Isolation and Chromosomal Localization of Highly Repeated DNA Sequences in Drosophila melanogaster

Abstract

The nuclear DNA of D. melanogaster contains DNA sequences that are repeated between ten and a hundred times more often than the next class of redundant DNA. This DNA, as a renatured duplex isolated on the basis of its renaturation kinetics, has a buoyant density of 1.691 (g/ml). In its native state it bands within the unique nuclear DNA peak (p = 1.701). These sequences have been localized by "in situ" hybridization in the chromocenter of the chromosomes of the salivary gland. The properties of centromeres are discussed in terms of the occurrence of repeated sequences at this locus.At present, our understanding of the organization of DNA sequences in the higher organism is very poor. The presence of multiple copies of certain sequences is one feature that has been established through the study of renaturation kinetics of denatured DNA (1, 2). Gene classes have been defined (1) The satellite DNAs of rodents represent a discrete set of sequences, with the highest extent of repetition (3-5) that is found in their genomes. The other repeated sequences belong to an intermediate class, with a continuous distribution of moderately repeated families, until a second discontinuity in the extent of repetitions presumably reveals sequences that represent single-copy DNA. Though representing 10% of the total DNA, the mouse satellite has a complexity of 300 base pairs. The basic unit probably contains a sequence of from 8 to 13 base pairs, distributed in some manner between sequences that have been derived by mutation from this basic sequence (6). There is evidence that this class of highly-redundant DNA is not transcribed (4). Recently, the mouse satellite DNA has been located in the centromeric heterochromatin of the metaphase chromosomes (7,8). This observation is consistent with the earlier demonstration that the mouse satellite DNA is associated with the less soluble fraction of metaphase chromosomes (9) (12), with the addition of a 4-hr digestion with 0.5 mg pronase per ml (self-digested for 2 hr) after the RNase and a-amylase treatments. As discussed in the results, cytoplasmic DNA contaminants were found in crude nuclear preparations (600 X g pellet). Nuclear DNA with less than 0.3% cytoplasmic DNA contamination was obtained from nuclei purified by sedimentation through 2.2 M sucrose for 1 hr at 45,000 X g at 4°C. Contamination with either bacterial or yeast nucleic acid was ruled out because 3H-labeled whole Escherichia coli and yeast cells added to the starting material did not add any labeled DNA to the final extracts. X 10-10 g sec2/cm5 was used to calculate (15) density differences; 99% E. coli [15N] DNA was used as a marker of buoyant density 1.727 ± 0.0004, relative to a primary standard, unlabeled E. coli DNA, assigned a buoyant density of 1.710. The cytoplasmic peaks contaminate this nuclear DNA sample by 3%.

Independent Control of Ribosomal Gene Replication in Polytene Chromosomes of Drosophila melanogaster

“…The second type of DNA is that found in the heterochromatin. It consists largely of repetitive satellite DNA (2)(3)(4)7). This DNA replicates very little or possibly not at all (2,6).…”

“…The heterochromatin, contrastingly, remains condensed during interphase, is consequently darkly staining in mitotic prophase, and contains very few genes. In addition to these genetic and cytological distinctions, the euchromatin consists mainly of unique DNA sequences, while most of the heterochromatin in diploid cells is highly repeated satellite DNA (2)(3)(4).…”

“…The heterochromatin, contrastingly, remains condensed during interphase, is consequently darkly staining in mitotic prophase, and contains very few genes. In addition to these genetic and cytological distinctions, the euchromatin consists mainly of unique DNA sequences, while most of the heterochromatin in diploid cells is highly repeated satellite DNA (2)(3)(4).During the formation of the giant polytene chromosomes of the Drosophila salivary gland, the differences between the euchromatin and heterochromatin become even more pronounced. The euchromatic arms proceed through a series of DNA replications resulting in as many as 1024 DNA fibers (5).…”

Novel Properties of Satellite DNA from Muskmelon

“…The kinetics of strand reassociation and the stability of these reformed duplexes are interpreted to mean that the multiple DNA segments are highly reiterated and are closely similar copies (2)(3)(4). Although its function is not known, some satellite DNA in mice (5) and insects (6,7) hybridizes primarily with the centromeric regions of chromosomes in cytological preparations. It is enriched in heterochromatin of guinea pig (8), mouse, crab (9) and calf (10), and in mouse nucleoli (11).…”

Repeated DNA Sequences in the Heterochromatic Y Chromosome of Adult Drosophila melanogaster

“…Recent reports (1)(2)(3)(4) have demonstrated a marked capacity of the centromeric regions of Drosophila chromosomes to form hybrid molecules with complementary RNA (c-RNA) transcribed from the rapidly reassociating, repetitious portion of the genome. All of these reports have used the technique of in situ hybridization, developed within the past few years by workers in several different laboratories.…”