猪LHβ亚基基因的单核苷酸多态性研究

本研究对猪LHβ亚基基因除上游调控区以外的序列进行了SSCP分析,结果发现在该基因的3个外显子和3′调控区内没有任何SNPs位点,仅在内含子1的1367位点和内含子2的1823位点发现2个多态性位点,并对这两个位点在二花脸、约克夏和长白猪3个群体中的基因频率进行了初步分析。 Study on the Single Nucleotide Polymorphisms of the Porcine LHβ Gene WANG Ai-hua1,LI Ning2,WU Chang-xin1 1.College of Animal Science and Technology,China Agricultural University,Beijing 100094; 2.National Laboratory of AgroBiotechnology,China Agricultural University,Beijing 100094 Abstract:SSCP analysis was commanded on the whole sequence of porcine LHβ gene except the upstream control region.No polymorphic site was found in the 3 exons and 3′control region.But there were 2 polymorphic sites in the 1367 site of intron 1 and 1823 site of intron 2.The gene frequencies of the 2 sites in the Erhualian,Yorkshire and Landrace populations were analyzed. Key words：LHβ gene;SNPs;intron;SSCP     

Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein β-5

By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein p-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DMA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Char-cot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two mis-sense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR anal     

云南傣族中所见的G6PD突变型

利用错配碱基PCR/酶切法,在云南傣族中发现ntl388 G→A、ntl376 G→T和nt392 G→T G6PD基因突变型。其中主要为1388突变(18/23)。此3种突变也见于华南地区的汉族中,而有别于非中国人的突变型。提示傣族与汉族可能有同一民族渊源。利用PCR-SSCP方法在不同外显子中发现3例未知突变,待进一步DNA序列测定定型。 Abstract:By using mis-matched PCR followed by endonuclease digestion,G6PD gene mutations nt1388G→A,nt1376G→T,and nt392G→T were found among Dai national minority in Yunnan Province.Among these mutations,18 out 23 were nt1388G→A mutation.These three types of mutation were also found in Han people in the southern China,and never reported in other ethnic groups worldwide.It implied that the Han and Dai people perhaps had the same ethnic origin.Mutations of three undefined cases were identified in different exons by PCR-SSCP method.The exact mutation point will be detected by DNA sequencing under further investigation.     

有和无甘油的聚丙烯酰胺胶在检测突变时的差别

有文献报道在非变性的聚丙烯酰胺中加入甘油可提高SSCP检测的灵敏度。我们的实验结果建议研究者在进行SSCP筛查未知突变时最好采用不加甘油的非变性的聚丙烯酰胺胶,这既省力省钱,又灵敏。在判读SSCP胶时,千万不要看到在双链带位置有一条比正常迁移率慢的带就判定为插入突变。此时要判定突变的性质,最好测序。 Abstract：It was reported that glycerol in the non-denatured SSCP polyacrylamide gel could increase the sensibility of detecting mutation. We detected the mutation of PKD 1 gene in the patients with autosomal dominant polycystic kidney disease.PCR com bined with SSCP(single-strained conformation polymorphism),the non-denatured 10% polyacrylamide gel without glycerol or 10% polyacrylamide gel with 5% glycerol and DNA sequencing method were used.Our results showed that four single strand b ands were found in the non-denatured polyacrylamide gel without glycerol while t wo single strand bands were found in the polyacrylamide gel with glycerol in the same patient.Sequence showed there is a deletion of G in one DNA molecular and a G→A substitution in another DNA molecular in the patient with abnormal shift SSCP bands.Therefore, our experiment suggested that non-denat ured polyacrylamide gel was better than the polyacrylamide gel with glycerol in detection mutation,and it will save labor and money.It also suggeste d that one basedeletion can cause a slow double-strand DNA following the normal double strand band,which was caused by the heterogeneous DNA molecule formed bet ween the normal DNA strand and the one base deletion DNA strand with the protrud ing base.Our results suggest that when judging mutation in SSCP gel,it is not re liable to decide that mutation is inversion according to slow mobility in the ge l,and when the characteristic of mutation need to be judged,it must be sequenced .     

