Frequently Asked Questions (FAQs)

Transcription

1 Frequently Asked Questions (FAQs) Q1. What is meant by Satellite and Repetitive DNA? Ans: Satellite and repetitive DNA generally refers to DNA whose base sequence is repeated many times throughout the genome of an organism. It is common in eukaryotes, accounting for about half of the total DNA, for example, in mammals. Its propotion varies in different organisms and can be divided into various types. While some of this DNA serves a useful purpose, but a significant proportion is of uncertain function, and may be junk, or selfish DNA. Though used quite often synonymously or interchangeably but the two are actually different. Q2. What is the difference between Satellite and Repetitive DNA? Ans. Satellite DNA referes to the serially repeated DNA sequences of one or a few nucleotides with a repeat length of up to 250 nucleotides that are not transcribed and commonly located in the heterochromatin associated with the centrometric regions of chromosomes. In fact, the term `satellite DNA` is derived

2 from the way in which repetitive DNA is prepared as a pure fraction separate from the rest of the DNA in a cell. Repetitive DNA refers to substrings of the genome that repeat multiple times & includes (a) satellite DNA and socalled (b) interspersed repeated DNA sequences. The latter are interspersed throughout the chromosomes in hundreds of thousands of individual copies, each about 300 nucleotides long; they are, unlike satellite DNA, transcribed. Moreover, different instances of the repeat element can have slightly different patterns. It is also highly prevalent in eukaryotes (organisms with a visible nucleus and cell structure, as opposed to bacteria) Which attributes of DNA make it the ideal genetic material? Q3. How does the proportion of repetitive DNA vary amongst organisms? Repetitive DNA Organism % Repetitive DNA Homo sapiens 21 % Mouse 35 % Calf 42 % Drosophila 70 % Wheat 42 % Pea 52 % Maize 60 % Saccharomycetes cerevisiae 5 % E. coli 0.3 % Ans. A major part of the nuclear genome of most plants is composed of different repetitive DNA elements. Studying these sequence elements is essential for our understanding of the nature and consequences of genome size variation between different

3 species, and for studying the large-scale organization and evolution of plant genomes. Q4. What is the difference between SINES and LINES? Ans. LINES and SINES are both the examples of dispersed repetitive DNA. SINES stand for short interspersed nuclear elements and LINES for Long interspersed nuclear elements. The best example of SINES is Alu family in humans. Their average length is about 280bp and they are repeated about 700,000 to 1000, 000 times in the genome occurring all over the place, even in introns. They propagate by transposition. LINES are also thought to propagate by transposition. e.g. LINE-1 (copy number of 60,000 to 100,000) is a type of non-viral retroelement, a transposon that can replicate and move around the genome by a process involving reverse transcription. The average length of LINE-1 element is 1.4kb Q5. What is the evidence for existence of repetitive DNA? The first evidence for repetitive DNA came from Density-Gradient analysis of eukaryotic DNA. Density-Gradient procedure was first used by Meselson & Stahl to demonstrate semi-conservative replication of E. coli chromosomes. The density of 6M CsCl is about 1.7 g/cm 3. If such a solution is centrifuged at a very high speed for

4 long enough period of time, density gradients will be established because of the equilibrium between: a. Sedimentation of the CsCl to the bottom of the centrifuge tube as a result of the centrifugal force, and b. Diffusion of the CsCl towards top of the centrifuge tube When the DNA of a prokaryote such as E.coli is isolated, fragmented and centrifuged to equilibrium in a CsCl density gradient, the DNA usually forms a single band in the gradient at a position where the CsCl density is equal to the density of DNA containing about 50% G- C base-pairs. It is important to note that DNA density increases with the increasing G-C content. However, CsCl-density gradient analysis of eukaryotic DNA usually reveals the presence of one large band of DNA (usually called the mainband DNA) and one to several small bands. These small bands of DNA are called satellite bands and the DNA embodied in them is referred to as Satellite DNA. The Satellite DNA upon isolation and analysis reveals repeating sequences of variable lengths in different organisms. Q6. How can we establish location of the satellite DNA in chromosomes? Ans. Chromosomal location of satellite DNA have been determined by a technique called in-situ hybridization, which usually involves

5 annealing single strands of isolated radioactive satellite DNA, or complementary RNA sequences synthesised using satellite DNA as a template, directly to denatured DNA in chromosome quash preparations. After washing out the non-hybridised radioactive material, the location of the satellite DNA sequences in chromosomes are determined by autoradiography. They are usually found in the heterochromatin region of chromosomes. It is important to note that a repetitive DNA sequence will be identified as satellite DNA only if the sequence has a base composition sufficiently different from that of the main-band DNA; only then it is recognized as a distinct band in the density gradient. DNA renaturation kinetics gives a more complete picture of repetitive DNA complexity and frequency. In fact, the time required for reassociation of a particular DNA sequence is inversely proportional to the number of times that sequence is present in the genome. Thus, highly repetitive DNA sequences will renature very rapidly (due to more number of collisions). Q7. Enumerate the important types of Repetitive DNA. Ans. The important types of Repetitive DNA include: Satellite DNA Minisatellite DNA Microsatellite DNA Transposable elements

