Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

新疆亚鳞木（比较种）角质层特征

报道了产自江苏宜兴晚泥盆世五通组新疆亚鳞木（比较种）Ｓｕｂｌｅｐｉｄｏｄｅｎｄｒｏｎ ｃｆ． ｘｉｎｊｉａｎｇｅｎｓｅＳｕｎ）的表皮角质层用荧光分析显示的特征．该种茎干表皮角质层覆于叶座及叶座间隔带,其中间隔带角质层厚于叶座．表皮细胞在间隔带与叶座表现特征不同．间隔带中部,表皮细胞呈纵长的多边形,其纵长方向与茎干延伸方向相同,细胞壁略有弯曲．间隔带靠近叶座之表皮细胞,细胞壁直,形状类似于前者;但大小仅为前者的１／２左右,且其纵长方向逐渐向叶座边缘偏转．叶座表皮细胞呈近等多边形,有胞间隙．该种茎表皮无气孔     

CCD imaging of the electrical activity in the leech nervous system

A single ganglion of the nervous system of the leechHirudo medicinalis was isolated. One or both roots emerging from each side of the ganglion were sucked into suction pipettes used either for extracellular stimulation or for recording the gross electrical activity. The ganglion was stained with the fluorescence voltage sensitive dye Di-4-Anepps. The fluorescence was measured with a nitrogen cooled CCD camera. Our recording system allowed us to measure in real time slow optical signals corresponding to changes in light intensity of at least 5. These signals were caused by the direct polarization of neuronal structures, the afterhyperpolarization or the afterdischarge induced by a prolonged stimulation. When images were acquired at fixed times, several of them could be averaged and optical signals of at least 2 could be reliably measured. These optical signals originated from well identified neurons, such as T, P and N sensory neurons. By taking images at different times and at different focal planes, electrical events could be followed at a temporal resolution of 50 Hz. The three dimensional dynamics of electrical events, initiated by a specific stimulation, was imaged and the spread of excitation among leech neurons was followed. When two roots were selectively stimulated, their neuronal interactions could be imaged and the linear and non-linear terms of the interaction could be characterized.     

多胚水稻品系APⅣ的多卵遗传行为分析

通过多胚水稻品系APⅣ与单胚水稻品种IR36、明恢77和龙特浦B正反杂交, 研究APⅣ的多卵遗传行为,表明APⅣ的多卵性状可能不是由孢子体基因型决定的,而是由雄配子体基因型决定,属配子体遗传的范畴。 Abstract:The inheritance of poly-eggs was investigated by crossing and reciprocally crossing APIV with monoembryonic rice variety IR36,Minghui No.77 and Longtepu B,respectively.It was suggested that the production of poly-eggs is probably controlled by gametophytic genotypes,rather than sporophytic ones.     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)     

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.     

贝氏隐孢子虫在珍珠鸡体内发育的扫描电镜观察

采用扫描电镜观察了贝氏隐孢子虫在珍珠鸡体内的发育。大量球形虫体镶嵌于气管和法氏囊微绒毛丛中。气管纤毛消失,微绒毛生发生融合。法氏囊粘膜表面可观察到宿主细胞突起,在突起的表面有数个虫体寄生。滋养体呈球状,平均大小为１．７μｍ。裂殖体拥有４个或８个香蕉状裂殖子。成熟大配子体大小为 ４.２ × ３．３μｍ,在其侧面可观察到锯齿状突起。偶尔能观察到卵囊,其表 面有一明显裂缝。虫体逸出后所留下的带虫空泡似弹坑状,根据其结构可将其分为两类,其中一类为裂殖子或小配子的形成场所,另一类为卵囊的形成场所。     

使用MUG培养基定量检测植物药中大肠杆菌时荧光猝灭现象的发生与克服方法

实验中观察到,用ＭＵＧ培养基对植物药中的大肠杆菌定量时多发生荧光猝灭现象,影响检测结果。本文对此现象产生的原因与克服方法进行了系统的考察,发现以一种简便的转接方法可排除植物药介质对菌检的干扰。该方法由两组检验系列构成,当怀疑正常稀释系列（第一系列）４０ｈ培养液的荧光结果可能因猝灭现象呈假阴性时,立即分别将该系列的１—３号管培养液以０．５ｍｌ的接种量转接入新鲜的ＭＵＧ培养基（第二系列）,重新培养２４ｈ,荧光猝灭现象即可克服。综合两系列的荧光、产气和吲哚三项生化特征得出检品中大肠杆菌含量。实际应用表明,此法能显著提高使用该培养基时菌检结果的可靠性。     

Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley-Liss, Inc.     

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S 1 during the transition. Correspondence to: Y. K. Levine