人β-NGF基因在大肠杆菌中的表达

目的 为 β NGF的基因制药选择一种新方法。 方法 直接从人血细胞基因组DNA中PCR扩增出 β NGF基因片段 ,将构建的重组体 pBV2 2 0 β NGF转化大肠杆菌DH5α,并将筛选的阳性克隆通过 4 2℃诱导表达。结果 经限制酶酶切电泳和DNA测序证实 ,确为 β NGF全长序列 (约 380bp) ;全菌经SDS PAGE电泳显示表达了 β NGF蛋白。结论 成功克隆了 β NGF基因全长序列并在大肠杆菌中获稳定表达。

人神经生长因子基因在大肠杆菌中的表达

目的 为进一步研究神经生长因子 (β- NGF)及其基因的结构及功能奠定基础。方法 自行设计合成一对特异引物 ,直接从人血细胞基因组 DNA中 PCR扩增出 NGF基因片段 ,克隆至 p GEM- T Easy载体 ,p GEM- T- NGF克隆片段再定向插入原核表达载体 p GEX- 5 T,构建的重组体 p5 TNF转化大肠杆菌 JM10 9并经IPTG诱导表达。结果 经限制性酶谱和 DNA测序确证 ,p GEM- T- NGF克隆片段为 β- NGF全长编码序列(380 bp) ;SDS- PAGE、分光光度计扫描和 Western印迹显示 ,p5 TNF转化菌有 40 .5× 10 3u特异区带 ,融合蛋白表达量为 5 0 3.2 mg/ L,占菌体可溶性蛋白总量 (7.4g/ L) 6 .8% ,该产物具 NGF抗原活性。结论 本研究成功克隆 β-NGF基因全长编码序列并在大肠杆菌中获稳定表达 ,同时证实国人 β-

Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells

AIM:To detect human papillomavirus(HPV) DNA in esophageal carcinoma(EC) 109 cells and investigate the relationship between HPV and EC.METHODS:Genomic DNA and total RNA from EC109 cells were isolated.HPV DNA was detected by polymerase chain reaction(PCR) with the general primer sets of My09/11 and GP5 +/6 + for the HPV L1 gene and type-specific primer sets for HPV18 E6 and HPV18 E6-E7.Reverse transcription(RT) of mRNA isolated from EC109 cells was performed to produce a cDNA.And then a PCR-based protocol for the amplification of papillomavirus oncogene transcripts was used to analyze HPV18 DNA and integrated transcripts of HPV18 in the chromosomes of EC109 cells.The final nested PCR products were cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration.RESULTS:HPV18 DNA was detected in EC109 cells by PCR using the general primer sets of My09/11 and GP5 +/6 + for HPV L1 and the type-specif ic primer sets for HPV18 E6 and E6-E7 to generate products of 450 bp,150 bp,335 bp and 944 bp,respectively.Approximately 600 bp of integrated HPV18-specific transcript was identified.The final nested PCR product of integrated HPV18 DNA was cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration.Sequence alignment showed that the HPV18 sequence from EC109 cells was identical to that of the encoded early protein E7-E1 of the standard HPV18 strain X05015,and another partial gene sequence was identical to a partial sequence of human chromosome 8.CONCLUSION:Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis.

Studies on rice seed quality through analysis of a large- scale T-DNA insertion population

A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was developed to systemi- cally study the rice seed quality control. Genome-wide analysis of the FST distribution showed that T-DNA insertions were positively correlated with expressed genes, but negatively with transposable elements and small RNAs. In addition, the recovered T-DNAs were preferentially located at the untranslated region of the expressed genes. More than 11 000 putative homozygous lines were obtained through multi-generations of planting and resistance screening, and measurement of seed quality of around half of them, including the contents of starch, amylose, protein and fat, with a nondestructive near-infrared spectroscopy method, identified 551 mutants with unique or multiple altered param- eters of seed quality. Analysis of the corresponding FSTs showed that genes participating in diverse functions, including metabolic processes and transcriptional regulation, were involved, indicating that seed quality is regulated by a complex network.

Nuclear pseudogenes of mitochondrial DNA as a variable part of the human genome

Novel pseudogenes homologous to the mitochondrial(mt) 16S rRNA gene were detected via different approaches. Eight pseudogenes were sequenced. Copynumber polymorphism of the mtDNA pseudogenes wasobserved among randomly chosen individuals, and evenamong siblings. A mtDNA pseudogene in the Ychromosome was observed in a YAC clone carrying onlyrepetitive sequence tag site (STS). PCR screening of human yeast artificial chromosome (YAC) libraries showedthat there were at least 5.7×105 hp of the mtDNA pseudogenes in each haploid nuclear genome. Possible involvement of the mtDNA pseudogenes in the variable part ofthe human nuclear genome is discussed.

