Tissue specificity of the decrease of cattle lymphocyte DNA methylation during chronic lymphoid leukemia]

It has been found that the content of m5C in the DNA preparations tested have been revealed. The DNAs from normal and leukemic lymphocytes of blood, lymphonodi and spleen differ in ther acceptor ability in the reaction of heterologous methylation in vitro, induced by DNA-methylase from Enterobacter cloacea in the presence of [3H-methyl]S-adenosyl methionine: the ratio of radioactivities in methylated cytosine and adenine residues (m5C/m6A) in leukemic lymphocyte DNA is much lower than in healthy animals' lymphocytes. The decrease in the methylation of DNAs from various lymphoid organs of animals with chronic lymphoid leukemia is well correlated with the impairment. No significant changes of the m5C level and the acceptor ability of the in vitro reaction of heterologous methylation of cow lymph lymphocyte DNA have been observed. The data obtained may be interpreted in terms of tissue (cell) specificity or differences in the degree of DNA methylation under conditions of chronic lymphoid leukemia. It is assumed that the changes in DNA methylation may underlie the disturbances in the regulation of activity of the leukemic cell genetic mechanisms.     

Changes in the methylated sequences and molecular population of wheat DNA during germination

The fractions of unique (Cot less than 405), moderately (Cot=0.13--405) and highly reiterated (Cot less than 0--0.13) sequences were isolated from DNA of wheat seeds and 3 day old seedlings, and GC content, amount of 5-methylcytosine and its distribution among various pyrimidine isostichs in the fractions isolated were studied. Different in Cot value DNA fractions from seeds or from seedlings are similar in GC content and in all other characteristics studied. Seed DNA differs from DNA of seedlings in the content of pyrimidine isostichs from the respective fractions of reiterated sequences. Pronounced differences in the amount of pyridmidine clusters with various base composition in the corresponding fractions of DNA from seeds and seedlings were found. These differences in the frequencies of respective pyrimidine clusters from DNA of seeds and seedlings may be considered as being a result of changes in the molecular population of wheat DNA on germination. The seed and seedling DNA differ significantly in the 5-methylcytosine content in the respective pyrimidine isostichs isolated from unique sequences. In the seedling DNA some other nucleotide sequences are to be methylated as compared to DNA of dormat seeds. Thus, on germination some changes occur in DNA methylation as well as in the genome organization.     

Effect of hydrocortisone on methylation and molecular population of DNA in rat liver]

The content of 5-methyl cytosine in rat liver DNA increases 1,7-fold 8 hours after intraperitoneal injection of hydrocortisone (5 mg per 100 g animal weight). The content of GC, physicochemical parameters (Tm, delta T, etc.) and DNA renaturation pattern did not show any changes. No changes were observed in the pattern of H3-thymidine incorporation into rat liver DNA: after hydrocortisone injection the radioactivity was found to be equally distributed in all isolated sequences of DNA, differing in the degree of reiteration (specific radioactivities of these DNA, fractions are very similar). Thus, the molecular population of DNA in liver cells remains unchanged, which suggests that the hormone-induced increase in the 5-methyl cytosine content is due to a change in the DNA methylation level. The methyation level of unique sequences (COt greater than 600), i. e. that of structural genes, does not undergo any essential changes. The reversible methylation of DNA regulated by hormones seems to be one of the mechanisms controlling gene activity.     

Changes in base composition and molecular population of wheat DNA on germination]

Germination of wheat seeds results in small changes of the GC content of total DNA (from 47.5 to 49.0 mole %): at the same time the amount of 5-methylcytosine in seeds 10 hours after wetting and at day 3 of germination significantly decrease (from 6.0 to 5.4 and 5.2 mole %, respectively). The wheat genome is methylated in non-uniform fashion: moderute repeats (less than a hundred copies, interval Cot = 0,12 . 10(2)-420) possess the maximal amount of 5-methylcytosine, while the unique sequences (Cot greater than 420) have the lowest 5-methylcytosine content. Methylation of highly reiterated sequences (Cot less than 0,8 . 10(-2) is similar to that of the total DNA. At day 3 of germination the amount of 5-methycytosine in all DNA fractions is lower as compared with these fractions isolated from DNA of dormant seeds. This is probably due to (1) diminution in the amount of reiterated sequences with high 5-methylcytosine content and (2) to lowering of DNA methylation level in germinating seeds. Changes in DNA methylation may be associated with the regulation of gene activity in the differentiating plant cells at various stages of ontogenesis.     

