航天诱变凤仙花院3突变株后代小孢子变化的研究

凤仙花种子经神舟4号飞船搭载后发现的院，突变株，从SP1代到SP3代小孢子的染色体数目和小孢子的大小进行了追踪研究，发现该突变株SP1代小孢子中的染色体数目变化大，从仅含1条染色体，到最多含28条染色体，小孢子之间的大小差异也大；SP2代小孢子中的染色体数目变化范围缩小，仅在4—9条之间；小孢子大小之间的差距也变小．在秋播条件下SP3代的变化趋势与SP2代基本一致，并对院，突变株后代小孢子内染色体数目及小孢子大小的变化趋势，原因进行了讨论．     

棉花自然单倍体植株小孢子发育的细胞学研究

本文对陆地棉洞庭一号自然雌雄不育株小孢子败育进行了细胞学观察,发现减数分裂中期Ⅰ染色体多分散,为单价体;后期Ⅰ时染色体分配不均,常有落后染色体出现;末期Ⅰ形成大小不等,数目各异的核及微核。减数分裂Ⅱ时,姊妹染色体分开,产生多个核,形成多分孢子。故由一个花粉母细胞产生的小孢子的数目不等(2—14个),大小各异。花粉粒的数目多,大小差异悬殊,有的无棘状突起。经过对减数分裂中染色体行为、花粉粒的大小、气孔保卫细胞中叶绿粒的数目和体细胞染色体数目的测定和分析,认为该株棉花为自然单倍体植株。     

重组真菌细胞色素P450nor2在酵母细胞中的表达

酿酒酵母（Saccharomyces cerevisiae）是一种理想的真核蛋白表达系统．将真菌细胞色素P450nor2基因亚克隆到酵母表达载体pAUR123中,构建重组表达质粒pAUR-P450nor2并转化酿酒酵母AH22,经Aureobasidin A筛选和菌落PCR鉴定得到阳性克隆．SDS-PAGE分析证实：重组的真菌细胞色素P450nor2在酵母细胞中实现了高表达．     

卫星搭载凤仙花种子诱发的变异及品种选育

研究了“神舟”4号搭载的凤仙花种子的细胞和形态学变异,在SP1代发现小孢子母细胞减数分裂时出现染色体断片、染色体桥和落后染色体。在院3突变株小孢子中的染色体数目呈现大量的非减半变化,其染色体数目为1～28条不等。小孢子的形状和大小变化明显,且小孢子的大小与所含染色体的数目呈正相关。花粉的大小和育性与减数分裂的异常有某种联系。在四分孢子期的小孢子数目有多分孢子,即小孢子数除4个外,尚含有5～10个不等,个别的有三分孢子。在SP2代发现子叶形态和数量的变化;真叶除披针形外,还有短圆形、长柳叶形;另外还发现顶花叶化和顶端叶呈蔷薇花状等。通过多代的选择,已选育出“西航”1号、“西航”2号凤仙花新品种。     

伪东方罂粟愈伤组织细胞核型研究

本文研究了伪东方罂粟（Ｐａｒａｖｅｒｐｓｅｕｄｏｏｒｉｅｎｔａｌｅ）愈伤组织细胞核型的变化特点，结果证明培养初期的愈伤组织细胞经常发生染色体的大量丢失．继代培养两个月的愈伤组织细胞染色体数目变化较大，培养３个月、４个月、５个月以后染色体数目减少到２１条时趋于稳定，经对各个时期细胞的２１条染色体测量分析，证明这些细胞不是真正的单倍性细胞．随着培养时间的延长，染色体的结构变化也越大．在每一个具有２１条染色体的细胞中发现都有一条较长的具中部着丝粒的染色体．     

rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用

根据酵母整合质粒的设计要求,PCR扩增特定的2．2kb rDNA片段,并以此替换酿酒酵母(Saccharomyces cereristae)整合载体YIp5的URA3片段;在此基础上,引入G418抗性基因KanMX和酵母磷酸甘油激酶(phosphoglycerale kinase,PGK)组成型强启动子和终止子序列(PGKp-t),构建适合酿酒酵母工业菌株高拷贝整合表达载体pYMIKP,以细菌木糖异构酶(xylose isomerase,XI)基因xy/A为目标基因,通过载体pYMIKP引入到酵母工业菌株NAN-27中,酵母转化子在非选择培养条件下,连续生长50世代质粒稳定性为99．72％,目标基因高拷贝重组菌的木糖异构酶比酶活是对照菌株的67．2倍,达到0．672U／mg蛋白,实现了外源基因在酿酒酵母工业菌株中的稳定高效表达。     

甲基亚硝基尿诱发鼠T细胞淋巴瘤的染色体变化

给RF/J品系小鼠注射甲基亚硝基尿(MNU,N-Nitroso-N-Methylutea)每周30mg/kg,共5周。全部小鼠都发生T细胞淋巴瘤。为了搞清楚我们在早期肿瘤发生与发展的研究中所观察到的染色体非随机变化的意义,我们仔细观察了其原发性淋巴瘤及其在裸鼠和同基因小鼠中连续传代培养细胞的染色体。结果表明,早期的绝大多数变异是染色体数目差异,仅少数为结构变化。虽然在全部小鼠中受累的染色体不完全相同,但是常常局限在某些染色体上,主要是X, l, 3, 4, 6, 10, 12和15染色体,其出现受累的频率在受检细胞中达5—47％。绝大多数发生变化的染色体在后来的传代培养中持续出现,但有少数丢失,亦有频率增加和出现新的变化等现象。这与其他鼠类白血病和淋巴细胞增生性疾病所观察到的结果是一致的。MNU有效地产生这些变化的能力而使其出现高度的组织特异性和有效的致癌作用,可能是其对T细胞染色体转化的优先选择作用所致。     

