Whole Genome Methylation Scanning Based on phi29 Polymerase Amplification.

Identifying differences in DNA methylation is critical to understanding how epigenetics influences gene expression during processes such as development. Here, we propose a method that employs a single, methylation-sensitive restriction endonuclease of choice, to produce discrete pools of methylated and unmethylated DNA from the same sample. A pool of restriction fragments representing unmethylated regions of the genome is first obtained by digestion with a methylation-sensitive endonuclease. The restriction-digested DNA is then concatamerized in the presence of stuffer-adaptor DNA, which prevents interference from originally unmethylated DNA by blocking the ends of the restriction fragments. The concatamerized DNA is amplified by phi29 polymerase to remove methylation marks, and again digested with the same endonuclease to produce a pool of DNA fragments representing methylated portions of the genome. The two pools of DNA fragments thus obtained can be analyzed by end-sequencing or hybridization to a genomic array. In this report we detail a proof of concept experiment that demonstrates the feasibility of our method.

The association of homeobox gene expression with stem cell formation and morphogenesis in cultured Medicago truncatula.

Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24-48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are "hijacked" for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed.

Full-sized HERV-K (HML-2) human endogenous retroviral LTR sequences on human chromosome 21: map locations and evolutionary history.

One of the evolutionary mechanisms for acquisition of novel functional sequences can be domestication of exogenous retroviruses that have been integrated into the germ line. The whole genome mapping of such elements in various species could reveal differences in positions of the retroviral integration and suggest possible roles of these differences in speciation. Here, we describe the number, locations and sequence features of the human endogenous retrovirus HERV-K (HML-2) long terminal repeat (LTR) sequences on human chromosome 21. We show that their distribution along the chromosome is not only non-random but also roughly correlated with the gene density. Amplification of orthologous LTR sites from a number of primate genomes produced patterns of presence and absence for each LTR sequence and allowed determination of the phylogenetic ages and evolutionary order of appearance of individual LTRs. The identity level and phylogenetic age of the LTRs did not correlate with their map locations. Thus, despite the non-random distribution of LTRs, they have apparently been inserted randomly into the chromosome relative to each other. As evidenced in previous studies of chromosomes 19 and 22, this is a characteristic of HERV-K integration.

Differences in HERV-K LTR insertions in orthologous loci of humans and great apes.

The classification of the long terminal repeats (LTRs) of the human endogenous retrovirus HERV-K (HML-2) family was refined according to diagnostic differences between the LTR sequences. The mutation rate was estimated to be approximately equal for LTRs belonging to different families and branches of human endogenous retroviruses (HERVs). An average mutation rate value was calculated based on differences between LTRs of the same HERV and was found to be 0.13% per million years (Myr). Using this value, the ages of different LTR groups belonging to the LTR HML-2 subfamily were found to vary from 3 to 50Myr. Orthologous potential LTR-containing loci from different primate species were PCR amplified using primers corresponding to the genomic sequences flanking LTR integration sites. This allowed us to calculate the phylogenetic times of LTR integrations in primate lineages in the course of the evolution and to demonstrate that they are in good agreement with the LTR ages calculated from the mutation rates. Human-specific integrations for some very young LTRs were demonstrated. The possibility of LTRs and HERVs involvement in the evolution of primates is discussed.