Transposons

Posted by admin on Jun 16, 2010 in Articles | 0 comments

                               INTRODUCTION TO TRANSPOSONS

                                                (Jumping Genes)

 Author: Waseem Ashfaq

            Transposons are pieces of DNA that can “jump” into novel positions in the genome. There are numerous different types of transposon which differ from each other in many ways. They have been exploited very extensively in molecular biology research. They can insert themselves into DNA without requiring any similar sequence to be present in themselves and their “target”. This makes them powerful mutagens, since their insertion into a gene will generally prevent the gene from producing a functional protein. As mutagens, their effect is not always completely random, as some do have preferences for certain sequences or types of chromatin, and others will transpose preferentially to adjacent regions rather than randomly throughout the genome. Generally, a minimum of two things are required for transposition.

             The first is a gene which encodes transposase enzyme and which can recognize the ends of the transposon, excise the transposon (or a copy of it) from its starting point, cut DNA randomly or semi-randomly elsewhere, and catalyze the movement of the transposon from its starting point to its new target. The transposase can be encoded either by the transposon itself (in which case the transposon is often referred to as being “autonomous”) or elsewhere – on another transposon, for example, or on a plasmid. Transposons which rely on a transposase which they do not themselves carry are called “non-autonomous”. They occur naturally, and any autonomous transposon can be converted to a non-autonomous one by removing the transposase gene from it. The second requirement for transposition are the sequences at the ends of the transposon, which are recognized by the transposase. This minimal requirement for an autonomous transposon is shown below. There are many variants on this theme! Transposons use many different mechanisms to jump around the genome, but they all boil down to two basic types: replicative and nonreplicative. Replicative transposons copy themselves when they transpose, and leave one copy at the original site while inserting the second copy elsewhere. Non-replicative transposons are excised from their starting position and inserted elsewhere in the genome.

             In replicative transposition a), the transposon makes a copy of itself which is inserted randomly in the target DNA. In non-replicative transposition b), the transposon excises from its original position (usually leaving a small change in the sequence at the excision point) and reinserts at random in the target DNA.

They may be:

·        cause mutations

·        Increase (or decrease) the amount of DNA in the genome.

            These mobile segments of DNA are sometimes called “jumping genes”. They were discovered by Barbara McClintock early in her career, for which she was awarded a Nobel prize in 1983. The chromosomal basis of heredity was already well established by the time McClintock began her graduate training in the Botany Department at Cornell University. Her experiments laid the groundwork for a serie of cytogenetic discoveries by the Cornell maize genetics group between 1929 and 1935. McClintock developed a method for using broken chromosomes to generate new mutations. Among the progeny of plants that had received a broken chromosome from each parent, she observed unstable mutations at an unexpectedly high frequency, as well as a unique mutation that defined a regular site of chromosome breakage. These observations so intrigued her that she began an intensive investigation of the chromosome-breaking locus. Within several years she had learned enough to reach the conclusion, published in 1948, that the chromosome-breaking locus did something unknown for any genetic locus: it moved from one chromosomal location to another, a phenomenon she called transposition.

              Transposons are discrete segments of DNA capable of moving through the genome of their host via an RNA intermediate in the case of class I retrotransposon or via a “cut-and-paste” mechanism for class II DNA transposons. Since transposons take advantage of their host’s cellular machinery to proliferate in the genome and enter new hosts, transposable elements can be viewed as parasitic or “selfish DNA”. However, transposons may have been beneficial for their hosts as genome evolution drivers, thus providing an example of molecular mutualism. Some transposable elements contain heat-shock like promoters and their rate of transposition increases if the cell is subjected to stress, thus increasing the mutation rate under these conditions, which might be beneficial to the cell.

           The evolution of transposons and their effect on genome evolution is currently a dynamic field of study. Transposons are found in all major branches of life. The study of transposable genetic elements and transposition became the central theme of her genetic experiments from the mid 1940s until the end of her active research career. This was incredulous at the time, DNA was believed to be stable and invariable. They may or may not have originated in the last universal common ancestor, or arisen independently multiple times, or perhaps arisen once and then spread to other kingdoms by horizontal gene transfer. While transposons may confer some benefits on their hosts, they are generally considered to be selfish DNA parasites that live within the genome of cellular organisms. In this way, they are similar to viruses. Viruses and transposons also share features in their genome structure and biochemical abilities, leading to speculation that they share a common ancestor.

