- Highly repetitive DNA (Simple sequence DNA) is the first component to reassociate and is equated with satellite DNA.
- Satellite DNA (Simple-sequence DNA) consists of many tandem repeats (identical or related) of a short basic repeating unit.
- A density gradient is used to separate macromolecules on the basis of differences in their density. It is prepared from a heavy soluble compound such as CsCl.
- A cryptic satellite is a satellite DNA sequence not identified as such by a separate peak on a density gradient; that is, it remains present in main-band DNA.
- In situ hybridization (Cytological hybridization) is performed by denaturing the DNA of cells squashed on a microscope slide so that reaction is possible with an added single-stranded RNA or DNA; the added preparation is radioactively labeled and its hybridization is followed by autoradiography.
- Heterochromatin describes regions of the genome that are highly condensed, are not transcribed, and are late-replicating. Heterochromatin is divided into two types, which are called constitutive and facultative.
- Euchromatin comprises all of the genome in the interphase nucleus except for the heterochromatin. The euchromatin is less tightly coiled than heterochromatin, and contains the active or potentially active genes.
- Highly repetitive DNA has a very short repeating sequence and no coding function.
- It occurs in large blocks that can have distinct physical properties.
- It is often the major constituent of centromeric heterochromatin.
Repetitive DNA is defined by its (relatively) rapid rate of renaturation. The component that renatures most rapidly in a eukaryotic genome is called highly repetitive DNA, and consists of very short sequences repeated many times in tandem in large clusters. Because of its short repeating unit, it is sometimes described as simple sequence DNA. This type of component is present in almost all higher eukaryotic genomes, but its overall amount is extremely variable. In mammalian genomes it is typically <10%, but in (for example) Drosophila virilis, it amounts to ~50%. In addition to the large clusters in which this type of sequence was originally discovered, there are smaller clusters interspersed with nonrepetitive DNA. It typically consists of short sequences that are repeated in identical or related copies in the genome.
The tandem repetition of a short sequence often creates a fraction with distinctive physical properties that can be used to isolate it. In some cases, the repetitive sequence has a base composition distinct from the genome average, which allows it to form a separate fraction by virtue of its distinct buoyant density. A fraction of this sort is called satellite DNA. The term satellite DNA is essentially synonymous with simple sequence DNA. Consistent with its simple sequence, this DNA is not transcribed or translated.
Tandemly repeated sequences are especially liable to undergo misalignments during chromosome pairing, and thus the sizes of tandem clusters tend to be highly polymorphic, with wide variations between individuals. In fact, the smaller clusters of such sequences can be used to characterize individual genomes in the technique of "DNA fingerprinting" (see 4.14 Minisatellites are useful for genetic mapping).
The buoyant density of a duplex DNA depends on its G·C content according to the empirical formula
ρ = 1.660 + 0.00098 (%G·C) g-cm–3
Buoyant density usually is determined by centrifuging DNA through a density gradient of CsCl. The DNA forms a band at the position corresponding to its own density. Fractions of DNA differing in G·C content by >5% can usually be separated on a density gradient.
When eukaryotic DNA is centrifuged on a density gradient, two types of material may be distinguished:
- Most of the genome forms a continuum of fragments that appear as a rather broad peak centered on the buoyant density corresponding to the average G·C content of the genome. This is called the main band.
- Sometimes an additional, smaller peak (or peaks) is seen at a different value. This material is the satellite DNA.
Satellites are present in many eukaryotic genomes. They may be either heavier or lighter than the main band; but it is uncommon for them to represent >5% of the total DNA. A clear example is provided by mouse DNA, shown in Figure 4.19. The graph is a quantitative scan of the bands formed when mouse DNA is centrifuged through a CsCl density gradient. The main band contains 92% of the genome and is centered on a buoyant density of 1.701 g-cm–3 (corresponding to its average G·C of 42%, typical for a mammal). The smaller peak represents 8% of the genome and has a distinct buoyant density of 1.690 g-cm–3. It contains the mouse satellite DNA, whose G·C content (30%) is much lower than any other part of the genome.
The behavior of satellite DNA on density gradients is often anomalous. When the actual base composition of a satellite is determined, it is different from the prediction based on its buoyant density. The reason is that ρ is a function not just of base composition, but of the constitution in terms of nearest neighbor pairs. For simple sequences, these are likely to deviate from the random pairwise relationships needed to obey the equation for buoyant density. Also, satellite DNA may be methylated, which changes its density.
Often most of the highly repetitive DNA of a genome can be isolated in the form of satellites. When a highly repetitive DNA component does not separate as a satellite, on isolation its properties often prove to be similar to those of satellite DNA. That is to say that it consists of multiple tandem repeats with anomalous centrifugation. Material isolated in this manner is sometimes referred to as a cryptic satellite. Together the cryptic and apparent satellites usually account for all the large tandemly repeated blocks of highly repetitive DNA. When a genome has more than one type of highly repetitive DNA, each exists in its own satellite block (although sometimes different blocks are adjacent).
Where in the genome are the blocks of highly repetitive DNA located? An extension of nucleic acid hybridization techniques allows the location of satellite sequences to be determined directly in the chromosome complement. In the technique of in situ hybridization, the chromosomal DNA is denatured by treating cells that have been squashed on a cover slip. Then a solution containing a radioactively labeled DNA or RNA probe is added. The probe hybridizes with its complements in the denatured genome. The location of the sites of hybridization can be determined by autoradiography (see Figure 19.19).Satellite DNAs are found in regions of heterochromatin. Heterochromatin is the term used to describe regions of chromosomes that are permanently tightly coiled up and inert, in contrast with the euchromatin that represents most of the genome (see 19.7 Chromatin is divided into euchromatin and heterochromatin). Heterochromatin is commonly found at centromeres (the regions where the kinetochores are formed at mitosis and meiosis for controlling chromosome movement). The centromeric location of satellite DNA suggests that it has some structural function in the chromosome. This function could be connected with the process of chromosome segregation.
An example of the localization of satellite DNA for the mouse chromosomal complement is shown in Figure 4.20. In this case, one end of each chromosome is labeled, because this is where the centromeres are located in M. musculus chromosomes.