YR-黏和染色体模型初探(上)

30nm螺线管是如何形成300nm染色线的?高度重复序列占人类第22号染色体DNA量的41.9％［1］,它们的功能是什么?全身着丝粒或弥散型着丝粒染色体是怎样形成的? 姐妹染色体由前期到中期为什么不分开?同源染色体联会是怎样形成的?联会复合体的中央区是什么?为什么通过花粉管会导入外源遗传物质?高等生命是怎样从原始生命进化而来的?在此,我们给出一个新的五级染色体模型：YR－黏和染色体模型。它不仅能解释以上问题,同时能解释多线染色体、灯刷染色体 以及一些经典遗传现象。 Abstract:How is the 300nm(nanometer)chromonema compacted with 30nm solenoid?What are the functions of repeat family sequence?How does the holocentromere or polycentromere chromosome form?What is the reason that non?separation of sister chromatid occur from prophase to metaphase?How do the synaptonemal complex(SC)occur?What are the compositions in the central region of SC?How to explain the transmission of heterologous?DNA by pollen tube pathway?How did the higher organism evolve from primitive forms?Hereon we provide a new five degree chromosome model-YR-cohesion chromosome model which will give a better answer to the questions above,as well as polytene chromosome,puff,and lampbrush chromosome and many other genetic phenomena.     

Sequence Signatures of Nucleosome Positioning in Caenorhabditis elegans

Our recent investigation in the protist Trichomonas vaginalis suggested a DNA sequence periodicity with a unit length of 120.9 nt, which represents a sequence signature for nucleosome positioning. We now extended our observation in higher eukaryotes and identified a similar periodicity of 175 nt in length in Caenorhabditis elegans. In the process of defining the sequence compositional characteristics, we found that the 10.5-nt periodicity, the sequence signature of DNA double helix, may not be sufficient for cross-nucleosome positioning but provides essential guiding rails to facilitate positioning. We further dissected nucleosome-protected sequences and identified a strong positive purine (AG) gradient from the 5′-end to the 3′-end, and also learnt that the nucleosome-enriched regions are GC-rich as compared to the nucleosome-free sequences as purine content is positively correlated with GC content. Sequence characterization allowed us to develop a hidden Markov model (HMM) algorithm for decoding nucleosome positioning computationally, and based on a set of training data from the fifth chromosome of C. elegans, our algorithm predicted 60%-70% of the well-positioned nucleosomes, which is 15%-20% higher than random positioning. We concluded that nucleosomes are not randomly positioned on DNA sequences and yet bind to different genome regions with variable stability, well-positioned nucleosomes leave sequence signatures on DNA, and statistical positioning of nucleosomes across genome can be decoded computationally based on these sequence signatures.     

SMC蛋白的结构和功能

近年来新发现的一类蛋白――染色体结构维持蛋白(SMC蛋白,structural maintenance of chromosome proteins)与染色体结构细胞周期性的动态变化紧密相关,它们参与有丝分裂染色体的集缩和分离、性染色体的剂量补偿效应、姐妹染色单体的内聚作用(cohesion)、遗传重组和DNA修复等过程。本文从生化特性和生物学功能两方面叙述了对SMC蛋白的研究。 Abstract：The newly discovered proteins, SMC (structural maintenance of chromosome) proteins, are associated with chromosome dynamics change in the cell cycle. They are involved in chromosome condensation, sister-chromatid cohesion, sex-chromosome dosage compensation, genetic recombination and DNA repair,etc. The current understanding of the biochemical properties and biological functions of SMC proteins is summarized in this paper.     

Further analyses of variation of ribosome DNA copy number and polymorphism in ciliates provide insights relevant to studies of both molecular ecology and phylogeny

Sequence-based approaches, such as analyses of ribosome DNA(rDNA) clone libraries and high-throughput amplicon sequencing, have been used extensively to infer evolutionary relationships and elucidate the biodiversity in microbial communities.However, recent studies demonstrate both r DNA copy number variation and intra-individual(intra-genomic) sequence variation in many organisms, which challenges the application of the rDNA-based surveys. In ciliates, an ecologically important clade of microbial eukaryotes, rDNA copy number and sequence variation are rarely studied. In the present study, we estimate the intraindividual small subunit rDNA(SSU r DNA) copy number and sequence variation in a wide range of taxa covering nine classes and 18 orders of the phylum Ciliophora. Our studies reveal that:(i) intra-individual sequence variation of SSU rDNA is ubiquitous in all groups of ciliates detected and the polymorphic level varies among taxa;(ii) there is a most common version of SSU rDNA sequence in each cell that is highly predominant and may represent the germline micronuclear template;(iii)compared with the most common version, other variant sequences differ in only 1–3 nucleotides, likely generated during macronuclear(somatic) amplification;(iv) the intra-cell sequence variation is unlikely to impact phylogenetic analyses;(v) the rDNA copy number in ciliates is highly variable, ranging from 103 to 106, with the highest record in Stentor roeselii. Overall,these analyses indicate the need for careful consideration of SSU r DNAvariation in analyses of the role of ciliates in ecosystems.     

黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

cGAS guards against chromosome endto-end fusions during mitosis and facilitates replicative senescence

As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.     

Cosntruction of the physical map of yeast（Saccharomyces cerevisiae）chromosome V~1

There are about 17 chromosomes in yeast Saccharomycescerevisiae.A middle sized chromosome,chromosome V,waschosen in this work for studying and constructing the physi-cal maps.Chromosome V from strain A364a was isolatedby pulsed-field gradient gel electrophoresis(PFGE).Gelslices containing chromosome V DNA were digestedwith two rare cutting enzymes,NotⅠand SfiⅠ,and three6-Nt recognizing enzymes,SmaⅠ,SstⅡ and ApaⅠ.Several strategies-partial or complete digestions,digestion with different sets of two enzymes,and hybrid-ization with cloned genetically mapped probes(CAN1,URA3,CEN5,PRO3,CHO1,SUP19,RAD51,RAD3)——were used to align the restriction fragments.There are 9,9,15,17,and 20 sites for NotⅠ,SfiⅠ,SmaⅠ,SstⅡ and ApaⅠrespectively in the map of the A364a chromosome V.Itstotal length was calculated to be 620 Kb(Kilo-bases).Thedistributions of the cutting sites for these five enzymesthrough the whole chromosome are not uniform.A comp-arison between the physical map and the genetic map wasalso made.     

染色体微切割、微分离、微克隆技术及其研究进展

自1981年染色体微切割及微克隆技术创建以来,该技术已广泛应用于人类及动植物遗传学、医学、进化学等研究领域,主要包括构建特定染色体或染色体区域的DNA文库、制备染色体描绘探针池以研究染色体重排和染色体进化等。本文对该技术的产生、发展及某些研究进展作一综述。 Abstract: Since chromosome microdissection and microcloning technique was developed in 1981,it has been wide ly used in genetics,medicine,evolution and other fields,mainly in establishing chromosome or chromosone specific region DNA libraries,preparing chromosome painting probe pools to study chromosome rearrangement and evolution.In the paper,the development and research progress of the technique are discussed.     

大麦基因组中的微卫星标记及其应用

微卫星是以少数几个核苷酸为单位多次串联重复的DNA序列,是一种简单序列重复(simple sequence repeats,SSR),两侧一般是保守序列。由于它具有多态性高、共显性、容易用PCR检测和结果稳定可靠等特点,因此是一种十分理想的分子标记。大麦的微卫星DNA随机分布于基因组中,平均每一个微卫星基因座有3～18个等位基因,最高可达37个。SSR标记已广泛用于分子遗传图谱的构建、遗传多样性研究、种质鉴定、主要性状基因的定位及分子标记辅助选择育种等。大多数SSR标记集中在着丝粒附近区域,1HL、5HL和6HS明显缺乏SSR标记。大麦的SSR标记还有待进一步的开发。 Microsatellite Markers and Applications in the Barley Genome FENG Zong-yun1,2,3,ZHANG Yi-zheng1,LING Hong-qing3 1.College of Life Sciences,Sichuan University,Chengdu 610065,China; 2.College of Agronomy,Sichuan Agricultural University,Ya'an 625014,China; 3.The State Key Laboratory of Plant Cell & Chromosome Engineering,Institute of Genetics & Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China Abstract:Microsatellites,also called simple sequence repeats (SSR),are simple,tandemly repeated DNA sequences with a repeat length of a few base pairs,and are very ideally used as molecular markers because of their abundance,high level of polymorphism,co-dominance and ease of assay with the polymerase chain reaction (PCR) by selecting primers as the conserved DNA sequences flanking the SSRs,as well as better stability.The experiments showed that SSRs are randomly distributed throughout the barley genome,and there are 3~18 alleles at a single SSR locus,up to 37 alleles/locus.SSR markers have being widely applied in the construction of molecular genetic map,the study of genetic diversity,the identification of germplasm,gene mapping for important traits and molecular marker-assisted selection.Meanwhile,most of markers are strongly clustered around the centromeric regions of all seven linkage groups.As a result of the clustering,genome coverage with SSRs remains incomplete with an obvious lack of markers on the long arms of chromosomes 1H and 5H and short arm of chromosome 6H.Therefore,it is very potential and necessary to further develop SSR markers in barley. Key words：barley;microsatellite marker;simple sequence repeats;genetic diversity;molecular mapping     

