Organelle DNA

Although majority of DNA in most eukaryotes is found in the nucleus,some DNA is present within the mitochondria also. Here, I have included the basics and importance of the Organelle/ Mitochondrial DNA which can help you in better understanding of the same.

1. Organelle dna Dr. Aurelian Jovita Alexander MDS Dept . of Oral and Maxillofacial Pathology

2. INTRODUCTION CELL MITOCHONDRIA OXIDATIVE PHOSPHORYLATION mtDNA ENDOSYMBIOTIC THEORY STRUCTURE MATERNAL INHERITANCE BOTTLENECK EFFECT LACK OF RECOMBINATION MUTATIONS

3. cell Latin   cella , meaning "small room“ Basic structural, functional and biological unit of all known living organisms.  “Building blocks of life“.  Robert Hooke in 1665. Organisms - Unicellular  Multicellular

4. MITOCHONDRION  Greek mitos , i.e. "thread" and   chondrion , i.e. "granule" Membrane-bound organelle.  “Cellular power plants“

5. history The first observations of intracellular structures that probably represent mitochondria were published in the 1840s Richard Altmann , in 1894 , established them as cell organelles and called them “ Bioblasts " The term “Mitochondria " itself was coined by Carl Benda in 1898

6. structure

7. Outer mitochondrial membrane Has a protein-to- phospholipid  ratio (about 1:1 by weight). Large numbers of integral proteins called Porins . ----form channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse. Larger proteins - Signaling sequence at their N-terminus binds to a large  multisubunit  protein called  translocase of the outer membrane. MER

8. Intermembrane space Perimitochondrial space. Conc. of small molecules such as ions and sugars here is the same as the  cytosol . Protein composition of this space is different. Protein that is localized to the intermembrane space in this way is  Cytochrome c.

9. Inner membrane The inner mitochondrial membrane contains proteins with five types of functions: [7]

10. 151 different polypeptides 1/5 of the total protein Rich in an unusual phospholipid ,  - Cardiolipin May help to make the inner membrane impermeable Proteins are ferried into the matrix via the  Translocase of the inner membrane (TIM) complex or OXA1 ( Oxidase assembly Ptn ).

11. cristae Expand the surface area of the IMM, enhancing its ability to produce ATP. These folds are studded with small round bodies known as F 1  particles or oxysomes .

12. matrix 2/3 of the total protein.  Hundreds of enzymes, special mitochondrial  ribosomes ,  tRNA , and several copies of the mitochondrial DNA genome.

13. Oxidative phosphorylation  Is the metabolic pathway in which the mitochondria in cells use their structure, enzymes, and energy released by the oxidation of nutrients to reform  ATP.

14. Complex I- NADH coenzyme Q oxidoreductase Complex II- Succinate Q oxidoreductase Complex III- Q Cytochrome C oxidoreductase Comples IV- Cytochrome C oxidase

15. Mitochondrial DNA  ( mtDNA  or  mDNA ) Present within the mitochondria of animals, plants, fungi, chloroplasts of plants.

16. In humans, mitochondrial DNA can be assessed as the smallest chromosome coding for 37 genes and containing approximately 16,600 base pairs.  Human mitochondrial DNA was the first significant part of the human genome to be sequenced. In most species, including humans, mtDNA is inherited solely from the mother

17. ORIGIN Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today's eukaryotic cells. This theory is called the  endosymbiotic theory.

18. ENDOSYMBIOTIC THEORY Greek: endon "within", syn "together" and biosis "living" The  endosymbiotic theory  states that several key organelles of eukaryotes originated as symbioses between separate single-celled organisms. According to this theory, mitochondria and plastids (e.g. chloroplasts), and possibly other organelles, represent formerly free-living bacteria that were taken inside another cell as an  endosymbiont .

19. Evidence suggest that the mitochondrion developed from  proteobacteria  (in particular,  Rickettsiales , the SAR11 clade , or close relatives) The chloroplast from  cyanobacteria .

20. ENDOSYMBIONT An  endosymbiont  is any organism that lives within the body or cells of another organism, i.e. forming an  endosymbiosis .

21. Each mitochondrion has 2-10 mtDNA . 100-10,000 separate copies of mtDNA are usually present per cell

22. MITOCHONDRIAL INHERITANCE   mtDNA is inherited from the mother (maternally inherited). An egg contains 100,000 to 1,000,000 mtDNA molecules, whereas a sperm contains only 100 to 1000 Degradation of sperm mtDNA in the fertilized egg Failure of sperm mtDNA to enter the egg

23. Ancestral information The maternal inheritance pattern of the mtDNA has important significance for ancestral studies.   By testing our own mtDNA , we are in fact able to indirectly read the mtDNA genetic code of our own maternal ancestors from thousands of generations ago.

