Content and localisation of 5-methylcytosine in DNA of healthy and wilt-infected cotton plants.

5-Methylcytosine has been found in all pyrimidine isopliths isolated from the DNA of cotton plants, but it localizes predominantly in tri- (about 52%) and dipyrimidine (about 22%) clusters. The 5-methylcytosine distribution by pyrimidine isopliths in DNA of cotton plants is specific and quite different from that in other plant and animal DNA studied. The total 5-methylcytosine content in DNA from wilt-infected cotton plants (2.3 mol %) is less than half that found in DNA from non-infected cotton plants (4.9 mol %). No other visible differences (G+C content, Tm, deltaT, s20,w, frequencies of pyrimidine clusters and others) in these DNA have been found. This suggests that in wilt-infected plants, no essential alteration in DNA sequence or molecular population takes place. As a result of wilt infection 5-methylcytosine completely disappears from dipyrimidine oligonucleotides of cotton plant DNA; its content decreases markedly in long pyrimidine clusters (heptaoligonucleotides and longer) and in C3, C2 T, CT2 fragments. Thus, DNA in wilt-infected plant cells is specifically undermethylated (demethylated). The induced alteration in DNA methylation may be considered one of the possible mechanisms for the specific distortion of gene activity of host cells and primary fungal pathogenic action on plants.     

Lipids of Cuscuta reflexa and changes in lipids of its host plants after infection

The lipid level (fresh weight basis) of Cuscuta reflexa Roxb. was related to the lipid content of the host plants Meilicago saliva L., Helianthus annuus L., Pisum sativum L. and Lantana camara L. Parasitizing by the dodder significantly increased the total lipid level of the hosts. The increase was mainly due to enhancement in the neutral lipid fraction.
The level of phospholipid in the parasite was always higher than in its hosts. Phospholidyl choline and phosphatidyl ethanolamine constituted about 65% of the total phospholipid of Cuscuta. This was followed by phosphatidyl inositol (ca 20%) and phosphatidyl glycerol (ca 12%). Phosphatidic acid constituted only ca 3% of the phospholipids of Cuscuta. Although the total phospholipid levels of various host plants were not affected as a result of the infection by Cuscuta, a significant decrease occurred in the levels of phosphatidyl eholine and phosphatidyl ethanolamine as well as marked increases in phosphatidyl inositol and phosphatidic acid. The infected tissue showed an increase in phospholipase D activity as compared with the controls. The results have been discussed in relation to changes in permeability of the infected tissue.     

Changes in abundance of native and introduced parasites (Hymenoptera: Braconidae), and of the target and non-target plant bug species (Hemiptera: Miridae), during two classical biological control programs in alfalfa.

High numbers of tarnished plant bugs [Lygus lineolaris (Palisot)], were once common in alfalfa, as was a low level of parasitism (9%) by the native Peristenus pallipes (Curtis). After the bivoltine European parasite Peristenus digoneutis Loan became well established, average parasitism of the first and second generations increased to 64%, and tarnished plant bug numbers dropped by 65%. This reduced host density eventually caused a decline in total parasitism by both parasite species to 22%. A few P. digoneutis also attacked the alfalfa plant bug, Adelphocoris lineolatus (Goeze), but did not reduce this pest or increase its parasitism rate. At another location, where P. digoneutis is not established, parasitism of first generation alfalfa plant bugs, an adventive (accidently introduced) pest, was increased to 21% by the introduced univoltine parasite, Peristenus conradi Marsh, and a slight reduction in the pest may have resulted. P. digoneutis did not parasitize the meadow plant bug, Leptopterna dolabrata (L.), an adventive pest of forage grasses, so did not affect this mirid or its parasite. Neither introduced parasite eliminated the native parasites of the tarnished or alfalfa plant bugs. The narrow host ranges of the braconid parasites of mirid nymphs are contrasted with the broad host range of the native tachinid parasite [Phasia robertsoni (Towns.)] of adult mirids. The major changes in mirid abundance and their mortality by parasites that slowly occurred during this 19-year study demonstrate the need for long-term field research, to adequately document and understand these complex interactions.     

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.     

