DNA hybrids stabilized by heterologies.

The double D-loop DNA hybrid contains four DNA strands following hybridization of two RecA protein coated complementary single-stranded DNA probes with a homologous region of a double-stranded DNA target. A remarkable feature of the double D-loop DNA hybrids is their kinetic stabilities at internal sites within linear DNA targets after removal of RecA protein from hybrids. We report here that heterologous DNA inserts in one or both probe strands affect the kinetic stability of protein-free double D-loop hybrids. DNA heterologies normally distort DNA-DNA hybrids and consequently accelerate hybrid dissociation. In contrast, heterologous DNA inserts impede dissociation of double D-loops, especially when the insert sequences interact with each other by DNA base pairing. We propose a mechanism for this kinetic stabilization by heterologous DNA inserts based on the hypothesis that the main pathway of dissociation of double D-loop DNA hybrids is a DNA branch migration process involving the rotation of both probe-target duplexes in the hybrids. Heterologous DNA inserts constrain rotation of probe-target duplexes and consequently impede hybrid dissociation. Potential applications of the stabilized double D-loops for gene targeting are discussed.     

Assessment of multiple displacement amplification in molecular epidemiology

Well-characterized epidemiological resources are generated with great effort, yet associated patient DNA samples can be limiting. The efficacy of the whole genome amplification (WGA) method, termed multiple displacement amplification (MDA), was assessed for detecting heterozygous sequence variants, mutation scanning, and PCR for challenging segments. Fifteen common polymorphisms from 10 genes located on 8 chromosomes were genotyped by direct sequencing of 300 PCR products from 115 high-quality MDA-amplified DNA samples extracted by different methods. The GC content of these analyzed segments ranges from 30% to 69%. Genotyping results demonstrate 100% accuracy. For heterozygotes, the relative intensity of peaks generated by the two alleles is highly similar for genomic and MDA-amplified genomic DNA, independent of GC content. In contrast, one of four heterozygous loci was mistyped when lower quality MDA-amplified DNA samples were used. The results of single-stranded conformation polymorphism (SSCP)-type of mutation scanningfor seven MDA-amplified DNA samples in four genes were concordant with the genomic DNA samples. PCR on MDA-amplified DNA was routinely successful for challenging 10- and 12-kb segments with GC content ranging from 30% to 80%, demonstrating that rather long segments, which are difficult to amplify with PCR, are amplified well with MDA. These results suggest that MDA is an effective method of WGA with utility in molecular epidemiology. Quality control of the MDA-amplified DNA is critical for high performance.     

Instability of Candida albicans hybrids.

Total cellular DNA content, determined by a colorimetric method, was used as an index of ploidy in Candida albicans. Mononucleate hybrids were formed by fusion of spheroplasts derived from diploid parent strains. Five hybrids, of six studied, were taken to be tetraploid on the basis of estimated DNA content. One hybrid was taken to be hexaploid or near-hexaploid. Selection for increased resistance to 5-fluorocytosine in the hybrids, which were heterozygous for resistance, resulted in isolation of variants which were of lower ploidy than the hybrids from which they originated. Variants were obtained which corresponded (in measured DNA content) to aneuploid, triploid, and diploid states. These results may form the basis of a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis of this asexual species.     

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Variability in DNA content to testis cells and sperm from F₁ hybrids between the laboratory mouse (M. muscullus) and the tobacco mouse (M. poschiavinus), has been determined by flow cytometry (FMC). The F₁ hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F₁ hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f₁ hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F₁ hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.     

Double heterozygous hemoglobin Q India/hemoglobin D Punjab hemoglobinopathy: Report of two rare cases

Cation exchange high performance liquid chromatography (CE HPLC) provides an excellent tool for accurate and reliable diagnosis of various hemoglobin (Hb) disorders. HbQ India is a rare alpha chain variant that usually presents in the heterozygous state. Its presence in double heterozygous state with HbD Punjab is extremely rare. The double heterozygosity for α and β chain variants leads to formation of abnormal heterodimer hybrids, which can lead to diagnostic dilemmas. We report two rare cases of double heterozygous HbQ India/HbD Punjab where the hybrid Hb was seen to elute at retention time similar to HbC on CE HPLC. The first case had unconjugated hyperbilirubinemia at presentation; while, the second case was asymptomatic.     

