S3: “Meet the S3: “Meet the Molecules”Molecules”

Nuclear genesNuclear genes ITS = ribosomal internal ITS = ribosomal internal transcribed spacertranscribed spacer

Glu = Polyphenolic adhesive protein Glu = Polyphenolic adhesive protein

Mitochondrial geneMitochondrial gene CO3 = Cytochrome c oxidase subunit CO3 = Cytochrome c oxidase subunit IIIIII

The moleculesThe molecules1.1. ITSITS = the internal transcribed spacer of the = the internal transcribed spacer of the

nuclearnuclear ribosomal genes ribosomal genes Ribosomal genes code for rRNARibosomal genes code for rRNA Spacer regions are transcribed but then removedSpacer regions are transcribed but then removed Region has restriction site polymorphism between speciesRegion has restriction site polymorphism between species

http://fp.bio.utk.edu/

mycology/Techniques/mt-what_dna.htm

DNADNARNARNA

Ribosome Ribosome (RNA + proteins)(RNA + proteins)

ITSITS

DNADNA

RNARNA

Ribosome Ribosome (RNA + proteins)(RNA + proteins)

http://fp.bio.utk.edu/mycology/Techniques/mt-what_dna.htm

http://fp.bio.utk.edu/mycology/Techniques/mt-what_dna.htm

(1809 bp total)(1809 bp total) (1341 bp total)(1341 bp total)

(303-321)(303-321)(1781-1799)(1781-1799)

ITSITS

Multiple copies in genome = many targets for PCRMultiple copies in genome = many targets for PCR

ITSITS

M. galloprovincialis (~959 bp) 

| 442 | 156 | 172 | 189 |   M. trossulus (~951 bp) 

| 197 | 240 | 156 | 169 | 189 |

ITSITS

Restriction sites and fragment lengths for Restriction sites and fragment lengths for the the MytilusMytilus ITS locus using ITS locus using Hha IHha I restriction enzymerestriction enzyme

ITS is under less selection ITS is under less selection more variable! more variable! Reference: Braby & Somero, 2005, Marine BiologyReference: Braby & Somero, 2005, Marine Biology

PCR product

M. galloprovincialis (~959 bp) 

| 442 | 156 | 172 | 189 |   M. trossulus (~951 bp) 

| 197 | 240 | 156 | 169 | 189 |

ITSITS

442240197

The moleculesThe molecules

2.2. Glu Glu == Polyphenolic adhesive protein - Polyphenolic adhesive protein - nuclearnuclear

Mefp-1 proteinMefp-1 protein One of the glues that attaches byssal threads One of the glues that attaches byssal threads

to rocksto rocks Number of repeats (and therefore gene length) Number of repeats (and therefore gene length)

varies between speciesvaries between species

protein

= repeat

http://biomaterials.bme.northwestern.edu/mussel.asp

GluGlu Adhesive proteins Adhesive proteins anchor byssal treads anchor byssal treads to hard surface to hard surface

At least five At least five different proteins different proteins

Many variants of each Many variants of each Most studied foot Most studied foot protein is Mefp-1protein is Mefp-1

The Mefp-1 gene codes The Mefp-1 gene codes for the polyphenolic for the polyphenolic adhesive proteinadhesive protein

GluGlu

http://biomaterials.bme.northwestern.edu/mussel.asp

Collagen-like threads with adhesive proteinsCollagen-like threads with adhesive proteins

http://biomaterials.bme.northwestern.edu/mussel.asp

GluGluDOPA groups (hydroxylated tyrosines) chelate metal ions on surfaceDOPA groups (hydroxylated tyrosines) chelate metal ions on surface

Novel uses for mussel Novel uses for mussel adhesivesadhesives

Safe, natural glue for Safe, natural glue for surgeriessurgeries

Coating for medical Coating for medical implantsimplants

Tooth coating for Tooth coating for decay preventiondecay prevention

Anti-biofouling for Anti-biofouling for boatsboats

But synthesis is But synthesis is difficultdifficult StickySticky Post-translational Post-translational modificationmodification

~110 kDa

Mefp-1 proteinMefp-1 protein

Decapeptide repeats contain multipleDecapeptide repeats contain multiple Lys = Lysine = electrostaticLys = Lysine = electrostatic Hyp = Hydroxyproline = “sticky”Hyp = Hydroxyproline = “sticky” DOPA = hydroxylated tyrosine = chelatorDOPA = hydroxylated tyrosine = chelator

Repeats vary in number with ≤ 80/proteinRepeats vary in number with ≤ 80/protein

Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr- DOPA-Lys

Mefp-1 geneMefp-1 gene

Glu primers amplifyGlu primers amplify 2 products in invader 2 products in invader M. galloprovincialisM. galloprovincialis

~ 300 and 500 bp (shown above)~ 300 and 500 bp (shown above) 1 product in native 1 product in native M. trossulusM. trossulus

~ 240 bp~ 240 bp

Number of repeats (and therefore gene Number of repeats (and therefore gene length) varies between specieslength) varies between species

2502 bp2502 bp

160160

474474 654654

The moleculesThe molecules

3. CO33. CO3 = Cytochrome c oxidase = Cytochrome c oxidase subunit III - subunit III - mitochondrial mitochondrial

One subunit of a large protein in the One subunit of a large protein in the electron transport chainelectron transport chain

Species level differences are observed Species level differences are observed after DNA sequencingafter DNA sequencing

http://www.bioeng.auckland.ac.nz/images/database/bioinformatics/mitochondria.gif

MitochondriaMitochondria Organelles involved in Organelles involved in oxidative respiration oxidative respiration to produce ATPto produce ATP

Derived from bacterial Derived from bacterial endosymbionts (about endosymbionts (about 1.2 billion years ago)1.2 billion years ago)

Have genome, a Have genome, a circular DNA molecule circular DNA molecule (as in modern (as in modern bacteria)bacteria)

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endosymbiosis.html

Mitochondrial DNA Mitochondrial DNA (mtDNA)(mtDNA)

Most ancestral genes lost Most ancestral genes lost or explanted to nucleusor explanted to nucleus

Remaining genes:Remaining genes: Proteins for respiration Proteins for respiration Ribosomal genes Ribosomal genes

Mitochondrial DNA Mitochondrial DNA qualities:qualities: A haploid genomeA haploid genome Non-Mendelian inheritanceNon-Mendelian inheritance Little or no repair, Little or no repair,

therefore, evolves ~ 4 x therefore, evolves ~ 4 x faster than nuclear DNAfaster than nuclear DNA

Useful for evolutionary Useful for evolutionary studies of closely studies of closely related speciesrelated species

Complex IComplex I Complex IIComplex II Complex IIIComplex III Complex IVComplex IV

SubunitsSubunits NADH NADH DehydrogenaseDehydrogenase

Cytochrome Cytochrome bcbc11

Succinate Succinate DehydrogenaseDehydrogenase

Cytochrome c Cytochrome c OxidaseOxidase

mtDNAmtDNA 77 11 00 33nDNAnDNA 3535 1010 44 1010TOTALTOTAL 4242 1111 44 1313

http://pages.slu.edu/faculty/kennellj/

Cellular respirationCellular respiration

http://dec4.ucdavis.edu/website/Bioenergetics/

3D structure of 3D structure of cytochromescytochromes