Synchronous synthesis of DNA in coleoptiles and the initial leaf of developing etiolated wheat shoots: the nature and correlation of nuclear and mitochondrial DNA syntheses

In etiolated coleoptiles and initial leaf of developing wheat shoots the DNA synthesis is periodical and synchronous. In the initial leaf each step of DNA synthesis results in a stepwise increase of DNA content and is doubled at the first three steps. During the leaf plane formation the synthesis of nuclear DNA (nDNA) is decreased, while that of mitochondrial DNA (mitDNA) continues in synchronous cycles. This is the cause of relative stabilization of DNA content per unit of leaf plane length. The DNA increase in this organ occurs due to synchronous synthesis of nDNA and mitDNA in intercalary meristem cells. In coleoptiles a marked replication of nDNA is observed at the first three steps of the synthesis; in each cycle nDNA synthesis precedes that of mitDNA. With completion of coleoptile formation the nDNA synthesis in it practically ceases, whereas that of mitDNA continues in synchronous cycles. MitDNA is non-methylated and its composition (56 mol.% GC) differs significantly from that of the newly synthesized nDNA (44 mol.% GC; 100 X m5C/(C + m5C) = 16-17%). It may be concluded that in various organs of wheat shoots the composition and methylation of newly synthesized DNA depend on the age of the shoot and on the ratio of nDNA/mitDNA syntheses.     

DNA methylation and cellular ageing.

Methylated cytosine (m5C) in DNA appears to be an important modulator of the expression of some genes. There are several lines of evidence that gradual loss of m5C is relevant to in vitro cellular ageing: m5C loss occurs during cell culture; m5C loss is detectable at an early stage of culture; m5C loss appears to be related to cell division not just duration in culture; the rate of m5C loss appears to be related to in vitro lifespan of the cell strain in question; and the total loss of m5C during an in vitro lifespan is significant by comparison with induced-changes in m5C levels which effect cell growth, or cause cell-death in culture. Progressive loss of m5C in dividing cells may thus produce the multi-step cell division "clock" which underlies the Hayflick phenomenon.     

Inhibition of DNA methylation by chemical carcinogens in vitro

A diverse range of ultimate chemical carcinogens inhibited the transfer of methyl groups from S-adenosylmethionine to hemimethylated DNA in a reaction catalyzed by mouse spleen methyltransferase. The formation of alkali-labile sites in DNA lessened its ability to accept methyl groups in vitro, but the methylation reaction was much less sensitive to thymine dimers or double-strand breaks. Carcinogens induced the formation of alkali-labile DNA lesions, but the degree of methyltransferase inhibition observed was greater than that expected for this damage alone. Certain carcinogens were also capable of direct modification and inactivation of the methyltransferase enzyme. Benzo(a)pyrene treatment of living BALB/3T3 A31 clone 1-13 but not C3H/10T1/2 clone 8 cells resulted in a 12% decrease in total 5-methylcytosine content of cellular DNA. Carcinogenic agents may therefore cause heritable changes in 5-methylcytosine patterns in certain cell types by a variety of mechanisms, including adduct formation, induction of apurinic sites and single-strand breaks and direct inactivation of DNA methyltransferase.     

Study of the methylation process and DNA methylase specificity in Shigella]

The nature and content of minor bases in DNA of 3 Shigella strains are investigated. DNAs from Shigella stutzeri 2, Sh. sonnei 1188 and Sh. sonnei 311 are found to contain 0.43, 0.56 and 0.45 mol.% of N6-methyladenine respectively. 5-methylcytosine (0.16 mol.%) is discovered in Sh. sonnei 311. Substrate specificity of adenine methylase from Sh. sonnei 1188 with respect to phage DNAs of different host modification is investigated. Recognition sites for guanine methylase of DDVI phage and for adenine methylase of Sh. sonnei 1188 turned to be different. DNA of DDII phage grown in Sh. stutzeri 2 cells does not accept methyl groups under the treatment with Sh. sonnei 1188 extracts, but it is methylated by Escherichia coli extract. Adenine methylases of Sh. sonnei 1188 and Sh. stutzeri 2 are suggested to be either the same enzyme, or enzymes, which recognition sites are partially overlapped.     

Correlation of the increase in DNA methylation and antioxidant activity of mouse liver nuclear lipids after administration of antioxidant and in Ehrlich ascites carcinoma

The content of 5'-methylcytosine in total DNA of mouse liver increases 2--2,5-fold 3 hrs after a single intraperitoneal injection of antioxidant (4-methyl-2,6-ditretbutylphenol) (20 or 60 mg per 1 kg of body weight) and makes up to 2--2.4 mol.%. The methylation of liver DNA is also increased more than 2-fold in Ehrlich ascite carcinoma. The DNA isolated from mouse liver after administration of antioxidant or during cancer growth markedly differs from liver DNA of intact animals in its CH3-accepting ability under in vitro methylation by the methylase complex from Enterobacter cloacea. The changes in DNA methylation in mouse liver under the effects of antioxidant and in Ehrlich ascite carcinoma are correlated with the changes in the antioxidant activity of liver nuclear lipids.     

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.     

Relationship of DNA methylation level to the presence of heterochromatin in mealybugs.

