利用酵母人工染色体(YAC)减数分裂同源重组构建人免疫球蛋白κ链基因组大片段

具有同源重叠区的酵母人工染色体(YAC)可以利用酵母细胞减数分裂进行同源重组,从而构建更大的人工染色体基因组,这对生命科学基础研究和生物技术应用研究有着非常重要的意义。本实验以两个含人免疫球蛋白κ链基因簇片段的YAC克隆为材料,通过酵母改型、异型接合、二倍体发孢、单孢子筛选和分子生物学鉴定等技术和方法,利用酵母菌减数分裂同源重组机制,构建了一条包含人的免疫球蛋白κ轻链32个Vκ基因、5个Jκ基因、Cκ基因、Eκ基因和κde基因的YAC重组体,长度约400kb。同时,本实验利用溶壁酶消化法获取单孢子重组体,代替了传统的显微分孢操作。使得利用酵母人工染色体减数分裂同源重组的技术更加简便可行。     

利用同源重组对酵母人工染色体左右臂进行多基因修饰

构建携带哺乳动物细胞筛选基因和酵母人工染色体(YAC)同源序列的载体,利用酵母中能够发生高频率同源重组的特点对YAC分别进行左、右臂修饰,依次将NEO、EGFP及PURO基因定点整合到YAC左右臂上。用营养缺陷筛选的方法排除酵母发生突变或随机整合等情况后,用PCR及Southern杂交方法证实各筛选基因定点整合于YAC两臂上,从而获得携带3个哺乳动物细胞筛选基因的YAC克隆。并且由此建立了通过同源重组将哺乳动物标记基因定点引入YAC左右臂的多基因修饰平台。     

兰州百合小孢子母细胞减数分裂异常现象的观察

对兰州百合(Lilium davidii var.unicolor)小孢子母细胞减数分裂异常进行了研究,发现存在不等二价体、同源染色体早分离、染色体桥、不均等分离、滞后染色体、核外染色体、微核等。分析了这些异常形成的可能机制及对正常小孢子形成的影响。人工花粉萌发实验表明：小孢子母细胞减数分裂异常是导致花粉败育的主要原因。认为兰州百合长期行无性繁殖引起染色体结构变异,导致减数分裂异常。     

扣囊复膜孢酵母BGL1基因在工业酒精酵母中的整合表达

构建了含有工业酿酒酵母自身GPD2启动子和终止子、扣囊复膜孢酵母b-葡萄糖苷酶基因(BGL1)和潮霉素选择性标记hyg的重组质粒pPIC-gpd-bgl-hyg, 通过酵母染色体同源重组, 将BGL1基因整合进入工业酒精酵母的染色体上。重组酵母可以在以纤维二糖为唯一碳源的培养基上生长, 48 h时b-葡萄糖苷酶酶活达到0.764 U/mL。在玉米浓醪酒精发酵实验中, 与宿主菌株相比, 重组酵母醪液中纤维二糖含量减少约80％, 达到了消耗醪液中纤维二糖含量的目的。     

扣囊复膜孢酵母BGLl基因在工业酒精酵母中的整合表达

构建了含有工业酿酒酵母自身GPD2启动子和终止子、扣囊复膜孢酵母β-葡萄糖苷酶基因（BGL1）和潮霉素选择性标记hyg的重组质粒pPIC-gpd-bgl-hyg,通过酵母染色体同源重组,将BGLl基因整合进入工业酒精酵母的染色体上。重组酵母可以在以纤维二糖为唯一碳源的培养基上生长,48h时β-葡萄糖苷酶酶活达到0.764U／mL。在玉米浓醪酒精发酵实验中,与宿主菌株相比,重组酵母醪液中纤维二糖含量减少约80％,达到了消耗醪液中纤维二糖含量的目的。     

