Global distribution of nearly identical phage-encoded DNA sequences

Phages, the most abundant biological entities on the planet, play important roles in biogeochemical cycling, horizontal gene transfer, and defining microbial community composition. However, very little is known about phage diversity or biogeography, and there has not yet been a systematic effort to compare the phages found in different ecosystems. Here, we report that T7-like Podophage DNA polymerase sequences occur in every major biome investigated, including marine, freshwater, sediment, terrestrial, extreme, and metazoan-associated. The majority of these sequences belong to a unique clade that is only distantly related to cultured isolates. Some identical T7-like phage-encoded DNA polymerase genes from this clade were >99% conserved at the nucleotide level in multiple different environments, suggesting that these phages are moving between biomes in recent evolutionary time and that the global genomic pool for T7-like phages may be smaller than previously hypothesized.     

DNA associated with nucleosomes in plants.

50 to 55% of tobacco and barley nuclear DNA is accessible to micrococcal endonuclease digestion. The DNA fragments resulting from a mild endonuclease treatment are multiples of a basic unit of 194 +/- 6 base pairs in tobacco and 195 +/- 6 base pairs in barley. After extensive digestion, a DNA fragment of approximately 140 base pairs is predominant. Hence the "extra-core" or "linker"-DNA is 55 base pairs long. Other fragments having 158 and less than 140 base pairs are present as well. Treatment with DNase I results in multiples of 10 bases when analysed under denaturating conditions. These results show that the general organization of the DNA within the nucleosomes is about the same in higher plants as in other higher eukaryotes.     

Clustering of identical oligomers in coding and noncoding DNA sequences.

We develop a quantitative method for analyzing repetitions of identical short oligomers in coding and noncoding DNA sequences. We analyze sequences presently available in the GenBank separately for primate, mammal, vertebrate, rodent, invertebrate and plant taxonomic partitions. We find that some oligomers "cluster" more than they would if randomly distributed, while other oligomers "repel" each other. To quantify this degree of clustering, we define clustering measures. We find that (i) clustering significantly differs in coding and noncoding DNA; (ii) in most cases, monomers, dimers and tetramers cluster in noncoding DNA but appear to repel each other in coding DNA. (iii) The degree of clustering for different sources (primates, invertebrates, and plants) is more conserved among these sources in the case of coding DNA than in the case of noncoding DNA. (iv) In contrast to other oligomers, we find that trimers always prefer to cluster. (v) Clustering of each particular oligomer is conserved within the same organism.     

Specific association of repetitive DNA sequences with major histocompatibility genes.

The DNA sequence organization of a 17.8-kilobase segment of porcine DNA, containing a functional major histocompatibility (MHC) gene, has been studied. The DNA flanking the MHC gene contains at least 10 distinct repetitive DNA sequence elements, each of which occurs only once within the 17.8-kilobase DNA segment. Their reiteration frequencies in the genome range from 10(2) to 10(4). The genomic organization of seven of these sequence elements has been examined; all are interspersed with other, unrelated DNA sequences. These seven repeated sequences are not generally associated in the genome. However, they appear to be nonrandomly linked in MHC-associated regions of the genome: at least two additional DNA segments containing MHC-homologous DNA also contain sequences homologous to DNA fragments bearing the seven different repeats. Of the seven sequences, four can be detected in splenic total RNA. These results suggest that these repeated elements are specifically associated with the MHC locus.     

Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity.

Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes. To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S. cerevisiae during transformation. We found that homeologous DNAs recombine efficiently. Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity. Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt). We suggest that these events occur by a conventional homologous recombination mechanism.     

Novel strategies to clone identical and distinct DNA sequences for several complex genomes

In this minireview I briefly describe the new methods suggested for cloning sequences identical by descent, homo-or hemizygously deleted, amplified or polymorphic, and compare them with the most efficient techniques developed earlier. The new methods include cloning of identical sequences (CIS), cloning of polymorphic sequences (COP), and cloning of deleted sequences (CODE). Although these methods are based on the same combination of biochemical techniques, their aims are different. These methods are fully complementary, and they may be combined to analyze a given object. If one aims to clone a disease gene responsible for familial cancer syndrome, these methods may be applied as follows. CIS can be used to identify the sequences identical by descent comparing the DNA obtained from affected or unaffected family members. COP can be used to find sequences that are different between affected and unaffected members, and CODE would be useful to compare tumor and normal (control) samples to isolate, deleted sequences (putative candidate tumor suppressor genes) and amplified sequences (putative oncogenes). The COP and CODE procedures can be applied to analyze the CpG islands, thus allowing direct candidate gene identification.     