Purified nuclear DNA from two mealybug species was analyzed for its 5-methylcytosine (m5C) content by reversed-phase high-pressure liquid chromatography. We observed that the percent m5C (percentage of cytosines which are methylated) varied between the two species, between males and females of the same species, and between lines with and without supernumerary B chromosomes. This is the first case of a sex-specific difference in overall DNA methylation level. In contrast to a recent report (Deobagkar et al., J. Biosci. [India] 4:513-526, 1982), we found no other modified bases in the DNA. Overall, the percent m5C in Pseudococcus obscurus was two to three times higher than in Pseudococcus calceolariae. In both species, the percent m5C in males was higher than in females, although only in P. calceolariae was the difference statistically significant (0.68 +/- 0.02 versus 0.44 +/- 0.04). The high m5C content in males was correlated with the presence of a paternally derived, genetically inactive set of chromosomes which is facultatively heterochromatic. The presence of constitutive heterochromatin, however, was associated with a lower m5C content. Thus, for example, the percent m5C in females of a P. obscurus line with heterochromatic B chromosomes (1.09 +/- 0.04) was significantly lower than that of a related line lacking such chromosomes (1.26 +/- 0.06). Our findings are discussed with respect to the possible relationship between DNA methylation and heterochromatization.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Slavery in plants: how the facultative hemi‐parasitic plant Rhamphicarpa fistulosa can completely dominate its host

The rain‐fed lowland rice weed Rhamphicarpa fistulosa (Rice Vampireweed) is a facultative root parasitic plant. Growth and reproduction of R. fistulosa benefit considerably from parasitism, but how this affects the host plant is not well established. We determined accumulation and partitioning of rice–parasite biomass in two pot experiments. First, rice (cv. IR64) was grown under eight R. fistulosa densities (15–1000 seeds per pot) with two sampling times. Next, 2 parasite densities (6 and 13 plants per pot) were combined with 9 destructive samplings. Infection increased host root: shoot ratios and decreased host plant height, leaf area and tiller number. Reductions in light interception were followed by reductions in light use efficiency, causing 22–71% losses in host plant biomass and 78–100% losses in host kernel production. Parasitism eventually caused a complete standstill of host plant growth, while the parasite managed to gradually increase its share in total host plant–parasite biomass up to 50–82%. This implies that ultimately the host plant was producing solely for the sake of the parasite. Due to its facultative nature, R. fistulosa may incorrectly be perceived as relatively harmless. Upon infection this Rice Vampireweed, however, turns into a genuine slave master, whereby it completely dominates its host.     

Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.     

Host preferences of introduced and native parasites (Hymenoptera: Braconidae) of phytophagous plant bugs (Hemiptera: Miridae) in alfalfa-grass fields in the northeastern USA

During an eight-year field study at two widely-separated locations (NJ & DE, USA), seven species of phytophagous mirid plant bugs were found in alfalfa and alfalfa-grass fields grown for animal forage. Five species of parasites were reared from these mirids. Two parasite species introduced from Europe killed significantly higher proportions of nymphs of the two important mirid pests of alfalfa than did native parasites. The introduced Peristenus digoneutis Loan parasitized an average of 31% of first and second generation tarnished plant bugs, Lygus lineolaris, a native insect; and the introduced Peristenus conradi parasitized 22% of first generation alfalfa plant bugs, Adelphocoris lineolatus, an introduced species. In addition, Peristenus pallipes significantly parasitized nymphs of Trigonotylus caelestialium (43%) and Leptopterna dolabrata (37%); both mirids are immigrant species. Because the parasite P. pallipes significantly attacked only these two non-native mirids, and is present in Europe, it also may be an accidental introduction to North America. A native wasp, Leiophron uniformis, heavily parasitized (49%) the native garden fleahopper, Halticus bractatus. A third native species, P. pseudopallipes, occasionally parasitized a few Lygus lineolaris in alfalfa at one location. Two other mirids, Stenotus binotatus and Megaloceroea recticornis, both accidently-introduced grass-feeding species, were not parasitized by native or introduced species. It is noteworthy that the effective host ranges of all the parasites in alfalfa-grass fields were limited: four of the five parasite species significantly parasitized only one of the seven mirid species, and the other parasite significantly parasitized only two mirids. Activity of the four common parasites was correlated with the mirids' host plant: three species principally parasitized alfalfa-feeding mirids, and one species principally parasitized grass-feeding mirids.     

Effect of tamponade on the nucleotide composition and cytosine methylation level of DNA in the heart muscle of dogs

Base composition and quantitative changes in the 5-methylcytosine (m5c) content in DNA isolated from male dogs' heart muscle under the acute tamponade have been studied. Due to the effect of tamponade statistically reliable decrease (15%) in m5c content in DNA has been found to take place in experiment in comparison with control. Possible mechanisms of DNA methylation decrease by cytosine are under discussion.