扣囊复膜孢酵母BGL1基因在工业酒精酵母中的整合表达

构建了含有工业酿酒酵母自身GPD2启动子和终止子、扣囊复膜孢酵母b-葡萄糖苷酶基因(BGL1)和潮霉素选择性标记hyg的重组质粒pPIC-gpd-bgl-hyg, 通过酵母染色体同源重组, 将BGL1基因整合进入工业酒精酵母的染色体上。重组酵母可以在以纤维二糖为唯一碳源的培养基上生长, 48 h时b-葡萄糖苷酶酶活达到0.764 U/mL。在玉米浓醪酒精发酵实验中, 与宿主菌株相比, 重组酵母醪液中纤维二糖含量减少约80％, 达到了消耗醪液中纤维二糖含量的目的。     

人基因组YAC分子克隆库的构建及DMD基因YAC克隆的筛选

以pYAC4为载体,以正常人白细胞和含4条X染色体的细胞株GM1414为DNA源构建成人基因组YAC(Yeast Artificial Chromosome,酵母人工染色体)分子克隆库,已得到原始克隆近2万个,插入DNA片段长度在400—1000kb,从其中选出一组YAC克隆,它们含有DMD基因全部DNA顺序。     

嗜热四膜虫SPO11基因缺陷型细胞株的基因表达变化分析

有性生殖的关键过程是通过减数分裂产生生殖细胞,而减数分裂的一个重要环节是进行基于DNA双链断裂的同源染色体重组。在同源染色体重组过程中,SPO11蛋白催化产生DNA双链断裂,从而起始同源染色体的重组。因此,研究SPO11基因缺失在减数分裂过程中所引起的基因表达变化有助于在转录组水平上了解该基因的作用。本研究通过对嗜热四膜虫(Tetrahymena thermophila)野生型和SPO11敲除细胞株在接合生殖时期2 h、3 h、4 h、5 h四个时间点的转录组进行高通量测序。通过差异表达基因分析和功能富集分析,发现SPO11基因敲除之后嗜热四膜虫在接合生殖时期2 h时,与DNA代谢过程和DNA复制相关基因的表达发生变化,推测SPO11基因敲除导致的减数分裂过程异常可能与DNA代谢过程和DNA复制相关。     

拟南芥同源四倍体减数分裂过程的分子细胞学分析

以拟南芥(Columbia生态型)二倍体(AA,2n=10)为材料,经0.2%秋水仙素处理和细胞学鉴定,成功获得拟南芥同源四倍体(AAAA,2n=20)。以二倍体为对照,通过对拟南芥同源四倍体减数分裂过程染色体行为的观察,以及减数分裂调控同源染色体联会与重组相关基因的定量PCR分析,研究结果表明,与二倍体相比,拟南芥同源四倍体叶片表皮细胞间气孔孔径显著增大,荚果变长,但气孔密度和结实率显著降低;在减数分裂过程中出现部分单价体和三价体,以及二价体和四价体等染色体配对构型;减数分裂期重组相关基因ZYP1表达水平降低,ASY1、DMC1、MRE11和SPO11-1表达水平均升高。因此,我们推断,伴随着多倍体化,与二倍体相比,多倍体植物减数分裂期染色体行为和相关基因表达都有一定改变,影响多倍体植物生殖发育以适应环境。     

糖多孢红霉菌同源片段长度与染色体重组率关系的研究

为了探索同源片段长度与糖多孢红霉菌染色体同源重组率的关系,化学合成或用重叠PCR合成带有突变位点、在突变位点两侧长度为（26bp+27bp）、（500bp+576bp）和（1908bp+1749bp）的同源序列,克隆于糖多孢红霉菌同源重组载体pWHM3后,分别构建了pWHM1113、 pWHM1116和 pWHM1119质粒。以PEG介导转化糖多孢红霉菌A226原生质体,3个质粒分别获得每皿30个、69个和170个转化子,但pWHM1113质粒不能与染色体有效整合,pWHM1116质粒与染色体整合率为转化子的2%,而pWHM1119质粒与染色体整合率达到转化子的19%。 pWHM1116和 pWHM1119质粒均可进行有效的染色体二次重组,将突变位位点引入染色体。因此,同源片段长度为（500bp+576bp）或更长时,可与糖多孢红霉菌染色体进行有效的单重组和双重组。     

Mitotic recombination of yeast artificial chromosomes.

Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.     

Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes

Yeast artificial chromosome (YAC) cloning systems have advanced the analysis of complex genomes considerably. They permit the cloning of larger fragments than do bacterial artificial chromosome systems, and the cloned material is more easily modified. We recently developed a novel YAC cloning system called transformation-associated recombination (TAR) cloning. Using in vivo recombination in yeast, TAR cloning selectively isolates, as circular YACs, desired chromosome segments or entire genes from complex genomes. The ability to do that without constructing a representative genomic library of random clones greatly facilitates analysis of gene function and its role in disease. In this review, we summarize how recombinational cloning techniques have advanced the study of complex genome organization, gene expression, and comparative genomics.     

Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae

Here, we describe a protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC). The method is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae between genomic DNA and a linearized TAR cloning vector containing targeting sequences homologous to a region of interest. Recombination between the vector and homologous sequences in the co-transformed mammalian DNA results in the establishment of a YAC that is able to propagate, segregate and be selected for in yeast. Yield of gene-positive clones varies from 1% to 5%. The entire procedure takes 2 weeks to complete once the TAR vector is constructed and genomic DNA is prepared. The TAR cloning method has a broad application in functional and comparative genomics, long-range haplotyping and characterization of chromosomal rearrangements, including copy number variations.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Detection of homologous recombination between yeast artificial chromosomes with overlapping inserts.

We have developed a system which facilitates the detection of recombination between Yeast Artificial Chromosomes (YAC's) carrying homologous inserts. The system consists of a classical YAC vector, a new YAC vector and two appropriately labelled yeast strains of opposite mating type. The new YAC vector differs in markers from the canonical YAC vector. To test whether homologous recombination takes place, phage lambda DNA was cloned in the two vectors to provide a region of homology. The two constructs were then introduced into yeast strains of opposite mating type in which the endogenous genes for the selective markers present in the vectors are not expressed. Artificial chromosomes obtained by meiotic recombination are detected in the spores resulting from the mating.     

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.     

Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.     

Aberrant recombination events in B cell lines derived from a kappa-deficient human.

We have analyzed the structure of Ig kappa chain genes in B cell lines derived from a human individual who cannot synthesize any kappa chains, and whose Igs all contain lambda chains (1). We have characterized secondary DNA recombination events at two kappa alleles which have undergone misaligned V-J recombinations. One such secondary recombination has joined the flanking sequences of a V kappa and a J kappa 2 gene segment as if it were the reciprocal product of a V-J kappa 2 recombination, and resulted in the displacement of the recombined VJ kappa 1 gene segments from the C kappa locus. The non-rearranged form of the V kappa fragment which had recombined with the J kappa 2 flank was cloned. Nucleotide sequencing of this fragment identified a V kappa gene that differed by at least 38% from all previously sequenced human V kappa genes. The other V-J kappa segment analyzed has undergone a secondary recombination at a different site from that described above, at a site within the intervening sequence between the J kappa and C kappa gene segments, similar to the location of secondary recombinations which have occurred in lambda + B cell lines from mice and humans (2,3). These results prove that multiple recombinations can occur at one J kappa-C kappa locus.     

Construction of a yeast artificial chromosome contig encompassing the human alpha 5(IV) collagen gene (COL4A5).

A PCR-based screening approach was used to isolate six yeast artificial chromosome (YAC) clones containing segments of the human alpha 5(IV) collagen gene (COL4A5). This gene is located at Xq22 and is known to be involved in the kidney disorder known as Alport syndrome (AS). By analyzing sequence-tagged sites, cDNA content, and rare-cutting restriction site patterns in these YAC clones, a contig that spans the entirety of the alpha 5(IV) gene was constructed. This contig may contain as much as 690 kb of DNA from the alpha 5(IV) locus. On the basis of the information obtained from these YAC clones, the genomic map and gene structure of the alpha 5(IV) gene have been refined. This study has also provided a valuable resource for subsequent studies of the alpha 5(IV) gene and its flanking DNA sequences.