ABSTRACT
OBJECTIVE: To screen genetic polymorphisms in the 5'-flank region of 8-hydroxyguanine DNA glycosylase( hOGG1) gene and analyze the characteristics of their genetic distribution in Han population of radon exposure area in Guangdong Province. METHODS: A simple random sampling method was used to select 60 subjects as radon exposure population. The genomic DNA samples were extracted from peripheral blood. The single nucleotide at-1721 nt-+ 164 nt locus of hOGG1 were screened using polymerase chain reaction( PCR) amplification,purification and direct sequencing for polymorphisms. Genetic characteristics of single nucleotide polymorphisms( SNP) in the study population were analyzed and compared with different populations reported in Hap Map data. RESULTS: The 5'-flank region of hOGG1 were amplified and sequenced in these 60 individuals( 120 chromosomes) of healthy Han Chinese in radon exposure area. Eight SNPs were identified by sequence alignment in the study population. Among them,there was 3 known polymorphisms and their minor allele frequencies( MAF) were-1493 G > A( 4. 2%),-834 G > C( 0. 8%) and-18 G > T( 3. 3%),respectively. The MAF of other 5 novel variations were-1455 G > A( 0. 8%),-1293 A > T( 23. 3%),-1187 C > A( 7. 5%),-337 C > A( 36. 7%) and-323 G > A( 0. 8%),respectively. The differences in the MAF distribution of-1493 G > A between the study population and the Hap Map-CEU were statistically significant( P < 0. 05). CONCLUSION: Eight SNPs and their genetic characteristics were screened and identified in the 5'-flank region of hOGG1 of Han Chinese population in radon exposure area. This result provides a basis for construction of polymorphism haplotypes and functional analysis for the target population.
ABSTRACT
Aim: Clone and characterize of the 5′- flanking region of the nitrate reductase (NR) gene derived from Dunaliella salina(D. salina). Methods : The genomic DNA from D. salina was respectively digested with BamHI, EcoRI, HindIII, Pst I, Sal I and Xba I. A genomic walking cassette was ligated to the ends of the digested DNA fragments, and then genomic walking libraries comprising BL, EL, HL, PL, SL and XL were constsucted. The 5′- flanking region of the NR gene from genomic walking libraries of D. salina was amplified by LA-PCR. The DNA sequences were analyzed with the software - Promoter Predictions. Isolated 5′-flanking regions fused to the GUS gene were tested for transient expression in the alga. Results: A single specific PCR product of about 1200bp in length from the HL library was generated. Also, several conserved motifs, such as CAAT-box, GAGA-box were found, which are related to regulation of transcription, and the putative binding sites of transcriptional factors such as EBP, EFII, NF-E1 and LV. BLAST showed that the DNA sequences shared high homology with 5′-upstream region of the NR gene from Dunaliella viridis. The isolated 5′-flanking regions were able to strongly drive GUS reporter gene expression, suggesting that it contains the promoter elements necessary for the transcription of the NR gene. The expression pattern of the GUS gene and the NR gene were similar, both ware induced by nitrate and repressed by ammonium. Conclusion: The cloned 5′- flanking sequences of NR gene derived from D. salina might be a specific promoter with the ability to“switch on or off” an expression of the heterologous gene in transgenic D. salina.
ABSTRACT
The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, i.e. astaxanthin up to 4%dry weight under stress conditions. Phytoene synthase is considered to be the first rate limiting enzyme in carotenoid biosynthesis pathway in H. pluvialis. The cDNA and genomic genes of phytoene synthase, i.e. psy from H.pluvialis were cloned and characterized.Result showed that psy had one open reading frame of 1 200 bp encoding a putative polypeptide of 400 amino acids which was interrupted by four introns. Phylogenetic analysis revealed that psy from green algae formed a monophyletic clade, and its closer relationship was higher plants. By using genomic walking approach, an approximate 1 kb 5′ flanking region ofpsy gene was cloned and a number of putative cis-regulatory elements were revealed. Fusing a 297 bp internal sequence (-297 to -1 bp from the translation initiation codon ofpsy) with the reporter gene, i.e. lacZ before attemptedintroducing the construct into the green alga via particle bombardment resulted in lacZ transient expression.
ABSTRACT
PURPOSE: We investigated whether TIGR/MYOC, a candidate gene for the primary open angle glaucoma(POAG) is also involved in the pathogenesis of normal tension glaucoma(NTG) and steroid-induced glau-coma(SIG). METHODS: Genomic DNA was extracted from the peripheral blood samples collected from 72 normal volunteers and 60 POAG, 47 NTG, 61 SIG patients. The genotype distribution of dinucleotide repeat polymorphism, (G-T)n microsatellite located 249 bp upstream of transcription start site was determined by direct sequencing of the Polymerase Chain Reaction(PCR) product. RESULTS: We found 6 alleles in the (G-T)n microsatellite of TIGR/MYOC ranging from 12 to 17, which differ slightly from that of previous reports. There was no obvious difference in the genotype distribution and allele frequency between the POAG group and the control group. However, a significant association of the microsatellite marker with SIG and, to a lesser extent, with NTG was observed. A significant increase in the frequency of (G-T)13/(G-T)13 genotype and a concomitant decrease in the frequency of (G-T)13/(G-T)14 genotype was seen in both the NTG and SIG group compared to that of the control group. In the SIG group, a significant decrease in the frequency of (G-T)14 allele was also observed compared to the control group, although the decrease did not contribute to the increase in the frequency of the allele. CONCLUSIONS: These findings suggest that a polymorphism in the 5 flanking region of the TIGR/MYOC is associated with patients with NTG and SIG.