S3: “Meet the Molecules”

1. S3: “Meet the Molecules” • Nuclear genes • ITS = ribosomal internal transcribed spacer • Glu = Polyphenolic adhesive protein • Mitochondrial gene • CO3 = Cytochrome c oxidase subunit III

2. DNA RNA Ribosome (RNA + proteins) The molecules • ITS = the internal transcribed spacer of the nuclear ribosomal genes • Ribosomal genes code for rRNA • Spacer regions are transcribed but then removed • Region has restriction site polymorphism between species http://fp.bio.utk.edu/mycology/Techniques/mt-what_dna.htm

3. DNA RNA Ribosome (RNA + proteins) ITS http://fp.bio.utk.edu/mycology/Techniques/mt-what_dna.htm

4. ITS (1781-1799) (303-321) (1809 bp total) (1341 bp total) ITS Multiple copies in genome = many targets for PCR http://fp.bio.utk.edu/mycology/Techniques/mt-what_dna.htm

5. ITS PCR product M. galloprovincialis (~959 bp) | 442 | 156 | 172 | 189 | M. trossulus (~951 bp) | 197 | 240 | 156 | 169 | 189 | • Restriction sites and fragment lengths for the Mytilus ITS locus using Hha I restriction enzyme • ITS is under less selection  more variable! • Reference: Braby & Somero, 2005, Marine Biology

6. ITS M. galloprovincialis (~959 bp) | 442 | 156 | 172 | 189 | M. trossulus (~951 bp) | 197 | 240 | 156 | 169 | 189 | 442 240 197

7. The molecules • Glu =Polyphenolic adhesive protein - nuclear • Mefp-1 protein • One of the glues that attaches byssal threads to rocks • Number of repeats (and therefore gene length) varies between species = repeat protein

8. Glu • Adhesive proteins anchor byssal treads to hard surface • At least five different proteins • Many variants of each • Most studied foot protein is Mefp-1 • The Mefp-1 gene codes for the polyphenolic adhesive protein http://biomaterials.bme.northwestern.edu/mussel.asp

9. Glu Collagen-like threads with adhesive proteins http://biomaterials.bme.northwestern.edu/mussel.asp

10. Glu DOPA groups (hydroxylated tyrosines) chelate metal ions on surface http://biomaterials.bme.northwestern.edu/mussel.asp

11. Novel uses for mussel adhesives • Safe, natural glue for surgeries • Coating for medical implants • Tooth coating for decay prevention • Anti-biofouling for boats • But synthesis is difficult • Sticky • Post-translational modification

12. Mefp-1 protein ~110 kDa • Decapeptide repeats contain multiple • Lys = Lysine = electrostatic • Hyp = Hydroxyproline = “sticky” • DOPA = hydroxylated tyrosine = chelator • Repeats vary in number with ≤ 80/protein

13. Mefp-1 gene 160 2502 bp 474 654 • Glu primers amplify • 2 products in invader M. galloprovincialis • ~ 300 and 500 bp (shown above) • 1 product in native M. trossulus • ~ 240 bp • Number of repeats (and therefore gene length) varies between species

14. The molecules 3. CO3 = Cytochrome c oxidase subunit III - mitochondrial • One subunit of a large protein in the electron transport chain • Species level differences are observed after DNA sequencing

15. Mitochondria • Organelles involved in oxidative respiration to produce ATP • Derived from bacterial endosymbionts (about 1.2 billion years ago) • Have genome, a circular DNA molecule (as in modern bacteria) http://www.bioeng.auckland.ac.nz/images/database/bioinformatics/mitochondria.gif

16. Mitochondrial DNA (mtDNA) • Most ancestral genes lost or explanted to nucleus • Remaining genes: • Proteins for respiration • Ribosomal genes • Mitochondrial DNA qualities: • A haploid genome • Non-Mendelian inheritance • Little or no repair, therefore, evolves ~ 4 x faster than nuclear DNA • Useful for evolutionary studies of closely related species http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endosymbiosis.html

17. http://pages.slu.edu/faculty/kennellj/ Cellular respiration

18. 3D structure of cytochromes http://dec4.ucdavis.edu/website/Bioenergetics/