基因组原位杂交的新进展及其在植物中的应用

基因组原位杂交 ( Genomic in situ hybridization GISH)是 2 0世纪 80年代末发展起来的一种原位杂交技术。它最初应用于动物方面的研究[1 ] ,但很快被植物方面所借用 ,并且使用频率高于动物方面的研究。它采用来自一个物种的总基因组 DNA作为标记探针 ,用另一物种的总基因组 DNA以适当的浓度进行封阻 ,在靶染色体上进行原位杂交。在封阻DNA和标记 DNA探针之间 ,封阻 DNA优先与一般序列杂交 ,剩下的特异性序列主要被标记探针所杂交。在此基础上 ,人们先后发展了荧光基因组原位杂交、多色基因组原位杂交和比较基因组原位杂交等技术 ,…     

荧光原位杂交技术在植物学中的应用

荧光原位杂匀技术（fluorescence in situ hybridization,FISH）是80年代末才发展起来的一种非放射性原位杂交技术。作为一种新型的细胞分子遗传学技术,目前已广泛应用于细胞遗传学、分子生物学等领域。本文简要综述了该技术的基本原理与特点及其在植物学中的应用,包括在异源染色质的鉴定、染色体物理图谱的构建和染色体RNA及植物基因组进货中的应用。     

荧光原位杂交技术在植物学中的应用

荧光原位杂交技术（fluorescence 〖WTBX〗in situ 〖WTBZ〗hybridization, FISH）是80年代末才发展起来的一种非放射性原位杂交技术。作为一种新型的细胞分子遗传学技术,目前已广泛应用于细胞遗传学、分子生物学等领域。本文简要综述了该技术的基本原理与特点及其在植物学中的应用,包括在异源染色质的鉴定、染色体物理图谱的构建和染色体RNA及植物基因组进化中的应用。     

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

A rapid and sensitive determination of bacterial rRNA by means of hybridization-competition

A rapid and sensitive liquid hybridization-competition method for the assay of in vitro synthesized Escherichia coli rRNA was developed. Hybridization with homologous DNA and with heterologous DNA from Proteus vulgaris was compared. Besides unfractionated DNA we used DNA enriched for the sites complementary to ribosomal RNA by chromatography on methylated albumin Kieselguhr. Hybridization with heterologous enriched DNA is the method of choice due to its low background and high hybridization efficiency.     

寡核苷酸探针制备的优化

目的:探讨寡核苷酸微阵列制备中适合使用的探针浓度、探针缓冲液pH值、离子强度、优化杂交条件。方法:选取人白细胞抗原DQA1位点,针对多态性集中的外显子2设计一对保守引物及16条特异性分型探针;分别用ddH2O和0.1、0.2mol/L碳酸盐缓冲液(pH=7)稀释探针至100μmol/L;选取合适的缓冲液浓度后,调碳酸盐缓冲液为5.0、6.0、7.0、8.0、9.0、10.0等6种pH值,选取最优pH值及离子强度,分别溶解探针至20、50、100、200μmol/L,比较上述不同条件的杂交结果。结果:用0.1mol/L、pH9的碳酸盐缓冲液溶解探针杂交效果最佳;探针浓度为20μmol/L时信号弱,其他浓度下无显著差别。结论:通过探针制备的优化可以提高杂交效率,探针浓度与杂交信号强度无明显正相关。     

Reiteration Frequency of Haemoglobin Genes in the Duck

Hybridization between duck DNA and duck haemoglobin mRNA shows that there is little or no reiteration of the haemoglobin genes     

Hybridization of rabbit cardiac tropomyosin with nonpolymerizable tropomyosin species

Rabbit cardiac tropomyosin was hybridized with its nonpolymerizable form, produced by treatment with carboxypeptidase A, and with a naturally occurring nonpolymerizable tropomyosin from horse platelets. Hybridization was achieved by heating equimolar mixtures to 60 degrees C in the presence of 10 mM dithiothreitol, followed by recooling. Samples of intact and carboxypeptidase-truncated tropomyosins treated in this way show lower viscosities at low ionic strength than predicted assuming random reformation of the coiled coils, suggesting that hybrids formed with one intact COOH-terminus are unable to polymerize normally. Hybridization of cardiac and platelet tropomyosins was detected by observation of the fluorescence of pyrene groups attached to cysteine residues on platelet tropomyosin.     

Hybridization and speciation

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near‐instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky–Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock‐on effects on speciation both within and outside regions of hybridization.     

