The use of heparin as a simple cost-effective means of controlling background in nucleic acid hybridization procedures.

The use of heparin as a simple, effective and cheap substitute for conventional methods of controlling background in hybridization procedures is described and illustrated with reference to the use of DNA probes in filter and in situ hybridization. Possible specific mechanisms for this heparin effect are discussed.     

水稻体细胞杂交研究进展

水稻是世界重要的粮食作物,全世界约有120个国家种植水稻。水稻的近缘或远缘种具有一些优良性状,如抗病、抗虫、抗逆等。将这些性状导入水稻,是科学家们所希望的,但因用普通杂交方法存在交配系统的不亲和性等难题而进展不大。随着组织培养技术的发展,特别是植物原生质体培养技术的日趋成熟,使得人类能够在细胞水平通过体细胞杂交方法实现遗传信息的重组。     

Factors influencing cDNA microarray hybridization on silylated glass slides

cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The aim of this paper is to provide more insight into the practical aspects of microarray construction and hybridization. The influence of the size, composition, and concentration of the spotted DNA fragments on the final hybridization signal and the effect of hybridization volume, sample concentration, and sample depletion have been tested and are discussed.     

Hybridization of <Emphasis Type="Italic">Motacilla alba</Emphasis> Linnaeus, 1758, and <Emphasis Type="Italic">M. (a.) personata</Emphasis> Gould, 1861, in the south of Siberia

The authors attempt to estimate the level of hybridization of Motacilla alba and M. (a.) personata in the area of their secondary contact in the south of Siberia. Processing of the materials in several museum collections (n = 424) and author’s own collections (n = 347) resulted in conclusion on the presence of introgression, in spite of limited hybridization. The data on pair composition and breeding success in the hybridization zone as well as data on habitat preferences are given. The possible reasons for the limited hybridization are discussed.     

Beyond Affymetrix arrays: expanding the set of known hybridization isotherms and observing pre-wash signal intensities

Microarray hybridization studies have attributed the nonlinearity of hybridization isotherms to probe saturation and post-hybridization washing. Both processes are thought to distort ‘true’ target abundance because immobilized probes are saturated with excess target and stringent washing removes loosely bound targets. Yet the paucity of studies aimed at understanding hybridization and dissociation makes it difficult to align physicochemical theory to microarray results. To fill the void, we first examined hybridization isotherms generated on different microarray platforms using a ribosomal RNA target and then investigated hybridization signals at equilibrium and after stringent wash. Hybridization signal at equilibrium was achieved by treating the microarray with isopropanol, which prevents nucleic acids from dissolving into solution. Our results suggest that (i) the shape of hybridization isotherms varied by microarray platform with some being hyperbolic or linear, and others following a power-law; (ii) at equilibrium, fluorescent signal of different probes hybridized to the same target were not similar even with excess of target and (iii) the amount of target removed by stringent washing depended upon the hybridization time, the probe sequence and the presence/absence of nonspecific targets. Possible physicochemical interpretations of the results and future studies are discussed.     

The Southern blot

This article is devoted to the recent advances made in the Southern blotting technique, which is used for the detection of gel-fractionated DNA molecules following transfer to a membrane. The latest practical improvements made to the techniques of Southern blotting, probe labeling, and hybridization are discussed. Among the most significant advances are: new membranes, transfer methods, probe labeling, and rapid hybridization. Detailed protocols show the application of these improvements in this widely used technique.     

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

Summary The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.     

Hybridization and speciation in the carabid beetles of the subgenusOhomopterus (Coleoptera, Carabidae, genusCarabus)

Natural hybridization among wingless carabid beetles of the subgenusOhomopterus (Carabidae, genusCarabus) is reviewed, and its significance in the evolution of this subgenus discussed. Natural hybridization occurs between parapatric species of similar size. Two case studies of natural hybridization suggest that natural hybridization could have affected the evolution of this subgenus in different ways. When there is a large difference in genital morphology between hybridizing species, interspecific copulation often results in genital injuries that causes mortality of copulating individuals, and hence reduces the fitness of hybridizing individuals greatly. In such a case, hybridization may be effective in maintaining the parapatric distribution of the two species, and in the long term, may promote reinforcement selection for traits which are effective in prezygotic reproductive isolation. When the morphological difference in genitalia is not so large as to cause genital injury, a hybrid population may be established at the intermediate zone between two parental species, provided that the immigration rates of the two species into the intermediate zone are small. Thus, natural hybridization may have contributed to both divergence and reticulate evolution in this subgenus.     

嫁接杂交与果树遗传的特殊性

在介绍近年来植物嫁接杂交研究新进展的基础上,着重阐述了嫁接杂交与果树遗传特殊性的关系。砧木中的遗传物质转移并整合到接穗生殖细胞和胚胎细胞的染色体组中,是杂种后代出现大量野生植株和果树遗传不遵守孟德尔定律的主要原因。还讨论了嫁接杂交在果树育种中的重要意义。 Abstract：Emphatically discusses the relationship between graft hybridization and the specificity of heredity in fruit trees on the basis of introducing the recent achievements in plant graft hybridization. We propose that genetic materials in rootstock being translocated and integrated into the genome of the germ cells and embryonic cells in scion are the main reasons why the majority of the hybrid seedlings have wild properties and the heredity of fruit trees violate Mendel’s laws of heredity. The potential of graft hybridization in fruit breeding are also discussed.     