24. STRUCTURE Circular, covalently closed, double-stranded DNA Each double-stranded circular mtDNA  molecule consists of 15,000-17,000 base pairs

25. Total 37 genes. Heavy strand- 28 genes Light strand- 9 genes. 13 are for proteins (polypeptides), 22 are tRNA and 2 are for the small and large subunits of  rRNA .

26. 16,569 base pairs. 13 sequences begin with ATG ( methionine ) codon , end with a stop codon , and are long enough to encode a polypeptide of more than 50 amino acids. Mammalian mtDNA , in contrast to nuclear DNA, lacks introns and contains no long noncoding sequences.

27. The location of each base pair in the mtDNA can be specified with an accession number according to its position in the mtDNA .    The position of any base pair in the mtDNA is designated by counting from “1″ clockwise around the mtDNA . 

28. Hypervariable region HVR1 is considered a "low resolution" region and HVR2 is considered a "high resolution" region

29. Most of the ancestral markers are found in the D-Loop.  The D-Loop is considered a non-vital part of the mtDNA because it does not have a useful biological function.  Thus, whenever a mutation occurs in this region ,the individual does not die and survives to pass the mutation along to future generations.  Over a period of thousands of years, many mutations accumulate in the D-Loop, but very little are found in the coding region. 

30. High copy number Present in high copy number in human cells. Extranuclear , cytoplasmic location of mtDNA . Easier to obtain mtDNA for analysis, Makes mtDNA the molecule of choice for analyzing ancient DNA and for certain forensic DNA applications.

31. Same property also complicates. Several levels at which the populations of mtDNA can be defined. Need not be identical – Heteroplasmy . 14% of the population. The overall homogeneity of mtDNA within individuals indicates that a substantial bottleneck in the number of mitochondria occurs early in oogenesis .

32. Bottleneck effect

33. MULTIPLE mt DNA MOLECULES Mitochondria are large enough to be seen under the light microscope Mitochondrial DNA ( mtDNA ) can be detected by fluorescence microscopy.

34. Time-lapse microscopy. Replicates through interphase .

35.  At mitosis each daughter cell receives approximately the same number of mitochondria. Total amount of mtDNA in a cell depends on the

36. Genes in mtDNA Exhibit Cytoplasmic Inheritance and Encode rRNAs , tRNAs , and Some Mitochondrial Proteins Studies of mutants in yeasts and other single-celled organisms first indicated that mitochondria exhibit  cytoplasmic inheritance and thus must contain their own genetic system.

37. A  mutant  is an organism or a new genetic character arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism.

38. Petite  yeast mutants exhibit structurally abnormal mitochondria and are incapable of oxidative phosphorylation .  Petite cells grow more slowly than wild-type yeasts and form smaller colonies  Genetic crosses showed that the  petite mutation does not segregate with any known nuclear gene or chromosome. Deletions of mtDNA .

39.  Haploid cells fuse to produce a diploid cell that undergoes meiosis, during which random segregation of parental chromosomes and mitochondria containing mtDNA occurs. Since yeast normally contain ≈50 mtDNA molecules per cell, all products of meiosis usually contain both normal and petite mtDNAs and are capable of respiration.

40. As these cells grow and divide mitotically , the cytoplasm (including the mitochondria) is randomly distributed to the daughter cells. Occasionally, a cell is generated that contains only defective petite mtDNA and yields a petite colony. Thus formation of such petite cells is independent of any nuclear genetic marker.

41. The entire mitochondrial  genome  from a number of different organisms has now been cloned and sequenced. rRNA . tRNA Proteins

42.  All mitochondrially synthesized polypeptides identified thus far (with one possible exception) are not complete enzymes but subunits of  multimeric  complexes used in electron transport or ATP synthesis. Most proteins localized in mitochondria, such as the mitochondrial RNA and DNA polymerases, are synthesized on cytoplasmic ribosomes and are imported into the  organelle.

