人类基因组3pter-p26区域的序列比较分析

对人类基因组3号染色体短臂的3pter-p26区域进行了初步分析, 序列组装后整个区域全长约为8.1 kb, 靠近端粒区还存在一个约20~30 kb的空洞. 序列比较分析表明: (ⅰ) 该区域的GC含量为38.5%, 为人类基因组所有近端粒区域最低; (ⅱ) 共含有42个基因, 平均大小为97.5 kb, 以前通过基因连锁分析定位于该区域的基因Cntn3存在定位错误, 应位于3p12.3; (ⅲ) 该区域散在重复序列活跃程度高于全基因组平均水平, 其中(TA)_n含量是全基因组平均水平的2倍; (ⅳ) 该区域在小鼠6号染色体104.1~112.4 Mb的位置有一个保守的同源序列; 通过染色体进化分析推断, 3pter-p26区域可能于最近的一次染色体重排之后才转移到染色体的末端, 其发生时间应在人与小鼠的基因组分离之后, 在这次重排过程中, 染色体断裂点附近的一些序列, 连同所包含的基因发生了丢失, 而丢失的基因总拷贝数却在基因组的其他地方得到了增加; (ⅴ) 3pter-p26区域较低的GC含量可能不利于保持染色体末端的稳定, 容易发生DNA丢失, 而这又可能是引发基因表达异常导致疾病的原因之一.     

PSIIP680 ChlaAP基因在水稻叶绿体基因组中的定位

本实验总结出一套水稻叶绿体DNA的提取方法,并获得清晰的叶绿体DNA限制性内切酶图谱。Southern杂交结果表明,菠菜PSIIP680ChlaAP基因探针与水稻叶绿体DNA的Pst-1,Pst-14,Pvu-2和Sal-1片段的部分顺序有较高的同源性。根据Hirai和赵衍的水稻叶绿体基因组物理图,可以确定该基因位于紧靠RuBPCaseLS基因,距反向重复区约26kb处。高等植物叶绿体基因组中这种基因排列方式还未见报道。     

小鼠p16~(INK4a)基因位点的结构和功能研究

p1 6INK4a基因的失活与多种肿瘤的发生和发展有联系。通过筛选小鼠基因组文库 ,获得长度为 1 4.5kb的p1 6INK4a基因组DNA片段。对上述 1 4.5kbDNA测序后进行生物信息学分析表明 :该片段包含 3个外显子 ,编码 1个由 1 68个氨基酸残基组成的多肽 ,其相对分子质量的理论计算值为 1 7941 ,有 7个可能的磷酸化位点 ,说明p1 6INK4a蛋白的功能可能受到磷酸化的调控。该DNA片段的非编码区分布着大量短散布元件、长散布元件和简单重复序列 ,这样的结构为转座和同源重组提供了结构基础 ,提示了部分肿瘤细胞中p1 6INK4a基因缺失的可能原因。对第一外显子序列与已发表的相应序列比较发现其DNA序列和所编码的多肽存在多态性     

用长PCR方法检测含有较大缺失或插入的DNA大片段

选择位于19q13.3上的人类肌张力蛋白激酶基因(myotonin protein kina se gene,MT-PK)为靶基因(基因全长为14kb),以G+C含量较高且含有1kb缺失或插入,由基因第8内含子中的Alu±1kb的5'端至第15外显子3'非编码区中的CTG重复序列3'端,即两者间的距离为5.3kb的DNA片段为待扩增靶序列,通过优化DNA聚合酶的组合和反应缓冲体系,点考查了含有Alu-1k b和Alu+1kb缺失或插入的MT-PK等位基因片段共扩增的长PCR方法。本方法可有效地同步扩增6.5kb和5.5kb两个等位基因片段,对6.5kb和5.5kb纯合等 位片段则达到了更有效的扩增。     

小鼠p16^INK4a基因位点的结构和功能研究

p16^INK4a基因的失活与多种肿瘤的发生和发展有联系。通过筛选小鼠基因组文库,获得长度为14.5kb的p16^INK4a基因组DNA片段。对上述14.5kbDNA 测序后进行生物信号学分析表明：该片段包含3个外显子,编码1个由168个氨基酸残基组成的多肽,其相对分子质量的理论计算值为17941,有7个可能的磷酸化位点,说明p16^INK4a蛋白的功能可能受到磷酸化的调控。该DNA片段的非编码区分布着大量短散布元件、长散布元件和简单重复序列,这样的结构为转座和同源重组提供了结构基因,提示了部分肿瘤细胞中p16^INK4a基因缺失的可能原因。对第一外显子离列与巳发表的相应序列比较发现其DNA序列和所编码的多肽存在多态性。     

