人类性染色体特异ＤＮＡ对三种鱼类染色体的描绘

染色体描绘是研究基因组进化的强有力手段之一,用人Ｘ和Ｙ染色体文库特异ＤＮＡ为探针,对３种硬骨鱼类－刺鳅、黄鳝和斑马鱼的有丝分裂中期裂染色体进行了描绘研究,结果表明,在这３种鱼类的染色体组中都发现有人Ｘ染色体特DN的同源片段,它们散布在几对同源染色体中,但用人Ｙ染色体ＤＮ描绘这３种鱼类染色体时,则没有检测出可见的同源片段。同时对从低等脊椎动物到人类的Ｘ染色体进化过程进行了进一步探讨。     

鱼类基因组结构研究——Ⅰ.染色体的限制性内切酶显带

本文报道了1种新的鱼类染色体研究方法——限制性内切酶显带技术。我们应用限制性内切酶Bsp631等分别对黄鳝和长春鳊的染色体进行显带处理,结果表明,Bsp631能使这两种鱼的染色体发生稳定的带纹分化:适度的处理产生多重的G-带状带型,而过度的处理则产生特殊的限制性内切酶抗性带型。根据显带结果分析,我们对鱼类染色体限制性内切酶显带的可行性和实用价值进行了讨论。     

东北梅花鹿染色体限制性内切酶显带

采用限制性内切酶Hae Ⅲ、Hind Ⅲ分别处理梅花鹿中期染色体标本,发现HaeⅢ处理标本21h左右,显出类似G带、C带带型,第1、4、11、18、27、28、31对染色体的结构异染色质区对HaeⅢ敏感而浅染,而HindⅢ处理标本18h左右能诱导梅花鹿中期染色体显出类似G带、C带,第2、6、15、18、29、32对染色体的结构异染色质区对HindⅢ较敏感而浅染,性染色体Y对HaeⅢ敏感呈浅染,性染色体X对HindⅢ敏感呈浅染。表明东北梅花鹿12对常染色体和两条性染色体结构异染色质在这两种酶处理时表现出异质性。     

弯曲菌染色体DNA限制性内切酶带型研究

从鸡、羊、鹅、鸭粪便中分离出的空肠弯曲菌,提取染色体DNA做酶切分析,得到几乎相同的带型;与从比利时引进的结肠弯曲菌及海鸥弯曲菌比较,带型完全不同;同时,从人胃粘膜中分离出的幽门螺旋菌,做染色体DNA酶切分析,得到与前者完全不同的带型。     

斑马鱼二价体制备与多重带显带的方法学探讨

采用碱性低渗结合高氯仿一步显带技术获得了斑马鱼二价体高分辨多重G带,所出现的带纹丰富,反差明显。采用地高辛为标记的以限制性内切酶AfuⅠ介导的原位切口平移技术使斑马鱼二价体上呈现出类G带的多重带,获得了明暗反差强烈的带纹结果。通过比较多个不同分裂相的多条染色体,发现带纹的分布具明显特征性．并且特征一致,带纹数目基本吻合。首次从方法学上对斑马鱼二价体的制备过程及多重带显带技术进行了分析、探讨和总结,力求将该技术程序化、系统化,使其具有可重复性和可操作性,并对显带的可能机理进行了讨论。     

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。     

Effect of Coriolus versicolor supplemented diet on innate immune response and disease resistance in kelp grouper Epinephelus bruneus against Listonella anguillarum

The effect of Coriolus versicolor extract supplemented diets on innate immune response and disease resistance in kelp grouper, Epinephelus bruneus against Listonella anguillarum, is reported. Kelp grouper were divided into four groups of 25 each and fed with C. versicolor enriched diets at 0% (control), 0.01%, 0.1%, and 1.0% level. After 30 days of feeding, all fish were injected interaperitoneally (i.p.) with 50 μl of L. anguillarum (4.7 × 10⁷ CFU) to investigate the immune parameters at weeks 1, 2, and 4. The reactive oxygen species and reactive nitrogen species production were significantly enhanced in fish fed with 0.1% and 1.0% supplementation diets from weeks 1-4 when compared to the non enriched diet fed and infected control. The phagocytic activity significantly increased with 0.1% and 1.0% diets on weeks 2 and 4. The leucocyte myeloperoxidase content, lysozyme activity, and total protein level significantly increased when fed with 0.1% and 1.0% supplementation diets from weeks 1-4. The cumulative mortality was 35% and 45% in 1.0% and 0.1% enriched diet fed groups whereas it was 55% and 80% in 0.01% and 0% groups respectively. The present results suggest that diets enriched with C. versicolor at 0.1% or 1.0% level positively enhance the innate immune system and affords protection from L. anguillarum.     

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.     