          Since excessive transposon activity can destroy a genome, many organisms seem to have developed mechanisms to reduce transposition to a manageable level. Bacteria may undergo high rates of gene deletion as part of a mechanism to remove transposons and viruses from their genomes while eukaryotic organisms may have developed the RNA interference (RNAi) mechanism as a way of reducing transposon activity. In the nematode Caenorhabditis elegans, some genes required for RNAi also reduce transposon activity.

          Interspersed Repeats within genomes are created by transposition events accumulating over evolutionary time. These Interspersed Repeats block gene conversion thereby catalyzing the formation of new genes. Transposons are therefore an evolutionary device promoting creation of new genes and protecting novel gene sequences from being overwritten by similar gene sequences through gene conversion.

There are three distinct types:

·  Class II Transposons consisting only of DNA that moves directly from place to place.

·   Class III Transposons; also known as Miniature Inverted-repeats Transposable Elements or MITEs.

·   Retrotransposons (Class I) that

           -first transcribe the DNA into RNA and then

          -use reverse transcriptase to make a DNA copy of the RNA to insert in a new location.

          Many transposons move by a “cut and paste” process: the transposon is cut out of its location (like command/control-X on your computer) and inserted into a new location (command/control-V). This process requires an enzyme — a transposase — that is encoded within some of these transposons. Transposase binds to:

·  both ends of the transposon, which consist of inverted repeats; that is, identical sequences reading in opposite directions.

·   a sequence of DNA that makes up the target site. Some transposases require a specific sequence as their target site; other can insert the transposon anywhere in the genome.

     After the transposon is ligated to the host DNA, the gaps are filled in by Watson-Crick base pairing. This creates identical direct repeats at each end of the transposon. Often transposons lose their gene for transposase; but as long as somewhere in the cell there is a transposon that can synthesize the enzyme, their inverted repeats are recognized and they, too, can be moved to a new location.

           Retrotransposons move by a “copy and paste” mechanism but in contrast to the transposons described above, the copy is made of RNA, not DNA.  The RNA copies are then transcribed back into DNA — using a reverse transcriptase — and these are inserted into new locations in the genome.  Many retrotransposons have long terminal repeats (LTRs) at their ends that may contain over 1000 base pairs in each. Like DNA transposons, retrotransposons generate direct repeats at their new sites of insertion. In fact, it is the presence of these direct repeats that often is the clue that the intervening stretch of DNA arrived there by retrotransposition. 42% of the entire human genome consists of retrotransposons.

            Retroviruses were first identified 80 years ago as agents involved in the onset of  cancer. More recently the AIDS epidemic has been shown to be due to the HIV retrovirus. In the early 1970s it was discovered that retroviruses had the ability to replicate their RNA genomes via conversion into DNA which became stably integrated in the DNA of the host cell. It is only comparatively recently that retroviruses have been recognized as particularly specialized forms of eukaryotic transposons. In effect they are transposons which move via RNA intermediates that usually can leave the host cells and infect other cells. The integrated DNA form (provirus) of the retrovirus bears a marked similarity to a transposon.

           The transposition cycle of retroviruses has other similarities to prokaryotic transposons, which suggest a distant familial relationship between these two types of transposon. Crucial intermediates in retrovirus transposition are extrachromosomal DNA molecules. These are generated by copying the RNA of the virus particle into DNA by a retrovirus-encoded polymerase called reverse transcriptase. The extra chromosomal linear DNA is the direct precursor of the integrated element and the insertion mechanism bears a strong similarity to “cut and paste” transposition.

            One family of transposons in the fruit fly Drosophila melanogaster are called P elements. They seem to have first appeared in the species only in the middle of the twentieth century. Within 50 years, they have spread through every population of the species. Transposons are also a widely used tool for mutagenesis of most experimentally tractable organisms.

             In bacteria, transposons can jump from chromosomal DNA to plasmid DNA and back, allowing for the transfer and permanent addition of genes such as those encoding antibiotic resistance (multi-antibiotic resistant bacterial strains can be generated in this way). Bacterial transposons of this type belong to the Tn family. When the transposable elements lack additional genes, they are known as insertion sequences. The most common form of transposon in humans is the Alu sequence. The Alu sequence is approximately 300 bases long and can be found between 300,000 and a million times in the human genome.