人类寿命揭秘

延年益寿,健康地生活一百岁、二百岁,甚至更久远,这是人类千百年来的梦想。与人类祖先22岁的平均寿命相比,目前70岁以上的平均寿命,不能不说是一大进步。医学家们认为,随着医学科学的发展,人类将不断认识和揭示自身生命的奥秘,从而进一步延长自身寿命。 本世纪初,运用现代科学技术,人类开始了缓慢而艰苦的探索进程。1908年,俄国医生伊尔娃·麦茨尼克夫首次揭示人体肠道中的“有害微生物”会导致生命衰退,它们寄生在人体结肠中,不断地产生一种特殊的有毒物质,这些物质随时间推移,促使人体逐渐衰老、退化、死亡。故而,他告诫人们经常饮用酸奶,以涤除肠胃中的杂质,保持人类脏器的清洁。

A hybrid neural network system for prediction and recognition of promoter regions in human genome

This paper proposes a high specificity and sensitivity algorithm called PromPredictor for recognizing promoter regions in the human genome. PromPredictor extracts compositional features and CpG islands information from genomic sequence,feeding these features as input for a hybrid neural network system (HNN) and then applies the HNN for prediction. It combines a novel promoter recognition model, coding theory, feature selection and dimensionality reduction with machine learning algorithm.Evaluation on Human chromosome 22 was ～66% in sensitivity and ～48% in specificity. Comparison with two other systems revealed that our method had superior sensitivity and specificity in predicting promoter regions. PromPredictor is written in MATLAB and requires Matlab to run. PromPredictor is freely available at http://www.whtelecom.com/Prompredictor.htm.

Nur-related receptor 1 gene polymorphisms and alcohol dependence in Mexican Americans

AIM： To investigate the association of polymorphisms of nur-related receptor 1 （Nurrl） and development of alcohol dependence in Mexican Americans. METHODS： Peripheral blood samples were collected from 374 alcoholic and 346 nonalcoholic Mexican Amer- icans; these two groups were sex- and age-matched. Sample DNA was extracted and genomic DNA was amplified by polymerase chain reaction. The -2922（C） 2-3 polymerase chain reaction products were digested with Sau96I, alleles of 1345（G/C）, and -1198（C/G） in the regulatory region as well as Ex＋132 （G/T/A/C） and Ex＋715（T/-） in exon 3 were studied by sequencing. RESULTS： The C2/C2, C2/C3, C3/C3 genotype distribu- tion of -2922（C） 2-3 was 34.4%, 38.2% and 27.5% in the nonalcoholic group compared to 23.3%, 51.2% and 25.4% in the alcoholic group （P = 0.001）. The C/C, C/G ,G/G genotype distribution of -1198（C/6） was 23.5%, 46.1% and 30.3% in the nonalcoholic group compared to 13.9%, 50.9% and 35.3% in the alcoholic group （P = 0.007）. However, the -1345 （G/C）, Ex3＋132（G/T/A/C） and Ex3＋715（T/-） alleles were not polymorphic in Mex- ican Americans, and all those studied had G/G, G/G and T/T genotype for these three alleles, respectively. The -2922（C） 2-3 did not show allele level difference be- tween alcoholic and nonalcoholic individuals, but -1198 （C/G） showed a significant allele frequency difference between alcoholic （39.3%） and nonalcoholic （46.6%） populations （P = 0.005）. Excluding obese individuals, significant differences were found at both genotypic and allelic levels for the -2922（C） 2-3 polymorphism （P = 0.000 and P = 0.049） and the -1198 （C/G） polymor- phism （P = 0.008 and P = 0.032） between nonobese alcoholics and nonobese controls. Excluding smokers, a significant difference was found only at the genotypic level for the -2922（C） 2-3 polymorphism （P = 0.037） between nonsmoking alcoholics and nonsmoking con- trols, but only at the allelic level for the -1198（C/G） polymorphism （P = 0.034）. CONCLUSION： Polymorphisms in the regulatory region of Nurrl are implicated in pathogenesis of alcohol de- pendence and the Nurrl/dopamine signaling pathway might be important for this dependence development in Mexican Americans.