Genetic distance estimates among single cross hybrids and correlation with specific combining ability and yield in corn double cross hybrids

The objective of the present study was to correlate the genetic distances (GD) of single cross hybrids with yield, heterosis and specific combining ability (SCA) in the double cross hybrid synthesis. For this, 10 single cross commercial hybrids were used from different companies, and all the possible double hybrids were synthesized by a complete dialell. The hybrids were assessed in 15 locations in the 2005/2006 agricultural season, using the randomized complete block design with three repetitions. DNA was extracted from the single cross hybrids and 20 simple sequence repeat primers were used, nine of which were linked to the quantitative trait loci. It was ascertained that the single hybrids were superior in general to the double cross hybrids and that yield was highly correlated with heterosis and SCA (r = 0.75 and 0.82, respectively). There was no significant correlation between yield and GD (r = 0.25), but this index was at the limit of significance. There was a medium correlation between GD and heterosis (r = 0.40) and GD and SCA (r = 0.38). The intergroup hybrids placed by genetic grouping were generally more productive than intragroup hybrids, and the hybrids with GD greater than 0.84 had the maximum heterosis and SCA. It was concluded that the markers were efficient in placing hybrids in different heterosis groups and were also useful in eliminating the most negative heterosis and SCA.     

Signatures of DNA flexibility, interactions and sequence-related structural variations in classical X-ray diffraction patterns

The theory of X-ray diffraction from ideal, rigid helices allowed Watson and Crick to unravel the DNA structure, thereby elucidating functions encoded in it. Yet, as we know now, the DNA double helix is neither ideal nor rigid. Its structure varies with the base pair sequence. Its flexibility leads to thermal fluctuations and allows molecules to adapt their structure to optimize their intermolecular interactions. In addition to the double helix symmetry revealed by Watson and Crick, classical X-ray diffraction patterns of DNA contain information about the flexibility, interactions and sequence-related variations encoded within the helical structure. To extract this information, we have developed a new diffraction theory that accounts for these effects. We show how double helix non-ideality and fluctuations broaden the diffraction peaks. Meridional intensity profiles of the peaks at the first three helical layer lines reveal information about structural adaptation and intermolecular interactions. The meridional width of the fifth layer line peaks is inversely proportional to the helical coherence length that characterizes sequence-related and thermal variations in the double helix structure. Analysis of measured fiber diffraction patterns based on this theory yields important parameters that control DNA structure, packing and function.     

The role of RNA and RNA-related proteins in the regulation of DNA double strand break repair pathway choice

DNA end resection is a critical step in the repair of DNA double strand breaks. It controls the way the lesion is going to be repaired, thus its regulation has a great importance in maintaining genomic stability. In this review, we focus in recent discoveries in the field that point to a modulation of resection by RNA molecules and RNA-related proteins. Moreover, we aim to reconcile contradictory reports on the positive or negative effect of DNA:RNA hybrids in the resection process.     

A new method for detection of small modifications in genomic DNA, applied to the human delta-beta globin gene cluster

Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single-stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single-stranded DNA. Hybrids were submitted to S1-nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the delta-beta globin gene cluster from the total genomic DNA of a beta 0-thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the beta-globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC----dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1-nuclease, which corresponds to an additional sequence d(T-A-T-A) in a 52 alternating purine-pyrimidine run, leading to a complex change from d[(A-T)7(T)7] to d[(A-T)11(T)3].     

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins.

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker 'A' motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW-K141R, inhibits replication from a T4 origin at which persistent RNA-DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double-strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA-DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW-K141R fusion, displays ssDNA-dependent ATPase activity and is able to unwind a branched DNA substrate.     

Simultaneous haploinsufficiency of Pten and Trp53 tumor suppressor genes accelerates tumorigenesis in a mouse model of prostate cancer

Tumor suppressor gene PTEN is important in the initiation and progression of human prostate carcinoma, whereas the role of TP53 remains controversial. Since Pten/Trp53 double conditional knockout mice show earlier onset and fast progression of prostate cancer when compared to Pten knockout mice, we asked whether heterozygosity of these two tumor suppressor genes was sufficient to accelerate prostatic tumorigenesis. To answer this question we examined prostatic lesion progression of Pten/Trp53 double heterozygous mice and a series of controls such as Pten heterozygous, Pten conditional knockout, Trp53 heterozygous and Trp53 knockout mice. Tissue recombination of adult prostatic epithelium coupled with embryonic rat seminal vesicle mesenchyme was used as a tool to stimulate prostatic epithelial proliferation. In our study, high-grade prostatic intraepithelial neoplasia (PIN) was found with high frequency at 8 weeks post-tissue recombination transplantation. PIN lesions in Pten/Trp53 double heterozygous mice were more severe than those seen in Pten heterozygous alone. Furthermore, morphologic features attributable to Pten or Trp53 loss appeared to be enhanced in double heterozygous tissues. LOH analysis of Pten and Trp53 in genomic DNA collected from high-grade PIN lesions in Pten heterozygous and Pten/Trp53 double heterozygous mice showed an intact wild-type allele for both genes in all samples examined. In conclusion, simultaneous heterozygosity of Pten and Trp53 accelerates prostatic tumorigenesis in this mouse model of prostate cancer independently of loss of heterozygosity of either gene.     