Computational analysis and prediction for exons of PAC579 genomic sequence

To isolate the novel genes related to human hepatocellular carcinoma (HCC), we se-quenced P1-derived artificial chromosome PAC579 (D17S926 locus) mapped in the minimum LOH (loss of heterozygosity) deletion region of chromosome 17p13.3 in HCC. Four novel genes mapped in this genomic sequence area were isolated and cloned by wet-lab experiments, and the exons of these genes were located. 0-60 kb of this genomic sequence including the genes of interest was scanned with five different computational exon prediction programs as well as four splice site recognition programs. After analyzing and comparing the computationally predicted results with the wet-lab experiment results, some potential exons were predicted in the genomic sequence by using these programs.     

Characters of DNA constitution in the rye B chromosome

We have used chromosome microdissection and microcloning to construct a DNA library of the entire B chromosome （B） of rye. New rye B-specific sequences have been screened from this pool, blasted with other sequences and analyzed to elucidate the characters of DNA constitution and the possible pathway of the origin of the rye B chromosome. We report the discovery of a new sequence that is specific to the rye B centromere.     

T-DNA Integration Category and Mechanism in Rice Genome

T-DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T-DNA integration mechanism. The structures of T-DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T-DNA right border occurred mainly on five nucleotides “TGACA” in inverse repeat (IR) sequence of 25 bp, especially on the third base “A”. However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T-DNA right border, which made the T-DNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T-DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T-DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T-DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T-DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position of T-DNA. Some small regions in the fight border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the T-DNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T-DNA integration. The fragments flanking the T-DNA fight border in transformed rice plants could derive from different parts of the inner T-DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T-DNA, in which the homologous area was comparatively easier to be disrupted. The structure of flanking sequences of T-DNA integrated in the rice chromosome presented various complexities. These complexities were probably a result of different patterns of recombination in the integrating process. Some types of possible integrating mechanism are detailed.     

Identification and Validation of a Major Quantitative Trait Locus for Slow-rusting Resistance to Stripe Rust in Wheat

Stripe (yellow) rust,caused by Puccinia striiformis Westend.f.sp.tritici Eriks (Pst),is one of the most important wheat (Triticum aestivum L.) diseases and causes significant yield losses.A recombinant inbred (RI) population derived from a cross between Yanzhan 1 and Xichang 76-9 cultivars was evaluated for resistance to wheat stripe rust strain CYR32 at both the seedling and adult plant stages.Four resistance quantitative trait loci (QTLs) were detected in this population,in which the major one,designated as Yrq1,was mapped on chromosome 2DS.The strategy of using the Brachypodium distachyon genome,wheat expressed sequence tags and a draft DNA sequences (scaffolds) of the D-genome (Aegilops tauschii Coss.) for the development of simple sequence repeat (SSR) markers was successfully used to identify 147 SSRs in hexaploid wheat.Of the 19 polymorphic SSRs in the RI population,17 SSRs were mapped in the homeologous group 2 chromosomes near Yrq1 region and eight SSRs were genetically mapped in the 2.7 cM region of Yrq1,providing abundant DNA markers for fine-mapping of Yrq1 and marker-assisted selection in wheat breeding program.The effectiveness of Yrq1 was validated in an independent population,indicating that this resistance QTL can be successfully transferred into a susceptible cultivar for improvement of stripe rust resistance.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

黄鳝激素敏感性脂肪酶基因Hsl染色体原位杂交定位

动物脂肪组织中的甘油三酯在数量上是最重要的储存能源。Hsl基因所编码的激素敏感性脂肪酶是一种多功能酶。它通过催化水解储存在脂肪组织中的甘油三酯,以及卵巢、肾上腺、睾丸和胎盘中的胆固醇酯,在机体能量供应和类固醇生成作用中发挥重要作用。本研究以放射性同位素和地高辛标记重组质粒pBS中所含猪Hsl基因作为探针,分别与Pst Ⅰ酶切的黄鳝基因组总DNA和有丝分裂染色体标本进行Southern杂交和荧光原位杂交(FISH)。结果显示,Southern杂交呈现一条带,片段长度约为11.5kb。同时,应用FISH定位Hsl基因于黄鳝5号染色体,相对着丝粒距离为78.35±1.26。该定位结果与应用“特定染色体DNA池”定位黄鳝Hsl基因结果相符,且定位结果更为精细。表明在淡水鱼类黄鳝基因组中存在Hsl基因,另一方面也首次提供黄鳝5号染色体上FISH杂交信息,从而为增加黄鳝染色体组中已知的遗传标记和建立高精度遗传图谱奠定基础。 Abstract:Adipose tissue triacylglycerols are the quantitatively most important source of stored energy in animals.Hormone-sensitive lipase encoded by hormone-sensitive lipase gene (Hsl) is a multifunctional enzyme that catalyzes the hydrolysis of triacylglycerol stored in adipose tissue and cholesterol esters in the adrenals,ovaries,testes and macrophages.Using pig Hsl gene inserted into pBS labeled by the radioactive isotope and the digoxigenin as the probes respectively,one band,11.5kb,has been shown to hybridized with total DNA of rice field eel digested with Pst Ⅰby Southern blotting and Hsl gene has been assigned to metaphase chromosome 5,at the position of 78.35±1.26 from the c entromere in rice field eel by fluorescent in situ hybridization (FISH).The mapping results are corresponding to that of “specific-chromosomal DNA pool”obtained by chromosome microisolation used to map gene and the mapping result is more accurate.The results of the study further illustrate the importance of the presence of Hsl gene in rice field eel genome and provide the first FISH mapping data for rice field eel chromosome 5.The current studies would advance the addition of known genetic markers and the construction of high resolution genetic map in rice field eel genome.     

