Rat serum albumin synthesis in variant rat hepatoma cells

Variant subclones of the rat hepatoma cell line FU5-5 have been isolated that are altered in their production of rat serum albumin. Three of these variants, isolated in a random screening, have been categorized as high, intermediate, and low producers. They secrete albumin into the culture medium at different rates: 16, 1.7, and 0.3 μg/mg cell protein/48 h. A fourth variant, isolated on the basis of altered morphology, secretes no detectable albumin. Unlike the albumin-producing variants, this null variant is also deficient in the level and inducibility of tyrosine aminotransferase activity. Albumin biosynthesis as determined in pulse-labeling experiments is affected similarly in the four variants, yielding albumin synthetic rates of 0.24, 0.035, 0.006, and < 0.002% of total protein synthesis. The translatable albumin messenger RNA content in these variants was measured using a rabbit reticulocyte lysate system. The null variant contains no detectable mRNA, and the three quantitative variants contain levels of translatable albumin messenger RNA corresponding to 0.07, 0.03, and 0.005% of total stimulated polypeptide synthesis. The highest producing variant contains less translatable albumin mRNA than expected on the basis of cellular biosynthetic measurements, suggesting a translation efficiency difference in this clone. Cell hybrids constructed by fusing the high-producing clone and the null variant produce little or no albumin. This extinction indicates that the null variant contains a diffusible regulatory factor capable of decreasing albumin gene expression. The relatively stable and discrete heritable phenotypic changes exhibited by these clones may serve as a model for similar changes that occur during hepatic differentiation.     

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.     

Hypothesis of the initiation of DNA methylation de novo and allelic exclusion by small RNA

We have discovered that 5'-CG-3' dinucleotide and 5'-CNG-3' trinucleotide are found in published sequences of small interfering RNA and microRNA more often than they should be found in a random sequence. This circumstance is evidence of an important biological purpose of 5'-CG-3' dinucleotides and 5'-CNG-3' trinucleotides in small RNA sequences. We suppose that small RNAs containing mentioned di- and trinucleotides participate in creation of chromatin marks of epigenetic information through high-specific search of DNA sequences liable to repression and through initiation of the methylation de novo of 5'-CG-3' and 5'-CNG-3' sites in DNA fragments, which appeared to be bound complementary with small RNA. Several genes can be inactivated simultaneously when they contain the motif which is recognized by small RNA. Allelic exclusion appears, to our opinion, as a result of initiation by small RNA of de novo DNA methylation of all alleles but one that exist in the cell. The predecessor of this small RNA is transcribed from the antiparallel allele chain. Those alleles are inactivated which antiparallel chain is less actively read by RNA-polymerase, which, as we suppose, releases DNA from attached to it small RNA in the process of transcribing. But the quantity of small RNA which is transcribed from just one allele is insufficient to overcome the level when the repression process of this allele de novo starts.     

Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome

Mammalian DNA methyltransferases methylate cytosine residues within CG dinucleotides. By statistical analysis of published data of the Human Epigenome Project we have determined flanking sequences of up to +/-four base-pairs surrounding the central CG site that are characteristic of high (5'-CTTGCGCAAG-3') and low (5'-TGTTCGGTGG-3') levels of methylation in human genomic DNA. We have investigated the influence of flanking sequence on the catalytic activity of the Dnmt3a and Dnmt3b de novo DNA methyltransferases using a set of synthetic oligonucleotide substrates that covers all possible +/-1 flanks in quantitative terms. Methylation kinetics experiments revealed a >13-fold difference between the preferred (RCGY) and disfavored +/-1 flanking base-pairs (YCGR). In addition, AT-rich flanks are preferred over GC-rich ones. These experimental preferences coincide with the genomic methylation patterns. Therefore, we have expanded our experimental analysis and found a >500-fold difference in the methylation rates of the consensus sequences for high and low levels of methylation in the genome. This result demonstrates a very pronounced flanking sequence preference of Dnmt3a and Dnmt3b. It suggests that the methylation pattern of human DNA is due, in part, to the flanking sequence preferences of the de novo DNA MTases and that flanking sequence preferences could be involved in the origin of CG islands. Furthermore, similar flanking sequence preferences have been found for the stimulation of the immune system by unmethylated CGs, suggesting a co-evolution of DNA MTases and the immune system.     

