三倍体草鱼染色体限制性内切酶带分析

本文报道运用Ｇｉｅｍｓａ染色、Ｃ－带显带、ＮＯＲ－显带和多种限制性内切酶消化对草鱼三倍体的染色体进行分析。其中,内切酶ＨａｅⅢ和ＨｉｎｄⅢ等使染色体产生Ｇ－带,其余内切酶产生与常规Ｃ－带不同的分化,即所谓修饰Ｃ－带。实验结果表明,限制性内切酶可使鱼类染色体产生多种带型,各种显带技术的结合使用,可探讨鱼类染色体及染色质结构与进化。     

限制性核酸内切酶Bsp 631特性的研究

11型限制性核酸内切酶的发现和应用,革 命性地推动了分子生物学和遗传工程的发展。 不管从酶本身的理论研究或从工具酶的应用研 究方面来说,都必须首先确定酶促反应系统的 最适条件以及各种因素对酶活性的影响,但是 目前关于这类酶促反应条件的确定,多数是根 据定性实验和经验,很少是根据定量实验作动 力学描述。 限制性核酸内切酶Bsp 631是我国自己分 离的菌种和发现的新酶Ci]。已测定其识别序列, 并确定它是Pst I酶的异源同功酶。根据我们 对这两个异源同功酶的比较研究,我们认为 Bsp 631比PsA有较多的优点：酶含量较高,且 比较稳定,易于纯化。因此,估计可用Bsp 631 取代PstIo但对Bsp 631酶性质的研究,国内 外尚未见报道。因此我们采用本实验室自己纯 化的Bsp 631酶,用定量测活的方法,对其最适 反应条件,二价阳离子及一价阳离子对其活性 的影响,以及酶的Km值等进行了初步的研究, 从而为今后对该酶的动力学研究结构分析及其 作为工具酶的应用提供了必要的资料。     

II型限制性核酸内切酶在真核生物染色体显带中的应用

限制性核酸内切酶（简称限制酶)能切割DNA双链。已知的限制酶有I, II和III型三类。II型限制酶能识别专一核普酸序列,并在识别序列内有固定切割点,是分子生物学研究和基因工程重要工具酶。1980年,以II型限制酶处理印度鹿染色体,沿染色体纵轴得到选择消化结果^[16]。目前,限制酶这一应用研究发展成称为限制性核酸内切酶显带（Restriction endonuclease banding）的最新显带技术,已应用于哺乳类、鸟类、两栖类、鱼类和昆虫等真核生物。限制酶显带与其他显带法相比有其特点,特别在揭示异染色质的异质性方面有其独到之处。本文介绍此项技术的发展与现状,并指出需要进一步探索的几个主要方面。     

用限制酶显带和CMA_3/DA/DAPI荧光染色研究中国小型白兔染色体结构异染色质

限制性内切酶(restriction endonuclease)是一类能识别并切开特定DNA序列的核酸内切酶,利用限制酶处理甲醇、冰醋酸固定后的哺乳动物中期染色体标本,再经Giemsa染色,能产生不同的特征性带纹,这是近几年发展的一种新的显带技术。目前,除用于人的中期染色体研究外,     

用限制性内切酶HaeⅢ进行绦虫染色体G分带的研究

限制性内切酶(简称限制酶)是一类能识别并切开特定DNA序列的核酸内切酶。近几年来,一些作者用限制酶处理人和小鼠的中期染色体,观察到特征性的带纹,并且认为限制酶的显带作用与染色体上DNA片段的选择性抽提有关。这一新技术的应用,不     

三种鱼mtDNA的限制性内切酶分析

鱼类线粒体DNA(mtDNA)的限制性酶切图谱分析,对于探讨鱼类的起源和演化等方面均有十分重要的意义。但是目前有关鱼类mtDNA酶切图谱的研究较少,这主要是受方法学的限制。为此我们改良了一种鱼类mtDNA的提取法,对鲤科的草鱼(Ctenopharyngodon idellus)、鲢鱼(Hypophthalmichthys molitrix)和鳙鱼(Aristi-chthys nobilis)的mtDNA进行了限制性内切酶酶切分析。     

芽孢杆菌中的限制性核酸内切酶

我们从66株国内收集的芽孢杆菌中筛选出10株菌株,它们含有限制性核酸内切酶活力。这些酶已经分别部分纯化并鉴定了其专一性。Bsp211Ⅰ、Bsp226Ⅰ及Bce71Ⅰ是Hae Ⅲ的异源同功酶,识别顺序是,Bsp211Ⅰ的切点如箭头所示。Bsp105Ⅰ、Bsp67Ⅰ、Bsp64Ⅰ、Bsp74Ⅰ及Bsp76Ⅰ和MboⅠ有相同的识别顺序:此顺序中腺嘌呤被甲基化后,Bsp105Ⅰ与MboⅠ一样不能切割,而Bsp67Ⅰ不论此顺序中腺嘌呤是否甲基化均能识别并切断,它们的切点如箭头所示。Bsp63Ⅰ及Bsp78Ⅰ是PstⅠ的异源同功酶,识别顺序是:Bsp63Ⅰ的切点如箭头所示。     

