Alexander Bolshoy

Large Retrotransposon Derivatives: Abundant, Conserved but Nonautonomous Retroelements of Barley and Related Genomes

Retroviruses and LTR retrotransposons comprise two long-terminal repeats (LTRs) bounding a central domain that encodes the products needed for reverse transcription, packaging, and integration into the genome. We describe a group of retrotransposons in 13 species and four genera of the grass tribe Triticeae, including barley, with long, ∼4.4-kb LTRs formerly called Sukkula elements. The ∼3.5-kb central domains include reverse transcriptase priming sites and are conserved in sequence but contain no open reading frames encoding typical retrotransposon proteins. However, they specify well-conserved RNA secondary structures. These features describe a novel group of elements, called LARDs or large retrotransposon derivatives (LARDs). These appear to be members of the gypsy class of LTR retrotransposons. Although apparently nonautonomous, LARDs appear to be transcribed and can be recombinationally mapped due to the polymorphism of their insertion sites. They are dispersed throughout the genome in an estimated 1.3 × 103 full-length copies and 1.16 × 104 solo LTRs, indicating frequent recombinational loss of internal domains as demonstrated also for the BARE-1 barley retrotransposon.

CURVATURE: software for the analysis of curved DNA

Software is presented to plot the sequence-dependent spatial trajectory of the DNA double helix and/or distribution of curvature along the DNA molecule. The nearest-neighbor wedge model is implemented to calculate overall DNA path using local helix parameters: helix twist angle, wedge (deflection) angle and direction (of deflection) angle. The procedures described proved to be very convenient as tools for investigation of a relationship between overall DNA curvature and its gel electrophoretic mobility. All parameters of the model had been estimated from experimental data. Using these wedge parameters the program takes, as input, any DNA sequence and calculates the likely degree of curvature at each point along the molecule. This information is displayed both graphically and in the form of simplified representations of curved double helices. The Software, CURVATURE, can thus be used to investigate possible roles of curvature in modulation of gene expression and for location of curved portions of DNA, which may play an important role in sequence-specific protein--DNA interactions.

Sequence-Dependent Kinks Induced in Curved DNA

In certain curved DNA fragments without AA dinucleotides, the gel retardation anomaly associated with curvature passes through a maximum with fragment length, indicating length (and electric field) dependent structural transitions in the DNA. We suggest that thermally induced stereochemical kinks in DNA are stabilized in the gel, thus relieving the effects of curvature. These kinks are shown to occur specifically at CA/TG and TA/TA stacks. Other physical and biological evidence points to frequent structural dislocations at CA and TA steps. These reversible sequence dependent kinks may therefore represent a novel class of structural protein-DNA recognition elements.

Sequence complexity profiles of prokaryotic genomic sequences: A fast algorithm for calculating linguistic complexity

MOTIVATION One of the major features of genomic DNA sequences, distinguishing them from texts in most spoken or artificial languages, is their high repetitiveness. Variation in the repetitiveness of genomic texts reflects the presence and density of different biologically important messages. Thus, deviation from an expected number of repeats in both directions indicates a possible presence of a biological signal. Linguistic complexity corresponds to repetitiveness of a genomic text, and potential regulatory sites may be discovered through construction of typical patterns of complexity distribution. RESULTS We developed software for fast calculation of linguistic sequence complexity of DNA sequences. Our program utilizes suffix trees to compute the number of subwords present in genomic sequences, thereby allowing calculation of linguistic complexity in time linear in genome size. The measure of linguistic complexity was applied to the complete genome of Haemophilus influenzae. Maps of complexity along the entire genome were obtained using sliding windows of 40, 100, and 2000 nucleotides. This approach provided an efficient way to detect simple sequence repeats in this genome. In addition, local profiles of complexity distribution around the starts of translation were constructed for 21 complete prokaryotic genomes. We hypothesize that complexity profiles correspond to evolutionary relationships between organisms. We found principal differences in profiles of the GC-rich and other (non-GC-rich) genomes. We also found characteristic differences in profiles of AT genomes, which probably reflect individual species variations in translational regulation. AVAILABILITY The program is available upon request from Alexander Bolshoy or at http://csweb.haifa.ac.il/library/#complex.

SEQUENCE COMPLEXITY AND DNA CURVATURE

A linguistic complexity measure was applied to the complete genomes of HIV-1, Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium, and to long human and yeast genomic fragments. Complexity values averaged over entire genomic sequences were compared, as were predicted average values of intrinsic DNA curvature. We found that both the most curved and the least complex fragments are located preferentially in non-coding parts of the genome. Analysis of location of the most curved and the simplest regions in bacteria showed that the low-complexity segments are preferentially located in close proximity to the highly curved sequences, which are, in turn, placed from 100 to 200 bases upstream to the start of the nearest coding sequence. We conclude that the parallel analysis of sequence complexity and DNA curvature might provide important information about sequence-structure-function relationship in genomes.

