From Genome to Life: Structural, Functional and Evolutionary Approaches Cargèse, Corsica July , 2002 Hypothetico-deductive science and the function of cytoplasmic genomes John F. Allen Lund University, Sweden, Plant Cell Biology-Plant Biochemistry

Popper, K.R. The Bucket and the Searchlight: Two Theories of Knowledge. Appendix to "Objective Knowledge. An Evolutionary Approach". Oxford University Press, Oxford KARL POPPER

Hypothetico-deductive science (After Popper) Problem in existing knowledge Proposed, tentative solution - Hypothesis Predictions - potential falsifications - explanations of existing knowledge - reasons for doing experiments: to test hypotheses New Problem OR Falsification of hypothesis

Mitochondria and Chloroplasts From: Goodsell, D. S. The Machinery of Life. Springer-Verlag, New York and Berlin, 1993

Problem Why Do Mitochondria and Chloroplasts Have Their Own Genetic Systems? Why do mitochondria and chloroplasts require their own separate genetic systems when other organelles that share the same cytoplasm, such as peroxisomes and lysosomes, do not? …. The reason for such a costly arrangement is not clear, and the hope that the nucleotide sequences of mitochondrial and chloroplast genomes would provide the answer has proved unfounded. We cannot think of compelling reasons why the proteins made in mitochondria and chloroplasts should be made there rather than in the cytosol. Molecular Biology of the Cell © 1994 Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson Molecular Biology of the Cell, 3rd edn. Garland Publishing

Proposed solutions (hypotheses) The “Lock-in” hypothesis. (Bogorad, 1975). In order for core components of multisubunit complexes to be synthesised, de novo, in the correct compartment. The evolutionary process of transfer of genes from organelle to nucleus is still incomplete. E.g. Herrmann and Westhoff, 2001: The partite plant genome is not in a phylogenetic equilibrium. All available data suggest that the ultimate aim of genome restructuring in the plant cell, as in the eukaryotic cell in general, is the elimination of genome compartmentation while retaining physiological compartmentation. The frozen accident. The evolutionary process of gene transfer was underway when something happened that stopped it. E.g. von Heijne, It’s all a question of hydrophobicity. The five-helix rule. (Anon) Some proteins (with co-factors) cannot be imported. (Anon) Redox control of gene expression (Allen 1993 et seq.) Vectorial electron and proton transfer exerts regulatory control over expression of genes encoding proteins directly involved in, or affecting, redox poise. This regulatory coupling requires co-location of such genes with their gene products; is indispensable; and operated continuously throughout the transition from prokaryote to eukaryotic organelle. Organelles “make their own decisions” on the basis of environmental changes affecting redox state. Why Do Mitochondria and Chloroplasts Have Their Own Genetic Systems?

Proposed solution (hypothesis) Why Mitochondria and Chloroplasts Have Their Own Genetic Systems Allen, J. F. (1993) J. Theor. Biol. 165,

BacteriumEndosymbiontBioenergetic organelle

Prediction Explanation of previous knowledge Distribution of genes for components of oxidative phosphorylation between mitochondria and the cell nucleus

IIIIIIIVATPase Matrix Inter-membrane space

Prediction Explanation of previous knowledge Distribution of genes for components of photosynthetic phosphorylation between chloroplasts and the cell nucleus

Race, H. L., Herrmann, R. G. and Martin, W. (1999) Trends Genet. 15,

Prediction Experimental results Redox control of mitochondrial and chloroplast gene expression

D + ascorbate D + dithiothreitol D + dithionite D, anaerobic L + DCMU L + DBMIB D + duroquinol D + ferricyanide Dark (D) Light (L) 35 S-methione labelling of newly synthesised proteins in pea leaf thylakoids

+ ferricyanide + ascorbate + dithiothreitol + dithionite + duroquinol + rotenone, malonate.,dicumarol + cyanide, SHAM pyruvate, malate control 35 S-methione labelling of newly synthesised proteins in pea leaf mitochondria Allen, C. A. et al Redox Report 1,

Allen, J. F. (1992) BBA 1098, Pfannschmidt, T. et al. (1999) Nature 397, Pfannschmidt, T. et al. (1999) IUBMB Life 48,

The mitochondrial theory of ageing Figure from: ALLEN, J F (1996) J. Theor. Biol. 180,

Problem The Mitochondrial Theory of Ageing predicts that offspring should inherit their mother’s acquired state of accumulated damage, but they evidently do not. Babies are not born at the age of their mothers. How can this be?

Proposed solution (hypothesis) Separate sexes allows specialisation of mitochondria either as genetic templates (female germ line) or as energy-transducing organelles performing oxidative phosphorylation (male germ line) ALLEN, J F (1996) J. Theor. Biol. 180,

Predictions Explanations of previous knowledge Mitochondria are maternally inherited Females have a time-limited reproductive activity - oocyte mitochondria become useless as genetic templates after a certain threshold of oxidative damage is reached Predictions (1996) now “previous knowledge” (since 1998). Somatic, reproductive cloning should produce inherited ageing of offspring. Dolly: = 11 Experimental predictions Many….

Postscript In silico veritas The bucket or the searchlight? Which is the better metaphor for scientific enquiry - the bucket or the searchlight?

The bucket

The bucket or the searchlight? Even the oceans of data from DNA- microarrays, genomics (and heter- omics) cannot be converted into knowledge or understanding by being scooped up, at random, as if in bucketfuls. Knowledge is more than information, and cannot be deduced from it.

The searchlight Allen, J. F. (2001) EMBO Reports 2,

The bucket or the searchlight? Every observation presupposes an object of attention. We select what we examine with care. Our intention, in selecting what to look for, is to compare the results of experiments with the predictions of our hypotheses, to see if the results and predictions agree. So we look, purposefully, to see if our experimental data resembles our expectations. This is where computers can help us.

References –1. Allen, J.F. (2001) Bioinformatics and discovery: induction beckons again. BioEssays 23, –2. Allen, J.F. (2001) In silico veritas. Data mining and automated discovery- the truth is in there. EMBO Reports 2, –3. Wilkins, A. S. (2001) Why the philosophy of science actually does matter. BioEssays 23, 1-2 – – And THANK YOU FOR LISTENING!