Influence of the quantity of nonspecific DNA and repeated freezing and thawing of samples on the quantification of DNA by the Light Cycler

Quantification of DNA in real-time using the Light Cycler is increasingly being used for the detection and follow-up of various infectious and other diseases. We evaluated the effect of two parameters, namely the presence of nonspecific DNA and prior repeated freezing and thawing on the accurate quantification of DNA extracts from the RH strain of Toxoplasma gondii by the SYBR Green I and the Hybridization Probe techniques. For both parameters, a high copy number sample containing 5x10(5) parasites/extract and a low copy number sample containing 100 parasites/extract were tested. Reliable quantification was possible in the presence of up to 200 ng of nonspecific DNA by the SYBR Green I technique and up to 1000 ng by the Hybridization Probe technique as compared to the company threshold of 50 and 500 ng, respectively. As tissue samples usually contain more than 200 ng of nonspecific DNA, the ideal choice is the Hybridization Probe technique. The stability of DNA extracts after repeated freeze-thaw cycles was found to be dependent on the volume in which they were stored. Samples stored in 100-microl total volumes were not stable after 3 freeze-thaw cycles, whereas those stored in 1-ml total volumes were stable after 14 freeze-thaw cycles.     

Detection of Epstein-Barr Viral Genome in Nonproductive Cells

Hybridization experiments have shown that EB viral DNA comprises between 0.06 and 1.6% of the total DNA of Burkitt lymphoma cells and established leucocyte cell lines.     

Limited hybridization between introduced and Critically Endangered indigenous tilapia fishes in northern Tanzania

Hydrobiologia - Hybridization between introduced and indigenous species can lead to loss of unique genetic resources and precipitate extinction. In Tanzania, the Nile tilapia (Oreochromis...     

Outbreeding Depression in Hybrids Between Spatially Separated Pink Salmon, Oncorhynchus gorbuscha, Populations: Marine Survival, Homing ability, and Variability in Family Size

Hybridization between distinct populations and introgression of nonnative genes can erode fitness of native populations through outbreeding depression, either by producing a phenotype intermediate to that of both contributing genomes (and maladapted in either population's environment) or by disrupting distinct coadapted complexes of epistatic genes. In salmon, fitness-related traits such as homing ability or family-size distribution may be eroded. We investigated geographically separated pink salmon populations in repeated trials in independent broodyears (odd and even). Hybrids were made between female Auke Creek (Southeast Alaska) pink salmon and Pillar Creek (Kodiak Island, ～1 000 km away) males; hybrids and their offspring were compared to offspring of control crosses of the same females with Auke Creek males. Parentage assignment from microsatellite analysis was used to improve estimates of survival and straying and to examine variation of family size. Hybridization reduced return rates of adults (a proxy for survival at sea) in the F₁ generation in the odd-year broodline (p < 0.0001) but not in the even-year broodline (p = 0.678). Hybridization reduced survival in both the odd- and even-broodyear F₂ (p < 0.005 and p < 0.0001). Hybridization did not appear to impair homing ability; weekly surveys revealed similar straying rates (～2%) by both hybrid and control fish into nearby (～1 km) Waydelich Creek in both generations in both trials. Hybridization did not increase the index of variability (σ²/μ) in family size. Decreased survival in the hybrid F₂ generation supports an epistatic model of outbreeding depression; nonepistatic effects may have contributed to reduced survival in the odd-broodyear F₁ hybrid fish. Outbreeding depression in hybrids of geographically separated populations demonstrates that introgression of nonnative fish can erode fitness, and should be recognized as a potential detriment of both aquaculture and management practices.     

Ribonucleotide Sequence Homology Among Avian Oncornaviruses

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between ³H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-0, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of ³H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as efficiently (100%) as by unlabeled AMV RNA. Hybridization between ³H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between ³H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between ³H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNAs. Hybridization between ³H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 to 26% by RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybridization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0 are different from the sequences shared by AMV and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Hybridization Between Neotropical Primates with Contrasting Sexual Dichromatism

International Journal of Primatology - Hybridization is relatively well documented among Old World primates, but poorly investigated among New World monkeys. We investigated hybridization between...     

In situ hybridization of nucleic acids

Hybridization of radioactive nucleic acid sequences can be carried out with DNA in cytological preparations. By autoradiography distinct DNA sequences can then be localized in eukaryotic chromosomes.     

Sugar-cane breeding in Java

Hybridization work and commercial introduction of many new clones into the sugar plantations of this island have resulted in a tenfold increase in sugar production on an acreage basis since 1840.     

Presence of Cell and Virus Specific Sequences in the Same Molecules of Nuclear RNA from Virus Transformed Cells

Hybridization studies show that nuclear RNA in transformed cells contains both host and virus specific sequences. This suggests virus-specific mRNA could be formed by selective cleavage of high molecular weight nuclear RNA.     

The Hybrid Origin of the Indochinese Gray Langur Trachypithecus crepusculus

International Journal of Primatology - Hybridization among formerly separated lineages is a common phenomenon in almost all groups of organisms, including primates. Indicators of hybridization...