水稻线粒体DNA酶切带型研究

水稻IR36线粒体DNA经6种限制酶酶切,用脉冲电泳和长距离琼脂糖凝胶电泳分离酶切片段,获得高分辨率的清晰带型。每组酶切片段加和测得水稻IR36线粒体基因组大小分别为227kb(HindⅢ)、253kb(EcoRⅠ)、253kb(XhoⅠ)、294kb(BamHⅠ)、239kb(SalⅠ)和283kb(xbal)采用9个来自水稻和玉米线粒体基因组的基因探针与酶切条带杂交发现,水稻线粒体基因组含有包括编码基因在内的重复顺序。     

青鳉p53基因克隆、结构分析及同源重组载体构建

应用“Long PCR”技术 ,用 6对 p5 3引物从青胚胎干细胞基因组DNA中扩增出 6个相互重叠的片段 ,其中最大的片段长达 4 5kb ,这 6个PCR片段覆盖了整个 p5 3基因。序列分析表明青p5 3基因长约 8 7kb ,由 11个外显子和 10个内含子组成。结构比较表明 ,青 p5 3基因在大小上与人和小鼠 p5 3基因存在较大差异。青p5 3基因的内含子 1仅为 0 85kb ,而人和小鼠p5 3基因的内含子 1则分别长达 10kb和 6kb ;青 p5 3基因的外显子 3(86bp)明显大于人和小鼠 p5 3基因的外显子 3(2 2bp) ;外显子 4 (170bp)比人 (2 80bp)和小鼠 (2 6 0bp)的外显子 4小 ;内含子 10 (3 5kb)则比人和小鼠内含子 10 (0 7kb和 0 9kb)大得多。用SVTK neo基因作正选择标记基因 ,用SVTK tk基因作负选择标记基因 ,用青 p5 3基因组片段作同源序列 ,构建了鱼类 p5 3基因同源重组载体。将此载体转染青胚胎干细胞 ,并经G4 18和Ganc药物选择后证明上述正、负选择标记基因在干细胞中能够有效表达 ,并提供对G4 18的抗性和对Ganc的敏感性。     

粘虫核型多角体病毒凋亡抑制基因的定位、序列和启动子结构

以AcNPV凋亡抑制基因p35为探针,与LsNPV DNA的限制性片段和LsNPV DNA EcoRV片段杂交,发现EcoRV 5.5kb片段有强烈的杂交信号.将此片段亚克隆后,测定了1244bp序列,发现一个完整的ORF,推导的302个氨基酸与AcNPV p 35蛋白有70.4%的氨基酸同源性,证明所测ORF为LsNPV的p 35基因.结构分析发现其5′端有早期基因启动子元件GC、ACGT和TATA box.有22 bp的顺向重复序列,包括由两个重叠的TATA box和上下游两个ACGT motif组成的两套启动子元件,这些结构特征与AcNPV的凋亡抑制基因十分相似.     

青Jiang p53基因克隆、结构分析及同源重组载体构建

应用“Long-PCR”技术,用6对p53引物从青Jiang胚胎干细胞基因组DNA中扩增出6个相互重叠的片段,其中最大的片段长达4.5kb,这6个PCR片段覆盖了整个p53基因。序列分析表明青Jiang p53基因长约8.7kb,由11个外显子和10个内含子组成。结构比较表明,青Jiang p53基因在大小上与人和小鼠p53基因存在较大差异。青Jiang p53基因的内含子1仅为0.85kb,而人和小鼠p53基因的内含子1则分别长达10kb和6kb;青Jiang p53基因的外显子3（86bp)明显大于人和小鼠p53基因的外显子3（22bp);外显子4（170bp)比人（280bp)和小鼠（260bp)的外显子4小;内含子10（3.5kb)则比人和小鼠内含子10（0.7kb和0.9kb)大得多。用SVTK－neo基因作正选择标记基因,用SVTK－tk基因作负选择标记基因,用青Jiang p53基因组片段作同源序列,构建了鱼类p53基因同源重组载体。将此载体转染青Jiang胚胎干细胞,并经G418和Ganc药物选择后证明上述正、负选择标记基因在干细胞中能够有效表达,并提供对G418的抗性和对Ganc的敏感性。     

