Molekulare Analyse des Kolumnar-Gens beim Apfel (Malus x domestica)

Das Kolumnarwachstum beim Apfel (Malus x domestica) geht auf eine in den frühen 1960er Jahren entdeckte Zufallsmutation zurück. Die daraus resultierende Sprossmutante ist von großem wirtschaftlichem Interesse, da diese sehr kompakte Wuchsform unter anderem zu einer enormen Ertragssteigerung durch eine hohe Pflanzdichte der Bäume führt. Das Ziel der Arbeit ist die Entschlüsselung der molekularen Ursache dieser Mutation, die bisher weitgehend ungeklärt ist. Die Analyse wurde durch die Erstellung einer Referenzsequenz der Co-Zielregion einer kolumnaren Apfelsorte sowie durch die Konstruktion eng gekoppelter molekularer Marker realisiert. Durch die Konstruktion von genomischen Apfel-BAC-Bibliotheken mit mehrfacher Genomabdeckung und die Erstellung geeigneter Sonden wurde die Co-Region kloniert und deren Sequenz bestimmt. In Kombination zu dieser klassischen positionellen Klonierungsstrategie wurden genomische Illumina „mate pair“-Bibliotheken erstellt, sequenziert und bioinformatisch analysiert, um die genomische Region vollständig zu annotieren. Somit wurde eine vollständige genomische Referenz der Co-Region einer kolumnaren Apfelsorte erstellt, die die Grundlage für weitere Analysen bildet. Auf Basis dieser Referenz konnte die Co-Mutation in Form der Integration des LTR-Retrotransposons Gypsy-44 im kolumnaren Chromosom an Position 18,79 Mbp auf Chromosom 10 lokalisiert werden. Darüber hinaus konnten Transposon-basierende molekulare Marker erstellt werden, die eine verlässliche Genotypisierung von Apfelbäumen in Bezug auf das Kolumnarwachstum ermöglichen und dies unabhängig von der verwendeten Apfelsorte. Der genaue Wirkmechanismus von Gypsy-44, der zur Ausprägung dieses extremen Phänotyps führt, ist bislang unklar. Zusammenfassend lässt sich sagen, dass die molekulare Ursache für das kolumnare Wachstum aufgeklärt werden konnte und zudem die ersten molekularen Marker erstellt wurden, die eine sortenunabhängige Differenzierung zwischen kolumnaren und nicht kolumnaren Apfelbäumen ermöglichen.

The quality of life and caregiving burden among caregivers of people with dementia in Hanoi, Bac Ninh and Hai Phong, Vietnam

This three phase study aimed to describe dementia carer's quality of life (QoL) and perceived burden, and explore the associations between family carer characteristics, burden and perceived QoL in Vietnam. Dementia carers in the capital, Hanoi, in Phase 1 (N= 153) and from Hanoi, Hai Phong and Bac Ninh in Phase 2 (N=347) completed questionnaires. Survey results showed dementia carers reported low QoL, predicted by high perceived burden. Other carer characteristics including age, gender, family income and perceived experience were significantly associated with QoL. Filial piety contributed to only a single domain of QoL. Phase 3 employed qualitative methods to explore the specific issues faced by daughter carers. Findings suggested that filial gratitude and positive aspects of the role may influence the caring experience of daughter carers. Further investigation of the specific support needs of general dementia carers, and daughter carers in particular, in Vietnam is warranted.

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): Molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

The black muntjac (Muntiacus crinifrons, 2n = 8 female/9 male) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled i

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.

