黑斑蛙核型、C-带及Ag-NORs 研究

本文采用外周血淋巴细胞培养法制备染色体标本,观察黑斑蛙的染色体标本,研究黑斑蛙的核型,C-带和Ag-NORs。研究结果表明：（1）黑斑蛙淋巴细胞染色体数目为2n=26,其中有5对大染色体和8对小染色体,核型是二型性核型;（2）分别对雌雄个体的中期分裂相进行观察,在第11号染色体长臂中部有明显的次缢痕,但变异核型次缢痕在第8号染色体长臂的中部;（3）在第5号染色体长臂上有一条明显的近端粒C-带;（4）第11号染色体是一对具有银染核仁形成区的同源染色体,且雌雄个体的银染位置相同。     

青蛙骨髓细胞染色体的制备及观察

动物细胞染色体的制备和观察是生物学教学中的一个基本实验内容。我们据有关资料,选用青蛙骨髓细胞作为染色体制备和观察的材料,可看到细胞分裂相清楚,染色体大而数目较少,并且有典型的中部着丝粒和亚中部着丝粒类型,其效果明显优于普遍使用的小白鼠骨髓细胞。现将我们的方法介绍如下: 1.动物选择与饲养健康成蛙,雌雄皆可,大小不作特殊要求。种类包括黑斑蛙(Rana nigromaculata)、金线蛙(R.plancyi plancyi)、虎纹蛙(R.tigrina rugulosa)以及泽蛙(R.limnocharis)、沼蛙(R.guentheri)     

小弧斑姬蛙的染色体组型及Ag-NORs研究

姬蛙属(Microhyla)为小型蛙类,我国姬蛙属现已知有6个种,本文研究了黄山小弧斑姬蛙(Microhyla heymonsi)的染色体组型和Ag-NORs,以期对姬蛙属的遗传、进化、系统分类等的研究提供有关的资料。 材料及方法 小弧斑姬蛙(10♂、13♀)捕自安徽黄山。以四肢骨骨髓细胞为材料。染色体标本的制备     

黑眶蟾蜍、黑斑蛙、中国雨蛙不同地理居群的染色体多样性

本文研究温州地区的黑眶蟾蜍、黑斑蛙、中国雨蛙的核型,分析了三个地理居群的黑眶蟾蜍、四个地理居群的黑斑蛙、三个地理居群的中国雨蛙核型.结果表明不同地理居群的同种蛙有相同的染色体数和核型模式.黑眶蟾蜍为2n=22,NF=44,核模式6+5;黑斑蛙为2n=26,NF=52,核模式5+8;中国雨蛙为2n=24,NF=48,核模式6+6.但同一种蛙的不同地理居群之间在SM数目和顺序、次缢痕或随体的位置等有所不同.说明不同地理居群的同种蛙的染色体具有丰富的多样性.     

四种无尾两栖类SCE分析

本文应用腹腔注射活性炭吸附1dUR的方法制成骨髓细胞SCE标本,测定出泽蛙、虎纹蛙、黑斑蛙和中华大蟾蛛的SCE值（雄雌）分别为7.81士0.42,8.02土0.45,4.76士0.28,4.54士0.32,8.79士0.34,8.38士0.42和7.42士0.48、7.91士0.46,表明无尾两栖类体细胞的SCE值是比较高的。本文还观察了SCE在染色体上的分布,结果表明SCE的分布与染色体的长度有一定的关系。     

黑眶蟾蜍、黑斑蛙、中国雨蛙不同地理居群的染色体多样性

本文研究温州地区的黑眶蟾蜍、黑斑蛙、中国雨蛙的核型,分析了三个地理居群的黑星期五蟾蜍、四个地理居群的黑斑蛙、三个地理居群的中国雨蛙核型。结果表明不同地理居群的同种蛙有相同的染色体数和核型模式。黑星期五蟾蜍为2n＝22,NF＝44,核模式6＋5;黑斑蛙为2n=26,NF=52,核模式5＋8;中国雨蛙为2n＝24,NF＝48,核模式6＋6。但同一种蛙的不同地理居群之间在SM数目和顺序、次缢痕或随体的位置等有所不同。说明不同地理居群的同种蛙的染色体具有丰富的多样性。     

应用BrdU-Hoechst33258-Giemsa技术对黑斑蛙性染色体的研究

黑斑蛙染色体数目为2n=26。利用血细胞培养和骨髓制片及BrdU-Hoechst 33258-Giemsa技术研究表明,黑斑蛙的性别决定为XY型。其第9染色体可能是性染色体,该染色体长臂近中部SRR区可能是与性别决定有关的区域。该区域在雌性中是同步复制的;在雄性中是非同步复制的,一个比另一个更晚复制。这一复制异态开始于中S期,终止于晚S期。     

壬基酚对雄性黑斑蛙生殖毒性的研究

通过研究壬基酚对雄性黑斑蛙(Rana nigromaculata)成体的精子和精巢的影响,探讨壬基酚对黑斑蛙的生殖毒性.用不同剂量的壬基酚对雄性黑斑蛙进行处理,对黑斑蛙的精巢系数、精子数、形态结构、畸形率和精巢显微结构等分别进行研究.结果表明,与对照组相比,随着壬基酚浓度的升高,染毒组黑斑蛙的精巢系数下降,精子数减少,精子畸形率明显增大;畸形精子主要表现为其头部出现肥大、弯曲和圆形等现象;精巢显微结构发生变化,表现为生精小管萎缩,生精细胞层次减少,间质区不明显.说明壬基酚对雄性黑斑蛙生殖系统具有毒性效应.     