Academician of Academia Sinica:Biographical Sketch of Prof.C.L.Tsou(Chenglu Zou)

C.L.Tsou( Chenglu Zou) was born on May 1 7,1 92 3in Jiangsu,China.He received his B.Sc.in1 945from the chemistry department of The National Southwest Associated University in Kunming andwas awarded a Ph.D.( Biochemistry) from the University of Cambridge,England in 1 951 .He was aResearch Associate then Professor and Head of Enzyme Division of the Shanghai Institute of Biochemistry,Academia Sinica.He moved to Beijing in1 970 to the Institute of Biophysics and was its Deputy Direc…     

Profile of Xiaofeng Cao

正Dr.Xiaofeng Cao obtained her B.S.degree in Applied Biochemistry from Peking University in 1988.She received her M.S.degree(supervised by Mr.Longfei Yan)in Biochemistry from China Agricultural University in 1991.From1993 to 1997,she carried out her Ph.D.study(supervised by Dr.Zhangliang Chen)in the National Laboratory of Protein Engineering and Plant Genetic Engineering in the College of Life Sciences,Peking University.From 1995 to     

Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein β-5

By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein β-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.     

人癌细胞线粒体DNA控制区序列特征分析

为了探讨癌细胞mtDNA控制区序列的变化特征, 采用PCR产物限制性片段长度多态性(PCR-RFLP)分析与直接测序相结合的方法,对比分析6株人癌细胞系、 6例癌患者及4例健康成人白细胞mtDNA控制区序列。发现第16519位T→C、16 534位A→G、46位T→G和49位A→C突变, 在癌细胞系和癌患者白细胞mtDNA中分别占50%(3/6)和33.3%(2/6), 健康成人白细胞mtDNA中未见此类型突变;第16 278位C→T突变,在癌细胞系mtDNA中占50%(3/6),显著高于正常人群mtDNA中此位点的多态性变异。表明癌细胞和癌患者白细胞mtDNA重链复制起点及其 相邻D环区的特征性突变可能与细胞癌变/或癌的易感性有关。 Abstract：　To explore the sequence feature of mitochondrial DNA(mtDNA) control region in human carcinoma cells, polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and direct sequence techniques were used to analyze the sequence of mtDNA control region of 6 human carcinoma cell lines versus white blood cells which from 6 tumor patients and 4 normal adults. The T to C mutation at np 16 519, A to G mutation at np 16 534, T to G mutation at np 46, and A to C mutation at np 49 was found in 50% (3/6 cases) of carcinoma cell lines and in 33.3%(2/6 cases) of tumor patients, but it was not found in normal adults. The C to T mutation at np 16 278 was found in 50%(3/6 cases) of carcinoma cell lines, it was significantly higher than that of the polymorphism of normal population. These findings suggest that the typical mutation in the starting area of heavy-strand replication and the first half of D-loop region might probably be associated with carcinogenesis or susceptibility of carcinoma.     

III型神经中丝蛋白基因与中国高度近视人群相关性的研究

为了检测peripherin基因（PRPH）的突变与高度近视的病因有无相关关系,采用PCR-SSCP检测180例中国人高度近视先证者及60例正常人中PRPH基因所有外显子有无突变;对有突变的外显子区域进行克隆测序。结果表明,分析180例高度近视先证者PRPH基因编码区9个外显子及其邻近内含子,分别发现有下列核苷酸改变：密码子21TTC→TTT(Phe21Phe、4/180),nt2138C→G（IVS3、1/180）,密码子277GCC→ACC（Ala277Thr、8/180）,密码子237CCA→TCA（Arg237stop、1/180）,密码子292GCG→GCA(Ala292Ala,1/180),密码子361CUG→CUC（Leu361Leu,12/180）,密码子369AAA→AAG（Lys369Lys,12/180）,nt3331G→C（IVS7、3/180）,其中GCC277ACC为错义突变（Ala277Thr）;CCA237TCA为无义突变（Arg237stop）;密码子361CUG→CUC,密码子369AAA→AAG属于同义突变并且相连锁。Ala277Thr突变尚存在于正常人群中（1/60）,亦存在于患者正常亲属中;Arg237stop仅见于一个常染色体隐性遗传家系的患者中,为杂合性突变。分析180例高度近视先证者PRPH基因,未发现致病突变,可排除PRPH基因与高度近视病因的相关性。在中国人群中PRPH基因有多种变异。 Variation of the Peripherin Gene in Chinese with or Without High Myopia LI Jiang1,ZHANG Qing-jiong1,FU Rong2,XIAO Xue-shan1,LI Jia-zhang3,ZHANG Feng-sheng4, LI Shi-qiang1,LI Wei5,LI Tuo3,JIA Xiao-yun1,GUO Li1,GUO Xiang-ming 1.Zhongshan Ophthalmic Center,Sun Yat-sen University,Guangzhou 510060,China; 2.Shenzhen Municipal People's Hospital,Shenzhen 518000,China; 3.Department of Opthalmolgy,The people's Hospital of Enshi Autonomous Prefecture,Enshi 445000,Hubei,China; 4.Chaoju Eye Hospital,Baotou,Inner Mongolia 014000,China; 5.Shenzhen 2nd People's Hopital,Shenzhen 518000,China Abstract:To analyze the relationship of the peripherin gene(PRPH,OMIM17071) mutations with high myopia,genomic DNA was collected from 180 probands with high myopia (≤-6.0 dipoters) and 60 unrelated persons without high myopia.The coding sequences of PRPH gene in 240 subjects were analyzed using exon-by-exon PCR-heteroduplex-SSCP analysis and sequencing.Variations at codon21TTC→TTT(Phe21Phe、4/180),nt2138C→G（IVS3、1/180）,codon277 GCC→ACC（Ala277Thr、8/180）,codon237 CCA→TCA （Arg237stop、1/180）,codon292CCG→CCA (Ala292Ala,1/180),codon361CUG→CUC（Leu361Leu,12/180）,codon369 AAA→AAG（Lys369Lys,12/180）,nt3331G→C（IVS7、3/180）were detected in a number of probands as indicated in the blanket.Of the 8 variations one( codon 277,G→A,Ala277Thr) is a missense mutation identified in 8 of the 180patients and one of 60 controls;The mutation of codon361 and codon 369were synonymous one and linkage each other;Another one(codon237,CCA→TCA,Arg237stop) is a heterozygous nonsense mutation identified in one patient with autosomal recessive inheritance mode population but not in the 60 normal controls.The others were synonymous mutations.Eight nucleotide variations were found in the PRPH gene.We found no evidence that mutations in the PRPH gene are responsible for the high myopia in Chinese. Key words：high myopia; peripherin gene; PCR-SSCP     

