Posted: Mon Apr 23, 2007 1:22 pm
We are not discussing retrotranslocated elements per se. The reconstruction is base on HERV-K proviruses.NewCreature wrote:OK I'm good with that Phoenix integrates in a petri dish with the exact signature of existing HERV-K elements. IT is not clear how the sequences would change in a living human. Many changes to the sequence would result from transposition and interaction with specific substrates, transposases, recombinases, and other cellular mechanisms. Many of the mutations visible in existing HERV-K sequnces could happen during reverse transcription to the RNA intermediary during retrotranslocation.
The signature may include these other elements, but we can differentiate between these different elements. Otherwise a reconstruction would not have been possible.
"To construct a consensus HERV-K(HML2) provirus, we assembled all of the complete copies of the 9.4-kb proviruses that are human specific (excluding those with the 292-nt deletion at the beginning of the env gene) and aligned their nucleotide sequence to generate the consensus in silico, taking for each position the most frequent nucleotide."(1)
Again please site the source of this information.NewCreature wrote:It would be unwise to generalize from one instance in the laboratory. Again it has been shown that a proviral element can insert in a specific place on a specific chromosome. Clearly the mechanism exists.BGoodForGoodSake wrote:Yes, I hope this clarifies insertion affinity in general.NewCreature wrote:The same sequence was not located in that cells DNA in the same spot as we find that sequence in the human genome.
We are not talking about activity after integration when discussing integration sites. Also retroviruses have not shown exact integration preference, in any experiments I know of. Additionally this phenemonen is not unique to this paper. I am certainly not generalizing from a single instance.(2)(3)(4)
Please site an instance of a retroviral integrase which is site specific to the level you have just described. The same for recombinases. Now, lets answer the question is this the general pattern we find among retroviruses?NewCreature wrote:Remember that shortly after integration a retrovirus becomes a provirus. There are site specific recombinases and integrases as well.
Also would not a virus which can target multiple integration sites have an advantage?
I don't think our contentions are in these areas, we are specifically focused on integration sites, and not on recombinatorial elements afterwards.NewCreature wrote:I agree but that is where the understanding gets cloudy. The propagation once inserted is a bit more fuzzy. This is a young field compared to many other areas of science, but we are gaining knowledge rapidly. IF we are patient perhaps many of our contentious points will be more clearly demonstrated in the coming years.BGoodForGoodSake wrote:What is meant by this, we have a fairly good understanding of how the viral material finds its way into the genome.
Is it not significant that the pattern of insertion by phoenix matches the patterns found in the human genome?NewCreature wrote:You asked how I can question the Viral Origin of HERV-K. It very well may be viral in origin. Phoenix simply took information that naturally exists in the human genome and packaged it in a retrovirus and inserted it through that mechanism. Nearly half of the human functioning genome is reverse transcribed. IT is becoming more and more clear that these are important elements of our life. Further I would point out again that most retroelements don't have naturally occurring exogenous retroviruses.
What is the significance that half the genome(your figure, not significant) resulted from retrotransposons?
From your knowledge of retroviruses, does the method of insertion (not to be confused with insertion into the genome) into the host cell matter in this discussion?
There may be some instances where ERV's are implicated in human related function, but certainly this does not apply to the majority of cases.
HERV-K activity for instance has been observed in teratocarcinoma.(5)
Finally why would you expect a homologous exogenous retrovirus if the conclusion is that this insertion occurred <5 million years ago?
The method by which the material was inserted into the host cell does not matter. Once inside the envelope material is no longer part of the insertion process.NewCreature wrote:I would ask a question in response. What observations demonstrate that HERV-K must be from a virus?
The methodology use to reconstruct the virus was purely statistical. (see below)
The signature of phoenix is identical to the pattern of HERV-K.
The distribution of HERV-K is consistent with that of viral infection and reinfection inter uterine.
Methodology used to reconstruct the viral RNA.
"To construct a consensus HERV-K(HML2) provirus, we assembled all of the complete copies of the 9.4-kb proviruses that are human specific (excluding those with the 292-nt deletion at the beginning of the env gene) and aligned their nucleotide sequence to generate the consensus in silico, taking for each position the most frequent nucleotide
...
Based on this in silico reconstruction, we then generated a molecular clone corresponding to the consensus DNA proviral sequence that we named Phoenix, using the related K108 and K109 proviruses (which we had previously cloned from a commercial human BAC library) as a backbone, and a commercial kit to introduce single nucleotide mutations at each position required to match the consensus."(1)
1. Dewannieux, M, Harper, F, Richaud, A, Letzelter, C, Ribet, D, Pierron, G & Heidmann, T. (2006) Genome Res 16, 1548—1556.
2. Boeke, J.D. and Stoye, J.P. 1997. Retrotransposons, endogenous retroviruses, and the evolution of retroelements. In Retroviruses (eds. J.M. Coffin, et al.), pp. 343—435. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
3.Brown, P.O. 1997. Integration. In Retroviruses (eds. J.M. Coffin, et al.), pp. 161—204. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
4. Bushman, F.D. 2003. Targeting survival. Integration site selection by retroviruses and LTR-retrotransposons. Cell 115: 135—138.
5. Dewannieux et al. Genome Res..2006; 16: 1548-1556