Defining the orientation of the tandem fusions that occurred during the evolution of Indian muntjac chromosomes by BAC mapping

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n=6 in the female and 7 in the male, the karyotypic evolution of which through extensive tandem fusions and several centric fusions has been well-documented by recent molecular cytogenetic studies. In an attempt to define the fusion orientations of conserved chromosomal segments and the molecular mechanisms underlying the tandem fusions, we have constructed a highly redundant (more than six times of whole genome coverage) bacterial artificial chromosome (BAC) library of Indian muntjac. The BAC library contains 124,800 clones with no chromosome bias and has an average insert DNA size of 120 kb. A total of 223 clones have been mapped by fluorescent in situ hybridization onto the chromosomes of both Indian muntjac and Chinese muntjac and a high-resolution comparative map has been established. Our mapping results demonstrate that all tandem fusions that occurred during the evolution of Indian muntjac karyotype from the acrocentric 2n=70 hypothetical ancestral karyotype are centromere-telomere (head-tail) fusions.

Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti)

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.

BAC及其转基因研究进展

本文综述了细菌人工染色体 (BacterialArtificialChromsomes ,BAC)的构建、物理图谱制作方法及其靶位精细操作策略的研究进展。同时 ,简要介绍了BAC转基因在基因功能以及基因表达与调控研究中的应用

Construction and characterization of a Lipotes vexillifer genomic DNA BAC library

We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexilliter genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species.

宁康霉素产生菌海洋地衣芽孢杆菌BAC-9912的诱变育种

宁康霉素(Nincomycin)产生菌BAC-9912经紫外线单因子和亚硝基胍化学试剂诱变后,获得两株高产菌株9912-7-2U(UV处理获得)和9912-2U-32N(NTG处理获得),其摇瓶效价分别为176.464×105u·L-1和217.808×105u·L-1。比出发菌株效价分别提高126.9%和180.0%。连续传代摇瓶效价稳定。初步建立了形态观察法和琼脂块大通量法两种初筛模型。

利用比较染色体涂色和BAC定位技术探讨麂属动物染色体的起源和演化

比较细胞遗传学研究发现物种的形成过程往往伴随着核型结构的变化，各种鹿属动物染色体的巨大差异正是很好的例证'赤魔的染色体被证明是由具有端着丝粒的祖先染色体经过多次的串联融合进化而来的。根据祖先染色体的形态和连接方式，可以将染色体串联融合主要分为三类：着丝粒一着丝粒融合，着丝粒一端粒融合和端粒一端粒融合。经典细胞遗传学，比较染色体涂色，卫星DNA序列和部分融合位点序列比较研究都支持串联融合假说。但串联融合的类型和分子机制迄今没有完全弄清楚。本论文旨在对魔属动物的串联融合类型和分子机制进行研究，分为两个部分：一、通过比较染色体涂色的方法，建立小鹿一林察一大额牛的比较染色体图谱，结合以前发表的结果来阐明鹿科（小魔）与牛科（大额牛），寮科（林磨）之间的核型关系；二、建立赤鹿的全基因组BAc文库，通过克隆定位的方法确立在赤魔的染色体进化过程中所发生的串联融合类型，并对其机制进行探讨。主要结果如下：利用小鹿的染色体特异探针与林寮，大额牛的染色体进行杂交，首次建立了小鹿一大额牛，小魔一林察的核型对比关系。结果显示小鹿的1-5、11号染色体均与林集和大额牛的多段染色体同源。结合高分辨的G带比较，发现小魔1-5和11号染色体上的与林爵和大额牛对应的所有同源片断在小鹿染色体上呈着丝粒一端粒连接排列。从而进一步证明小鹿的1-5和11号染色体是由2n=70的祖先核型通过染色体的着丝粒一端粒串联融合方式进化而来的。建立了我国第一个赤魔全基因组BAC文库，该文库由124800个克隆组成。大部分插入片段的大小在100-145kb之间，片段的平均大小为12＜0kb。克隆定位显示克隆的分布与小鹿染色体大小成正比关系，说明整个文库没有染色体的偏倚，文库为整个基因组的6倍，覆盖率几乎为100％。通过赤鹿BAC克隆在小魔和赤鹿染色体上的精确定位，使我们得以准确判断小鹿染色体在赤鹿染色体同源片段上的连接方式，并确定串联融合的类型。结果进一步支持赤鹿的核型是由一个2n=70祖先核型通过着丝粒一端粒的融合方式进化而来的。