Transposable element

Transposable elements (TEs) are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's genome size. Barbara McClintock's discovery of these jumping genesearly in her career earned her a Nobel prize in 1983. TEs make up a large fraction of the C-value of eukaryotic cells. They are often considered "junk DNA". In Oxytricha, which has a unique genetic system, they play a critical role in its development. They are also very useful to researchers as a means to alter DNA inside a living organism. Classification: Transposable elements are only one of several types of mobile genetic elements. They are assigned to one of two classes according to their mechanism of transposition, which can be described as either "copy and paste" (for class I TEs) or "cut and paste" (for class II TEs). Class I (Retrotransposons): They copy themselves in two stages, first from DNA to RNA by transcription, then from RNA back to DNA byreverse transcription. The DNA copy is then inserted into the genome in a new position. Reverse transcription is catalyzed by a reverse transcriptase, which is often coded by the TE itself. Retrotransposons behave very similarly to retroviruses, such as HIV. There are three main orders of retrotransposons (other orders are less abundant): Those with long terminal repeats (LTRs): encode reverse transcriptase, similar to retroviruses; LINEs: encode reverse transcriptase, lack LTRs, transcribed by RNA polymerase II; SINEs: do not code for reverse transcriptase, transcribed by RNA polymerase III. Retroviruses can be considered as TEs. Indeed, after entering a host cell and converting their RNA into DNA, retroviruses integrate this DNA into the DNA of the host cell. The integrated DNA form (provirus) of the retrovirus is viewed as a particularly specialized form of eukaryoticretrotransposon, which is able to encode RNA intermediates that usually can leave the host cells and infect other cells. The transposition cycle of retroviruses also has similarities to that of prokaryotic TEs. The similarities suggest a distant familial relationship between these two TEs types. Class II (DNA transposons): By contrast, the cut-and-paste transposition mechanism of class II TEs does not involve an RNA intermediate. The transpositions are catalyzed by various types of transposase enzymes. Some transposases can bind non-specifically to any target site, while others bind to specific sequence targets. The transposase makes a staggered cut at the target site producing sticky ends, cuts out the DNA transposon and ligates it into the target site. A DNA polymerase fills in the resulting gaps from the sticky

ends and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the TE excision by transposase). The duplications at the target site can result in gene duplication, which plays an important role in evolution Not all DNA transposons transpose through a cut-and-paste mechanism. In some cases a replicative transposition is observed in which transposon replicates itself to a new target site (e.g. Helitron (biology)). Cut-and-paste TEs may be duplicated if transposition takes place during S phase of the cell cycle when the "donor" site has already been replicated, but the "target" site has not. Both classes of TEs may lose their ability to synthesise reverse transcriptase or transposase through mutation, yet continue to jump through the genome because other TEs are still producing the necessary enzymes. Hence, they can be classified as either "autonomous" or "non-autonomous". For instance for the class II TEs, the autonomous ones have an intact gene that encodes an active transposase enzyme; the TE does not need another source of transposase for its transposition. In contrast, non-autonomous elements encode defective polypeptides and accordingly require transposase from another source. When a TE is used as a genetic tool, the transposase is supplied by the investigator, often from an expression cassette within a plasmid.

GENETIC MARKERS; APPLICATIONS OF DNA TECHNOLOGY

I. What are genetic markers? II. Restriction fragment length polymorphisms. III. Variable number of tandem repeats. IV. Single nucleotide polymorphisms. V. Gene therapy. VI. Transgenic organisms. I. Genetic markers are inherited variations that can be used to understand genetic events. They have a number of applications. A. In order to be useful as a marker, the inherited variations must be polymorphic, i.e. there must be two or more common variations in the population under study. Monomorphic sites/genes have only one common form and therefore are not useful as markers. B. A genetic marker can be a functional gene. However, it is not the function that is of interest but the variations in DNA structure that are reflected in functional differences between alleles. For example, blood groups can be used in gene mapping or to resolve issues of identity, paternity, or who committed a crime. The biological function of blood groups is not related to these applications. C. A genetic marker can also be a segment of DNA that has no known function or that is known not to have a function. So long as that DNA varies among homologous chromosomes, it can serve as a genetic marker that is transmitted according to Mendelian rules for codominant alleles. D. There are some dozens of polymorphic functional genes that are useful as markers. By contrast, there are thousands of polymorphic DNA sites that are useful as markers.