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconiosis workers

To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers＇ pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods： A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results： No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% （29/31） in resistant strains, mainly concentrated in codon 531 （51.6%, 16/31） and 526 （32.26%, 10/31）. Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn＇t reported by internal and overseas scholars. Conclusion： The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.

Associations between interleukin-1 polymorphisms and gastric cancers among three ethnicities

AIM:To investigate the associations between interleukin(IL)-1B and IL-1RN polymorphisms and gastric cancers among the Tibet,Hui and Han ethnicities.METHODS:Genomic DNA was extracted from peripheral blood of 210,205,and 202 healthy volunteers and from 155,158,and 197 gastric cancer patients from the Tibet,Hui,and Han populations,respectively.Polymorphisms in IL-1B and IL-1RN were analyzed by denaturing high-performance liquid chromatography.RESULTS:Carriers of the IL-1B-31 CC genotype had an increased risk of intestinal type gastric cancer [odds ratio(OR) = 2.17,P = 0.037] in the Tibet ethnicity.Carriers of the IL-1B 2/L genotype had an increased risk of both intestinal and diffuse types of gastric cancer(OR = 2.08,2.31,P = 0.007,0.016,respectively) in the Hui ethnicity.In the Han population,carriers of the IL-1B-31 CC,IL-1B-511CT,TT genotypes had increased risk of intestinal type gastric cancer(OR = 2.51,2.74,5.66,P = 0.005,0.002,0.000,respectively).CONCLUSION:IL-1B and IL-RN genotypes may differentially contribute to gastric cancer among the Tibet,Hui,and Han ethnicities in the Qinghai area of China.

Study on polymorphism ofArtemisia capillaris by using RAPD for Terengganu population in Malaysia

Artemisia capillaris is a herbaceous aromatic and therapeutic plant. The genetic variability among individuals of Artemisia capillaris from state of Terengganu, Malaysia was examined by using the random amplified polymorphic DNA （RAPD） technique to assess the polymorphism at the species level, The samples from differences regional in Terengganu State. The genomic DNA was extracted from the samples leaves using Sarkosyl method. The results produced by the machine showed clear RAPD banding pattern. Fifty-seven oligonucleotide primers were screened and five primers were selected （OPA 04, OPA 09, OPA 16, OPA 17 and OPA 18） to amplify DNA from five samples of Artemisia capillaris from State of Terengganu, Malaysia. A total of 135 RAPD fragments （RAPDs） with all polymorphic fragments （100%） with size ranging from 250--3000 bp were scored from the population. Genetic distance for samples ranges from 0.0000 to 0.320000. For similarity index samples ranges from 0.0000 to 0.7547.

DNA sequences homologous to hepatitis C virus(HCV) in the extrachromosomal circular DNA in peripheral blood mononuclear cells of HCV-negative subjects

Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.

Nanopore DNA sequencing:Are we there yet?

Nanopores have been studied as a unique DNA sequencing technology that can quickly read long stretched DNA sequences. A DNA molecule could pass through a nanopore in a speed of microsecond per base and even faster. With this speed, a human genome can potentially be sequenced by one nanopore in 〈1 h. In contrast to next- generation DNA sequencing （NGS）, the nanopore sequencing is enzyme free without need of sample amplification due to its single-molecule nature. The nanopore sequencing has been envisioned as a new generation of DNA sequencing technology in the post-NGS era. This progress focuses on status quo of the nanopore DNA sequencing and discusses the opportunities and challenges in this rapidly growing field.

Mathematical and computational analysis of CRISPR Cas9 sgRNA off-target homologies

Revolutionary in scope and application, the CRISPR Cas9 endonuclease system can be guided by 20-nt single guide RNA （sgRNA） to any complementary loci on the double- stranded DNA. Once the target site is located, Cas9 can then cleave the DNA and introduce mutations. Despite the power of this system, sgRNA is highly susceptible to off-target homologous attachment and can consequently cause Cas9 to cleave DNA at off- target sites. In order to better understand this flaw in the system, the human genome and Streptococcus pyogenes Cas9 （SpCas9） were used in a mathematical and computational study to analyze the probabilities of potential sgRNA off-target homologies. It has been concluded that off-target sites are nearly unavoidable for large-size genomes, such as the human genome. Backed by mathematical analysis, a viable solution is the double-nicking method which has the promise for genome editing specificity. Also applied in this study was a computational algorithm for off-target homology search that was implemented in Java to confirm the mathematical analysis.