Molecular and cytological evidence of S-adenosyl-L-homocysteine as an innocuous undermethylating agent in vivo.

Undermethylating agents are frequently used in the study of DNA methylation. However, the drugs of choice to induce experimental DNA undermethylation, 5-azacytidine and 5-aza-2'-deoxycytidine, are known to be cytotoxic. We report on S-adenosyl-L-homocysteine (SAH) as an alternative agent for inducing undermethylation of human DNA in vivo. The molecular and cytological evidence presented in this paper clearly suggests that SAH could be even more efficient than the cytidine analogs; in addition, SAH does not exhibit the secondary toxic effects that cripple the usefulness of cytidine analogs.

S-adenosyl-L-homocysteine: a non-cytotoxic hypomethylating agent.

The cytotoxic effect caused by the hypomethylating agent S-adenosyl-L- homocysteine (SAH) was compared with that of two drugs commonly used to induce DNA hypomethylation, 5-azacytidine and 5-aza-2'-deoxycytidine. Two in vitro cytotoxicity tests, the tetrazolium MTT assay and the intracellular lactate dehydrogenase (LDH) activity test, suggest that SAH induces hypomethylation without causing any cytotoxic effect. We propose the use of SAH as a non-cytotoxic agent which may be more suitable for inducing experimental DNA hypomethylation.

Induction of G- and R-banding in human chromosomes by the demethylating agent S-adenosyl-L-homocysteine.

In this report we describe the procedure of growing human lymphocytes with the demethylating agent S-adenosyl-L-homocysteine (SAH). After this treatment, which is not toxic for cell survival, both R- and G-banding were obtained by new experimental procedures: R-bands have been directly demonstrated with the GC-specific fluorochrome chromomycin A3 without the necessity of any AT-specific counterstaining agent; simultaneous G-banding and active nucleolar organizer regions have been obtained by silver impregnation of chromosomes and subsequent Giemsa staining. These results suggest a possible relationship between local differences in DNA methylation and the determination of the banded chromosome structure.

Cytogenetic analysis of a human familial 15p+ marker chromosome.

An extreme variant of a human 15p+ marker chromosome is described in a human family. The short arm of this variant is greatly enriched in ribosomal RNA genes, and there are up to three secondary constrictions potentially active for rRNA transcription. These sites are hypomethylated, whereas the rest of rDNA is highly methylated, as shown by the isoschizomer pair HpaII-MspI. This chromosome variant is similar to others previously described for chromosomes 14 and 22, but some differences have been found after C-banding and after treatments with AluI and Sau3AI.

Longitudinal differentiation of the human Yq heterochromatin as revealed by the restriction enzyme TaqI.

The restriction endonuclease TaqI cleaves DNA at TCGA sites which are very common in human satellite DNAs. However, this enzyme was not used successfully up to now to digest constitutive heterochromatin of human chromosomes, where those highly repetitive DNAs are preferentially located. In this work, we show that TaqI is able to cut and extract DNA from the major heterochromatic regions on chromosomes 1, 9, 15, and 16 which appear as unstained gaps. Yq heterochromatin displays moderate digestion along its entire length but a middle region can be distinguished which is usually more affected. Complete digestion of Yq heterochromatin can be achieved when this block has been previously undercondensed by treating cell cultures with the cytidine analog, 5-azacytidine. Thus, it may be deduced that some factors related to chromatin organization might be involved in the action of TaqI. These results come to reinforce previous data about heterogeneity of Yq heterochromatin, and allow us to subdivide it into three different regions according to their differential response to TaqI digestion.

Visualization of R-bands in human metaphase chromosomes by the restriction endonuclease MseI.

Human metaphase chromosomes were treated with the restriction endonuclease MseI, which cuts DNA at TTAA sequences. This enzyme preferentially cuts and extracts DNA from G-bands and thus is the first restriction endonuclease allowing direct R-band visualization. Specific patterns ranging from R+C-like to C-like banding can be induced, depending on the concentration of the enzyme. At intermediate concentrations, only a subset of R-bands are produced, corresponding to GC-rich bands that are especially resistant to heat denaturation (so-called T-bands). These results suggest that compositional differences between chromosomal regions determine the different rates of cleavage by MseI, not only between R- and G-bands but also among different R-bands.

Cryptic variants of acrocentric human chromosomes as analysed by restriction endonucleases.

Ten phenotypically normal human individuals have been analysed by in situ treatments with restriction endonucleases in order to obtain a better characterization of some cryptic variants of acrocentric chromosomes. Treatments with AluI, NdeII and Sau3AI confirm the existence of two cryptic amplified regions on the short arms of both one chromosome 15 and one chromosome 22, in one female. These amplifications seem to be of different origin involving the nucleolar organizer region of chromosome 15 and the satellite of chromosome 22.