卫星搭载凤仙花诱发的院3突变株后代小孢子的染色体数目变化研究

凤仙花种子经"神舟"4号卫星搭载后,在SP1代中发现一个减数分裂异常和小孢子内染色体数目非减半突变株,本文对其后代的花色变化和染色体数目变化进行了研究。发现粉红花和红花突变系小孢子染色体在7条左右,而蓝紫色突变系则平均在10以上,二者有较大差异。在花粉粒大小方面则有相似的结果。     

红豆杉离体培养染色体数变异与细胞多核现象

分别对在固体培养基上静止培养和在摇瓶、１０Ｌ及１００Ｌ生物反应器内悬浮培养的红豆杉茎尖和根尖离体细胞进行染色体数目观察,发现其染色体数目主要类型和亲本株相同,为２ｎ＝２４,且存在较大比例的染色体数目变异的细胞．还发现,离体培养红豆杉细胞有丝分裂间期存在多核现象．认为染色体数目的变异可能是红豆杉离体培养细胞紫杉醇产量不稳定的主要原因．     

棕色田鼠减数分裂及精细胞染色体数目的研究

以棕色田鼠精母细胞为研究对象，观察减数分裂各时期染色体特征性变化，特别对精细胞染色体构成及数目进行了研究．结果表明，ｘ型和ｙ型精细胞均显示了染色体数目的多态性（２ｎ＝２４，２５，２６），可以认为这是造成棕色田鼠体细胞染色体数目多态性的原因之一．     

Mitochondrial DNA repairs double-strand breaks in yeast chromosomes

The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.     

Molecular cloning of human telomeres in yeast

Telomeres are the DNA sequences found at the ends of linear chromosomes. They define the boundaries of the genetical and physical maps of such chromosomes and so are particularly important for the complete mapping of large genomes that is now being attempted. Telomeres have been intensively studied in the yeast Saccharomyces cerevisiae and in ciliated protozoa: in these organisms the telomeric DNA consists of arrays of tandemly repeated short sequences in which one strand is guanosine-rich and oriented 5' to 3' towards the chromosome end. The conservation of these structural features is reflected in the observation that telomeric DNA from a variety of protozoa will function as telomeres on artificial linear mini-chromosomes in yeast. Tandem arrays of the sequence TTAGGG have been identified at the telomeres of humans and other mammals and also of trypanosomes. This indicates that the structural features of telomeres are conserved between higher and lower eukaryotes and implies that human telomeric DNA could function in yeast. I have used this idea to develop a strategy to isolate a specific human telomere as a molecular clone in yeast and have devised a simple and effective way of cloning other human telomeres and their associated sequences.     

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.

Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase.     

Chromosomal evolution in Saccharomyces

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 10(8) years ago. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces 'sensu stricto' complex. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.     

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.     

Transposable mating type genes in Saccharomyces cerevisiae.

A functional copy of the alpha mating type gene of Saccharomyces cerevisiae has been cloned by transformation in yeast. Using the Southern Blotting procedure it has been shown that three distinct genetic loci implicated in mating type interconversion (HML, HMR and MAT) contain sequences homologous to the clone fragment. The restriction fragment associated with each locus exhibits a characteristic size which can be correlated with the mating type allele present at that locus. The characteristic size difference between the a and alpha genetic elements made it possible to demonstrate that the homothallic interconversion of mating types in this yeast occurs by DNA rearrangement as proposed in the 'cassette hypothesis'.     

Size variation in chromosomes from independent cultured isolates of Plasmodium falciparum

The complexity of the life cycle of the protozoan malaria parasite Plasmodium falciparum has hindered genetic analysis; even the number of chromosomes in P. falciparum is uncertain. The blood stages of rodent malaria parasites are haploid and hybridization with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results). A novel approach to karyoptic and linkage analysis in P. falciparum has been provided recently by the technique of pulsed-field gradient (PFG) gel electrophoresis, which allows the fractionation of DNA molecules of 30-3,000 kilobases (kb), a range including the sizes of intact chromosomal DNA molecules from eukaryotes such as yeast and trypanosomatids. We describe here the fractionation by PFG electrophoresis of chromosomal DNA molecules from P. falciparum into at least seven discrete species which vary in size by up to 20% between different isolates. Several genes for P. faciparum antigens which contain repetitive sequences are located on different chromosomes. Surprisingly, two of the chromosomes seem to contain the same sequences.     

DNA sequences of telomeres maintained in yeast

Telomeres, the ends of eukaryotic chromosomes, have long been recognized as specialized structures. Their stability compared with broken ends of chromosomes suggested that they have properties which protect them from fusion, degradation or recombination. Furthermore, a linear DNA molecule such as that of a eukaryotic chromosome must have a structure at its ends which allows its complete replication, as no known DNA polymerase can initiate synthesis without a primer. At the ends of the relatively short, multi-copy linear DNA molecules found naturally in the nuclei of several lower eukaryotes, there are simple tandemly repeated sequences with, in the cases analysed, a specific array of single-strand breaks, on both DNA strands, in the distal portion of the block of repeats. In general, however, direct analysis of chromosomal termini presents problems because of their very low abundance in nuclei. To circumvent this problem, we have previously cloned a chromosomal telomere of the yeast Saccharomyces cerevisiae on a linear DNA vector molecule. Here we show that yeast chromosomal telomeres terminate in a DNA sequence consisting of tandem irregular repeats of the general form C1-3A. The same repeat units are added to the ends of Tetrahymena telomeres, in an apparently non-template-directed manner, during their replication on linear plasmids in yeast. Such DNA addition may have a fundamental role in telomere replication.     

Microtubule-motor activity of a yeast centromere-binding protein complex.

During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.     

Genome linking with yeast artificial chromosomes

The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.