from the overnight culture with plasmid DNA extraction kit.Agarose gel electrophoresis was used to identify the size of plasmid according their band compared with the marker approximately. PCR amplification with plasmid as template was used to determine the target gene in selective recombinant. Double digestion was used to identify the plasmid to prevent selective plasmid from double cloning with target gene. We selected 3 recombinants of genomic DNA, 3 recombinants of brain cDNA, and 5 recombinants of hepatic cDNA, and committed these recombinants to SinoGenoMax Co., Ltd. for sequencing.By the consequence of sequencing, we found it was 100% consensus of 3 recombinants of genomic DNA, 3 recombinants of brain cDNA and 1 recombinant of hepatic cDNA with rattus Hrh1 genomic DNA (gi: 62750804), But only 99.7% consensus with mRNA (gi: 220770), with 4 bases difference. To our surprise, there were 5 base mutations within 4 recombinants of hepatic cDNA, at 237, 928, 1041, 1210, and 1342 locus. After the triplet code of amino acid was analyzed, we found the mutations at 928 and 1342 locus can lead to the change of translation amino acid with that valine replaces methionine at 928 locus and methionine replaces valine at 1342 locus. According to the report of cSNP of Rattus Hrh1 gene at NCBI site, only the C/T polymorphism (rs8155549) at locus number 237 coincides with our result. There was no relevant report about the rest 4 loci mutations. It is suggested that (1) there are new cSNPs in rattus Hrh1 gene; and (2) there may be mutate at RNA level, when Hrh1 gene transcripts in different organs (e.g. brain or liver) of the rat, that is also called RNA editing.In experiment part II, we mainly researched the influences of different anticoagulant to the purity and yield of DNA extracted from whole blood genome, especially subsequent PCR amplification. Because, in experiment part I, we extracted genomic DNA from blood anticoagulated with heparin sodium, yield and purify of genomic DNA, detected by ulraviolet spectrophotometry and agarose gel electrophoresis, had no problem. But the result of PCR amplification was not stable and satisfied. Referring to involved information and after excluding many factors, we speculated that anticoagulant in blood may impact PCR amplification efficiency. Considering EDTA as chelant can inhibit the Mg2+ to catalyze PCR, we didn't choose it in this experiment, but use sodium citrate and heparin sodium as anticoagulant, to anticoagulate arterial blood and venous blood of rat respectively, and then perform the comparison.We designed 3 pairs of primers: Primers I amplifiesβ-actin (span across intron, 1104 bp); Primers II amplifies part of Hrh1 gene, 468bp; And primers III amplifies full length of Hrh1 gene, 1477 bp. From the result of experiments, we can make the conclusion that when genomic DNA from blood containing anticoagulant heparin sodium is extracted, the anticoagulant will inhibit PCR amplification efficiency, especially to amplify large size fragment. Heparin sodium inhibited amplification efficiency slightly while small size fragment was amplified. According to the experimental results, we extracted genomic DNA from blood with sodium citrate as anticoagulant, in order to guarantee the stability of sequent experiment.Part III of the experiments was cSNP detection in rattus Hrh1, to confirm our conclusion in experiment part I. We selected 13 healthy SPF SD rats, 7 males and 6 females, and phlebotomized the rats from jugular vein. Genomic DNA was extracted from whole blood by KI method. The purity and concentration of DNA samples were detected by the methods of ultraviolet spectrophotometry and agarose gel electrophoresis. PrimeSTAR? HS DNA Polymerase can be used to amplify full length of rattus Hrh1 gene coding sequence accurately. The product of PCR after agarose gel electrophoresis detection was purified and recovered with gel extraction kit. Purified product was committed to SinoGenoMax Co., Ltd. for sequencing.After studying the sequencing peak map, we discovered that there are 4 cSNPs in Rattus Hrh1 entire coding sequence which contains 1461 bp: 237 C/T polymorphism, 928 A/G polymorphism, 1041 C/T polymorphism, and 1342 G/A polymorphism. The statistical frequency of 4 cSNPs in 13 samples was analyzed by SPSS 15.0. Up to April 2009, the numbers of cSNP reported in rattus Hrh1 coding sequence at NCBI site are 3: (1) rs8153540 (102 A/C polymorphism);(2) rs8155549 (237 C/T polymorphism);and (3) rs8153541 (1404 C/G polymer- phism)。In the 4 cSNPs which we discovered, only cSNP 237 coincided with rs8155549 reported by NCBI, and the rest of other 3 cSNPs were not reported at NCBI site at the same time. We did not found the polymorphism about rs8153540 and rs8153541 which NCBI reported.In the 4 cSNPs, albeit alter triplet code changed by 237 C/T and 1041 C/T polymorphism will not change the coding amino acid. However, 928 A/G polymorphism and 1342 G/A polymorphism are missense mutation, and the polymorphism can alter the amino acid coded by changing triplet code。We applied Vector NTI Suite 10.0 and Lasergene 7.0 to analyze the homo sapience Hrh1 gene DNA sequence and mRNA sequence, Rattus norvegicus Hrh1 DNA sequence and mRNA sequence, and Mus Musculus Hrh1 DNA sequence and mRNA sequence, and discovered that the occurring region of cSNPs in Human Hrh1 gene, rattus Hrh1 gene and mus Hrh1 gene are resemble each other, although the numbers of human cSNPs in Hrh1 gene are much more than the others. By predicting the structure of Hrh1 gene coding protein, we found that their structures are very similar: all of them have two 7-transmem- brance-α-helix regions in the peptide chain, between 45-255AA and 390- 470AA.We predicted the secondary structure of Hrh1 protein peptide chain by Lasergene 7.0. The 928 and 1342 cSNPs can change the amino acid 310AA and 448AA in translated peptide chain. According to the secondary structure, alteration of amino acid can change the structure ofαregion and hydrophilicity. The result indicated that the activity of translated protein may be changed, resulting from amino acid mutation induced by the alteration of first structure of coding peptide chain, and finally impact the function and characteristic of receptor protein.For establish the approach to detect the cSNPs in rattus Hrh1 gene, which can carry out the experiments involved with pharmacogenetics and pharmacogenomics, in experiment part IV, we explored ASA (alleles specific amplification) PCR to detect the 928 and 1342 cSNPs. Sample rat 1 (928 A/G and 1342 G/A), rat 2 (928 G/G and 1342 A/A), and rat 8 (928 A/A and 1342 G/G), which already confirmed sequence of Hrh1 gene in experimental part III, were chose as detectable sample.Primers designed for ASA PCR are keys to the detection. Primers were designed by Vector NTI Suite 10.0 in accordance with 928(A/G) and 1342(G/A) polymorphisms. Primers of two positions were designed homologous base probe at 3'terminus, and the base next to 3'terminus probe was designed as mismatch. The genomic DNA was extracted from whole blood, the entire coding sequence of rattus Hrh1 gene was amplified, and the PCR product was purified and recoveried. The PCR product was diluted as template for ASA PCR later. Recombinant plasmid Blood 1 (928 A and 1342 G), and Liver 1 (928 G and 1342 A), for which, the sequences were confirmed in experimental part I, were chose as positive control.Experimental consequence showed that result of PCR coincide with our expectation, and proved that the probe primers for ASA PCR are qualify, and the system of ASA PCR reaction is reliable. Our results laid the experimental foundation for genotyping rat involved with Hrh1 gene and researching the mutant phenotype of Hrh1 gene, by the methods of pharmacogenetics and pharmacogenomics about human Hrh1 gene and its express product-histamine H1 receptor in the future.