1. Chapter 21 Extranuclear genes A variegated mosaic of Euonymus fortunei. Green(normal) and albino tissue caused by mixture of two chloroplast DNA types

2. Key concepts Components of energy-producing systems Organelle translation component Nucleus-encoded polypeptides Active proteins In organelle Chloroplast and mitochondria : Their own unique “chromosome” of genes Organelle DNA and their phenotype : maternally inherited Sorting-out process “Dihybrid” organelle mixture  Recombination can be detected Organelle genes Most organelle-encoded polypeptides

3. Eukaryotic organism DNA Nuclear chromosome Mitochondria chromosome (mtDNA) Chloroplast chromosome (cpDNA) Genes in Mitochondria : Oxidative phosphorylation Chloroplast : Photosynthesis ATP Content : small Human nuclear chromosome : 3,000,000 Kb (100,000 genes) Human mtDNA : 17Kb (37 genes)

4. Structure of a typical animal cell

5. Origin of extranuclear genes Generally believed Lost Integration Fully dependent on the organelle genes for their normal function cf.) The yeast, ‘Saccharomyces cerevisiae’ Most modern eukaryotic cells Most modern eukaryotic cells Without mitochondria obtain energy from fermentation ‘Endosymbionts’

6. Structure of organelle chromosomes Organelle DNA fraction preparation Organelle DNA fraction preparation Standard Recombination DNA technology Standard Recombination DNA technology Several organelle chromosomes – “fully sequenced” Several organelle chromosomes – “fully sequenced” Functions of organelle gene Functions of organelle gene Mutation analysis & homology searching Mutation analysis & homology searching *Protocol for studying

7. Structure of organelle chromosomes Overall organization Difference with nuclear chromosomes Difference with nuclear chromosomes Circular form Circular form by restriction mapping & electron microscope by restriction mapping & electron microscope Not in the highly condensed form Not in the highly condensed form Not in a euchromatic state Not in a euchromatic state

8. Structure of organelle chromosomes How many copies? Nuclear chromosome : 1 copy / cell (haploid) Nuclear chromosome : 1 copy / cell (haploid) 2 copy / cell (diploid) 2 copy / cell (diploid) Organelle chromosome : x00~x,000 copy/ cell Organelle chromosome : x00~x,000 copy/ cell * regulation of copy number is relatively loose * regulation of copy number is relatively loose ex.) Chloroplast : Leaf cells of the garden beet ex.) Chloroplast : Leaf cells of the garden beet ~ 40 chloroplast / cell ~ 40 chloroplast / cell 4 ~ 8 nucleoids / chloroplast 4 ~ 8 nucleoids / chloroplast (nucleoid : Specific heavily DNA area in chloroplast) (nucleoid : Specific heavily DNA area in chloroplast) 4 ~ 18 cpDNA molecules / nucleoid 4 ~ 18 cpDNA molecules / nucleoid MAX : 40 x 8 x 18 = 5760 copy of cpDNA / cell MAX : 40 x 8 x 18 = 5760 copy of cpDNA / cell Chlamydomonas Chlamydomonas 1 chloroplast / cell 500 ~ 1500 cpDNA molecules 1 chloroplast / cell 500 ~ 1500 cpDNA molecules

9. How many copies? ex.) Mitochondria : haploid yeast 1 ~ 45 mitochondria / cell 1 ~ 45 mitochondria / cell 10 ~ 30 nucleoids / mitochondria 10 ~ 30 nucleoids / mitochondria 4 ~ 5 mtDNA molecules / nucleoid 4 ~ 5 mtDNA molecules / nucleoid MAX : 45 x 30 x 5 = 6750 MAX : 45 x 30 x 5 = 6750 Fluorescent staining of a cell of Euglena gracilis. Nucleus Mitochondria Nucleoid Human 2 ~ 10 mtDNA mol. / mitochondria 2 ~ 10 mtDNA mol. / mitochondria Structure of organelle chromosomes

10. Mitochondrial genomes Structure of organelle chromosomes Maps of yeast and human mtDNAs Green : exons and uninterrupted genes Red : tRNA genes Yellow : URFs (unassigned reading fames)