6 LINES, SINES and other retrosequences High copy number genes (e.g. ribosomal genes, histone genes) Multifamily member genes (e.g. hemoglobin, immunoglobulin) Q.8. What are the transposable elements. Give some examples of from eukaryotes. Ans. Transposable elements, also referred to as jumping genes, are those elements of the genome which change their position from one place to another. The important examples from eukaryotes include : Maize - Ac-Ds = Activator (encodes a transposase), Dissociation (encodes an enzyme that promotes chromosome breakage). Drosophila melanogaster - P-element = most famous because of its use as a vector to insert foreign DNA into Drosophila. Causes hybrid dysgenesis when crossed between strains. Many organisms - Tc1/Mariner elements. The Mariner element family is exceptionally widespread in animals (from nematodes to mice) and are particularly common

7 in insects (humans have a mariner related element but it is not active). Q.9. What are the attributed functions of highly repetitive DNA Ans. Being located genetically inactive heterochromatin region of the chromosomes, the functions of highly repetitive DNA are almost unknown. Some attributed functions of this DNA include: Structural or organizational roles in chromosomes Involvement in chromosome pairing during meiosis Involvement in crossing over or recombination Protection of important structural genes such as histones, rrna, or ribosomal protein genes The validity of these postulated roles is by and large questionable and needs to be ascertained by robust and focussed experiments. Some people even think that the functions of repetitive DNA are more than meets the eye and are likely to be unravelled in future course of time. Repetitive DNA, with different selective pressures from those acting on genes and evolutionarily successful multigene modules, may

8 show extensive differences in sequence motifs and abundance even between closely related species. The repetitive DNA in the genome is also important for evolutionary, genetic, taxonomic and applied studies. Q.10. Highlight the relatively well-defined functions of highly repetitive DNA Ans. A few repetitive sequences are known to have well defined functions. The telomeric sequences, added at the ends of most plant and animal chromosomes, allow a linear replication unit to be maintained, protect chromosome ends and overcome the 'end replication problem'. The 18S-5.8S- 25S and 5S rrna gene loci, clustered at a small number of sites, encode the structural RNA components of ribosomes. Mobile DNA sequences - such as transposons and retrotransposons - make up a high proportion of most plant and animal genomes. A major class, the retroelements, encode the proteins necessary for their own reverse transcription and integration, and sometimes represent 50% of the genome. As a result of their transcription into RNA, reverse transcription into DNA and integration into the genome, they have a dispersed distribution along chromosomes. Notably, telomeres, rdna and retroelement sequences are all ancient - they are found in all animals and plants, and might be considered as early derivatives of the 'RNA-world' from which DNA-based organisms evolved.

9 Q.11. What is the difference between Minisatellite and Microsatellite DNA Ans. Minisatellite DNA is usually repeated times in the genome and is about bp long. Its units range from bp, averaging about 20. It is located generally in euchromatic region of the chromosome. Its examples include (a) DNA fingerprints which are tandemly repeated but often in dispersed clusters. They are also called VNTRs (variable number tandem repeats) (b) Human 33.1 minisatellite (62 bp) and (c) Human 33.5 minisatellite (17 bp). Microsatellite DNA is generally repeated about times in the genome with units of 2-4 bp (mostly 2). It is located generally in the euchromatin. Its examples include most useful marker for population level studies. Q.12 What is the precise role of DNA renaturation kinetics in the studies of repetitive DNA Ans. DNA renaturation kinetics gives a more complete picture of repetitive DNA complexity and frequency. In fact, the time required for reassociation of a particular DNA sequence is inversely proportional to the number of times that sequence

10 is present in the genome. Thus, highly repetitive DNA sequences will renature very rapidly (due to more number of collisions). Q. 13. Briefly describe retroelements as repetitive sequences. Ans. A major class, the retroelements, encode the proteins necessary for their own reverse transcription and integration, and sometimes represent 50% of the genome. As a result of their transcription into RNA, reverse transcription into DNA and integration into the genome, they have a dispersed distribution along chromosomes. Notably, telomeres, rdna and retroelement sequences are all ancient - they are found in all animals and plants, and might be considered as early derivatives of the 'RNA-world' from which DNA-based organisms evolved. Q. 14. How can we distinguish repetitive DNA in eukaryotes? Ans. A major class, the retroelements, encode the proteins necessary for their own reverse transcription and integration, and sometimes represent 50% of the genome. As a result of their transcription into RNA, reverse transcription into DNA and integration into the genome, they have a dispersed

11 distribution along chromosomes. Notably, telomeres, rdna and retroelement sequences are all ancient - they are found in all animals and plants, and might be considered as early derivatives of the 'RNA-world' from which DNA-based organisms evolved. Q. 15. Why is studying of repetive DNA important? Ans Studying repetive DNA sequence elements is essential for our understanding of the nature and consequences of genome size variation between different species, and for studying the large-scale organization and evolution of plant genomes.repeats contain some novel, though hidden and yet unraveld, functions.