限制性内切酶EcoRI诱发CHO细胞染色体畸变的细胞动力学研究

一些限制性内切酶能够诱发CHO细胞染色体畸变已经不同实验所证实［11,4.5.77。有作者提出 限制性内切酶的致染色体畸变作用类似于电离辐射,即这种作用是非S期依赖性的闻。最近,又 有作者详细研究了Alul对处于细胞周期各个时相的染色体的作用〔17o Alul识别序列为4个碱基 对,产生平切末端,而对另一大类识别序列为6个碱基对,产生粘性末端的限制酶则尚无研究。为 此,我们以CHO细胞为材料,观察了EcoRI对处于细胞周期不同时相染色体的效应。现将结果报 告如下。     

芽孢杆菌中的限制性核酸内切酶

我们从66株国内收集的芽孢杆菌中筛选出10株菌株,它们含有限制性核酸内切酶活力。这些酶已经分别部分纯化并鉴定了其专一性。Bsp211Ⅰ、Bsp226Ⅰ及Bce71Ⅰ是HaeⅢ的异源同功酶,识别顺序是■,Bsp211Ⅰ的切点如箭头所示。Bsp105Ⅰ、Bsp67Ⅰ、Bsp64Ⅰ、Bsp74Ⅰ及Bsp76Ⅰ和MboⅠ有相同的识别顺序:■。此顺序中腺嘌呤被甲基化后,Bsp105Ⅰ与MboⅠ一样不能切割,而Bsp67Ⅰ不论此顺序中腺嘌呤是否甲基化均能识别并切断,它们的切点如箭头所示。Bsp63Ⅰ及Bsp78Ⅰ是PstⅠ的异源同功酶,识别顺序是:■,Bsp63Ⅰ的切点如箭头所示。     

黄鳝二价体上微量DNaseⅠ敏感性和限制性内切酶原位切割分析

采用DNase Ⅰ超敏感性分析和限制性内切酶介导的原位切口平移技术(Restriction Enzyme Nick Translation,RE-NT)对黄鳝二价体基因组结构进行了分析研究。已知DNaseⅠ超敏感性与潜在活性基因分布密切相关。结果表明,经DNaseⅠ介导的原位切口平移处理,在黄鳍二价体上可展现类D带带型,而由限制性内切酶介导的原位切口平移结果显示,AluⅠ和MspⅠ均在黄鳝二价体上诱导产生类G带带型,HpaⅡ和HaeⅢ则优先切割5号二价体上一特定区域,诱导出一段由标记信号所构成的类C带,对上述结果进行了分析和讨论。     

Distribution of restriction endonucleases among some entomopathogenic strains of Bacillus sphaericus

The restriction enzyme Bsp TI, an isoschizomer of Hae III (recognition site GGCC), has been detected in eight strains of serotype 5a5b and two serotype 3 strains of the entomopathogenic bacterium Bacillus sphaericus . Strains from other serotypes contained the enzymes Bsp TII and Bsp TIII, which digested pBR322 DNA into similar banding patterns after agarose gel electrophoresis but differed in their susceptibility to methylation of the substrate. Strains from serotypes 9, 25 and 26a26b were lacking in restriction enzyme activity. There was little correlation between phage typing and restriction enzyme activity, suggesting that restriction and modification are not responsible for phage specificity among entomopathogenic B. sphaericus strains.     

The pattern of restriction enzyme-induced banding in the chromosomes of chimpanzee,gorilla, and orangutan and its evolutionary significance

Summary The pattern of banding induced by five restriction enzymes in the chromosome complement of chimpanzee, gorilla, and orangutan is described and compared with that of humans. The G banding pattern induced by Hae III was the only feature common to the four species. Although hominid species show almost complete chromosomal homology, the restriction enzyme C banding pattern differed among the species studied. Hinf I did not induce banding in chimpanzee chromosomes, and Rsa I did not elicit banding in chimpanzee and orangutan chromosomes. Equivalent amounts of similar satellite DNA fractions located in homologous chromosomes from different species or in nonhomologous chromosomes from the same species showed different banding patterns with identical restriction enzymes. The great variability in frequency of restriction sites observed between homologous chromosome regions may have resulted from the divergence of primordial sequences changing the frequency of restriction sites for each species and for each chromosomal pair. A total of 30 patterns of banding were found informative for analysis of the hominid geneaalogical tree. Using the principle of maximum parsimony, our data support a branching order in which the chimpanzee is more closely related to the gorilla than to the human.     

Chromosome banding in Amphibia

Fixed metaphase chromosomes of several species of Amphibia were treated with various restriction endonucleases and subsequently stained with Giemsa. Metaphases of man and chicken were examined in parallel under the same experimental conditions for comparison. The restriction enzymes always induce subsets of the C-banding patterns present in the amphibian karyotypes. The heterochromatic regions can be either resistant or sensitive to the restriction enzyme. The modified C-banding patterns revealed by different restriction endonucleases in the karyotype of the same species can be either extremely dissimilar or almost completely congruent. Correspondingly, the action of the same restriction enzyme on the karyotypes of different species may vary greatly. There is only rarely a correlation between the type of C-banding patterns produced by different restriction endonucleases and their specific base pair recognition sequences. In contrast to mammalian and avian chromosomes, restriction enzymes induce no multiple G-banding patterns in amphibian chromosomes. This is attributed to the difference in organization of the DNA in the genomes of poikilothermic vertebrates. The possible mechanisms of restriction endonuclease banding and the various uses of this technique for amphibian chromosomes are discussed.     