Fast quantification of nucleic acid hybrids by affinity-based hybrid collection.

A hybridization technique for the quantification of nucleic acids is described. In the method a probe pair is allowed to form hybrids with the target nucleic acid in solution. One of the probes has been modified with an affinity label, by which the formed hybrids can be isolated after the reaction. Streptavidin-agarose was used to capture hybrids containing biotinylated DNA. The hybrids were measured using radioiodine as label on the second probe. The rate of the hybridization reaction in solution is fast, allowing the whole procedure to be carried out in 3 h. The method is quantitative with a detection limit of 4 X 10(5) molecules (0.67 attomoles) target DNA. The test is insensitive to impurities in biological samples, which are analyzed without purification of the target DNA. Non-isotopic measurement of the hybrids can also be applied. In this case the hybrids are bound to microtitration wells and detected spectrophotometrically by peroxidase-catalyzed colour development.     

Use of a DNA probe for mapping by electron microscopy the ribosomal sequences in ribosomal RNA precursors from duck cells

This report describes the use of purified ribosomal DNA to map by electron microscopy the relative positions of the 18 S and 28 S RNA regions within the duck rRNA precursor and their relationship to the non-conserved portions of the precursor molecule. By repeated fractionation of the total DNA, based on the relative reassociation rates of the DNA sequences with different degrees of repetition, a fraction of the rapidly renaturing DNA was obtained which comprised only 6% of the total DNA, but contained 71% of the rRNA cistrons. Further purification of the rDNA was achieved by saturation hybridization with rRNA and separation of the rRNA-rDNA hybrids by banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained which had a buoyant density of 1.805 g/cm³, an RNA to DNA ratio of 1.01, and a base composition for the RNA present in the hybrid identical to that of an equimolar mixture of 18 S and 28 S rRNA. The final yield of rDNA isolated by this procedure is 32%. When the purified rDNA was annealed with a mixture of 18 S and 28 S rRNA and the hybrids spread for electron microscopy, they appeared as two distinct populations with a number-average length of 0.62 ± 0.13 μm and 1.37 ± 0.18 μm, respectively. Likewise, hybrids between the rRNA precursor, isolated from duck embryo fibroblasts, and the rDNA appeared as structures containing two duplex regions of lengths 0.60 ± 0.11 μm and 1.38 ± 0.15 μm, separated from each other by a single-stranded region appearing as a large bush: this represents a portion of the precursor molecule not conserved during processing of the parent molecule. From these observations a model of the structure of the duck rRNA precursor is proposed.     

A base-pairing model of duplex formation. I. Watson-Crick pairing geometries

We present a base-pairing model of oligonucleotide duplex formation and show in detail its equivalence to the nearest-neighbor dimer methods from fits to free energy of duplex formation data for short DNA-DNA and DNA-RNA hybrids containing only Watson-Crick pairs. For completeness, the corresponding RNA-RNA parameters are included. In this approach, the connection between rank-deficient polymer and rank-determinant oligonucleotide parameter sets for DNA duplexes is transparent. The method is generalized to include RNA-DNA hybrids where the rank-deficient model with 11 dimer parameters in fact provides slightly improved predictions relative to the standard method with 16 independent dimer parameters (DeltaG mean errors of 4.5 and 5.4%, respectively).     

酿酒酵母单倍体与双倍体原生质体的融合*

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

酿酒酵母单倍体与双倍体原生质体的融合

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

Analysis of the introgression of Solanum bulbocastanum DNA into potato breeding lines

Somatic hybrids have been obtained between potato and Solanum bulbocastanum PI 245310, a Mexican diploid (2n=2x=24) species. Through restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses it was found that the somatic hybrids contain each chromosome of the diploid parent and that the synteny of RFLP markers noted with tomato, potato and S. brevidens is largely maintained in S. bulbocastanum. RFLP analyses of BC1 progeny of two different hybrids indicated that a substantial number of markers were either lost or were heterozygous, in marked contrast with results previously noted with S. brevidens. A RAPD map for all 12 chromosomes of S. bulbocastanum was prepared and marker transmission was followed in three BC2 populations. Results with chromosomes 3, 8 and 10 from these populations are compared.     

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.