11. Some of the proteins: ~ oxidative phosphorylation tRNA, rRNAs, some proteins: ~ mitochondrial protein synthesis (some genes are still encoded in nucleus) mRNA is translated outside the mitochondria on cytosolic ribosomes Mitochondrial genomes Structure of organelle chromosomes Synthesized proteins are transported into the mitochondria Complete system is assemble in mitochondrial inner membrane Function The mitochondrial respiratory chain. Purple : mtDNA-encoded subunits Red : Nuclear DNA-encoded subunits

12. Mitochondrial genomes Structure of organelle chromosomes  tRNA of mitochondria : ‘more wobble’ And codon assignments is different with nuclear codon with different species 25 yeast, 22 human tRNA in mitochondria at least 32 tRNA : for nucleus-derived mRNA tranlation tRNA

13. Mitochondrial genomes Structure of organelle chromosomes The genetic code of the human mitochondria. 22 tRNA types by the 22 boxes that do not contain stop codons Differ with nuclear code

14. Mitochondrial genomes Structure of organelle chromosomes Yeast The intron in several mitochondrial gene The intron in several mitochondrial gene Ex.) Subunit I of cytochrome oxidase - 9 introns nuclear gene - rare intron The existence of unassigned reading frames (URFs) within the yeast intron * URF - sequences that have correct initiation codons & are uninterrupted by stop codons * some URF - important in the splicing out of the introns themselves at the RNA level  Specifying proteins Human Much smaller & more compact than yeast mtDNA Much smaller & more compact than yeast mtDNA >> much less spacer DNA

15. Chloroplast genomes Structure of organelle chromosomes cpDNA : 120 ~ 200Kb in different species ex.) Liverwort Marchantia : 136 genes 4 kinds of rRNA 31 kinds of tRNA 90 proteins (20: photosynthesis & electron-transport functions) half the chloroplast genome: relate with translational function Large inverted repeats of virtually all species of plants (the sequences of the repeats are same!) Like mtDNA, cpDNA cooperates with nuclear DNA to provide subunits for functional proteins

16. Small single copy region Large single copy region Counterclickwisely transcribe Clockwisely transcribe Inverted repeat region

17. Organelle mutations mtDNA : base-pair-substitution rate x10 compare to nuclear DNA Neurospora : Obligate aerobe - can’t survive without functional mt Yeast : can obtain ATP by fermentation - drastically deleted genotypes can survive Small colonies caused by petites - deletion of part or even all of the mtDNA mit mutation - point mutations in some electron-transport proteins typical mutant phenotype : energy deficiency & slow growth or sickly appearance cf.) antibiotics resistance : by mutation in rRNA or ribosomal protein-encoding gene (streptomycin, erythoromycin)

18. Deleted DNA region of petite is amplified through tandem duplication to provide the normal size Organelle mutations Human : Several diseases caused by mtDNA mutation (mitochondrial cytopathies) >> effected organ - high energy demanding organ (muscle, nerves)

19. Map of human mtDNA showing loci of mutations leading to cytopathies. Single letter are one-letter abbreviations for amino acids. ND, NADH dehydrogenase; COX, cytochrome oxidase; 12S and 16S, ribosomal RNAs Organelle mutations Point mutations or Large deletions

20. Organelle mutations Model for producing a deletion by crossing-over in a direct repeat Mutations in nuclear genes can result in white leaves Cannot photosythesize -> phenotypically similar to cpDNA mutation, but they are inherited in a strict Mendelian manner, whereas cpDNA mutation inherited in the non-Mendelian manner

21. Inheritance of organelle genes and mutations The genetic fate of an organelle DNA mutation Inheritance Expression; many copies Cytoplasmic segregation; division of heteroplasmon (cytohet) (cytohet) Maternal inheritance; Between heterogametes

22. Inheritance of organelle genes and mutations Expression of organelle mutations Possibility Suppressive organelle mutations (not mean as suppressor) : outreplicate the wild-type organelle genomes within a cell Petite mutation Wild-type cells Fused Petite Phenotype Petite-causing mitochondria (increased) (deletion of mt genes while duplication of replication origins, etc..) Yeast Neurospora : abn (abnormal) mutation (rapid express mut mt gene even in 1/10,000 ratio of mutants to wt in heteroplasmic mycelium) Human : in post-mitotic cells; different parts of the body contain different proportions smooth muscle 4% liver 14% heart 40% kidney 40% skeletal muscle 50% frontal lobe of brain 44% cerebellum of brain 14% Threshold effect!

