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: > [email protected]). > > 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 <[email protected]>: >> 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 <[email protected]> >>> >>> 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 >> >> Email: [email protected] >> Tel: +33 4 76 20 77 68 >> Fax: +33 4 76 20 71 99 >> Skype: hartdarren >> Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex >> 9, France >> ********************************************************************** > > > > -- > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: [email protected] > ******************************************* -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: [email protected] *******************************************