            Mariner-like elements are another prominent class of transposons found in multiple species including humans. The mariner transposon was first discovered by Jacobson and Hartl in drosophila1. This type-II trasposable element is known for its uncanny ability to be transmitted horizontally in many species. There are an estimated 14 thousand copies of mariner in the human genome composing of 2.6 million base pairs. These characteristics of the mariner transposon have inspired the science fiction novel titled, “The Mariner Project.”

 References:

 1.    McClintock, B. (June 1950). “The origin and behavior of mutable loci in maize”. Proc Natl Acad Sci U S A. 36 (6): 344–55.

2.    Rubin GM, Spradling AC (October 1982). “Genetic transformation of Drosophila with transposable element vectors”. Science 218 (4570): 348–53.

3.    Wilson MH, Coates CJ, George AL (January 2007). “PiggyBac transposon-mediated gene transfer in human cells”. Mol. Ther. 15 (1): 139–45.

4.    Mandal, P.K. & Kazazian, H.H., Jr. SnapShot: Vertebrate transposons. Cell 135, 192-192 e1 (2008).

5.    Kidwell, M.G. (2005). “Transposable elements.”. in (ed. T.R. Gregory). The Evolution of the Genome. San Diego: Elsevier. pp. 165–221. ISBN 0-12-301463-8. 

6.    Craig NL, Craigie R, Gellert M, and Lambowitz AM (ed.) (2002). Mobile DNA II. Washington, DC: ASM Press. ISBN 978-1555812096. 

7.    Lewin B (2000). Genes VII. Oxford University Press.. ISBN 978-0198792765. 

 

Contact :      waseemishfaq@gmail.com

 

 

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RNA Interference

Posted by admin on Jun 8, 2010 in Articles | 0 comments

RNA interference (RNAi) or double-stranded RNA (dsRNA) is a system within living cells that helps to control which genes are active and how active they are. siRNAs were first discovered by David Baulcombe’s group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants1 and later independently identified in wide variety of eukaryotic organisms. These dsRNAs are rapidly processed into short RNA duplexes of 21 to 28 nucleotides in length, which then guide the recognition and ultimately the cleavage of complementary single-stranded RNAs, such as messenger RNAs or viral genomic/antigenomic RNA (Fig. 1). According to their origin or function, naturally occurring small RNA have been described: short interfering RNAs (siRNA), repeat-associated short interfering RNA (rasiRNA or shRNA) and microRNA (miRNA). RNA interference has many biological functions – it is a vital part of the immune response against viruses and also downregulates gene expression by transcriptional silencing of genes or upregulates promoting by RNA activation. Finally, artificial introduction of long dsRNA or siRNA has been adopted as a tool to inactivate gene expression, both in cultured cells and in living organisms.http://www.biosyn.com/TEWdetail.aspx?TEWid=180

A biochemical understanding of the RNAi pathway was crucial to realizing that dsRNAs shorter than 30 base pairs (bp) could be used to trigger an RNAi response in mammals. Tuschl and colleagues showed that transfection of mammalian cells with short RNAs could induce the sequence-specific RNAi pathway, and so overcame the barrier to the use of RNAi as a genetic tool in mammals2. The impetus to use siRNAs and other small RNAs in mammalian cells also came from the long-standing view that protein kinase receptor (PKR) activation3 and similar responses were not effectively triggered by short dsRNAs. Following the initial reports, it took a remarkably short period of time for siRNAs triggers to be adopted as a standard component of the molecular biology toolkit. siRNAs can be introduced into mammalian cells using a variety of standard transfection methods. The strength and duration of the silencing response is determined by several factors: on a population basis, the silencing response is affected mainly by the overall efficiency of transfection, which can be addressed by optimizing conditions. In each cell, silencing depends on the amount of siRNA that is delivered and on the potential of each siRNA to suppress its target, or its potency. Even a relatively impotent siRNA can silence its target provided that sufficient quantities of the siRNA are delivered. However, essentially ‘forcing’ the system by delivering large amounts of reagent is likely to lead to numerous undesired effects.

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