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii

Dinolflagellate is one of the primitive eukaryotes,whose nucleus may represent one of the transition stages from prokaryotic nucleoid to typical eukaryotic nucleus,Using selective extraction together with embeddment-free section and whole mount electron microscopy,a delicate nuclear matrix filament network was shown,for the first time,in dinoflagellate Crypthecodinium cohnii nucleus,Chromosome residues are connected with nuclear matrix filaments to form a complete network spreading over the nucleus,Moreover,we demonstrated that the dinoflagellate chromosome retains a protein scafflod after the depletion of DNA and soluble proteins.This scaffold preserves the characterstic morphology of the chromosome.Two dimensional electrophoreses indicated that the nuclear matrix and chromosome scaffold are mainly composed of acidic proteins.Our results demonstrated that a framework similar th the nuclear matrix and chromosome scaffold in mammalian cells appears in this primitive eukaryote,suggesting that these structures may have been originated from the early stages of eukaryote evolution.     

鹰科四种鸟类线粒体DNA的差异和分子进化关系的研究

采用聚合酶链反应,从雀鹰、大蔫、秃鹰、灰脸焉鹰4种鸟类中分别扩增出线粒体DNA细胞色素b基因,并测定出1086bp的碱基序列,它们之间的序列差异在10.31%～16.57%之间.DNA一级序列数据显示,这4种鸟类DNA序列变异丰富,MEGA1.01数据软件构建了4种鸟类的分子系统树,与化石资料和形态学研究结果相吻合。 Abstract:Using well-known PCR techniques,we amplified and sequenced a 1086 basepair fragment of the Cytochrome-b DNA sequence from 4 species of raptors in China.Primers based on the published gallus domesticus sequence were used to generate initial sequences that were subsequently used to design raptor sepecific primers for internal sequencing.Relationship were estimated using Parsmony analysis and boot strapping.Levels of mitochondrial DNA divergence ranged from 10.31%～16.57% among species of raptors and 17.59%～18.60% betweem raptors and the outgroups.Our data showed that the variation rate of mitochondrial DNA Cytochrome b is in line with the fossil record of geological age.     

一个水稻重复序列的分析与定位

在利用PCR简并引物扩增水稻NBS-LRR类抗病基因同源序列的研究中,克隆了一个大小为560 bp左右的重复序列,命名为DH17。序列分析和同源性比较发现,该序列包含352 bp的重复单位,与已报道的OS48和TrsA等重复单位序列进行比较,差异多低于5%, 具有很高的同源性,因此为同一重复序列家族。分子杂交表明,该序列在籼型品种"窄叶青8号"（ZYQ8）中以大量的串联拷贝存在,拷贝数显著高于粳型品种"京系17"（JX17）。利用ZYQ8和JX17组配的DH群体,通过 RFLP分析,直接将DH17的大量串联拷贝定位于ZYQ8的12号染色体长臂末端区域。 Abstract:A repeated sequence with a length of 560 bp,termed as DH17,was obtained during PCR amplification of rice NBS-LRR homologues.A repeated unit of 352 bp in the DH17 fragment was revealed through sequence analysis and comparison,which has a high homology with the known sequences of OS48 and TrsA,and belongs to the same repeat family.Southern hybridization displayed that there are higher DH17 copies in the genome of an indica variety,ZYQ8,than that in the genome of japonica variety,JX17.The tandom repeated DH17 sequence was mapped on the long arm end of chromosome 12 through RFLP analysis of a double haploid population derived from ZYQ8 and JX17 using DH17 as a probe.