High-level expression of DNA architectural factor HMGA2 and its association with nucleosomes in human embryonic stem cells

The state of chromatin in human embryonic stem (hES) cells is a key factor determining stem cell identity. The non-histone chromatin-associated factor HMGA2 has been studied mostly in the mouse where its function seems critical for embryonic cell growth and adipocytic cell differentiation. Here we show that HMGA2 is highly expressed in two undifferentiated human embryonic stem cell lines at a level of at least 10(5) copies per individual stem cell. Interestingly, expression is further upregulated by a factor of three at day 7 of embryoid body formation, before it quickly drops to or below the level found in undifferentiated cells. We also show that HMGA2 is stably associated with inter- and metaphase hES cell chromatin, and that up to 12 HMGA2 protomers stably associate in vitro with a single nucleosome core particle of known atomic structure. Our data lend support to the possibility that HMGA2 interacts with nucleosomes in a way that imposes a global effect on the state of ES cell chromatin, which may contribute to the establishment of both ES cell identity and the initiation of specific differentiation programs.     

Asynchronous distance between homologous DNA sequences

The distance between homologous DNA sequences of two species is proposed to be -1/4 ln[det(P)], where P is the conditional probability matrix specifying the proportions of the various nucleotides in the second sequence, corresponding to each of the four nucleotides in the first sequence. A probability model is described which supports this choice of distance. Distance measures based on a constant evolutionary rate assumption are described and compared with the proposed measure. Sampling properties of both types of distance are examined and we conclude by applying the distance measures to mitochondrial DNA sequences of the hominoids.     

Analysis of DNA associated with nucleosomes in pea chromatin

Micrococcal nuclease digestion of chromatin from ungerminated and 48 h-germinated pea embryos yields DNA fragments which are multiples of basic units of 194–195 base pairs. Extensive digestion produces a core particle of 145 base pairs. Deoxyribonuclease I gives rise to fragments which are multiples of 10 bases upon analysis on denaturing gels. These values are comparable with those found for other plant materials. These results indicate that gross changes in nucleosomal organization do not accompany the onset of germination.     

Ion-induced DNA structure change in nucleosomes

Physical methods have been used to study calcium binding to the nucleosome core particle. Equilibrium dialysis of Ca2+ and spectroscopic analysis of a Ca2+ analogue show that the ion binds tightly to the particles, resulting in a significant change of DNA circular dichroism. This suggests that base stacking may be altered as a result of Ca2+ binding. In the presence of Ca2+, the absorbance and fluorescence properties of methylene blue (MB), a DNA-specific intercalator, confirm that the dye binds tightly to nucleosomes by intercalation. However, secondary changes occur which suggest that the MB binding site is altered as a result of Ca2+ binding. Triplet state anisotropy decay and triplet lifetime quenching both show that in the Ca2+-nucleosome complex, methylene blue is capable of wobbling over a substantial angular range at its binding site. To explain these data, it is proposed that Ca2+ binding to nucleosomes causes DNA to fold by means of a series of sharp bends (kinks). The properties of bound MB are best explained if it is presumed that the intercalator binds tightly to such kinked sites in the nucleosome. On the basis of these observations, we discuss the possibility that multivalent ion concentration in the nucleus is high enough that the smooth to kinked helix equilibrium may be near to its midpoint. Near such a midpoint, the secondary structure of DNA in the nucleosome might prove to be sensitive to effector molecule binding and to site-specific variation of DNA or histone composition within genes.     