人癌细胞线粒体DNA控制区序列特征分析

为了探讨癌细胞mtDNA控制区序列的变化特征, 采用PCR产物限制性片段长度多态性(PCR-RFLP)分析与直接测序相结合的方法,对比分析6株人癌细胞系、 6例癌患者及4例健康成人白细胞mtDNA控制区序列。发现第16519位T→C、16 534位A→G、46位T→G和49位A→C突变, 在癌细胞系和癌患者白细胞mtDNA中分别占50%(3/6)和33.3%(2/6), 健康成人白细胞mtDNA中未见此类型突变;第16 278位C→T突变,在癌细胞系mtDNA中占50%(3/6),显著高于正常人群mtDNA中此位点的多态性变异。表明癌细胞和癌患者白细胞mtDNA重链复制起点及其 相邻D环区的特征性突变可能与细胞癌变/或癌的易感性有关。 Abstract：　To explore the sequence feature of mitochondrial DNA(mtDNA) control region in human carcinoma cells, polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and direct sequence techniques were used to analyze the sequence of mtDNA control region of 6 human carcinoma cell lines versus white blood cells which from 6 tumor patients and 4 normal adults. The T to C mutation at np 16 519, A to G mutation at np 16 534, T to G mutation at np 46, and A to C mutation at np 49 was found in 50% (3/6 cases) of carcinoma cell lines and in 33.3%(2/6 cases) of tumor patients, but it was not found in normal adults. The C to T mutation at np 16 278 was found in 50%(3/6 cases) of carcinoma cell lines, it was significantly higher than that of the polymorphism of normal population. These findings suggest that the typical mutation in the starting area of heavy-strand replication and the first half of D-loop region might probably be associated with carcinogenesis or susceptibility of carcinoma.     

Detection of three separate DNA polymorphisms in the human lipoprotein lipase gene by gene amplification and restriction endonuclease digestion.

A rapid detection method was developed for DNA polymorphisms in the human lipoprotein lipase (LPL) gene. The examined polymorphisms include an A-C transversion in the 5'-region of intron 3, a T-G transversion that occurs within a Hind III site of intron 8, and the previously described C-T transition that causes a Pvu II polymorphism in intron 6. Gene fragments encompassing each polymorphic site were amplified by the polymerase chain reaction (PCR) and digested with an appropriate restriction enzyme whose recognition site was either naturally affected by the polymorphism or artificially created with a mismatched PCR-primer. According to the digestion profiles, genotypes were unambiguously distinguished. With this method, respective allelic frequencies were determined for 50 or 70 normal subjects. The procedure will facilitate LPL genotyping in the large population.     

