Brain nuclear DNA survives cardiac arrest and reperfusion.

Iron-mediated peroxidation of brain lipids is known to occur during reperfusion following cardiac arrest. Since in vitro damage to DNA is caused by similar iron-dependent peroxidation, we tested whether free radical damage to genomic DNA also develops during reperfusion following cardiac arrest and resuscitation. Genomic DNA was isolated from the cerebral cortex in (i) normal dogs, (ii) dogs subjected to a 20-min cardiac arrest, and (iii) dogs resuscitated from a 20-min cardiac arrest and then allowed to reperfuse for 2 or 8 h. DNA strand nicks were evaluated by in vitro labeling of newly created 3' and 5' termini. DNA base damage was evaluated utilizing reaction with piperidine prior to labeling of 5' termini. The 3' DNA termini were labeled before and after digestion with exonuclease III, and the 5' DNA termini were labeled before and after treatment with piperidine. In vitro experiments with genomic DNA damaged by oxygen radicals verified that these labeling methods identified radical damage. In the experimental animal groups, terminal incorporation and electrophoretic mobility of brain nuclear DNA are not significantly changed either by 20 min of complete brain ischemia or during the first 8 h of reperfusion. We conclude that genomic DNA is not extensively damaged during cardiac arrest and early reperfusion, and therefore such DNA damage does not appear to be an important early aspect of the neurologic injury that accompanies cardiac arrest and resuscitation.     

Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction

The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.     

Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes.     

Superhelical duplex destabilization and the recombination position effect

The susceptibility to recombination of a plasmid inserted into a chromosome varies with its genomic position. This recombination position effect is known to correlate with the average G+C content of the flanking sequences. Here we propose that this effect could be mediated by changes in the susceptibility to superhelical duplex destabilization that would occur. We use standard nonparametric statistical tests, regression analysis and principal component analysis to identify statistically significant differences in the destabilization profiles calculated for the plasmid in different contexts, and correlate the results with their measured recombination rates. We show that the flanking sequences significantly affect the free energy of denaturation at specific sites interior to the plasmid. These changes correlate well with experimentally measured variations of the recombination rates within the plasmid. This correlation of recombination rate with superhelical destabilization properties of the inserted plasmid DNA is stronger than that with average G+C content of the flanking sequences. This model suggests a possible mechanism by which flanking sequence base composition, which is not itself a context-dependent attribute, can affect recombination rates at positions within the plasmid.     

Restriction site tagged (RST) microarrays: a novel technique to study the species composition of complex microbial systems

We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.     

BIS 1, a major component of the cereal genome and a tool for studying genomic organization

We report the cloning and characterization of an element (BIS 1) in barley. Related sequences were also found in wheat and rye genomes. BIS 1-related sequences may be some of the more frequent of the complex repeats in the barley genome, and their dispersion throughout the barley genome suggests that they are capable of both mobility and amplification. BIS 1 sequences have been used to study the gross structure of the barley genome. These studies indicate that the genome may be formed from larger genomic structures of tens of kilobases which may be repeated. Surprisingly, these studies also show that these large genomic structures also occur in similar proportions and at similar size distribution in the wheat genome.     

