Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are — at least in species with large genomes — intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.     

Laser Microdissection (LM): Applications to plant materials

Abstract

In recent years, Laser Microdissection (LM), which has been widely used in animal biology, has been adapted to plant tissues. This short paper focuses on some technical aspects concerning plant sample preparation for LM technology, with particular attention to the application of gene expression and proteomics studies. Examples derived from the use of different LM methods are reported and their applications to plant–microbe interactions are explored.     

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.     

钉螺神经系统的显微解剖

作者于1988至1990年,对采自南京的133号日本血吸虫的中间宿主钉螺的神经系统进行了显微解剖研究。其神经系统由神经中枢和外周神经组成。前者由8对和两个不成对的神经节组成,神经节之间存在着3种联合和7种连索;后者主要由47对和18条不对称的神经组成;嗅检器位于鳃的基部。     

Optical isolation of individual mitochondria ofPhysarum polycephalum for PCR analysis

Summary We attempted to amplify a specific region of mitochondrial DNA (mtDNA) using the polymerase chain reaction (PCR) from fewer than ten mitochondria isolated individually by microdissection or use of an optical tweezer. We selected preliminarily isolated mitochondria fromPhysarum polycephalum as the model materials and tried to amplify the mtDNA region corresponding to the specific mitochondrial plasmid of this true slime mould. For separation of a few mitochondria from the mitochondrial population, we initially used a destruction method in which excluded mitochondria were disrupted by a UV laser. However, mtDNA was still amplified, although weakly, from mitochondria that had been destroyed by the UV laser. Therefore, we used an optical tweezer to trap individual mitochondria and separate them from the others. The required number of mitochondria were separated from the mitochondrial suspension through a narrow canal of isolation buffer and used directly for PCR amplification. The results showed that the mtDNA could be amplified from at least 9 mitochondria trapped by the optical tweezer.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EDTA ethylenediaminetetraacetic acid - mtDNA mitochondrial DNA - PCR polymerase chain reaction     

通过微切一扩增建立植物(蚕豆Vicia faba)染色体区段特异性基因文库研究初探

以蚕豆（Viciafaba,2n=12）根尖为材料,采用改良方法制备染色体标本,在光镜下切割分离一段大M染色体核仁组织区（NOR）特定区段（约合0．9pgDNA）,通过单一引物一聚合酶链式反应法（SingleUniquePrimer-PCR）随机扩增微切DNA后,获得近60μgDNA。经琼脂糖电泳分析测定扩增产物分子片段大小介于200-900bp。以地高新（Digoxigenin）标记蚕豆总体DNA,作为探针与扩增产物进行Southern杂交,证实扩增得到的DNA与蚕豆DNA同源,来自做切染色体。部分扩增产物经ECORI酶切后,连人经同酶切割后的pUC18载体质粒,转化大肠杆菌（EcoliJM109）。琼脂糖电泳分析得到的部分克隆,得知插人子长度介于0．25-0．9kb。本文将用于动物材料的单一引物一聚合酶链式反应法应用于植物染色体的微切微扩增,并作了一定程度简化,初步建立起一套包括微切、扩增、检测和克隆的便捷、经济的实验室制备植物染色体区域特异性基因文库的方法。     

An Exchange Assay for the Measurement of Cell Nuclear Estrogen Receptors in Microdissected Brain Regions

An exchange assay is described for the measurement of nuclear estrogen receptors (ERn) in microdissected brain regions. The distribution of ERn in the hypothalamus and amygdala of the rat 1 h after an injection of estradiol (E) is presented. Combining the exchange assay with a previously described method for measurement of cytosol estrogen receptors (ERc) in microdissected brain samples, gonadectomized male and female rats were compared for ERc and ERn. While ERc concentrations tended to be higher in females than in males in all regions of the hypothalamus, with a significant sex difference in the arcuate-median eminence, no sex difference in ERn concentrations was observed after E injection. These results suggest that ERc measurements alone are not sufficient to establish the capacity of the E receptor system: ERn measurements are also necessary to establish the relationship between receptor levels and physiologic estrogen responsiveness.     

黑麦1R染色体的微切微克隆研究

显微分离出黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体,用ＣｏｈｅｓｉｖｅａｄａｐｔｅｒｓｓｉｎｇｌｅｐｒｉｍｅｒＰＣＲ（ＣＡＳＰＰＣＲ）方法进行体外扩增,以ＤＩＧ１１ｄＵＴＰ标记扩增产物为探针,进行Ｓｏｕｔｈｅｒｎ分子杂交,结果表明扩增产物来自黑麦１Ｒ染色体。用１／１０体积的连接物转化Ｅ．ｃｏｌｉＤＨ５α,获得１００００多个重组菌落。经酶切分析,克隆子的插入片段为２５０～５００ｂｐ,为进一步筛选１Ｒ染色体的分子标记打下了基础