中国人群脂蛋白脂肪酶基因突变与高脂血症的相关性研究

为进行脂蛋白脂肪酶基因突变与中国人群高脂血症的相关性研究,采用单链构象多态性分析结合DNA序列测定的方法,对386例(其中108例高脂血症患者,278例正常对照)中国人群进行突变筛查。结果发现1个新的沉默突变L103L,1个错义突变P207L,3个剪接突变Int3/3′-ass/C(-6)→T和普遍存在的S447X多态性,其中发生在高脂血症组的P207L杂合子为亚洲首报,并对先证者的家系进行了研究,认为P207L是家族性高脂血症的病因之一,而在正常对照组中也有发现的Int3/3′-ass/C(-6)→T,对以往研究认为其是高脂血症易患因素的观点提出了相反的报告,对于普遍认为有益的多态性位点S447X,进一步研究认为其对于正常人群,特别是健康男性的保护作用更强。结论:脂蛋白脂肪酶基因变异与高脂血症的相关性十分复杂多样,大规模的人群筛查具有重要意义。     

Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low HDL-cholesterol levels

The lipoprotein lipase (LPL) enzyme plays a major role in lipid metabolism, primarily by regulating the catabolism of triglyceride (TG)-rich lipoprotein particles. The gene for LPL is an important candidate for affecting the risk of atherlosclerosis in the general population. Previously, we have shown that the HindIII polymorphism in intron 8 of the LPL gene is associated with plasma TG and HDL-cholesterol variation in Hispanics and non-Hispanic whites (NHWs). However, this polymorphism is located in an intron and hence may be in linkage disequilibrium with a functional mutation in the coding region or intron-exon junctions of the LPL gene. The aim of this study was to initially screen the LPL coding region and the intron-exon junctions by single-strand conformation polymorphism (SSCP) analysis for mutation detection in a group of 86 individuals expressing the phenotype of high TG/low HDL, followed by association studies in a population-based sample of 1,014 Hispanics and NHWs. Four sequence variations were identified by SSCP and DNA sequencing in the coding region of the gene, including two missense mutations (D9N in exon 2 and N291S in exon 6), one samesense mutation (V108V in exon 3), and one nonsense mutation (S447X in exon 9). Multiple regression analyses, including these four mutations and the HindIII polymorphic site, indicate that the association of the HindIII site with plasma TG (P=0.001 in NHWs and P=0.002 in Hispanics) and HDL-cholesterol (P=0.007 in NHWs and P=0.127 in Hispanics) is independent of all other LPL variable sites examined. These observations reinforce the concept that the intronic 8 HindIII site is functional by itself and provide a strong rationale for future comprehensive functional studies to delineate its biological significance.     

氧化LDL的不同组分在抑制巨噬细胞释放NO中的作用

研究表明：氧化ＬＤＬ（Ｏｘ－ＬＤＬ）可抑制ＬＰＳ诱导的巨噬细胞ＮＯ释放,而正常ＬＤＬ和乙酰化ＬＤＬ则无抑制作用。用谷胱甘肽地的酶模拟物ｅｂｓｅｌｅｎ清除Ｏｘ－ＬＤＬ上的脂氢过氧化物对其抑制作用没有影响。Ｏｘ－ＬＤＬ的蛋白组分对ＮＯ释放也没有影响,而脂质组分则有抑制作用。ＬＤＬ脂质的主要成分亚油酸和卵磷脂无论单独还是共同氧化后对ＮＯ释放都没有影响,而亚油酸和胆固醇一起氧化后则对ＮＯ的释放有很强的抑制     

Molecular basis of familial chylomicronemia: mutations in the lipoprotein lipase and apolipoprotein C-II genes.

The molecular basis of familial chylomicronemia (type I hyperlipoproteinemia), a rare autosomal recessive trait, was investigated in six unrelated individuals (five of Spanish descent and one of Northern European extraction). DNA amplification by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis allowed rapid identification of the underlying mutations. Six different mutant alleles (three of which are previously undescribed) of the gene encoding lipoprotein lipase (LPL) were discovered in the five LPL-deficient patients. These included an 11 bp deletion in exon 2, and five missense mutations: Trp 86 Arg (exon 3), His 136 Arg (exon 4), Gly 188 Glu (exon 5), Ile 194 Thr (exon 5), and Ile 205 Ser (exon 5). The Trp 86 Arg mutation is the only known missense mutation in exon 3. The other missense mutations lie in the highly conserved "central homology region" in close proximity with the catalytic site of LPL. These and other previously reported missense mutations provide insight into structure/function relationships in the lipase family. The missense mutations point to the important role of particular highly conserved helices and beta-strands in proper folding of the LPL molecule, and of certain connecting loops in the catalytic process. A nonsense mutation (Arg 19 Term) in the gene encoding apolipoprotein C-II (apoC-II), the cofactor of LPL, was found to underlie chylomicronemia in the sixth patient who had normal LPL but was apoC-II-deficient.     