Standard karyotype of sea trout (Salmo trutta morpha trutta) based on replication banding patterns

Replication banding patterns have been obtained from in vivo treatment of Salmo trutta morpha trutta chromosomes using a modification of the 5-BrdU technique and in kidney cultures using the fluorochrome photolysis Giemsa (FPG) staining method. Each chromosome pair was identified in the karyotype based on the banding pattern, chromosome size, and centromere position. The standard karyotype of sea trout has been proposed. Similarities of replication, and restriction enzyme banding patterns of chromosomes 8 and 9, and of chromosomes 11 and 12, are discussed. Fluorescence in situ hybridization was used to determine the location of telomeric sequences.     

Morphological and biochemical effects of endonucleases on isolated mammalian chromosomes in vitro

Endonuclease digestion of isolated and unfixed mammalian metaphase chromosomes in vitro was examined as a means to study the higher-order regional organization of chromosomes related to banding patterns and the mechanisms of endonuclease-induced banding. Isolated mouse LM cell chromosomes, digested with the restriction enzymes AluI, HaeIII, EcoRI, BstNI, AvaII, or Sau96I, demonstrated reproducible G- and/or C-banding at the cytological level depending on the enzyme and digestion conditions. At the molecular level, specific DNA alterations were induced that correlated with the banding patterns produced. The results indicate that: (1) chromatin extraction is intimately involved in the mechanism of endonuclease induced chromosome banding. (2) The extracted DNA fragments are variable in size, ranging from 200 bp to more than 4 kb in length. (3) For HaeIII, there appears to be variation in the rate of restriction site cleavage in G- and R-bands; HaeIII sites appear to be more rapidly cleaved in R-bands than in G-bands. (4) AluI and HaeIII ultimately produce banding patterns that reflect regional differences in the distribution of restriction sites along the chromosome. (5) BstNI restriction sites in the satellite DNA of constitutive heterochromatin are not cleaved intrachromosomally, probably reflecting an inaccessibility of the BstNI sites to enzyme due to the condensed nature of this chromatin or specific DNA-protein interactions. This implies that some enzymes may induce banding related to regional differences in the accessibility of restriction sites along the chromosome. (6) Several specific nonhistone protein differences were noted in the extracted and residual chromatin following an AluI digestion. Of these, some nonhistones were primarily detected in the extracted chromatin while others were apparently resistant to extraction and located principally in the residual chromatin. (7) The chromatin in constitutive heterochromatin is transiently resistant to cleavage by micrococcal nuclease.     

限制酶AluI显带和CA_3/DA/DAPI染色表达的家猪染色体结构异染色质

采用限制酶AluI显带、CA_(?)/DA/DAPI荧光染色和常规C带技术研究了家猪染色体着丝粒结构异染色质,结果表明:着丝粒结构异染色质至少可被区分为3类,并且在染色体组内各有其特异的染色体分布。将家猪染色体DA/DAPI荧光带和限制酶AluI显带与人类染色体比较,发现家猪13—18号端着丝粒染色体显带特征与人染色体1,9、16、Y一致。提示家猪13—18号端着丝粒区结构异染色质存在与人类随体DNA相似的DNA组成。     

Heteromorphic sex chromosome system with an exceptionally large Y chromosome in a catfish Steindachneridion sp. (Pimelodidae)

The chromosomes and banding patterns of Steindachneridion sp., a large catfish (Pimelodidae), endemic to the Igua?u River, Brazil, were analyzed using conventional (C-, G-banding) and restriction enzyme banding methods. The same diploid number (2n = 56) as in other members of the genus and the family was found but the karyotype displayed an XX/XY sex chromosome system. The X chromosome was the smallest submetacentric, while the Y was the largest chromosome in the karyotype. Meiotic analysis showed 27 autosomal bivalents plus one heteromorphic XY bivalent during spermatogenesis. Sex chromosomes had no particular pattern after C-banding but G- and restriction enzyme bandings showed specific banding characteristics. The present finding represents the first report of a well-differentiated and uncommon sex chromosome system in the catfish family Pimelodidae.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands, C-bands, and in situ hybridization.

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.     

AluI and HaeIII restriction enzyme banding patterns of Macaca fuscata and Cercopithecus aethiops sabaeus chromosomes

AluI and HaeIII restriction endonuclease banding patterns were analyzed in Macaca fuscata and Cercopithecus aethiops sabaeus chromosomes. AluI produced C-negative bands in both species of monkeys, while HaeIII induced the appearance of C-negative bands on Macaca chromosomes and of simultaneous G + C bands on Cercopithecus metaphases.