在变铅青链霉菌基因组中定域克隆外源DNA的置换性载体及通过反选择获得重组体的模式方法

ΦHAU3^R是变铅青链霉菌66中对噬菌体ΦHAU3显示抗性的基因,已从基因组中获得分离。将基因组中邻近于该基因两侧的一个3.5kb和另一个3.8kb的DNA片段分别以其在染色体上的天然取向插入到一个由pIJ101衍生的质粒pIJ653上,构建成pHZ806。然后在pHZ806上对应于pIJ101复制子的区域中插入一个spc/str抗性基因,同时在3.5kb和3.8kb片段之间插入一个潮霉素抗性基因(hyg),衍生出一个新质粒pHZ808。由于pHZ808中不具有完整的pIJ101复制功能区,所以它不能在链霉菌中复制。然而,在该质粒3.5kb和3.8kb片段之间插入的任何DNA片段,在导入到变铅青链霉菌中后都可借助于3.5kb和3.8kb两个片段与内源染色体的同源区域所发生的双交换而稳定地整入到内源染色体的特定区域(3.5kb和3.8kb片段之间),同时置换出染色体上的ΦHAU3-R基因。发生了这种基因置换的重组子菌株会对噬菌体ΦHAU3变得敏感,这种反选择方法可用来浓缩和初选携带定域插入片段的重组子。已利用潮霉素抗性基因(hyg)作为一个模式基因片段阐明了这种载体和这种在染色体上定域克隆外源基因片段的方法学和适用性。同时,用pHZ808作载体克隆另外的基因片段时还有另一个优越性：hyg可作为报告基因一同参与外源基因片段的定域整合,携带插入片段的重组子除了对噬菌体ΦHAU3显示敏感性以外,还对潮霉素显示抗性。     

DNA sequence comparative analysis of the 3pter-p26 region of human genome

Most proterminal regions of human chromosomes are GC-rich and gene-rich. Chromosome 3p is an exception. Its proterminal region is GC-poor, and likely to lose heterozygosity, thus causing a number of fatal diseases. Except one gap left in the telomeric position, the proterminal region of human chromosome 3p has been completely sequenced. The detailed sequence analysis showed: (i) the GC content of this region was 38.5%, being the lowest among all the human proterminal regions; (ii) this region contained 20 known genes and 22 predicted genes, with an average gene size of 97.5 kb. The previously mapped gene Cntn3 was not found in this region, but instead located in the 74 Mb position of human chromosome 3p; (iii) the interspersed repeats of this region were more active than the average level of the whole human genome, especially (TA)n, the content of which was twice the genome average; (iv) this region had a conserved synteny extending from 104.1 Mb to 112.4 Mb on the mouse chromosome 6, which was 8% larger in size, not in accordance with the whole genome comparison, probably because the 3pter-p26 region was more likely to lose neocleitides and its mouse synteny had more active interspersed repeats.     

DNA sequence comparative analysis of the 3pter-p26 region of human genome

Most proterminal regions of human chromosomes are GC-rich and gene-rich. Chromosome 3p is an exception. Its proterminal region is GC-poor, and likely to lose heterozy-gosity, thus causing a number of fatal diseases. Except one gap left in the telomeric position, the proterminal region of human chromosome 3p has been completely sequenced. The detailed sequence analysis showed: (i) the GC content of this region was 38.5%, being the lowest among all the human proterminal regions; (ii) this region contained 20 known genes and 22 predicted genes, with an average gene size of 97.5 kb. The previously mapped gene Cntn3 was not found in this region, but instead located in the 74 Mb position of human chromosome 3p; (iii) the interspersed repeats of this region were more active than the average level of the whole human genome, especially (TA)n, the content of which was twice the genome average; (iv) this region had a conserved synteny extending from 104.1 Mb to 112.4 Mb on the mouse chromosome 6, which was 8% larger in size, not in accordance with the whole genome comparison, probably because the 3pter-p26 region was more likely to lose neocleitides and its mouse synteny had more active interspersed repeats.     

Comparative genomic sequencing reveals a strikingly similar architecture of a conserved syntenic region on human chromosome 11p15.3 (including gene ST5) and mouse chromosome 7