Preferred positions of AA and TT dinucleotides in aligned nucleosomal DNA sequences.

Multiple alignment of 118 nucleosomal DNA sequences by maximizing simultaneously match of AA dinucleotides and match of TT dinucleotides results in a pattern of the dinucleotide distributions which is characteristic of the nucleosomal DNA sequences. The AA dinucleotides are found to be distributed symmetrically relative to the TT dinucleotide distribution, around the middle point of the nucleosomal DNA sequence. The distances between major peaks of the distributions are multiples of about 10.4 bases. The peaks of the TT distribution are shifted by 6 bases downstream from the peaks of the AA distribution.

Dinucleosome DNA of Human K562 Cells: Experimental and Computational Characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.

ENVIRONMENTAL INFLUENCES ON DNA CURVATURE

DNA curvature plays an important role in many biological processes. To study environmental influences on DNA curvature we compared the anomalous migration on polyacrylamide gels of ligation ladders of 11 specifically-designed oligonucleotides. At low temperatures (25 degrees C and below) most of the sequences exhibited a degree of anomalous migration. Increased temperature had a significant effect on the anomalous migration (curvature) of some sequences but limited effects on others; at 50 degrees C only 1 sequence migrated anomalously. Mg2+ had a strong influence on the migration of certain sequences, whilst spermine enhanced the anomalous migration of a different set of sequences. Sequences with a GGC motif exhibited greater curvature than predicted by the presently-used angles for the nearest-neighbour wedge model and are especially sensitive to Mg2+. The data have implications for models for DNA curvature and for environmentally-sensitive DNA conformations in the regulation of gene expression.

Involvement of DNA curvature in intergenic regions of prokaryotes

It is known that DNA curvature plays a certain role in gene regulation. The distribution of curved DNA in promoter regions is evolutionarily preserved, and it is mainly determined by temperature of habitat. However, very little is known on the distribution of DNA curvature in termination sites. Our main objective was to comprehensively analyze distribution of curved sequences upstream and downstream to the coding genes in prokaryotic genomes. We applied CURVATURE software to 170 complete prokaryotic genomes in a search for possible typical distribution of DNA curvature around starts and ends of genes. Performing cluster analyses and other statistical tests, we obtained novel results regarding various factors influencing curvature distribution in intergenic regions, such as growth temperature, A+T composition and genome size. We also analyzed intergenic regions between converging genes in 15 selected genomes. The results show that six genomes presented peaks of curvature excess larger than 3 SDs. Insufficient statistics did not allow us to draw further conclusion. Our hypothesis is that DNA curvature could affect transcription termination in many prokaryotes either directly, through contacts with RNA polymerase, or indirectly, via contacts with some regulatory proteins.

Adaptive microclimatic structural and expressional dehydrin 1 evolution in wild barley, Hordeum spontaneum, at ‘Evolution Canyon’, Mount Carmel, Israel

‘Evolution Canyon’ (ECI) at Lower Nahal Oren, Mount Carmel, Israel, is an optimal natural microscale model for unravelling evolution in action highlighting the twin evolutionary processes of adaptation and speciation. A major model organism in ECI is wild barley, Hordeum spontaneum, the progenitor of cultivated barley, which displays dramatic interslope adaptive and speciational divergence on the ‘African’ dry slope (AS) and the ‘European’ humid slope (ES), separated on average by 200 m. Here we examined interslope single nucleotide polymorphism (SNP) sequences and the expression diversity of the drought resistant dehydrin 1 gene (Dhn1) between the opposite slopes. We analysed 47 plants (genotypes), 4–10 individuals in each of seven stations (populations) in an area of 7000 m2, for Dhn1 sequence diversity located in the 5′ upstream flanking region of the gene. We found significant levels of Dhn1 genic diversity represented by 29 haplotypes, derived from 45 SNPs in a total of 708 bp sites. Most of the haplotypes, 25 out of 29 (= 86.2%), were represented by one genotype; hence, unique to one population. Only a single haplotype was common to both slopes. Genetic divergence of sequence and haplotype diversity was generally and significantly different among the populations and slopes. Nucleotide diversity was higher on the AS, whereas haplotype diversity was higher on the ES. Interslope divergence was significantly higher than intraslope divergence. The applied Tajima D rejected neutrality of the SNP diversity. The Dhn1 expression under dehydration indicated interslope divergent expression between AS and ES genotypes, reinforcing Dhn1 associated with drought resistance of wild barley at ‘Evolution Canyon’. These results are inexplicable by mutation, gene flow, or chance effects, and support adaptive natural microclimatic selection as the major evolutionary divergent driving force.