福建大头蛙的核型及带型分析

利用骨髓细胞蒸气固定法制备染色体标本,研究了福建大头蛙(Limnonectesfujianensis)黄山居群的核型、C 带和Ag NORs。结果表明,福建大头蛙核型为2n =2 2 =2 0M 2SM ,NF =44,次缢痕位于No 1 0q ;各染色体均有着丝粒C 带,3p、9q出现插入型C 带;Ag NORs位于1 0q。     

福建大头蛙五个地理居群核型多样性

采用骨髓细胞蒸汽固定法制备染色体标本,研究了产于安徽、浙江、江西、福建和湖南5个地理居群福建大头蛙的核型。结果表明：5居群样本的细胞染色体数均为2n=22,NF=44,核型模式为7＋4。福建居群11对染色体全为中部着丝粒染色体;其它4个居群中的NO．3为亚中部着丝粒染色体,其余为中部着丝粒染色体;各居群中NO．3和NO．7的相对长度和臂比值存在显著或极显著差异。显示不同地理居群福建大头蛙的核型具有丰富的多样性。     

Isolation, structural characterization, and bioactivity of a novel neuromedin U analog from the defensive skin secretion of the Australasian tree frog, Litoria caerulea

We report the isolation of a novel bioactive peptide, neuromedin U-23 (NmU-23), from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. The primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy and site-directed antiserum immunoreactivity as SDEEVQVPGGVISNGYFLFRPRN-amide (M(r) 2580.6). A synthetic replicate of frog NmU-23 displaced monoradioiodinated rat NmU-23 from uterine membranes in a dose-dependent fashion indistinguishable from nonisotopically labeled rat NmU-23. In a rat uterine smooth muscle strip preparation, synthetic frog NmU-23 produced dose-dependent contractions identical to porcine NmU-25. However, in a preparation of human urinary bladder muscle strip, the synthetic frog peptide was more potent than porcine NmU-25 in eliciting contraction and produced desensitization of the preparation to the latter peptide. This report demonstrates that the defensive skin secretion of a frog contains a novel peptide exhibiting a high degree of primary structural similarity to the endogenous vertebrate peptide, NmU, and that this frog skin analog displays biological activity in mammalian tissues.     

葫芦科植物幼嫩子房壁制片技术的优化及其染色体倍性鉴定

该研究利用黄瓜、甜瓜、西瓜和西印度黄瓜这几种葫芦科植物的幼嫩子房壁作为材料进行染色体制片,探索子房材料的样品大小、预处理时间和酶解时间对染色体制片的影响及其优化,并用该制片方法对黄瓜候选单倍体植株的子房壁进行倍性鉴定和荧光原位杂交实验。结果发现:(1)黄瓜、甜瓜、西瓜和西印度黄瓜的幼嫩子房壁最佳预处理时间分别为1 h 30 min、1 h、55 min和45 min,子房长度为0.2～1 cm,子房壁材料切成边长为1～1.5 mm小块,酶解时间为1 h 10 min～1 h 20 min时,用该优化制片方法均可观察到较多的分裂相。(2)利用该方法鉴定结果显示,葫芦科植物黄瓜、甜瓜、西瓜和西印度黄瓜的染色体分别为14、24、22和24条,黄瓜候选单倍体植株的体细胞染色体数为7条。(3)将该制片方法获得的染色体装片用于荧光原位杂交结果显示,在二倍体黄瓜染色体中有3对明亮的45S rDNA杂交信号和1对5S rDNA杂交信号,而单倍体黄瓜中相应信号数量均减半;在甜瓜、西瓜和西印度黄瓜中均有2对45S rDNA杂交信号和1对5S rDNA杂交信号。研究认为,利用葫芦科植物子房壁作为制片材料,不仅可以获得良好的分裂相,还具有易于取材、制片效率高等优点,因此子房壁制片法是研究植物染色体数目和鉴定倍性的有效方法,且该制片方法也适用于进一步的荧光原位杂交分析。     