23. Random drift : the frequency of mtDNA types can rise and fall entirely on the basis of chance Inheritance of organelle genes and mutations Expression of organelle mutations certain type of mutation of mitochondria recognize potential energy deficiency start replication faster

24. Inheritance of organelle genes and mutations Cytoplasmic segregation the production of mutant and wild-type descendant cells of a heteroplasmon The heterokaryon test is used to detect extranuclear inheritance in filamentous fungi. Not to mix between sg + and sg - Fused Heterokaryon test

25. Inheritance of organelle genes and mutations Maternal inheritance Mating Reciprocal crosses of poky (slow growing) and normal Neurospora. Female : the parent contributing most of the cytoplasm of the progeny cells Nuclear locus - 1:1 Mendelian ratio Poky determinants Poky female x wild-type male Wild-type female x poky male All progeny poky All progeny wild type Maternal inheritance of poky Neurospora

26. Inheritance of organelle genes and mutations Maternal inheritance Leaf variegation in Mirabilis japalpa, the four-o’clock plant. Maternal inheritance of chloroplast pigments in plants 1909 Carol Correns; Varigated expression of chloroplast in leaf Normal flowers  mating; maternal inheritance

27. Inheritance of organelle genes and mutations Maternal inheritance Maternal inheritance of chloroplast pigments in plants A model explaining the results of the Table 21-1.

28. Inheritance of organelle genes and mutations Maternal inheritance Uniparental inheritance in Chlamydomonas reinharditi. Chlamydomonas reinharditi. Pair of flagella and the large single chloroplasts Streptomycin-sensitive mutant (sm-s) Mating type gene (mat) sm-r mat + x sm-s mat - all progeny sm-r sm-s mat + x sm-r mat - all progeny sm-s Uniparental inheritance

29. Inheritance of organelle genes and mutations Uniparental inheritance of mitochondrial mutations in budding yeast The life cycle of baker’s yeast (Saccharomyces cerevisiae). Ex) uniparental inheritance in neutral petites mtDNA petite x wild-type  all wild-type progeny N.B. (suppressive petites) suppressive petites x wild-type  petite progeny in proportions that correlate with the degree of suppessiveness Drug-resistant mutation and mit point mutations clearly show uniparental inheritance pattern Next slide figure mtDNA inheritance; not dependent on mating type  uniparental inheritance Thus, petites are atypical example of uniparentality In mtDNA inheritance 

30. Inheritance of organelle genes and mutations Uniparental inheritance of mitochondrial mutations in budding yeast The special inheritance pattern : certain drug-resistant phenotypes in yeast. ery R and ery S : erythromycin resistance, sensitivity, respectively Drug resistancy >>Uniparental inheritance Nuclear genes, mating-type alleles a and  >> Mendelian pattern 1:1 Maternal inheritance of human cytopathies. Usually mtDNA deletion are de novo in origin and are not inherited maternally But, various point mutations: inherited maternally (ex., MERRF mutation)

31. Recombination of extranuclear DNA Recombination in mitochondrially “dihybrid” heteroplasmons mitochondrial fusion crossing-over-like process Drug-registance alleles (ery R = erythromycin re.; spi R = spiramycin re.) Demonstrate this process by drug resistance Recombination

32. Cytoplasmic male sterility The use of cytoplasmic male sterility to facilitate the production of hybrid corn. Male sterility in plant - cytoplasmical base - maternally inherited Double-cross hybrids Prevent self-pollination  Prevent self-pollination Larger and more vigorous!

33. Mitochondria and aging Wear-and-tear theory : one of the theories of the mechanism of aging damage Repair repair machines Do not fully repair Machine can’t function Die Aging process Mitochondria - Reduction in oxidative phosphorylation Accumulation of deletion and point mutations mtDNA A agesB agesA ages Ex.. Mitochondria replacment: Age dependent correlation in oxidative phosphorylation

34. Origin of extranuclear genes Structure of organelle chromosomes - overall organization - how many copies? - mitochondrial genomes - chloroplast genomes Organelle mutations Inheritance of organelle genes and mutations - expression of organelle mutations - cytoplasmic segregation - maternal inheritance Recombination of extranuclear DNA Cytoplasmic male sterility Mitochondria and aging