In most higher organisms, DNA is modified after synthesis by the enzymatic conversion of many cytosine residues to 5-methylcytosine. For several years, control of gene activity by DNA methylation has been recognized as a logically attractive possibility, but experimental support has proved elusive. However, there is now reason to believe, from recent studies, that DNA methylation is a key element in the hierarchy of control mechanisms that govern vertebrate gene function and differentiation.

https://science.sciencemag.org/content/sci/210/4470/604.full.pdf

DNA methylation and gene function

Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI.--In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI.--Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The cma-banding pattern appears to be similar to the pattern found by R-banding procedures.

Reverse fluorescent chromosome banding with chromomycin and DAPI.

A small portion of the cytosine residues in the DNA of higher eukaryotes as well as in that of many lowe eukaryotes if methylated. The resulting 5-methylcytosine residues occur in specific in the DNA, usually adjacent to guanine residues on the 3' side. This methylation of eukaryotic DNA has been proposed to function in many ways, including control of transcription, maintenance of chromosome structure, repair of DNA, establishment of preferred sites for mutation, oncogenic transformation, and, in certain systems, protection of DNA against enzymatic degradation.

https://science.sciencemag.org/content/sci/212/4501/1350.full.pdf

5-Methylcytosine in eukaryotic DNA

Publisher Summary This chapter discusses the functional aspects of satellite DNA and heterochromatin. Despite all attempts to formulate simple rules governing satellite evolution, it is now clear that each case so far analyzed has brought with it its own claims for generalization, none of which have proven sufficiently all-embracing. One initial hypothesis on satellite evolution was that satellites wax and wane with amazing rapidity in evolutionary terms, so that closely related species differ drastically in amount or type of satellite. However, improved methods of DNA sequencing have led to the suggestion that closely related species appear to modulate their satellites from a common library. Because some kinds of heterochromatin are now known to contain satellite DNA and because a large literature exists on the properties of heterochromatin, it is necessary to consider the relationships between heterochromatin and satellite DNA in some detail. The term “heterochromatin” was initially used to define chromosomes or chromosome segments which did not uncoil at mitotic telophase and so maintained a condensed or heteropycnotic state throughout interphase and into the subsequent prophase of the next division cycle.

Functional aspects of satellite DNA and heterochromatin.

Of the chronic mental disabilities of childhood, autism is causally least well understood. The former view that autism was rooted in exposure to humorless and perfectionistic parenting has given way to the notion that genetic influences are dominant underlying factors. Still, identification of specific heritable factors has been slow with causes identified in only a few cases in unselected series. A broad search for genetic and environmental influences that cause or predispose to autism is the major thrust of the South Carolina Autism Project. Among the first 100 cases enrolled in the project, abnormalities of chromosome 15 have emerged as the single most common cause. The four abnormalities identified include deletions and duplications of proximal 15q. Other chromosome aberrations seen in single cases include a balanced 13;16 translocation, a pericentric inversion 12, a deletion of 20p, and a ring 7. Candidate genes involved in the 15q region affected by duplication and deletion include the ubiquitin-protein ligase (UBE3A) gene responsible for Angelman syndrome and genes for three GABA(A) receptor subunits. In all cases, the deletions or duplications occurred on the chromosome inherited from the mother.

Autism and maternally derived aberrations of chromosome 15q

The H-Y locus is on the short arm of the human Y chromosome in most individuals but on the long arm in at least one of 17 individuals with structural abnormalities of the Y.

Mapping the locus of the H-Y gene on the human Y chromosome.

The marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Down's syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15

Human metaphase chromosomes were photooxidized in the presence of methylene blue, a process that destroys guanine residues in DNA. Indirect immunofluorescence showed that such chromosomes reacted with a cytosine-specific antibody revealing a consistent fluorescent banding pattern by which each chromosome could be identified. The observed fluorescent patterns were the reverse of those produced in formamide-denatured chromosomes treated with an antibody specific for adenine and of the patterns obtained with quinacrine and with Giemsa staining by the G-banding techniques. The patterns were identical to Giemsa R-banding patterns. The chromosome banding patterns, therefore, appeared to reflect DNA base composition, indicating the feasibility of a combined chemical-immunochemical investigation of the chemical organization of chromosomes.

R. R. Schreck

Papers

Mapping the locus of the H-Y gene on the human Y chromosome.

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15

Chromosome Structure as Revealed by a Combined Chemical and Immunochemical Procedure

The use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation.

Human chromosome structure as revealed by an immunoperoxidase staining procedure