Genome redundancy and plasticity within ancient and recent Brassica crop species

The crop species within the genus Brassica have highly replicated genomes. Three base 'diploid' species, Brassica oleracea , B. nigra and B. rapa , are likely ancient polyploids, and three derived allopolyploid species, B. carinata , B. juncea and B. napus , are created from the interspecific hybridization of these base genomes. The base Brassica genome is thought to have hexaploid ancestry, and both recent and ancient polyploidization events have been proposed to generate a large number of genome rearrangements and novel genetic variation for important traits. Here, we revisit and refine these hypotheses. We have examined the B. oleracea linkage map using the Arabidopsis thaliana genome sequence as a template and suggest that there is strong evidence for genome replication and rearrangement within the base Brassicas, but less evidence for genome triplication. We show that novel phenotypic variation within the base Brassicas can be achieved by replication of a single gene, BrFLC , that acts additively to influence flowering time. Within the derived allopolyploids, intergenomic heterozygosity is associated with higher seed yields. Some studies have reported that de novo genomic variation occurs within derived polyploid genomes, whereas other studies have not detected these changes. We discuss reasons for these different findings. Large translocations and tetrasomic inheritance can explain some but not all genomic changes within the polyploids. Transpositions and other small-scale sequence changes probably also have contributed to genomic novelty. Our results have shown that the Brassica genomes are remarkably plastic, and that polyploidy generates novel genetic variation through gene duplication, intergenomic heterozygosity and perhaps epigenetic change.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 665–674.     

A novel method to calculate the G+C content of genomic DNA sequences.

The base composition of a DNA fragment or genome is usually measured by the proportion of A+T or G+C in the sequence. The G+C content along genomic sequences is usually calculated using an overlapping or non-overlapping sliding window method. The result and accuracy of such an approach depends on the size of the window and the moving distance adopted. In this paper, a novel windowless technique to calculate the G+C content of genomic sequences is proposed. By this method, the G+C content can be calculated at different "resolution". In an extreme case, the G+C content may be computed at a specific point, rather than in a window of finite size. This is particularly useful to analyze the fine variation of base composition along genomic sequences. As the first example, the variation of G+C content along each of 16 yeast chromosomes is analyzed. The G+C-rich regions with length larger than 5 kb sequences are detected and listed in details. It is found that each chromosome consists of several G+C-rich and G+C-poor regions alternatively, i.e., a mosaic structure. Another example is to analyze the G+C content for each of the two chromosomes of the Vibrio cholerae genome. Based on the variations of the G+C content in each chromosome, it is shown that some fragments in the Vibrio cholerae genome may have been transferred from other species. Especially, the position and size of the large integron island on the smaller chromosome was precisely predicted. This method would be a useful tool for analyzing genomic sequences.     

Metabolically 35S-labeled recombinant calmodulin as a ligand for the detection of calmodulin-binding proteins

We have developed a simplified procedure for the production of metabolically labeled calmodulin. We used bacterial clones (Escherichia coli) that were found to express VU-1 calmodulin, a calmodulin that is fully active with a variety of calmodulin-regulated enzymes. VU-1 calmodulin was labeled with sulfur-35 in bacteria maintained in a sulfur-free medium. Calmodulin was then purified by chromatography on phenyl-Sepharose. Under these conditions, the specific activity of the proteins was 150 to 400 cpm/fmol of calmodulin. To demonstrate the utility of this labeled VU-1 calmodulin, we examined the calmodulin-binding proteins in aortic myocyte preparation from Day 0 and Day 15 cultures by using both the gel and the nitrocellulose overlay protocols. The results showed that calmodulin-binding proteins are easily detected by the two procedures and that the profile of these target proteins changed in myocyte with time in culture. While most of these calmodulin-binding proteins have not been identified, the relative mobility on SDS-PAGE gels suggests that myosin light chain kinase (Mr approximately 137,000) was detected by these methods. We demonstrated here that the nitrocellulose overlay was faster than the gel overlay and that this technique can be useful for the study of calmodulin-binding proteins.     

Detection of polymorphism in the Trypanosoma cruzi TcP2β gene family by single strand conformational analysis (SSCA)

Single strand conformation analysis (SSCA) is a technique that has been used to detect point mutations. We explored its usefulness in the analysis of four different members of the Trypanosoma cruzi TcP2β gene family and its suitability for detection of polymorphism in different parasite strains. The availability of primers covering a 97-bp sequence at the 5′ end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic conditions: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4°C. Shifts in mobility were radically dependent on the migration condition. Both 97-bp and 321-bp amplicons were best resolved at 4°C, without glycerol. Amplification products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the cloned genes. The results herein demonstrate the usefulness of SSCA to differentiate forms of a complex protozoan gene family, and to scan its polymorphic nature. Furthermore, due to the remarkable sensitivity of the technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project     

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.     

