Chapter 21 Extranuclear genes

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 • 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 Components of energy-producing systems Organelle translation component Active proteins In organelle Nucleus-encoded polypeptides

3. Nuclear chromosome Mitochondria chromosome (mtDNA) Chloroplast chromosome (cpDNA) Eukaryotic organism DNA 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 Structure of a typical animal cell

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

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

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

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

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

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

11. Structure of organelle chromosomes Mitochondrial genomes Function 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 Synthesized proteins are transported into the mitochondria Complete system is assemble in mitochondrial inner membrane The mitochondrial respiratory chain. Purple : mtDNA-encoded subunits Red : Nuclear DNA-encoded subunits

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

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

14. Structure of organelle chromosomes Mitochondrial genomes Yeast 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 less spacer DNA

15. Structure of organelle chromosomes Chloroplast genomes 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. Counterclickwisely transcribe Clockwisely transcribe Small single copy region Inverted repeat region Large single copy region

17. Organelle mutations mtDNA : base-pair-substitution rate x10 compare to nuclear DNA typical mutant phenotype : energy deficiency & slow growth or sickly appearance cf.) antibiotics resistance : by mutation in rRNA or ribosomal protein-encoding gene (streptomycin, erythoromycin) 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

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

22. Inheritance of organelle genes and mutations Petite mutation Wild-type cells Petite Phenotype Fused Petite-causing mitochondria (increased) (deletion of mt genes while duplication of replication origins, etc..) Expression of organelle mutations Possibility • Suppressive organelle mutations (not mean as suppressor) • : outreplicate the wild-type organelle genomes within a cell 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. Inheritance of organelle genes and mutations Expression of organelle mutations • Random drift : the frequency of mtDNA types can rise and fall • entirely on the basis of chance • 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 Heterokaryon test Fused Not to mix between sg+ and sg- The heterokaryon test is used to detect extranuclear inheritance in filamentous fungi.

25. Inheritance of organelle genes and mutations Mating Maternal inheritance Maternal inheritance of poky Neurospora Poky determinants 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 All progeny poky All progeny wild type Poky female x wild-type male Wild-type female x poky male

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

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. 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 Chlamydomonas reinharditi. Pair of flagella and the large single chloroplasts

29. Inheritance of organelle genes and mutations Uniparental inheritance of mitochondrial mutations in budding yeast mtDNA inheritance; not dependent on mating type  uniparental inheritance Ex) uniparental inheritance inneutral 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 Thus, petites are atypical example of uniparentality In mtDNA inheritance  Drug-resistant mutation and mit point mutationsclearly show uniparental inheritance pattern The life cycle of baker’s yeast (Saccharomyces cerevisiae). Next slide figure

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. eryR and eryS : erythromycin resistance, sensitivity, respectively Nuclear genes, mating-type alleles a and  >> Mendelian pattern 1:1 Drug resistancy >>Uniparental inheritance 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 Recombination Demonstrate this process by drug resistance Drug-registance alleles (eryR= erythromycin re.; spiR= spiramycin re.)

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

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

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