De novo mutation within the intron-exon junction in the PiZ allele of the alpha-1-antitrypsin gene

Summary A proband homozygous for the PiZ allele of the alpha-1-antitrypsin gene was found to be a heterozygous carrier of the additional nucleotide substitution (C-T) within the intron IV-exon V junction (position 9955 in intron IV, 3bp upstream of its 3-splice site). This mutation was not found in DNA from either the PiZ heterozygous parents or the PiZ homozygous brother of proband.     

Lipoprotein lipase genotypes for a common premature termination codon mutation detected by PCR-mediated site-directed mutagenesis and restriction digestion.

We have developed a procedure for the determination of a common mutation in exon 9 of the human lipoprotein lipase (LPL) gene. The mutation is due to a C-G transversion which creates a premature termination codon (Ser447-Ter) and results in a truncated LPL molecule lacking the C-terminal dipeptide SER-GLY. The mutation can be detected by polymerase chain reaction (PCR) amplification of exon 9 using a modified 3' amplimer that produces a 140 bp product containing a site for the restriction enzyme Hinf-1 in the presence of the mutation (G allele). The G allele was in strong linkage disequilibrium with a Hind-III restriction fragment length polymorphism (RFLP) allele in intron 8. Genotype determinations for the mutation can be performed by PCR amplification of genomic DNA, digestion with Hinf-1, and analysis of the products by polyacrylamide gel electrophoresis. The allelic frequency of the Ser447-Ter mutation in normal male Caucasian controls was 0.11. The frequency of the mutation was lower in a group of subjects with primary hypertriglyceridemia compared to normolipidemic controls.     

Partial gene duplication involving exon-Alu interchange results in lipoprotein lipase deficiency.

Major structural rearrangements are uncommon causes of mutation in human genetic diseases. We have previously described that a significant proportion of unrelated patients of western European descent who are deficient in lipoprotein lipase (LPL) activity have a major structural rearrangement in the LPL gene. Here we report the detailed characterization of this mutation. We show that this rearrangement is due to a duplication of approximately 2 kb which results from juxtaposition of intron 6 to a partially duplicated exon 6. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint in intron 6 is associated with the simple repeat found at the 3' end of an Alu element. The breakpoint within exon 6 shows no homology to this simple repeat. This result both suggests that this interchange arose as a nonhomologous recombination event and shows that such events resulting in duplication which occur in normal gene evolution may also lead to genetic disease. Cloning of the junction fragment has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with LPL deficiency.     

宫颈癌患者核糖核酸酶抑制因子基因突变的分析

目的通过对宫颈癌患者血液核糖核酸酶抑制因子(ribonuclease inhibitor,RI)基因RNH的突变分析,探讨RNH基因与肿瘤生长的关系。方法针对RNH全基因序列设计21对引物,PCR扩增后采用SSCP对突变进行检测。结果正常人和宫颈癌患者均扩增出相应的21个条带,未发现异常;经过SSCP分析未发现所收集的18例宫颈癌患者血液中的RNH基因有突变发生。结论尚不能肯定RNH基因的突变是否与肿瘤无关,也不能肯定其他肿瘤没有发生突变。需缩小检测片段进一步实验。     

乳腺癌的p53免疫组织化学和PCR-SSCP研究

为了明确p53突变与乳腺癌临床特征的关系,研究了50例浸润性乳腺导管癌中p53免疫组织化学检测与临床指标肿瘤大小、淋巴结转移情况及病人年龄的关系,并探讨p53免疫组织化学结果与PCR-SSCP检测结果的关系及意义.发现p53免疫组织化学检测阳性与肿瘤大小及淋巴结转移关系密切(P＜0.05);1例p53免疫组织化学检测阳性病例PCR-SSCP检测为杂合突变,1例p53免疫组织化学检测阴性病例为PCR-SSCP检测p53纯合缺失.我们的结果提示免疫组织化学检测阳性并不一定有p53突变,而阴性则可能有p53基因缺失,临床上结合两种检测可提供更准确的p53状况的参考资料.