Natural hybridization and conservation

This review deals with natural hybridization, an important subject in conservation biology. Natural hybridization is defined as the secondary contact between two populations that have evolved separately over a long period of time. This process is uncommon in terms of the total number of individuals involved, but is much less unusual if we consider the number of species that hybridize. Thus, natural hybridization may be an important process in the shaping of the evolutionary trajectories of many plant and animal species. The possible consequences of natural hybridization, which can either promote or prevent evolutionary divergence between taxa and will involve many ecological factors, are analysed here. I question whether natural hybridization poses always a problem in conservation and try to answer when conservation biologists and managers do have a responsibility to take decisions. Several examples of hybridization related to management strategies are also discussed. In conclusion, I believe that it is impossible to provide conservation managers with a simple handbook explaining how to proceed in cases of hybridization––each case is unique and should be analyzed individually. The only advice is that the more we know about hybridization and the factors involved, the better we will be able to assess each situation, to establish the possible consequences and even to estimate the probability of success of any particular conservation strategy.     

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.     

Plaque-based competitive hybridization

The authors have developed a simple, cost-saving experimental design, plaque-based competitive hybridization (PBCH), for genome-wide identification of genes differentially expressed in different tissues. PBCH offers advantages in comparison with other methods used in comparative genomics by combining the principles of differential hybridization with the subtractive hybridization. PBCH is particularly advantageous when libraries with few differences are to be analyzed. The authors demonstrate the use of PBCH by identifying 3 genes, up-regulated in the developing velvet antler of red deer (Cervus elaphus): ApoD, C011A2, and S100a1. The fidelity and sensitivity of PBCH is also shown: 1 specific clone among a library sample of 15,000 can be recognized. Possibilities for further utilizations are discussed.     

Biotinylated oligonucleotides as probes in the method of molecular hybridization

The chemical approaches to the preparation of non-radioactive and biotinylated probes based on synthetic oligonucleotides and their applicability to the molecular hybridization method are discussed.     

Long tandem arrays of complex repeat units in Chironomus telomeres.

A cloned 340-bp DNA fragment excised by EcoRI from the Chironomus pallividittatus genome has been localized to the telomeres by in situ hybridization as well as to connectives between telomeres. No hybridization was observed in other regions of the chromosomes. Another cloned EcoRI fragment, 525 bp long has also been studied. This represents a partial duplication of the 340-bp sequence. Genomic blot hybridization experiments show that the 340-bp sequence is a representative monomeric unit of tandemly repeated arrays which account for 1.2% of the Chironomus genome, on average 300 kb per telomere. The repeat unit contains two types of subrepeats each present twice per repeat unit. Northern blot hybridization experiments show that the telomere-associated sequences are transcribed into a discrete RNA species approximately 20 kb in size. The evolution of this telomere-associated DNA is discussed.     

Research advances in animal distant hybridization

Distant hybridization refers to crosses between two different species or higher-ranking taxa that enables interspecific genome transfer and leads to changes in phenotypes and genotypes of the resulting progeny. If progeny derived from distant hybridization are bisexual and fertile, they can form a hybrid lineage through self-mating, with major implications for evolutionary biology, genetics, and breeding. Here, we review and summarize the published literature, and present our results on fish distant hybridization. Relevant problems involving distant hybridization between orders, families, subfamilies, genera, and species of animals are introduced and discussed, with an additional focus on fish distant hybrid lineages, genetic variation, patterns, and applications. Our review serves as a useful reference for evolutionary biology research and animal genetic breeding.     

Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells

The present review evaluates methods for electron microscopic immunocytochemistry and in situ hybridization, using post-embedding techniques and colloidal gold as a label. Special emphasis is given to double labeling immunocytochemistry and double in situ hybridization and to their combined application on the same specimen. Brief guidelines are presented for fixation, embedding media, the use of polyclonal and monoclonal antibodies and nucleic acid probes. Conditions for labeling and binding of antibody and nucleic acid probes to the target and protocols for direct and indirect immunodetection are discussed. Combinations of direct and indirect immunodetections in multiple labeling experiments are summarized.     

The demonstration of natural hybridization between two swallowtail species Parnassius nomion and Parnassius bremeri (Lepidoptera, Papilionidae) using RAPD-PCR technique]

Genetic evidence for interspecific hybridization between Parnassius nomion and Parnassius bremeri in nature is presented. To demonstrate hybridization between these species, RAPD analysis was used. By testing 25 decamer primers, three and two diagnostic markers were revealed for P. nomion and P. bremeri, respectively. Out of 28 animals examined, 4 were shown to be interspecific hybrids. According to the distribution of diagnostic markers, the interspecific hybrids were intermediate with regard to the parental species. Ecological and biological characteristics of two swallowtail species that promote their hybridization in nature are discussed.     

Interaction of chromosomal and plasmid DNA in Acidithiobacillus ferrooxidans strains adapted to different oxidation substrates

Restriction analysis of plasmids pTFK1 and pTFK2 of the Acidithiobacillus ferrooxidans strain TFBk was carried out, and the sizes of these plasmids were determined (13.5 and 30 kb, respectively). A macrorestriction map was built for plasmid pTFK1. DNA-DNA hybridization revealed that the plasmids contained homologous nucleotide sequences. Plasmid pTFK2 labeled with 32P was used as a probe for Southern hybridization with blots of XbaI-generated fragments of the chromosomal DNA of A. ferrooxidans strains grown on a medium containing Fe2+ or adapted to different oxidation substrates. Low-intensity hybridization signals were observed for many fragments of the chromosomal DNA of the strains studied. In the process of adaptation to new oxidation substrates, the localization of bands producing the low-intensity hybridization signals changed in a number of cases. Certain fragments of the chromosomal DNA of the strains adapted to different oxidation substrates produced strong hybridization signals with pTFK2. The data obtained are discussed in terms of the possible role of IST elements and plasmids in the adaptation of A. ferrooxidans to new energy substrates, microevolution, and strain polymorphism.     

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.