Polar Head Group Decarboxylation and Methylation of Phospholipids: An Alternate Route for Phosphatidylcholine Formation in Cultured Neuronal Cells

Decarboxylation of phosphatidylserine (SPG) and methionine-dependent, stepwise methylation of phosphatidylethanolamine (EPG) to form phosphatidylcholine (CPG) were examined in monolayer cultures of rat cerebral cells. Ethanolamine, monomethylaminoethanol, or dimethylaminoethanol nitrogenous bases (N-bases) added to culture medium at millimolar level result each in synthesis of the corresponding phospholipid via a de novo pathway at initial rates of 0.18, 0.30, and 0.36 nmol/h/micrograms DNA, respectively. Addition of methyl-labeled methionine to culture medium at tracer levels or at millimolar concentration enabled measurements of the rates of phospholipid methylation from EPG phosphatidylmonomethylaminoethanol (Me1EPG) and phosphatidyldimethylaminoethanol (Me2EPG) precursors. At tracer doses, the rates of methylation from the above respective phospholipids are 0.45, 1.17, and 1.70 pmol/h/micrograms DNA. At 1 mM methionine, synthesis of CPG proceeds from [14C]EPG or [14C]Me2EPG at initial rates of 8 and 17 pmol/h/micrograms DNA, respectively. Although the latter phospholipid analog can be generated from its monomethyl precursor, methylation of EPG does not result in the accumulation of Me2EPG, suggesting two segregated and metabolically distinct pathways. In the presence of N-bases, of the total [3H]serine incorporated into cellular phospholipids 30-36.5% of labelled SPG is converted into decarboxylation products. The decarboxylation and methylation routes contribute a significant portion of choline from endogenous sources, most likely through conversion of SPG.     

镉胁迫下萝卜基因组DNA甲基化敏感扩增多态性分析

应用甲基化敏感扩增多态性(MSAP)技术分析了重金属镉(cd)胁迫处理后萝卜基因组DNA甲基化程度的变化。结果表明,经50、250和500mg/L CdCl_2处理后,MSAP比率分别为37%、43%和51%,均高于对照(34%);全甲基化率(双链C~mCGG)分别为23%、25%和27%,而其对照为22%,表明重金属CdCl_2胁迫后,某些位点发生了重新甲基化。萝卜叶片DNA中总甲基化水平的增加与CdCl_2处理浓度呈显著正相关。甲基化变异可分为重新甲基化、去甲基化、不定类型以及与对照相同的甲基化模式等类型,Cd胁迫处理引起的植株基因组DNA甲基化程度的提高主要是重新甲基化。     

Rates of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vivo

The absolute rate of cholesterol acquisition from de novo synthesis and from receptor-dependent and receptor-independent low-density lipoprotein (LDL) uptake was determined in the adrenal glands of the rat, hamster and rabbit under in vivo conditions. The rate of incorporation of [3H]water into cholesterol in the adrenal gland was much higher in the hamster (1727 nmol/h per g) and rabbit (853 nmol/h per g) than in the rat (71 nmol/h per g). Assuming that 23 atoms of 3H are incorporated into the cholesterol molecule during its biosynthesis, the absolute rates of cholesterol synthesis were then calculated to equal 59, 29 and 2.4 micrograms/h per g of adrenal gland in the hamster, rabbit and rat, respectively. Rates of LDL-cholesterol uptake were measured using a primed continuous infusion of [14C]sucrose-labeled homologous LDL (total LDL transport) and methylated human LDL (receptor-independent LDL transport). The rate of total LDL-cholesterol uptake in the adrenal gland was much higher in the rabbit (227 micrograms/h per g) than in the rat (18 micrograms/h per g) or hamster (6 micrograms/h per g). In all three species LDL uptake was mediated largely (greater than 93%) by receptor-dependent mechanisms. In terms of total cholesterol acquisition, the hamster adrenal gland derived 10-times more cholesterol from de novo synthesis than from LDL uptake, whereas the converse was true in the rabbit. Rates of de novo synthesis and LDL-cholesterol uptake were both low in the rat adrenal gland, which is known to derive cholesterol mainly from circulating high-density lipoproteins. Thus, the adrenal gland acquires cholesterol for hormone synthesis from at least three different sources and the quantitative importance of these sources varies markedly in different animal species, including man.     