Physical association of two simple alkylators to some DNA sequences

Molecular mechanics calculations have been used to determine the preferred physical association sites of the known alkylating agent dimethyl aziridinium ion (Az⁺) and a CH prototype test probe with B-form, tetrameric DNA sequences. Electrostatic interactions are most important in determining these preferential physical association sites. In turn, the intermolecular energy minima depend on the charge distribution assigned to the DNA sequence. However, for three reported DNA charge distributions, only two distinct sets of energy minima were obtained for the CH-like ion interacting with (G-C)₄, (A-T)₄, and [(G-C)·(A-T)]₂ deoxyribonucleic acids. These minima correspond to physical association geometries in which the CH-like ion is near known alkylation sites. The results of the Az⁺ … [(G-C)·(A-T)]₂ interaction are virtually identical to those found for the CH-like ion. Aqueous solvation energetics have little effect on the physical association of Az⁺ with [(G-C)·(A-T)]₂.     

Strong nucleosomes of mouse genome including recovered centromeric sequences

Recently discovered strong nucleosomes (SNs) characterized by visibly periodical DNA sequences have been found to concentrate in centromeres of Arabidopsis thaliana and in transient meiotic centromeres of Caenorhabditis elegans. To find out whether such affiliation of SNs to centromeres is a more general phenomenon, we studied SNs of the Mus musculus. The publicly available genome sequences of mouse, as well as of practically all other eukaryotes do not include the centromere regions which are difficult to assemble because of a large amount of repeat sequences in the centromeres and pericentromeric regions. We recovered those missing sequences using the data from MNase-seq experiments in mouse embryonic stem cells, where the sequence of DNA inside nucleosomes, including missing regions, was determined by 100-bp paired-end sequencing. Those nucleosome sequences, which are not matching to the published genome sequence, would largely belong to the centromeres. By evaluating SN densities in centromeres and in non-centromeric regions, we conclude that mouse SNs concentrate in the centromeres of telocentric mouse chromosomes, with ~3.9 times excess compared to their density in the rest of the genome. The remaining non-centromeric SNs are harbored mainly by introns and intergenic regions, by retro-transposons, in particular. The centromeric involvement of the SNs opens new horizons for the chromosome and centromere structure studies.     

Kinetic differences in cardiac myosins with identical loop 1 sequences

The kinetics of nucleotide turnover vary considerably among isoforms of vertebrate type II myosin, possibly due to differences in the rate of ADP release from the nucleotide binding pocket. Current ideas about likely mechanisms by which ADP release is regulated have focused on the hyperflexible surface loops of myosin, i.e. loop 1 (ATPase loop) and loop 2 (actin binding loop). In the present study, we investigated the kinetic properties of rat and pig beta-myosin heavy chains (beta-MHC) in which we have found the sequences of loop 1 (residues 204-216) to be virtually identical, i.e. DQSKKDSQTPKG, with a single conservative substitution (rat E210D pig). Pig myocardium normally expresses 100% beta-MHC, whereas rat myocardium was induced to express 100% beta-MHC by surgical thyroidectomy and subsequent treatment with propylthiouracil. Slack test measurements at 15 degrees C yielded unloaded shortening velocities of 1.1 +/- 0.8 muscle lengths/s in rat skinned ventricular myocytes and 0.35 +/- 0.05 muscle lengths/s in pig skinned myocytes. Similarly, solution measurements at the same temperature showed that actin-activated ATPase activity was 2.9-fold greater for rat beta-myosin than for pig beta-myosin. Stopped-flow methods were then used to assess the rates of acto-myosin dissociation by MgATP both in the presence and absence of MgADP. Although the rates of MgATP-induced dissociation of acto-heavy meromyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was approximately 3.8-fold faster for rat beta-myosin (135 s(-)(1)) than for pig beta-myosin (35 s(-)(1)). ATP cleavage rates were nearly 30% faster for rat beta-myosin. Thus, whereas loop 1 appears from other studies to be involved in nucleotide turnover in the pocket, our results show that loop 1 does not account for large differences in turnover kinetics in these two myosin isoforms. Instead, the differences appear to be due to sequence differences in other parts of the MHC backbone.     

Association of DNA polymerase with nucleosomes from mammalian cell chromatin

More than half of the DNA polymerase beta in mouse ascites cell chromatin was found to be associated with monomeric nucleosomal particles (produced by micrococcal nuclease treatment of chromatin). Almost all nuclear DNA polymerase activity in lymphocytes was found to be associated with nucleosomes. The nucleosome-associated enzyme was mainly DNA polymerase beta in chromatin from resting and mainly DNA polymerase alpha in chromatin from concanavalin-A-stimulated lymphocytes.