A possible bio-assay for chromosome movement in isolated mitotic apparatus

We isolated mitotic apparatus (MA) from sea urchin zygotes using several methods. The isolated MA were injected into nucleated frog eggs or into enucleated frog eggs, and 24 h later we determined whether or not the eggs had undergone normal cleavage. Normal cleavage occurred in about 50% of the cases when MA isolated in glycerol-dimethyl sulphoxide were injected into either nucleated or enucleated frog eggs. Likewise for MA isolated initially in hexylene glycol and transferred immediately into glycerol-dimethyl sulphoxide. No cleavage occurred when MA isolated in hexylene glycol (and stored in hexylene glycol) were injected into frog eggs. We discuss two possible interpretations of the results. In one interpretation cleavage of the frog eggs is a bioassay, measuring the ability of the isolated MA to support chromosome movement. In the other interpretation the isolated MA contribute only nuclear material and cleavage initiation factors, and there is no chromosome movement in the isolated MA per se.     

In situ chromosome preparation technique for simultaneous cytogenetic and immunocytochemical studies on cell cultures of solid tumors

Summary Immunophenotyping of cultured cancer cells requires intact antigenic structures; these are mostly destroyed by conventional chromosome preparation techniques. Thus, the simultaneous cytogenetic and immunocytochemical characterization of solid tumor cells appears unfeasible. Here, we describe a novel method that allows in situ chromosome preparation from monolayer cultures of solid tumor cells without affecting their immunological features. Using this technique, it is possible to achieve detailed cytogenetic data including chromosome banding together with the demonstration of cytoplasmic and nuclear antigens within the same tumor cell.     

A rapid method for preparation of sarcolemma from frog skeletal muscle

A rapid method for the preparation of sarcolema from frog skeletal muscle has been described. The purified cell segments were transparent and devoid of contractile material. The Na⁺, K⁺ -ATPase and 5'-nucleotidase activities in sarcolemma purified by this method were comparable to those reported for sarcolemmal preparations purified by density gradient centrifugation. The preparation also possessed acid phosphatase, alkaline phosphatase and K⁺ -activated, ouabain-sensitive p-nitrophenyl phosphatase activities. The cholesterol to phospholipid ratio of the sarcolemma was 0.33, indicating its high purity; further, the preparation was free from mitochondria and contractile proteins.     

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.     

The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement

At anaphase, the linkage betweeh sister chromatids is dissolved and the separated sisters move toward opposite poles of the spindle. We developed a method to purify metaphase and anaphase chromosomes from frog egg extracts and identified proteins that leave chromosomes at anaphase using a new form of expression screening. This approach identified Xkid, a Xenopus homolog of human Kid (kinesin-like DNA binding protein) as a protein that is degraded in anaphase by ubiquitin-mediated proteolysis. Immunodepleting Xkid from egg extracts prevented normal chromosome alignment on the metaphase spindle. Adding a mild excess of wild-type or nondegradable Xkid to egg extracts prevented the separated chromosomes from moving toward the poles. We propose that Xkid provides the metaphase force that pushes chromosome arms toward the equator of the spindle and that its destruction is needed for anaphase chromosome movement.     

Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.     

Increase of intracellular pH in secreting frog gastric mucosa

A method of estimation of pH in frog gastric mucosa by measuring the apparent creatine kinase equilibrium was studied. In a resting, in vitro preparation of frog stomach the intracellular pH was found to increase linearly with an increase in the serosal pH. This increase was also accompanied by an increase in the apparent equilibrium constant of the creatine kinase reaction. A similar increase was found when the resting mucosa was stimulated with histamine plus theophylline. During this procedure the total content of adenine nucleotides and creatine plus creatine phosphate remained constant.     

Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (<Emphasis Type="Italic">Pelophylax esculentus</Emphasis> complex)

Globalization and increasing human impact on natural aquatic systems have facilitated the movement of species and the establishment of nonindigenous species enhancing hybridisation opportunities between naturally allopatric species. In this review, we focus on a special case of natural hybrid speciation and the consequences of recent anthropogenic hybridisation in the water frog complex (Pelophylax esculentus complex), which consists of two parental species, Pelophylax lessonae and Pelophylax ridibundus and a hybrid taxon. The hybrid water frogs reproduce hybridogenetically and eliminate the genome of the syntopic water frog species. Although the actual cause triggering chromosome exclusion remains elusive, it has been proposed that chromosome elimination takes place prior to meiosis and may involve enzymatic degradation of the discarded genome. Translocations of water frogs in Western Europe have become frequent the last decade leading to rapid expansion of the range of the marsh frog P. ridibundus. Subsequent hybridisation of the exotic P. ridibundus may dramatically affect the viability and maintenance of hybrid water frog populations throughout Europe. Interestingly, the impact of this introduced species may differ depending on their geographic origin, which defines the ability to induce genome elimination. This may result in fertile or sterile hybrids, making global conservation guidelines challenging. We predict a severe genetic and ecological impact of nonindigenous P. ridibundus prompting for strict conservation measures to reduce species translocations and for studies on the geographic origin of exotic frog species.