Effects of gamma-irradiation on the infectivity and chromosome aberration of Clonorchis sinensis

Effects of gamma irradiation on the worm survival and chromosomal aberration of Clonorchis sinensis were studied. The metacercariae irradiated with various amounts of gamma radiation (ranging from 5 Gy to 50 Gy) were fed to rats, and the effects were compared with those of non-irradiated controls. Recovery rates of adult worms in irradiated groups were reduced gradually as increasing of the irradiation doses. No worm was recovered from rats which were fed with 50 Gy irradiated metacercariae. The chromosome number was 2n = 56 in all worms from all experimental groups. However, the groups irradiated with 20 Gy, 25 Gy or 30 Gy showed variations in the chromosome number, depending on different cells in the same individual. Radiation doses used in this study did not appear to induce chromosome aberrations, however, irradiation with 30 Gy showed slightly reduced chromosome size.     

Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae

An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5 mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10 mM Tris–HCl pH 7.0, 10 mM MgCl₂, 1 mM dithiothreitol at 37 °C. XorKII was easily inactivated by heating at 65 °C for 5 min, but retained most of the original activity after incubation at 37 °C for 24 h.     

An improved method for chromosome banding after fluorescence in situ hybridization: Use of a tetramethylrodhamine-conjugated BrdU antibody

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.Abbreviations BAT BrdU antibody technique - DABCO 1,4 diazobicyclo-(2.2.2)octane - FITC fluorescein isothiocyanate - FPG fluorochrome plus giemsa - PHA phytohemagglutinin - RBA R-banding BrdU acridine - TRITC tetramethylrhodamine isothiocyanate     

Optimization of melting analysis with TaqMan probes for detection of KRAS,NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔT_m (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.     

Sequencing and analysis of a 63 kb bacterial artificial chromosome insert from the Wolbachia endosymbiont of the human filarial parasite Brugia malayi.

Wolbachia endosymbiotic bacteria are widespread in filarial nematodes and are directly involved in the immune response of the host. In addition, antibiotics which disrupt Wolbachia interfere with filarial nematode development thus, Wolbachia provide an excellent target for control of filariasis. A 63.1 kb bacterial artificial chromosome insert, from the Wolbachia endosymbiont of the human filarial parasite Brugia malayi, has been sequenced using the New England Biolabs Inc. Genome Priming System() transposition kit in conjunction with primer walking methods. The bacterial artificial chromosome insert contains approximately 57 potential ORFs which have been compared by individual protein BLAST analysis with the 35 published complete microbial genomes in the Comprehensive Microbial Resource database at The Institute for Genomic Research and in the NCBI GenBank database, as well as to data from 22 incomplete genomes from the DOE Joint Genome Institute. Twenty five of the putative ORFs have significant similarity to genes from the alpha-proteobacteria Rickettsia prowazekii, the most closely related completed genome, as well as to the newly sequenced alpha-proteobacteria endosymbiont Sinorhizobium meliloti. The bacterial artificial chromosome insert sequence however has little conserved synteny with the R. prowazekii and S. meliloti genomes. Significant sequence similarity was also found in comparisons with the currently available sequence data from the Wolbachia endosymbiont of Drosophila melanogaster. Analysis of this bacterial artificial chromosome insert provides useful gene density and comparative genomic data that will contribute to whole genome sequencing of Wolbachia from the B. malayi host. This will also lead to a better understanding of the interactions between the endosymbiont and its host and will offer novel approaches and drug targets for elimination of filarial disease.     

Reduced Rates of Sequence Evolution of Y-Linked Satellite DNA in Rumex (Polygonaceae)

One characteristic of sex chromosomes is the accumulation of a set of different types of repetitive DNA sequences in the Y chromosomes. However, little is known about how this occurs or about how the absence of recombination affects the subsequent evolutionary fate of the repetitive sequences in the Y chromosome. Here we compare the evolutionary pathways leading to the appearance of three different families of satellite-DNA sequences within the genomes of Rumex acetosa and R. papillaris, two dioecious plant species with a complex XX/XY₁Y₂ sex-chromosome system. We have found that two of these families, one autosomic (the RAE730 family) and one Y-linked (the RAYSI family), arose independently from the ancestral duplication of the same 120-bp repeat unit. Conversely, a comparative analysis of the three satellite-DNA families reveals no evolutionary relationships between these two and the third, RAE180, also located in the Y chromosomes. However, we have demonstrated that, regardless of the mechanisms that gave rise to these families, satellite-DNA sequences have different evolutionary fates according to their location in different types of chromosomes. Specifically, those in the Y chromosomes have evolved at half the rate of those in the autosomes, our results supporting the hypothesis that satellite DNAs in nonrecombining Y chromosomes undergo lower rates of sequence evolution and homogenization than do satellite DNAs in autosomes.[Reviewing Editor: DR. Jerzy Jurka]