P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.[1]
The name was first suggested by evolutionary biologist Margaret Kidwell, who, together with James Kidwell and John Sved, researched hybrid dysgenesis in Drosophila. They referred to strains as P of paternal and M of maternal if they contributed to hybrid dysgenesis in this reproductive role.[2]
The P element encodes for an enzyme known as P transposase. Unlike laboratory-bred females, wild-type females are thought also to express an inhibitor to P transposase function, produced by the very same element. This inhibitor reduces the disruption to the genome caused by the movement of P elements, allowing fertile progeny. Evidence for this comes from crosses of laboratory females (which lack the P transposase inhibitor) with wild-type males (which have P elements). In the absence of the inhibitor, the P elements can proliferate throughout the genome, disrupting many genes and often proving lethal to progeny or rendering them sterile.
P elements are commonly used as mutagenic agents in genetic experiments with Drosophila. One advantage of this approach is that the mutations are easy to locate. In hybrid dysgenesis, one strain of Drosophila mates with another strain of Drosophila, producing hybrid offspring and causing chromosomal damage known to be dysgenic. Hybrid dysgenesis requires a contribution from both parents. For example, in the P-M system, where the P strain contributes paternally and M strain contributes maternally, dysgenesis can occur. The reverse cross, with an M cytotype father and a P mother, produces normal offspring, as it crosses in a P x P or M x M manner. P male chromosomes can cause dysgenesis when crossed with an M female.
The P element is a class II transposon, and moves by a DNA-based "cut and paste" mechanism. The recognition sequence comprises four exons separated by three introns.[3] Complete splicing of the introns produces the transposase enzyme, while alternative partial splicing of introns 1 and 2, leaving only intron 3 in the mRNA transcript, encodes the P element repressor. The complete, autonomous P element encodes a transposase enzyme, which recognizes the 31-bp terminal inverted repeats at either end of the P element and catalyzes P element excision and re-insertion. The complete element is 2,907 bp in length; non-autonomous P elements contain an internal deletion of varying length which abolishes transposase production, but such elements can still be mobilized if a functional transposase is encoded elsewhere in the genome. P element insertion and subsequent excision necessarily leaves behind 8-bp direct repeats at the excision site; thus the presence of such repeats is indicative of previous P element activity.
All P elements have a canonical structure containing 31-bp terminal inverted repeats and 11-bp internal inverted repeats located at the THAP domain of the transposase. The shortest and longest P elements are non-autonomous elements. The longest P elements encode transposase needed for transposition. The same sequence that encodes the transposase also encodes a suppressor of transposition, which accumulates in the cytoplasm during the development of cells. Thus, in a cross of a P or M male with a P female, the female cytoplasm contains the suppressor, which binds to any P elements and prevents their transposition.
Hybrid dysgenesis refers to the high rate of mutation in germ line cells of Drosophila strains resulting from a cross of males with autonomous P elements (P Strain/P cytotype) and females that lack P elements (M Strain/M cytotype). The hybrid dysgenesis syndrome is marked by temperature-dependent sterility, elevated mutation rates, and increased chromosomal rearrangement and recombination.
The hybrid dysgenesis phenotype is affected by the transposition of P elements within the germ-line cells of offspring of P strain males with M strain females. Transposition only occurs in germ-line cells, because a splicing event needed to make transposase mRNA does not occur in somatic cells.
Hybrid dysgenesis manifests when crossing P strain males with M strain females and not when crossing P strain females (females with autonomous P elements) with M strain males. The eggs of P strain females contain high amounts of a repressor protein that prevents transcription of the transposase gene. The eggs of M strain mothers, which do not contain the repressor protein, allow for transposition of P elements from the sperm of fathers. In P strain females, the repressors are found in the cytoplasm. Hence, when P strain males fertilize M strain females (whose cytoplasm contain no repressor), the male contributes its genome with the P element but not the male cytoplasm leading to P strain progeny.[3]
This effect contributes to piRNAs being inherited only in the maternal line, which provides a defense mechanism against P elements.[4]
The P element has found wide use in Drosophila research as a mutagen. The mutagenesis system typically uses an autonomous but immobile element, and a mobile nonautonomous element. Flies from subsequent generations can then be screened by phenotype or PCR. Naturally-occurring P elements contain coding sequence for the enzyme transposase and recognition sequences for transposase action. Transposase regulates and catalyzes the excision of a P element from the host DNA, cutting at the two recognition sites, and then reinserting randomly. It is the random insertion that may interfere with existing genes, or carry an additional gene, that can be used for genetic research.
To use this as a useful and controllable genetic tool, the two parts of the P element must be separated to prevent uncontrolled transposition. The normal genetic tools are DNA coding for transposase with no transposase recognition sequences so it cannot insert and a "P Plasmid". P Plasmids always contain a Drosophila reporter gene, often a red-eye marker (the product of the white gene), and transposase recognition sequences. They may contain a gene of interest, an E. coli selectable marker gene, often some kind of antibiotic resistance, an origin of replication or other associated plasmid "housekeeping" sequences.
There are two main ways to utilise these tools:
The inserted gene may have damaged the function of one of the host's genes. Several lines of flies are required so comparison can take place and ensure that no additional genes have been knocked out.
Possible mutations:
See main article: Enhancer trap. The hijack of an enhancer from another gene allows the analysis of the function of that enhancer. This, especially if the reporter gene is for a fluorescent protein, can be used to help map expression of the mutated gene through the organism, and is a very powerful tool. It is a useful tool for looking at gene expression patterns (temporally and spatially).
These methods are referred to as reverse genetics. Reverse genetics is an approach to discover the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing
Once the function of the mutated protein has been determined it is possible to sequence/purify/clone the regions flanking the insertion by the following methods:
See main article: article and Inverse PCR.
The process of cutting, self ligation and re cutting allows the amplification of the flanking regions of DNA without knowing the sequence. The point at which the ligation occurred can be seen by identifying the cut site of [enzyme 1].