Survival of only the shortest - an extracellular darwinian experiment. by Alexander Borowac

Transcription

1 Survival of only the shortest - an extracellular darwinian experiment by Alexander Borowac

2 Historical notes Example survival of only the shortest Qb virus Experiment Results

3 Charles Darwin ( ) Historical notes ( 1860s ) Survival of the fittest Herbert Spencer ( philosopher and biologist )

4 Example: Survival of the biggest t

5 Example: Survival of the biggest shortest

6 What size are we interested in? Giraffe ( ~ 5 m ) Mouse ( ~ 6 cm) Flea (~ 1mm) E.coli ( ~ 1 μm) Bacteriophage Qβ (~ 25nm) q=gr%c3%b6%c3%9fe&form=hdrs C2#view=detail&id=D05E8CDE46DBD C9D14EBDCB1DD1AA8AAEA4DED68& selectedindex=2

7 What size are we interested in? Giraffe ( ~ 5 m ) Mouse ( ~ 6 cm) E.coli ( ~ 1 μm) Flea (~ 1mm) Bacteriophage Qβ (~ 25nm) q=gr%c3%b6%c3%9fe&form=hdrs C2#view=detail&id=D05E8CDE46DBD C9D14EBDCB1DD1AA8AAEA4DED68& selectedindex=2

8 Sol Spiegelman * 1914 New York City; 1983 Professor of microbiology at the University of Illinoiss Over 350 papers. Worked a lot on RNA / DNA RNA/DNA Hybridization in Viruses, Famous experiment: Spiegelman's Monster man

9 Bacteriophage Qβ - Virus 25nm thick Single stranded RNA (surrounded by protein-coat) Host: E.coli tic_rna3-qb_virus.htm

10 Bacteriophage Qβ - genome nucleotides Codes four proteins A1 : extension coat protein (one / virus ) A2 : maturation protein ( lytic function in the host cell and lysis of host cell ) Coat protein B1: Qβ Replicase ( RNA dependet RNA polymerase ) tic_rna3-qb_virus.htm

11 The Problem How can a virus based on RNA complete its life cycle in a cell dominated by DNA? - Spiegelman assumed reverse transcription enzymes, but could find no evidence of a "reverse transcriptase in E.coli - RNA-dependent polymerase specific to Qβ-RNA (when RNA bases and strands of Qβ-RNA were combined with this polymerase, the RNA replicate) - Spiegelman reasoned that the RNA strand injected into its host must serve directly as its own "translator" for directing its replication.

12 an extracellular darwinian experiment from 60s

13 Transfer serial method Transfer serial method extracted Qβ-RNA 1 st 2 nd 3 th 74 th time reaction tubes ( 0.25ml ) RNA dependent Qβ RNA polymerase (Q β replicase) 40μg 32 P nucleotides ( 32 P -> appraise the results ) puffer (ph)

14 How to create a selection pressure? time pressure S. Spiegelman reduced the reaction time after a few tubes to attain an selection pressure arathon-ohne-zeitdruck/

15 Time pressure 35 C 10min min 5min 14 20min 15min S.Spiegelman qbeta pnas 1967

16 Time pressure After the 4 th transfer, Qβ was not able to replicate in E.coli anymore S.Spiegelman qbeta pnas 1967

17 First results First results Qβ RNA still able for replication in vitro Synthetic Qβ - RNA : Spiegelman s monster Non infectious after 4 th transfer Reactions became faster still replicated RNA available

18 How to determine the size? 218 nucleotides S.Spiegelman qbeta pnas 1967

19 Second results Second results Qβ - RNA string became shorter : original 3600 nucleotides to 218 nucleotides A1 : extension coat protein A2 : maturation Coat protein 74 steps B1: Replicase ( remains to replicate RNA) 17% medical.net/health/what-is- RNA.aspx

20 Only the shortest RNA string survived in this darwinian experiment

21 Summary Assumption of Qβ-replicase Extracellular darwinian experiment Original Qβ - RNA with about 4000 nucleotides became a very short 218 nucleotide chain Only information for Qβ relpicate remains

22 Sources: ( ) QB_Virus.htm( ) An extracellular darwinian experiment with a self-duplicating nucleic acid molecule, S. Spiegelman et al., pnas 1967 A relicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication

23 Thank you