Nuclear DNA changes within Helianthus annuus L.: variations in the amount and methylation of repetitive DNA within homozygous progenies

Complex alterations in the redundancy and methylation of repeated DNA sequences were shown to differentiate the nuclear genome of individuals belonging to single progenies of homozygous plants of the sunflower. DNA was extracted from seedlings obtained from seeds collected at the periphery of flowering heads (P DNA) or from seedlings obtained from seeds collected in their middle (M DNA). Three fractions of repeated sequences were isolated from genomic DNA: a highly repetitive fraction (HR), which reassociates within an equivalent Cot of about 2 × 10^-1, and two medium repetitive fractions (MR1 and MR2) having Cot ranges of about 2 × 10^-1-2 and 2-10², respectively. Denaturation kinetics allowed different sequence families to be recognized within each fraction of repetitive DNA, and showed significant differences in sequence redundancy to occur between P and M DNA, particularly as far as the MR2 fraction is concerned. Most DNA sequence families are more represented in P DNA than in M DNA. However, the redundancy of certain sequences is greater in the latter than in the former. Each repetitive DNA fraction was hybridized to Southern blots of genomic P or M DNA which was digested to completion by three pairs of isoschizomeric restriction endonucleases which are either insensitive or sensitive to the methylation of a cytosine in the recognition site. The results obtained showed that the repetitive DNA of H. annuus is highly methylated. Clear-cut differences in the degree of methylation of P and M DNA were found, and these differences were particularly apparent in the MR2 fraction. It is suggested that alterations in the redundancy of given DNA sequences and changes in their methylation patterns are complementary ways to produce continuous genotypic variability within the species which can be exploited in environmental adaptation.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2     

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

Background  

Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.     

Deoxyribonucleic acid sequence organization in the genome of the dinoflagellate Crypthecodinium cohnii

Details of the general DNA sequence organization in the dinoflagellate Crypthecodinium cohnii have been obtained by using hydroxylapatite binding experiments, S1 nuclease digestion .and electron microscopy of reassociated DNA. It has been found that roughly half of the genome is made up of unique sequences interspersed with repeated sequence elements with a period of approximately 600 nucleotides. This class represents roughly 95% of the total number of interspersed unique elements in the genome. The remaining 5% are uninterrupted by repeated sequences for at least 4000 nucleotide pairs. The interspersed repeated elements are narrowly distributed in length with 80% under 300 nucleotide pairs in length. About half of the repeated DNA (20-30% of the genome) is not interspersed among unique sequences. The close spacing of the short repeats interspersed throughout much of the genome is consistent with the occurrence of the huge network structures observed in the electron microscope for low Cot reassociation of moderately long fragments. An unusual class of heteroduplexes was detected in the electron microscope which is believed to derive from the reassociation of repeated sequences from different families which are frequently found adjacent to one another in different locations in the genome. The occurrence of this novel arrangement of repeated sequences may reflect the unusual organization of the dinoflagellate nucleus. However, in most respects the sequence arrangement in this unicellular alga is very typical of higher plants and animals.     

Interspecies variability in the organization of repeated sequences of the genus Hordeum

Seven barley species have been compared for organization of repeated sequences. Quantitative variation of repeated DNA fractions is demonstrated, though the total amount of sequences (reassociation up to Cot=10) in most cases does not vary. The repeats are divided into four groups by the mode of interspecific variability, with the help of dot and blot hybridization of the genomes under study with cloned highly repeated sequences of Hordeum vulgare. The first group contains the pHv7161 family of the most conservative sequences. The second group comprises moderately changing repeats. The third group includes highly variable Hind III repeats of Hordeum genomes, and the fourth group is represented by pHv7191 family of repeats that are highly amplified in H. vulgare genome. Comparative analysis of content and organization of highly repeated sequences in genome helps to clarify phylogenetic relationships in the genus and can be used for prediction of successfullness of interspecific hybridization.     