43. MATERNAL INHERITANCE Advantage of mtDNA . Trace the lineage. Very rare- Paternal (Schwartz and Vissing , 2002)

44. PATERNAL INHERITANCE Mussels Member of bivalvia mollusca Drosophilla , mouse , Bird Hybrids

45. Maternal inheritance pattern of mt dna

46. Products of Mitochondrial Genes Are Not Exported All RNA transcripts of mtDNA and their translation products remain in the mitochondrion. All mtDNA -encoded proteins are synthesized on mitochondrial ribosomes . Mitochondria encode the rRNAs that form mitochondrial ribosomes , although all but one or two of the ribosomal proteins (depending on the species) are imported from the  cytosol .

47. tRNAs Eukaryotes- tRNAs encoded by mtDNAs . Parasitic protozoan Trypanosoma brucei  and in ciliated protozoa- Nuclear DNA

48.  RNA and protein compositions, their size, and their sensitivity to certain antibiotics differ in both. Mitochrial and Bacterial Ribosome Cytoplasmic Ribosome Chloramphenicol   Cycloheximide

49. Mitochondrial Genetic Codes Differ from the Standard Nuclear Code The genetic code used in animal and fungal mitochondria is different from the standard code used in all prokaryotic and eukaryotic nuclear genes. Mitochondria Codon Standard Code: Nuclear-Encoded Proteins Mammals Drosophila Neurospora Yeasts Plants UGA Stop Trp Trp Trp Trp Stop AGA, AGG Arg Stop Ser Arg Arg Arg AUA Ile Met Met Ile Met Ile AUU Ile Met Met Met Met Ile CUU, CUC, CUA, CUG Leu Leu Leu Leu Thr Leu

50. Lack of recombination Recombination? Process by which two DNA molecules exchange genetic information, resulting in the production of a new combination of alleles.

51. DOES NOT UNDERGO RECOMBINATION! Initially papers claimed evidence for recombination , 1999-2000. Phylogenetic /statistical studies used faulty data and/or questionable statistical methods Reanalysis No significant results

52. Further studies on linkage disequilibrium and distance in large data sets of complete mtDNA sequences found no evidence for recombination, although excess of homoplasmic sites were detected. Linkage Disequilibrium?? Non-random association of alleles at two or more loci, that descend from single, ancestral chromosomes. It is necessary to refer to this as  gametic phase disequilibrium or simply  gametic disequilibrium  because it is described through DNA recombination.

53. Homoplasmic sites? Homoplasmy  is a term used in genetics to describe a mammalian cell whose copies of mtDNA are all identical.   Homoplasmic mtDNA copies may be normal or mutated. Most mutations are heteroplasmic  - only occurring in some copies of mtDNA But it has been discovered that homoplastic mtDNA mutations may be found in human tumours .

54. However, a case of observed recombination in human mtDNA was reported in the only known human with both maternal and paternal mtDNA . Recombination between the maternal and the paternal mtDNA occurred in approximately 0.7% of the total mtDNA in the patient’s muscle tissue.

55. MUTATION RATE The mutation rate of mtDNA is several orders of magnitude higher than that of nuclear genes. An estimated rate of 0.017 × 10 −6 substitutions per site per year for the whole genome excluding the control region. However, in the two hypervariable regions (HVR I and HVRII ) of the non coding control region, the rate is even higher.

56. Phylogenetics The study of evolutionary relationships among groups of organisms , which are discovered through molecular sequencing data and morphological data matrices. Phylogenetic comparisons, based on either interspecific or intraspecific comparisons were done. Pedigree chart   A diagram that shows the occurrence and appearance or phenotypes of a particular gene or organism and its ancestors from one generation to the next. Direct observations of mtDNA mutations in families or deep-rooting Pedigrees were done.

57. Within the control region the mutation rate is very heterogeneous, with some “mutational hot spots” mutating four to five times as fast as the average site. Some claim, Recombination.

58. Mutations in Mitochondrial DNA All cells have mitochondria, yet mutations in mtDNA only affect some tissues. Those most usually affected are, Tissues that have a high requirement for ATP produced by oxidative phosphorylation . Tissues that require most or all of the mtDNA in the cell to synthesize functional mitochondrial proteins.

59. Heteroplasmy and threshold effect A minimum critical number of mutant mtDNAs is required to cause mitochondrial dysfunction in a particular organ or tissue and mitochondrial disease in an individual (threshold effect).

60. Mitotic segregation At cell division, the proportion of mutant mtDNAs in daughter cells may shift and the phenotype may change accordingly.

61. Maternal Inheritance

62. Over 150 point mutations and innumerable large scale rearrangements have been associated with a bewildering variety of diseases.

