Definitions

microinject

P element

A P element is a transposon that is present in the fruit fly Drosophila melanogaster and is used widely for mutagenesis and the creation of genetically modified flies used for genetic research. The P element gives rise to a phenotype known as hybrid dysgenesis.

P elements seem to have first appeared in the species only in the middle of the twentieth century. Over the last fifty years, they have spread through every wild population, so that only older laboratory stocks lack them.

Characteristics

The P element is a class II transposon, which means that its movement within the genome is made possible by a transposase. The complete element is 2907 bp and is autonomous because it encodes a functional transposase; non-autonomous P elements which lack a functional transposase gene due to mutation also exist. Non-autonomous P elements can still move within the genome if there are autonomous elements to produce transposase. The P element can be identified by its terminal 31-bp inverted repeats, and the 8 bp direct repeats its movement into and out of DNA sequence produces.

Hybrid dysgenesis

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 chromosome rearrangement and recombination.

The hybrid dysgenesis phenotype is effected 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.

P element in molecular biology

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
  • Recognition sequences for transposase action

Transposase is an enzyme that regulates and catalyzes the excision of a P element from the host DNA, cutting at two recognition sites, and then reinserts 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 therefore:

  • DNA coding for transposase with no transposase recognition sequences so it cannot insert.
  • A "P Plasmid"

P Plasmids always contain:

  • A Drosophila reporter gene, often a red-eye marker (the product of the white gene).
  • Transposase recognition sequences.

And may contain:

Methods of usage

There are two main ways to utilise these tools:

Fly transformation

  1. Microinject the posterior end of an early-stage (pre-cellularization) embryo with DNA coding for transposase and a plasmid with the reporter gene, gene of interest and transposase recognition sequences.
  2. Random transposition occurs, inserting the gene of interest and reporter gene.
  3. Grow flies and cross to remove genetic variation between the cells of the organism. (Only some of the cells of the organism will have been transformed. By breeding only the genotype of the gametes is passed on, removing this variation).
  4. Look for flies expressing the reporter gene. These carry the inserted gene of interest, so can be investigated to determine the phenotype due to the gene of interest.

It is important to note that 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.

Insertional mutagenesis

  1. Microinject the embryo with DNA coding for transposase and a plasmid with the reporter gene and transposase recognition sequences (and often the E. coli reporter gene and origin of replication, etc.).
  2. Random transposition occurs, inserting the reporter gene randomly. The insertion tends to occur near actively transcribed genes, as this is where the chromatin structure is loosest, so the DNA most accessible.
  3. Grow flies and cross to remove genetic variation between the cells of the organism (see above).
  4. Look for flies expressing the reporter gene. These have experienced a successful transposition, so can be investigated to determine the phenotype due to mutation of existing genes.

Possible mutations:

  1. Insertion in a translated region => hybrid protein/truncated protein. Usually causes loss of protein function, although more complex effects are seen.
  2. Insertion in an intron => altered splicing pattern/splicing failure. Usually results in protein truncation or the production of inactive mis-spliced products, although more complex effects are common.
  3. Insertion in 5' (the sequence that will become the mRNA 5' UTR) untranslated region => truncation of transcript. Usually results in failure of the mRNA to contain a 5' cap, leading to less efficient translation.
  4. Insertion in promoter => reduction/complete loss of expression. Always results in greatly reduced protein production levels. The most useful type of insertion for analysis due to the simplicity of the situation.
  5. Insertion between promoter and upstream enhancers => loss of enhancer function/hijack of enhancer function for reporter gene.† Generally reduces the level of protein specificity to cell type, although complex effects are often seen.

Enhancer trapping
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.

Other usage of P elements

These methods are referred to as reverse genetics.

Secondary mobilisation

If there is an old P element near the gene of interest (with a broken transposase) you can remobilise by microinjection of the embryo with coding for transposase or transposase itself. The P element will often transpose within a few kilobases of the original location, hopefully affecting your gene of interest as for 'Insertional Mutagenisis'.

Analysis of mutagenesis products

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:

Inverse PCR

Main article: Inverse PCR

  1. Isolate the fly genome.
  2. Undergo a light digest (using an enzyme [enzyme 1] known NOT to cut in the reporter gene), giving fragments of a few kilobases, a few with the insertion and its flanking DNA.
  3. Self ligate the digest (low DNA concentration to ensure self ligation) giving a selection of circular DNA fragments, a few with the insertion and its flanking DNA.
  4. Cut the plasmids at some point in the reporter gene (with an enzyme [enzyme 2] known to cut very rarely in genomic DNA, but is know to in the reporter gene).
  5. Using primers for the reporter gene sections, the DNA can be amplified for sequencing.

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].

Plasmid rescue

  1. Isolate the fly genome.
  2. Undergo a light digest (using an enzyme [enzyme 1] known to cut in the boundary between the reporter gene and the E. coli reporter gene and plasmid sequences), giving fragments of a few kilobases, a few with the E. coli reporter, the plasmid sequences and its flanking DNA.
  3. Self ligate the digest (low DNA concentration to ensure self ligation) giving a selection of circular DNA fragments, a few with the E. coli reporter, the plasmid sequences and its flanking DNA.
  4. Insert the plasmids into E. coli cells (eg. by electroporation).
  5. Select plasmids for the E. coli selectable marker gene. Only successful inserts of plasmids with the plasmid 'housekeeping' sequences will express this gene.
  6. The gene can be cloned for further analysis.

References

  • Leland Hartwell et al.. 2004. Genetics - From Genes to Genomes 2nd Edition. McGraw-Hill
  • Engels, W. R. P Elements in Drosophila

External links

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