Evolution of the gonosomal heterochromatin of Microtus agrestis: rapid amplification of a large, multimeric, repeat unit containing a 3.0-kb (GATA)₁₁-positive, middle repetitive element

Abstract

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have cloned and characterized a 2,999-bp (GATA)_n-positive sequence, following Haelll digestion, that is confined to the noncentromeric heterochromatin of the X chromosome. The cloned element exhibits an accumulation of certain oligomers, which are scattered throughout its entire length, and several copies of Chi-related sequence motifs, which are thought to be implicated in recombination. The latter might have been responsible for the extensive amplification of homologous genomic elements. The sequence has been amplified to a copy number of 1–2 × 10⁴ within the genome of M. agrestis. In contrast to many satellite DNAs, which are thought to be an inevitable constituent of constitutive heterochromatin, the sequence exhibits a tissue-specific methylation pattern and is organized, not as a simple tandem array, but as a component of an extremely large, multimeric, higher-order repeat unit with a length of over 20 kb. This higher-order repeat accounts for at least 15–30 % of the gonosomal heterochromatin in M. agrestis. Sequences homologous to pMAHAE2 are abundant in the genomes of all Microtus species. The copy number varies from ∼100 per diploid genome in M. arvalis, M. oeconomus, and M. cabrerae to –500 per diploid genome in M. guentheri and up to 1–2 × 10⁴ in M. agrestis. Our molecular data indicate that the sequences of the pMAHAE2 family probably arose during the evolution of the common ancestor of Microtus and have subsequently been amplified extensively in the X chromosomes of M. agrestis in the phylogenetically very short period of less than 1 million years.

A repeat DNA sequence from the Y chromosome in species of the genus Microtus

“…In the same way, MS2, MS3 and MS4 repeated DNA sequences are located on the heterochromatin of both sex chromosomes in several Microtus species and absent in others, showing also an autosomal distribution in some of them . Finally, the repeated DNA sequence pMAHAE2 from M. agrestis is present in the heterochromatic blocks of both sex chromosomes of this species (Kalscheuer et al 1996).…”

“…Thus, the AT content of MSAT-160, MSAT-21 and MSAT-2750 ranges between 58 and 64% (Modi 1992, 1993b, Ivanov & Modi 1996, Ferna¤ ndez et al 2001, Shevchenko et al 2002. The repeat DNA sequence pMAHAE2, located exclusively on the gonosomal heterochromatin of the giant sex chromosomes from M. agrestis, has a high AT content (65%) (Kalscheuer et al 1996). The heterochromatin of the sex chromosomes of M. agrestis is enriched also in L1 (pMAECO-14) and non-L1 (pMA11/3) retroposons which are 61% and 58% AT-rich, respectively (Neitzel et al 1998(Neitzel et al , 2002.…”

Copy and paste: the impact of a new non-L1 retroposon on the gonosomal heterochromatin of <i>Microtus agrestis</i>

“…The plasmid DNA of pMA11/31 served as a probe. A repetitive DNA, pMAHAE2, which is not transcribed, was utilized as a negative control (Kalscheuer et al, 1996), and mouse rRNA served as a positive control. To ensure that the hybridization signals were specific for DNA-RNA hybrids and not an unspecific binding of the probe to chromatin, we treated half of the slides with 20 ng/ml RNase A for 2 h at 37°C, followed by dehydration by passage through an ethanol series.…”

“…The techniques for Southern and Northern hybridization were adopted from Maniatis et al (1982), as described by Kalscheuer et al (1996). For Southern hybridization, the cloned probe was labeled with a random-priming labeling kit (Boehringer Mannheim); for Northern hybridization, the probe was labeled by nick-translation.…”

“…We have cloned several interspersed middle repetitive elements. One element, pMAHAE2, is confined to the constitutive heterochromatin, shows a tissue-specific methylation pattern, and is transcriptionally inactive (Kalscheuer et al, 1996). Another L1 element (pMAECO14) also accumulates in the constitutive heterochromatin but, in addition, is interspersed at autosomal locations and transcribed in a spontaneously transformed cell line C3 (Neitzel et al, 1998).…”