Complementary DNA-DNA hybridization in Drosophila

Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.On leave from the Dipartimento di Biologia, II Università di Roma Tor Vergata, Rome, Italy     

Non-radioactive detection of single-copy DNA-DNA hybrids

A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.     

The future of DNA-DNA hybridization studies

Summary This article draws on many vertebrate examples to assess the future of DNA-DNA hybridization studies. I first discuss whether applications of the method have reached the point of diminishing returns, or rather the start of a great leap forward, in our evolutionary understanding. Vertebrate groups whose relationships are especially likely to be illuminated include parrots, pigeons, bats, pinnipeds, mammalian carnivores, frogs, and rodents. There are at least two reasons why classifications based on DNA-DNA hybridization may prove to differ from classifications based on particular character, whether these be noncoding DNA sequences or protein sequences or anatomical characters. Because evolutionary relationships can now be deduced independently of anatomical characters, this should permit a renaissance in comparative anatomical studies of adaptation. The origin of major functional shifts from changes in a small fraction of the genome is illustrated by polar bears, sea otters, warblers, vultures, and especially by humans.     

热分析方法及裂解气相色谱在树木分类上的试探性研究

热分析是在程序控制温度下自动连续跟踪物质的物理性质与温度关系的一类技术。尤其是差热分析和热重法与其他手段联合,可用于化合物稳定性、分解机理和相转变等方面的研究。由于种子是一个复杂化合物的有机体,我们期望发现各不同种子化合物之间在成份上和组成方式上的共性和差异性,从热化学的角度对木本植物进行热化学分类。我们选用针叶类两个科(松科、杉木科)的种子进行了DTA、TG、550℃裂解气相色谱分析;300℃逸出成份分析,发现松科种子DTA图上在240±5℃有一个共同的吸热峰,而杉木则出现在320±5 ℃。TG曲线同科的不同种之间非常相似,逸出成份分析和裂解气相色谱分析同科的不同种之间也具共同特征,且又显示出一定的差异性。这种分类方法是可靠的,且可为树木(或植物)分类学提供更有价值的信息。     

Identification of Listeria using molecular DNA-DNA hybridization

The DNA-DNA hybridization method was used to study 4 species of bacteria of the genus Listeria. Concerning the DNA homology, L. monocytogenes strains may be divided into several species (in particular, the pathogenic forms may be isolated into independent taxon), in correlation with their biochemical and serological properties. Most of the studied strains of this species exhibit a high level of DNA homology--65-100%. Bacteria of the L. grayi and L. murrayi species are closely related to each other (90% of DNA homology), the reasonable suggestion being to unite them into a single species. L. denitrificans has 7% of DNA homology with the DNA of the other three species suggesting that it should be excluded from the genus Listeria.     

DNA-DNA hybridizations support ungulate ancestry of Cetacea

Recent morphological data on Pakicetus spp. and Basilosaurus spp. fossils suggest that cetaceans (whales, dolphins and porpoises) originate from carnivorous Mesonychid land mammals (Condylarthra) and made a gradual transition from land to sea in early Eocene (Gingerich et al. 1983; 1990). On the other hand, there is convincing evidence that Artiodactyla and Perissodactyla have evolved from Condylarthra (Van Valen 1978, Carrol 1988). Therefore, the Pakicetus and Basilosaurus data suggest a close genetic relationship between cetaceans and ungulates. An approach based on molecular genetics was used in this study to test the morphological hypothesis. Liver samples of two Delphinoidea species were obtained from animals caught in a Peruvian gillnet fishery. ³²P- or ³⁵S-labeled single copy nuclear genomes (scn-DNA) of the two cetacean species were hybridized each with unlabeled total DNA of various cetaceans, ungulates and other mammals including primates, rodents, lagomorphs and carnivores. The T_median (T_m) and T_mode of all melting curves, used as a measure of the DNA-DNA hybrids stability, clearly show a greater sequence similarity — and thus a lower genetic distance — between cetaceans and ungulates than between cetaceans and other mammals.     

Isolation and characterization of DNA-DNA and DNA-RNA.

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.     

DNA-DNA hybridization in the systematics of Streptomyces.

The DNA relatedness among strains in several different phenotypically defined Streptomyces species clusters was evaluated. It was found that the data from DNA-relatedness studies do not necessarily agree with the clustering generated using numerical taxonomic techniques. A study of the morphologically heterogeneous 'S. cyaneus' cluster showed that morphological criteria traditionally used to classify and identify Streptomyces species still have value, since strains in DNA-relatedness cluster groups were also similar morphologically (i.e., they had similar spore color, surface properties, and sporophore morphology). An evaluation of DNA relatedness among strains in the S. violaceusniger and S. lavendulae clusters indicated that, if anything, the genus is underspeciated, based on the number of single-member clusters observed. A study of strains of the sweet potato pathogen, S. ipomoea, collected in various locations in the United States and Japan indicated, not surprisingly, that all of the strains belong to the same species.     

Evaluation of membrane filter methods for DNA-DNA hybridization

Three membrane filter methods for DNA hybridization were compared. In all of them filter-fixed DNA is spontaneously released during incubation. The effect depends largely on the temperature: 40 to 85% of DNA is released at 70 to 80 C; at higher temperatures most of the DNA is released and heterologous DNA relatedness values are unreliable. Only the dimethylsulfoxide filter method (Legault-Démare et al., 1967) can be recommended for the entire temperature and % GC range. The methods of Denhardt (1966) and of Warnaar and Cohen (1966) are not recommended for hybridizations of DNA containing more than 50% GC. Impure DNA yields too low DNA relatedness values.     

Mechanism of spontaneous DNA-DNA interaction of homologous linear duplexes

Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.     

The DNA-DNA spacing in gemini surfactants-DOPE-DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n=2-12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L(α)(C) in the studied range of molar ratios CnGS12/DOPE in the temperature range 20-60°C. The DNA-DNA distance (d(DNA)) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE=0.35 and 0.4mol/mol) lead to the linear dependence of d(DNA) vs. n correlating with the interfacial area of the CnGS12 molecule.