Quasispecies

From: Jacob Keller
Date: 24 January 2012 18:25

Whoops--I meant to change the subject line, so if you want to reply,
please use this one not to perturb the original post.

JPK


> Inspired by the recent post about "quasispecies:"
>
> I have been bothered recently by the following problem: why do species
> of genetic uniformity exist at all (or do they?)? This first came up
> when I saw a Nature paper describing live bacteria extracted from a
> supposedly 250-million-year-old salt crystal whose 16S RNA was 99%
> identical to marismortui bacteria (ref below). What? Are the bacteria
> the same now as 250 million years ago? But there is a further
> question: given the assumptions of evolution, why should there be any
> bacterium whose genome is the same as any other, assuming that
> equivalent codons are really equivalent (or at least roughly so), and
> that even at the protein level, there is such a thing as "neutral
> drift?" After all, we even see in our lab cultures that they (at least
> e coli) mutate fairly frequently, so why is there such a thing as "e
> coli" at all, at least at the nucleotide level? I don't think we
> usually say that each bacterial species is totally optimized in all
> its features, do we? Even assuming that every single protein must be
> just so, shouldn't there be as many species of e coli as there are
> possible genomes encoding the same protein set, i.e. some extremely
> large number? Why is there any uniformity at all? Or IS there--maybe
> the bacteria too are only quasispecies...? And maybe also...
>
> JPK
>
>
>
>
> Nature 407, 897-900 (19 October 2000) | doi:10.1038/35038060; Received
> 15 November 1999; Accepted 4 July 2000
>
> Isolation of a 250 million-year-old halotolerant bacterium from a
> primary salt crystal
>
> Russell H. Vreeland1, William D. Rosenzweig1 & Dennis W. Powers2
>
> Department of Biology, West Chester University, West Chester,
> Pennsylvania 19383 , USA
> Consulting Geologist, Box 87, Anthony, Texas 79821, USA
> Correspondence to: Russell H. Vreeland1 Correspondence and requests
> for materials should be addressed to R.H.V. (e-mail: Email:
> rvreeland@wcupa.edu).
>
> Top of page
> Bacteria have been found associated with a variety of ancient
> samples1, however few studies are generally accepted due to questions
> about sample quality and contamination. When Cano and Borucki2
> isolated a strain of Bacillus sphaericus from an extinct bee trapped
> in 25–30 million-year-old amber, careful sample selection and
> stringent sterilization techniques were the keys to acceptance. Here
> we report the isolation and growth of a previously unrecognized
> spore-forming bacterium (Bacillus species, designated 2-9-3) from a
> brine inclusion within a 250 million-year-old salt crystal from the
> Permian Salado Formation. Complete gene sequences of the 16S ribosomal
> DNA show that the organism is part of the lineage of Bacillus
> marismortui and Virgibacillus pantothenticus. Delicate crystal
> structures and sedimentary features indicate the salt has not
> recrystallized since formation. Samples were rejected if brine
> inclusions showed physical signs of possible contamination. Surfaces
> of salt crystal samples were sterilized with strong alkali and acid
> before extracting brines from inclusions. Sterilization procedures
> reduce the probability of contamination to less than 1 in 10 9.
>
> 2012/1/24 Darren Hart <hart@embl.fr>:
>> I think the explanation is this:
>> The source is natural viral RNA which is a mixture of naturally mutated
>> sequences (e.g. flu forms such a quasispecies)
>> See:
>> http://www.virology.ws/2009/05/11/the-quasispecies-concept/
>>
>> The pooled RNA has an average sequence that you see when you sequence the
>> pooled cDNA (individual mutations are hidden by the averaging effect of
>> having many sequences present).
>>
>> But when you clonally separate DNA molecules by transformation (1 plasmid
>> enters 1 cell to yield 1 colony), you see each individual molecule
>> represented 100% in the sequencing chromatogram from the plasmid DNA that
>> you have isolated from colonies.
>>
>> This is effect is commonly observed when sequencing influenza virus isolates
>> from patients. It will have nothing to do with the E. coli strain. You can
>> avoid it completely by using gene synthesis.
>>
>> Darren
>>
>>
>>
>> 2012/1/24 Rubén Sánchez Eugenia 
>>>
>>> Dear everyone,
>>>
>>> I am trying to clone a viral protein in the E. Coli BSJ strain and i am
>>> having some problems.
>>>
>>> I start from the viral RNA carrying out a reverse transcription and PCR
>>> (RT-PCR) to obtain the protein cDNA. When I sequence this cDNA to check for
>>> mutations, there are no mutations. So the RT-PCR works fine.
>>>
>>> Then, I digest the cDNA and I ligate it with a pET plasmid to transform
>>> the E. Coli BSJ strain. I get recombinant colonies (checked by colony-PCR)
>>> but when I sequence them I get various mutations (aprox. 2 miss-sense) on
>>> the inserted cDNA. Furthermore, these mutations are different among
>>> different transformations and even among colonies of the same plate (in the
>>> same transformation).
>>>
>>> Maybe these mutations are produced by the cell (because of the lack of
>>> mutations in the cDNA) but these E. Coli clonning strains are supposed to be
>>> "optimized" to prevent the insertion of mutations. So I have no idea about
>>> what may be the problem.
>>>
>>> I hope you could help me. Thank you.
>>>
>>> Best regards,
>>>
>>> --
>>> ---------------------------------------------------
>>> Rubén Sánchez
>>>
>>>
>>
>>
>>
>> --
>> **********************************************************************
>> Dr. Darren Hart,
>> Team Leader
>> High Throughput Protein Lab
>> Grenoble Outstation
>> European Molecular Biology Laboratory (EMBL)
>> **********************************************************************
>> www.embl.fr/research/unit/hart/index.html
>>
>> For funded access to ESPRIT construct screening via EU FP7 PCUBE:
>> http://tinyurl.com/ydnrwg4
>>
>
>
> --