Archive for June 2011
Agrobacterium tumefaciens is a bacterium which, with the help of the Ti megaplasmid, manipulates plants into providing it with a safe and nutritious environment. The discovery of this interaction has been important not only for understanding the natural history of the bacterium, but for its use as a tool to transform plants. This has both allowed better understanding of plant biology and allowed genetic modification for agriculture.
The bacterium was first isolated by Erwin Smith. He was investigating a daisy with Crown Gall disease and found he could isolate a bacterium which produced a slimy exudate. He inoculated healthy plants with the bacterium and found that they developed tumours. This property gave the bacterium its species name – the tumour causer.
In the 1930s, Braun characterised the bacterium further. He took a tumorous plant and heat killed the bacteria by incubating it at 65° C for three days. The plant cells survived and so cells from the tumour could be grafted onto healthy plants. They continued to divide and formed new tumours. This indicated that the bacteria had passed some determinant to the plants, the Tumour Inducing Principle, rather than constantly producing some chemical to cause the tumours.
Research somewhat stalled here until the growth of molecular biology in the 1970s. Gel electrophoresis allowed the detection of plasmids in bacteria at this time, and scientists attempted to isolate them from Agrobacterium. They found that strains causing Crown Gall disease had a 250 kb megaplasmid. This property gave the plasmid its name the Ti plasmid (tumour inducing).
What is transferred?
Mary Dell Chilton prepared RNA from tumour cells, which she labelled and used to probe restriction fragments from the Ti plasmid. They hybridised, demonstrating that DNA is present in plant cells and transcribed into RNA.
Forward genetic mutant screens helped to shed light on this transferred (T-) DNA. Normally infected plants form tumours, this is due to high expression of two hormones. Cytokinin and auxin. In tms mutants less auxin was produced triggering shoot production. Conversely in tmr mutants less cutokinin was produced causing roots to form. When these loci were cloned it was shown that they coded for proteins involved in the biosynthesis of their respective hormones.
Another fascinating feature of the T-DNA is that is codes for the synthesis of a protein synthesing a particular opine. The exact opine depends on which strain of bacterium is involved. The non-transferred portion of the Ti plasmid codes in part for the metabolism of the respective opine. This means that any bacterium which loses the plasmid will no longer be able to benefit from resources produced by the plant.
What triggers DNA transfer?
In a mutant screen a series of virulence genes were identified. They were named vir A-G. These were probed by transforming the bacterium with a merodiploid reporter vector.
Analysis showed that virA was expressed constitutively, but that the other vir genes required plant washate for their activation. The inducers involved were wound phenolic compounds – acetylsyringone was found to be especially powerful. This allowed the discovery of an elegant regulatory mechanism involving a positive feedback loop. virA and virG are needed to activate the operon but do so only upon activation by a wound phenolic compound. This increases the rate of virG expression from a very small level and so in turn allows faster activation of the operon. This allows the virulence genes to be expressed in an all-or-nothing fashion.
What is the mechanism of transfer
Mary Dell Chilton sequenced the regions on either side of the T-DNA and found a 25bp repeat in direct orientation. Patty Zambryski showed that these borders were crucial for virulence. They attempted a Southern blot with a probe for T-DNA on induced Agrobacterium cells and found that they could identify this DNA even if they did not denature their sample before blotting, indicating the T-DAN was single stranded. They proposed a conjugation like mechanism. virD1D2 forms a nuclease which cuts at the borders. It is coated in the virE2 product and targeted to the plant through a type IV secretion system (the same type used by Heliobacter pylori). Once inside the plant importins target the protein DNA complex to the nucleus where it inserts.
Each of us has around 10^13 cells in our body, a mutation in just one of these can lead to a cancer. Thus it is crucially important that each cell has an advanced system for the detection of DNA damage and the control of appropriate response measures. These may be repair, via a number of pathways dependent on the type of damage, or senescence or even apoptosis when damage is severe.
Extent of damage
As many as one million lesions may occur per day in a single human cell. Damage can occur from endogenous sources, such as reactive oxygen species produced by metabolism. But it also created exogenously, by ultraviolet light and other ionising radiation, mutagens and viruses.
These can cause
- Oxidation of bases
- Alkylation of bases
- Hydrolysis – e.g. deamination, depurination, depyrimidation
- Formation of ‘bulky adducts’
- Mismatched bases
Every time a cell undergoes division its telomeres shorten. When these reach the Hayflick limit, the cell goes into senescence, an irreversible dormant state.
- Pyrimidine dimers, which are formed by UV irradiation, are repaired by photolyase on its activation by UV (photoreactivation).
- Guanine methylation is reversed by methyl guanine methyl transferase
- Some C and A methylation can also be directly reversed
- The adaptive response in E.coli, upon alkylation of DNA backbone. Ada suicidally transfers alkyl groups onto its cysteine residues (not catalysis, stoichiometric). Methylation of ada -> activation of itself, + other alkylation responsive genes.
Single strand damage
- Base-excision repair (BER) after oxidation, alkylation, hydrolysis or deamination
Damaged base is removed by DNA glycosylase
AP endonuclease recognises “missing tooth” and cuts phosphodiester bond, allowing resynthesis by DNA polymerase. Then ligase seals backbone.
- Nucleotide excision repair (NER) recognises bulky lesion s which distort the helix (e.g. thymine dimers). Requires Uvr system in E. coli, = UvrABC + DNA Helicase II. UvrA-UvrB scans DNA, UvrA recognises distortions, leaves complex to be replaced by UvrC. UvrB cleaves bond downstream of DNA damage while UvrC cleaves upstream. Helicase II breaks hydrogen bonds to remove excised segment. The gap is filled by DNA Pol I.
- DNA mismatch repair (MMR) is strand-specific, the daughter strand is recognised as the one to be corrected. Three proteins are involved mutS, mutH and mutL. mutS forms a dimer that recognises the daughter strand’s mismatched base and binds, mutS recruits mutL dimers. MutH binds to hemimethylated sites, and is activated by mutL. It nicks the daughter strand and recruits DNA helicase II. MutSHL then travels behind the helix along the single strand it creates towards the mismatch. An exonuclease digests the tail left behind. The process ends past the mismathed site and PolIII can then repair the daughter strand.
Double strand breaks
- NHEJ – DNA Ligase IV uses microhomology to rejoin two ends (also used in VDJ recombination. Ku?
- Micro-homology mediated end joining (MMEJ) uses the Ku protein too
- Homologous recombination-uses sister chromatid/ homolgous chromosome