The effects of aflatoxin B1 on growth hormone regulated gene-1 and interaction between DNA and aflatoxin B1 in broiler chickens during hatching

Many types of aflatoxin cause problems for both public and animal health. Aflatoxin B₁ (AFB₁) is the most toxic and commonly encountered fungal toxin that appears in poultry feed and in feeds stored under unsuitable conditions. AFB₁ decreases feed quality, egg production and fertility of hatching eggs. Also, AFB₁ alters the development of embryos by infecting eggs. We investigated using sequence analysis the changes caused by different concentrations of AFB₁ on the promoter sequences of the growth hormone regulated gene-1 (GHRG-1) in chick embryo at 13, 17, 19 and 21 days incubation. DNA isolated from the liver of chick embryos treated with different concentrations of AFB₁ was separated using agarose gel electrophoresis to detect apoptosis, and DNA interaction with AFB₁ was investigated using plasmids to detect changes in electrophoretic mobility and their effects on DNA. Base changes of the promoter sequences of GHRG-1 in 5 ng/egg, 15 ng/egg and 40 ng/egg doses of AFB₁ were increased on day 19 compared to base changes of the same AFB₁ doses on day 13. We also found that AFB at different concentrations changed the mobility of DNA by binding to it, and that high doses of AFB1 destroyed DNA. The DNA interaction study using plasmid demonstrated that AFB₁ at high doses was bound to plasmid DNA, slowed its mobility and inhibited restriction cuts.     

The dinitrophenyl group as a selective label in high-performance liquid chromatography of peptides

1-Fluoro-2,4-dinitrobenzene can be used to selectively label histidine, tyrosine, and cysteine residues in maleylated proteins. The usefulness of the resulting chromophores for peptide mapping by high-performance liquid chromatography was demonstrated with the lectin from sainfoin (Onobrychis viciifolia). The 2,4-dinitrophenyl (Dnp) label also can be used in a hydrophobic modulation approach as the mobility of a labeled model peptide changes considerably when its Dnp group is removed by thiolysis. Application of the method for checking sequences obtained by DNA or amino acid methods was shown by experiments with Viciae lectins. The probable cleavage site that generates the pea lectin's beta-chain from the alpha-beta precursor was identified and the sequence differences between the lentil and pea lectin beta-chains were examined.     

Comparative genomic hybridization as a new method for detection of genomic imbalance

Comparative Genomic Hybridization (CGH) is a molecular cytogenetic analysis that allows identification of genomic changes by comparing the copy number of DNA sequences in cells of tested tissue and the reference specimen. CGH is based on competitive suppressive in situ hybridization of two differently labeled DNA probes (tested and reference, karyotypically normal, fluorochrome-labeled DNAs) with metaphase chromosomes of a healthy subject. First described by Kallioniemi et al. in 1992, the CGH assay has been widely used for identification and characterization of both numerical and structural chromosome abnormalities in cells of different tissues at various pathological conditions in humans, especially in tumor diseases. We discuss the specific features and quality control of comparative genomic hybridization, its advantages and limitations in detection of genomic imbalance and the prospects for development of this technology.     

In situ random primer extension of metaphase chromosomes

We have developed a technique of random primer extension of fixed chromosomes that is applicable to both mouse and man. Human chromosomes are not homogeneously labeled with this technique; those regions corresponding to R-bands appear to be more sensitive than those identified as G-bands, whereas centromeric regions are not labeled. These results not only corroborate specific structural differences between distinct regions of mammalian genomes but also open up the possibility of assays with specific primers to test whether primer extension is useful for the identification of genes and families of sequences on chromosomes.     

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.     

A simple and rapid technique for identification of large numbers of individual mosquitoes using DNA hybridization

A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.