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.     

Biodegradation of p-nitrophenol in an aqueous waste stream by immobilized bacteria.

Microbiological analyses of activated sludge reactors after repeated exposure to 100 mg of p-nitrophenol (PNP) per liter resulted in the isolation of three Pseudomonas species able to utilize PNP as a sole source of carbon and energy. Cell suspensions of the three Pseudomonas sp., designated PNP1, PNP2, and PNP3, mineralized 70, 60, and 45% of a 70-mg/liter dose of PNP in 24, 48, and 96 h, respectively. Mass-balance analyses of PNP residues for all three cultures showed that undegraded PNP was less than 1% (less than 50 micrograms); volatile metabolites, less than 1%; cell residues, 8.4 to 14.9%; and water-soluble metabolites, 1.2 to 6.7%. A mixed culture of all three PNP-degrading Pseudomonas sp. was immobilized by adsorption onto diatomaceous earth biocarrier in a 1.75-liter Plexiglas column. The column was aerated and exposed to a synthetic waste stream containing 629 to 2,513 mg of PNP per liter at flow rates of 2 to 15 ml/min. Chemical loading studies showed that the threshold concentration for acute toxicity of PNP to the immobilized bacteria was 2,100 to 2,500 mg/liter. Further studies at PNP concentrations of 1,200 to 1,800 mg/liter showed that greater than 99 and 91 to 99% removal of PNP was achieved by immobilized bacteria at flow rates of 10 and 12 ml/min, respectively. These values represent hydraulic retention times of 48 to 58 min and PNP removal rates of 0.99 to 1.1 mg/h per g of biocarrier at 25 degrees C under optimal conditions. This study shows the successful use of immobilized bacteria technology to remove high concentrations of PNP from aqueous waste streams.     

Biodegradation of p-nitrophenol in an aqueous waste stream by immobilized bacteria

Microbiological analyses of activated sludge reactors after repeated exposure to 100 mg of p-nitrophenol (PNP) per liter resulted in the isolation of three Pseudomonas species able to utilize PNP as a sole source of carbon and energy. Cell suspensions of the three Pseudomonas sp., designated PNP1, PNP2, and PNP3, mineralized 70, 60, and 45% of a 70-mg/liter dose of PNP in 24, 48, and 96 h, respectively. Mass-balance analyses of PNP residues for all three cultures showed that undegraded PNP was less than 1% (less than 50 micrograms); volatile metabolites, less than 1%; cell residues, 8.4 to 14.9%; and water-soluble metabolites, 1.2 to 6.7%. A mixed culture of all three PNP-degrading Pseudomonas sp. was immobilized by adsorption onto diatomaceous earth biocarrier in a 1.75-liter Plexiglas column. The column was aerated and exposed to a synthetic waste stream containing 629 to 2,513 mg of PNP per liter at flow rates of 2 to 15 ml/min. Chemical loading studies showed that the threshold concentration for acute toxicity of PNP to the immobilized bacteria was 2,100 to 2,500 mg/liter. Further studies at PNP concentrations of 1,200 to 1,800 mg/liter showed that greater than 99 and 91 to 99% removal of PNP was achieved by immobilized bacteria at flow rates of 10 and 12 ml/min, respectively. These values represent hydraulic retention times of 48 to 58 min and PNP removal rates of 0.99 to 1.1 mg/h per g of biocarrier at 25 degrees C under optimal conditions. This study shows the successful use of immobilized bacteria technology to remove high concentrations of PNP from aqueous waste streams.     

Tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation

A cis-acting methylation center that signals de novo DNA methylation is located upstream of the mouse Aprt gene. In the current study, two approaches were taken to determine if tandem B1 repetitive elements found at the 3' end of the methylation center contribute to the methylation signal. First, bisulfite genomic sequencing demonstrated that CpG sites within the B1 elements were methylated at relative levels of 43% in embryonal stem cells deficient for the maintenance DNA methyltransferase when compared with wild type embryonal stem cells. Second, the ability of the B1 elements to signal de novo methylation upon stable transfection into mouse embryonal carcinoma cells was examined. This approach demonstrated that the B1 elements were methylated de novo to a high level in the embryonal carcinoma cells and that the B1 elements acted synergistically. The results from these experiments provide strong evidence that the tandem B1 repetitive elements provide a significant fraction of the methylation center signal. By extension, they also support the hypothesis that one role for DNA methylation in mammals is to protect the genome from expression and transposition of parasitic elements.     

Short TpA-rich segments of the zeta-eta region induce DNA methylation in Neurospora crassa

The mechanisms that establish DNA methylation in eukaryotes are poorly understood. In principle, methylation in a particular chromosomal region may reflect the presence of a "signal" that recruits methylation, the absence of a signal that prevents methylation, or both. Experiments were carried out to address these possibilities for the 1.6 kb zeta-eta (zeta-eta) region, a relict of repeat-induced point mutation (RIP) in the fungus Neurospora crassa. The zeta-eta region directs its own de novo methylation at a variety of chromosomal locations. We tested the methylation potential of a nested set of fragments with deletions from one end of the zeta-eta region, various internal fragments of this region, chimeras of eta and the homologous unmutated allele, theta (theta), and various synthetic variants, integrated precisely in single copy at the am locus on linkage group (LG) VR or the his-3 locus on LG IR. We found that: (1) the zeta-eta region contains at least two non-overlapping methylation signals; (2) different fragments of the region can induce different levels of methylation; (3) methylation induced by zeta-eta sequences can spread far into flanking sequences; (4) fragments as small as 171 bp can trigger methylation; (5) methylation signals behave similarly, but not identically, at different chromosomal sites; (6) mutation density, per se, does not determine whether sequences become methylated; and (7) neither A:T-richness nor high densities of TpA dinucleotides, typical attributes of methylated sequences in Neurospora, are essential features of methylation signals, but both promote de novo methylation. We conclude that de novo methylation of zeta-eta sequences does not simply reflect the absence of signals that prevent methylation; rather, the region contains multiple, positive signals that trigger methylation. These findings conflict with earlier models for the control of DNA methylation, including the simplest version of the collapsed chromatin model.     

Consequences of increasing or decreasing plasma FSH concentrations during the preovulatory period in Romney ewes

Romney ewes were infused with ovine FSH (NIADDK-oFSH-16) for 48 h from the initiation of luteolysis with cloprostenol. Doses of 2.5 or 5 micrograms/h which partly or completely prevented the normal preovulatory decline in plasma FSH concentrations caused a significant increase in mean ovulation rates. Ovulation rates were not increased significantly if the FSH (5 micrograms/h) was infused for only 20 h starting from the initiation of luteolysis or 24 h later. Infusion of a less potent and relatively impure preparation of FSH (i.e. FSH-P) at 0.5 mg/h for 48 h after cloprostenol treatment also increased the mean ovulation rate significantly. However, if the FSH-P was given for only the first 24 h, or if the start of the infusion was delayed for more than 12 h, mean ovulation rates were not increased significantly. Infusion of LH (NIADDK-oLH-25, 5 micrograms/h) for 48 h from the initiation of luteolysis decreased the mean ovulation rate significantly. Administration of bovine follicular fluid to suppress plasma FSH concentrations below normal during the first 24 h after cloprostenol injection did not delay oestrus. However, oestrus was delayed by approximately 2 days if plasma FSH concentrations were reduced by bovine follicular fluid 24 h after the initiation of luteolysis. As ovulation rate increased, the mean weight of individual corpora lutea of each ewe decreased. In ewes with a single ovulation, most corpora lutea weighed greater than 600 mg, but as the ovulation rate increased the proportion of corpora lutea present weighing less than 400 mg rose steadily.(ABSTRACT TRUNCATED AT 250 WORDS)     

CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia

Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.     

Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites

We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.     

Rat and human mammary tissue can synthesize choline moiety via the methylation of phosphatidylethanolamine.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.     

In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b

The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3' long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.