II. Some of the techniques developed for DNA manipulation are used to detect DNA variations known as restriction fragment length polymorphisms (RFLPs). A. Some one per thousand base pairs (bp = nucleotide pairs) varies in the population, i.e. instead of an AT pair; there might be a TA, GC, or CG pair. Often these are polymorphic. B. Some of these variants involve a sequence susceptible to attack by a restriction enzyme. The DNA of one sequence may be cut by a certain restriction enzyme; the other sequence is not a target for this restriction enzyme and is not cut. Since there will be flanking restriction sites, the first form will generate two DNA fragments; the second only one. C. The fragments can be separated on the basis of size by means of electrophoresis and detected in Southern blots. D. The "trait" that is expressed is variation in length of fragments of DNA (restriction fragments); thus the name restriction fragment length polymorphism. Most restriction sites are not polymorphic, however. E. These DNA variations are transmitted strictly by Mendelian rules as codominant traits and make excellent genetic markers. III. Repetitive sequences of DNA are especially useful genetic markers. A. There are many DNA sequences of unknown function that are repeated hundreds or even hundreds of thousands of times in the genome. Some are typically scattered throughout the genome; others are repeated in tandem and may occur at only a few sites. B. At a tandem repeat site, the number of repeats varies widely in the population, although the repeat number is usually well preserved during transmission. Therefore each different repeat number can be treated as a separate "allele" and the site can be treated as a highly polymorphic site with multiple alleles. Such a site is known as a VNTR (variable number of tandem repeats) site. C. Minisatellites have a repeating unit of 100-200 nt. 1. A restriction enzyme that cleaves on either side of the VNTR region generates fragments that differ in length. 2. These "alleles" are detected by Southern blots, using probes that bind to the repeating unit. D. Microsatellites have repeating units of 2-4 nt. 1. Because the overall length of the VNTR region is small, PCR can be used to amplify the entire region by selecting primers that flank the VNTR region. 2. The resulting fragments are separated by electrophoresis and do not require a probe for detection. IV. Single nucleotide polymorphisms (SNP's) are variations in the nucleotide present at a specific position on a chromosome. These are becoming especially important as markers because (1) they are very stable, i.e. have very low mutation rates; and (2) they can be amplified with PCR for testing. V. The techniques developed for gene cloning are the basis for most approaches to gene therapy.

A. Functional versions of the defective genes are cloned into viral vectors that introduce the desired gene into cells of the affected person. B. In ex vivo procedures, cells (usually bone marrow cells) of the patient are removed and infected with a vector into which has been inserted the functional gene. The modified cells are injected back into the patient, where they are encouraged in various ways to flourish. This approach is useful for conditions in which the primary defect is in the lymphocytes or is in blood metabolites that are in contact with the lymphocytes. E.g. ADA in severe combined immunodeficiency (Adenosine deaminase, mutation in chromosome 20). C. In in vivo procedures, the viral vector is introduced into the patient, where it invades target cells, carrying along the correct version of the gene. E.g. adenovirus vector and CFTR, the gene that is defective in cystic fibrosis. VI. Genes can be inserted into other species to create transgenic organisms. Several distinctive procedures exist for different organisms. A. In plants, genes are incorporated into a virus called Ti that can infect plants in general and that integrates into and is transmitted as part of the host DNA. E.g. FlavrSavr tomatoes. B. In mice, DNA is injected directly into a zygote, which is then implanted into a foster mother. During the early cell divisions, the injected DNA frequently is incorporated into chromosomes and becomes part of the genome of the descendent cells. The mouse that develops from that zygote is a chimera, i.e. some of the cells are modified and some are not. However, individual gametes produced by that mouse either have the new DNA or they do not. Therefore, some offspring of that mouse will come from zygotes that are modified, and the offspring will not be chimeras. C. In knockout mice, the DNA that is injected into the zygote is engineered so that it is inserted in the homologous site in the host DNA, causing the host gene to become nonfunctional. In this way, mice can be bred that have specific genes mutated to nonfunctional forms. Many recessive genes of great interest in humans have been "knocked out" in mice, creating the mouse equivalents of human diseases. E.g. cystic fibrosis.

Viral Retrotransposons
general structure: LTR - terminal direct repeats 100-1000 bp contain transcription control elements rest of element - encodes 1 or two open reading frames orf(s) have a gag-pol arrangement gag - NTP binding pol - protease, RNAse H, reverse transcriptase modules these features resemble, in sequence and organization, genes

found in retroviruses (and pararetroviruses) The origins of much highly-repetitive DNA reversion - low frequency (compared with Ac); presumably via recombination at LTRs insertion generates 4-6 bp duplications high copy number (>100/genome) expression characteristics - not well understood transposition properties - not well studied incomplete elements - analogous to non-autonomous TIR elements (incomplete orfs) transposition mechanism - presumed to be via reverse transcription: mRNA DNA Recombine Non-viral retro-elements Derived from reverse transcription of endogenous RNAs in the cell Source of many highly-repetitive DNA classes (Alu, LINEs, ) [polIII transcripts will accumulate as repetitive DNA because promoters are within the RNA coding region for these genes] transposition mechanism - presumed to be via reverse transcription: mRNA DNA recombine