Interspersion and transcription of repeated sequences of rat DNA.

Repetitive rat DNA reassociated to Cot=0.1 and deprived of "foldback" sequences showed close interspersion with unique sequences. As measured by electron microscopy, the average length of repetitive segments was about 600 +/- 400, and of unique segments 1800-3600 base pairs. Pyrimidine tracts over 80 nucleotides in length were found mainly in foldback and repetitive fractions. Oligo(dT) tracts, 20-30 bases in length prevailed in the DNA fraction reassociated to Cot=0.1. Repetitive and unique DNA fractions were annealed to Millipore filters and hybridized with hnRNA. Up to 1.6% of repetitive DNA reassociated to Cot=0.05 showed base complementarity with hnRNA, whereas the comparative figures for DNA reassociated to Cot=10 and for the unique fraction were 0.8% and 0.3% respectively. When hybridization of hnRNA was carried out in solution in vast DNA excess, no hybrid formation with repetitive sequences reassociated to Cot=0.1 was observed, although hybridization with DNA reassociated to Cot=10 was noticeable.     

DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.     

Isolation and identification of lymphocytic and myelogenous leukemia-specific sequences in genomes of gibbon oncornaviruses

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) “COM,” (ii) “LYM” or “MYE,” (iii) “UNI,” and (iv) “UND.” The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.     

Isolation and characterization of the highly repeated fraction of the banana genome

Although the nuclear genome of banana (Musa spp.) is relatively small (1C approximately 610 Mbp for M. acuminata), the results obtained from other sequenced genomes suggest that more than half of the banana genome may be composed of repetitive and non-coding DNA sequences. Knowledge of repetitive DNA can facilitate mapping of important traits, phylogenetic studies, BAC-based physical mapping, and genome sequencing/annotation. However, only a few repetitive DNA sequences have been characterized in banana. In this work, we used DNA reassociation kinetics to isolate the highly repeated fraction of the banana genome (M. acuminata 'Calcutta 4'). Two libraries, one prepared from Cot 相似文献   

The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.).

We report the results of a study on the effectiveness of Cot filtration (CF) in the characterization of the gene space of bread wheat (Triticum aestivum L.), a large genome species (1C = 16,700 Mb) of tremendous agronomic importance. Using published Cot data as a guide, 2 genomic libraries for hexaploid wheat were constructed from the single-stranded DNA collected at Cot values > 1188 and 1639 M x s. Compared with sequences from a whole genome shotgun library from Aegilops tauschii (the D genome donor of bread wheat), the CF libraries exhibited 13.7-fold enrichment in genes, 5.8-fold enrichment in unknown low-copy sequences, and a 3-fold reduction in repetitive DNA. CF is twice as efficient as methylation filtration at enriching wheat genes. This research suggests that, with improvements, CF will be a highly useful tool in sequencing the gene space of wheat.     

Molecular analysis of the genomic organization of the Hymenoptera Diadromus pulchellus and Eupelmus vuilleti

The genomic organization of two parasitic wasps was analyzed by DNA reassociation. Cot curves revealed a pattern with three types of components. A highly repetitive DNA, accounting for 15 to 25% of the genome, was identified as satellite DNA. The moderately repetitive DNA corresponds to 26 to 42% of the genome in both species, and shows large variations in complexity, repetitive frequency and a number of sub-components between males and females. These variations are seen as resulting from DNA amplification during somatic and sexual differentiation. Dot blot analyses show that such DNA amplifications concern several types of structural and regulatory genes. The presence of repeated mobile elements was studied by the Roninson method to compare the repeated sequence patterns of Diadromus pulchellus and Eupelmus vuilleti with those of Drosophila melanogaster. The occurrence and organization of mobile elements in these Hymenoptera differ from those of the neighboring order of Diptera. The repetitive and unique components define very large genomes (1 to 3 × 10⁹ base pairs). The genomic organization in Parasitica appears to be an extreme drosophilan type. We propose that the germinal genome of these parasitic wasps is primarily composed of satellite DNA blocks and very long stretches of unique sequences, separated by a few repeated and/or variously deleted, interspersed elements of each mobile element family.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.