Comparative genomics is a superior way to identify phylogenetically conserved features like genes or regions involved in gene regulation. The comparison of extended orthologous chromosomal regions should also reveal other characteristic traits essential for chromosome or gene function. In the present study we have sequenced and compared a region of conserved synteny from human chromosome 11p15.3 and mouse chromosome 7. In human, this region is known to contain several genes involved in the development of various disorders like Beckwith-Wiedemann overgrowth syndrome and other tumor diseases. Furthermore, in the neighboring chromosome region 11p15.5 extensive imprinting of genes has been reported which might extend to region 11p15.3. The analysis of approximately 730 kb in human and 620 kb in mouse led to the identification of eleven genes. All putative genes found in the mouse DNA were also present in the same order and orientation in the human chromosome. However, in the human DNA one putative gene of unknown function could be identified which is not present in the orthologous position of the mouse chromosome. The sequence similarity between human and mouse is higher in transcribed and exon regions than in non-transcribed segments. Dot plot analysis, however, reveals a surprisingly well-conserved sequence similarity over the entire analyzed region. In particular, the positions of CpG islands, short regions of very high GC content in the 5' region of putative genes, are similar in human and mouse. With respect to base composition, two distinct segments of significantly different GC content exist as well in human as in the mouse. With a GC content of 45% the one segment would correspond to "isochore H1" and the other segment (39% GC in human, 40% GC in mouse) to "isochore L1/L2". The gene density (one gene per 66 kb) is slightly higher than the average calculated for the complete human genome (one gene per 90 kb). The comparison of the number and distribution of repetitive elements shows that the proportion of human DNA made up by interspersed repeats (43.8%) is significantly higher than in the corresponding mouse DNA (30.1%). This partly explains why the human DNA is longer between the landmark genes used to define the orthologous positions in human and mouse.     

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.     

A systematic method to identify genomic islands and its applications in analyzing the genomes of Corynebacterium glutamicum and Vibrio vulnificus CMCP6 chromosome I

MOTIVATION: Some genomic islands contain horizontally transferred genes, which play critical roles in altering the genotypes and phenotypes of organisms, and horizontal gene transfer has been recognized as a universal event throughout bacterial evolution. A windowless method to display the distribution of genomic GC content, the cumulative GC profile, is proposed to identify genomic islands in genomes whose complete genome sequences are available. Two new indices are proposed to assess the codon usage bias and amino acid usage bias in genomic islands. RESULTS: A 211 kb genomic island (CGGI-1) has been identified in the genome of Corynebacterium glutamicum, and three genomic islands VVGI-1, VVGI-2 and VVGI-3, with lengths 167, 40 and 33 kb, respectively, have been identified in the genome of Vibrio vulnificus CMCP6 chromosome I. The CGGI-1 is flanked by two approximately 500 bp direct repeats, and utilizes a Val-tRNA as the integration site. For the VVGI-1 and VVGI-2, each has an integrase gene at 5' junction. All the identified genomic islands show unusual GC content, codon usage and amino acid usage, compared with the rest of the genomes. In addition, it is found that genomic islands are fairly homogenous in terms of GC content variation. An index, h, to quantify the homogeneity of GC content for genomic islands is proposed, and it is shown that h is less than 0.1 for all the genomic islands analyzed. The cumulative GC profile, as well as various indices to assess the codon usage bias, amino acid usage bias and homogeneity of the genomic islands, will be useful in the analysis of other genomes. AVAILABILITY: Programs used in this work and numerical results are available upon request.     

The distribution of genes in the Drosophila genome

In the present work we show that in the Drosophila genome (which covers a 37-51% GC range at a DNA size of approx.50kb) a linear correlation holds between GC (or GC(3)50kb) genomic sequences embedding them. This correlation allows us to position the two compositional distributions of (a) coding sequences, and (b) of long DNA segments relative to each other and to calculate gene concentration across the compositional range of the Drosophila genome. Using this approach, we show that gene concentration increases with increasing GC of the regions embedding the genes, reaching a 7-fold higher level in the GC-richest regions compared with the GC-poorest regions. The gene distribution of the Drosophila genome is, therefore, similar to (although less striking than) that of the human genome, whereas it is very different from those of the Arabidopsis genome, which has about the same size as the Drosophila genome.     

Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene

p36 is a major substrate of both viral and growth factor receptor associated protein kinases. This protein has recently been named calpactin I heavy chain since it is the large subunit of a Ca2(+)-dependent phospholipid and actin binding heterotetramer. The primary structure of p36 has been determined from analysis of cloned cDNA. The protein contains 338 amino acids, has an approximate molecular weight of 39,000, and is comprised of several distinct domains, including four 75 amino acid repeats. From two overlapping cosmid clones isolated from different mouse genomic liver libraries, the complete intron/exon structure of the p36 gene was determined and the 5' and 3' noncoding regions of the gene were analyzed. The coding and 3' untranslated region of the p36 gene contains 12 exons which range in size from 48 to 322 base pairs (bp) with an average size of 107 bp. The repeat structures found at the protein level are not delineated by single exons, but the N-terminal p11-binding domain is encoded by a single exon. Structural mapping of the gene demonstrated that the lengths of the first two introns in the coding region are together approximately 6 kilobases (kb), while the other introns range in size from 600 to 3600 bp with an average size of 1650 bp. The p36 gene is at least 22 kb in length and has a coding sequence of approximately 1 kb, representing only 4.5% of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.