Posts Tagged "Target"

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: Imgenex Launched the Psuppressoradeno Construction Kit for Adenovirus Mediated Gene Knockdown

Posted by admin on May 15, 2010 in Articles | 0 comments

RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has been observed in all eukaryotes, from yeast to mammals. The RNAi pathway is thought to be an ancient mechanism for protecting the host and its genome against viruses and rogue genetic elements that use double-stranded RNA (dsRNA) in their life cycles. They have also been shown to play a role not only in mRNA and dsRNA stability/degradation, but also in regulation of translation, transcription, chromatin structure, and genome integrity. In plants and animals, RNA silencing has been adapted to play a critical role in regulation of cell growth and differentiation using a class of small RNAs. In the RNA interference process, the dsRNAs get processed into 20-25 nucleotide (nt) small RNAs by an RNase III-like enzyme called Dicer. Then, the small RNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The small RNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. The small RNAs that provide target specificity to the silencing machinery includes short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and microRNAs (miRNAs) and is distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two distinct strands of perfectly base-paired RNA, while miRNAs originate from a single, long transcript that forms imperfectly base-paired hairpin structures. siRNAs were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA; however, endogenous sources of siRNAs have now been recognized. The endogenous siRNAs are derived from repetitive sequences within the genome, and are termed repeat-associated siRNAs, or rasiRNAs. miRNAs were discovered through their critical roles in development and cellular regulation, and represent a large class of evolutionarily conserved RNAs. miRNAs have always been recognized as being of endogenous origin. RNA interference has emerged as a natural mechanism for silencing gene expression over the past decade. This ancient cellular antiviral response can be harnessed to allow specific inhibition of the function of any chosen target genes, including those involved in causing diseases such as cancer, AIDS, and hepatitis. It is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Last but not the least the technology can be harnessed as a novel therapeutic agent and is suitable for combating viral diseases, cancers and inflammatory diseases.

Imgenex (San Diego) recently launched the pSuppressorAdeno construction kit for adenovirus mediated gene knockdown. The kit provides the ability to infect a broad range of cell types, including many primary cell lines as well as dividing and nondividing cells, according to a company official. The kit also offers the flexibility to validate sequences using the nonviral expression plasmid prior to construction of adenoviruses, notes Sujay K. Singh, Ph.D., president and CEO of Imgenex, which markets plasmid-based RNA interference (RNAi) products. “One of the greatest advantages is the ability of recombinant adenovirus vectors to reduce gene expression both in vitro and in vivo,” he adds. RNAi, initially considered a bizarre attribute of petunias and later a gene-silencing mechanism in worms, is creating a stir as one of the hottest new technologies in molecular biology. It is revolutionizing the field of functional genomics.

For more information about“RNA interference” please visit www.imgenex.com/rna_interference.php />

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How Short Is Short In Rnai Research

Posted by admin on Apr 29, 2010 in Articles | 0 comments

The original work of Mello and Fire from the Univ. of Massachusetts demonstrated in C. elegans, that gene expression is controlled by RNA interference (RNAi) . The initial patent applications filed by the Univ. of Massachussetts, with both Mello and Fire as the inventors, and covering this use of RNAi, clained only dsRNA longer than 25 bp’s. Later on, work done in mamalians showed that the long ds RNA was capable of inducing the release of IFN and other pro-inflammatory cytokines, causing dangerous reactions in the animals we tested. We now know that long dsRNA is recognized in the endosomes by TLRs, inducing an undesirable immune response in the animals; a situation that created a new challenge in the study of RNAi. Subsequently other researchers, such as Tuschl et.al. now at Rockefeller Univ, realized that shorter dsRNAi fragments of 25bp of smaller, while still capable of inhibiting the expression of a targeted gene, failed to induce an innate immunity response. In other words, the mammalian system is still capable of utilizing the diced dsRNA produced by the enzyme Dicer, which normally chops down the long dsRNA to sizes of 21-23 nts with 2 bases overhanging at the 3’ends of each strand. These 2 bases overhanging in dsRNA suggests that perhaps Dicer cleaves the long dsRNA in a fashion analogous to restriction enzymes. This short dsRNA can then interact with the RISC complex, where the guide strand is prepared and readied up to base pair with the target mRNA for its cleavage.

The RNAi situation is a good example of the unexpected in science. Although at the time of the initial discovery it was hard to predict that very small fragments of RNA could be pivotal in such important newly found mechanism, currently, even shorter dsRNA fragments, e.g. 15-18 bps’ are being tested. These new third generation modifiers such as LNA’s, UNA’s and others, because of their size have significant therapeutic potential.

There is a significant amount of ongoing research to elucidate the fine details of this novel gene control mechanism, including but not limited to studies of how miRNA precursors are transported to specific compartments of the cell, as these events may play important roles in the processing of the precursor by Dicer to render the active mature form of dsRNA.

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