63.  KSS involves a triad of the already described Chronic Progressive External Opthalmoplegia , as well as bilateral pigmentary retinopathy, and cardiac conduction abnormalities. Dominant and recessive forms of PEO can be caused by genetic mutations in the  ANT1 ,  POLG ,  POLG2  and  PEO1  genes. KSS Kearns-Sayre syndrome

64. Progressive myoclonic epilepsy " Ragged Red Fibers " - clumps of diseased mitochondria accumulate in the subsarcolemmal region of the muscle fiber and appear as "Ragged Red Fibers" when muscle is stained with modified  Gömöri trichrome stain short stature hearing loss lactic acidosis exercise intolerance poor night vision MERRF Myoclonus epilepsy and ragged red fibers

65. Normal Diseased

66. Caused by a maternally-inherited mutation at position 8344 in the mitochondrial genome in over 80% of cases. This point mutation disrupts the mitochondrial gene for  tRNA -Lys and so disrupts synthesis of proteins essential for oxidative phosphorylation . Many genes are involved. These include: MT-TK MT-TL1 MT-TH MT-TS1 MT-TS2 MT-TF

67. Increased acidity in the blood can lead to vomiting, abdominal pain, extreme tiredness (fatigue), muscle weakness, loss of bowel control, and difficulty breathing. Some of the genes ( MT-ND1 ,  MT-ND5 ) affected in MELAS encode proteins that are part of  NADH dehydrogenase  (also called complex I). Other genes ( MT-TH ,  MT-TL1 , and  MT-TV ) encode mitochondrial specific transfer RNAs ( tRNAs ). MELAS mitochondrial encephalomyopathy,lactic acidosis, stroke -like episodes

68. Mutations in the  MT-ATP6  gene cause neuropathy, ataxia, and retinitis pigmentosa . The  MT-ATP6  gene provides instructions for making a protein that is essential for normal mitochondrial function. NARP Neuropathy, ataxia, retinitis pigmentosa

69. The most common of these mutations is found in 10 to 20 percent of Leigh syndrome and occurs in  MT-ATP6 , a gene that codes for a protein in the last complex of the oxidative phosphorylation chain,  ATP synthase , an enzyme that directly generates ATP. MILS Maternally inherited Leigh syndrome

70. LHON is usually due to one of three pathogenic mitochondrial DNA point mutations. These mutations are at nucleotide positions 11778  G  to  A , 3460  G  to  A  and 14484  T  to  C , respectively in the ND4, ND1 and ND6 subunit genes of complex I of the oxidative phosphorylation  chain in mitochondria. LHON Leber hereditary optic neuropathy

71. Due to single rearrangements To point mutations in genes affecting protein synthesis in toto , and to a protein coding gene

72. mtDNA -related disorders fall into two major groups: Those due to mutations in genes involved in mitochondrial protein synthesis Those due to mutations in genes encoding individual proteins of the respiratory chain

73. Laboratory results-Lactic Acidosis, muscle biopsy- RRF, and muscle biochemistry

74. ORAL DISEASES Recurrent oral ulcers Haplogroups G1 and H were found significantly more abundant in ROU patients than in healthy persons.

75. Oral cancer and Precancerous Lesions Real time Quantitavie PCR, To detect and quantify mtDNA with the 4,977-bp deletion in the histologically defined specified cell groups. The proportion of 4,977-bp deleted mtDNA in all oral lesions is higher than normal and consistently decreased during cancer progression from precancer to primary cancer. This suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis.

76. Squamous cell carcinoma The mitochondrial D-Loop was also analysed . Three mutation hotspots were observed in the D-Loop at nt 146, 152 and 186, Two of which ( nt 146 and 152) have also been implicated in oesophageal SCC, another smoking-related cancer.

77. Inheritance of single mt DNA mutations Single mtDNA deletions are neither inherited from the mother nor transmitted to the progeny and disorders due to single mtDNA deletions, KSS, PEO, and PS, are almost always sporadic. Attributed to bottleneck phenomenon. Few cases- maternal transmission.

78. Frequency of mt dna related diseases Among the most common genetic disorders and a major burden for society. reviewed by Schaefer et al Nuclear and mtDNA Mutations- The minimum prevalence is at least 1 in 5,000 Study in England- Only mtDNA mutations- 6.57/100,000 , with a particularly high prevalence of LHON

79. ROS-Generating MitochondrialDNA Mutations Can RegulateTumor Cell Metastasis Many chemical carcinogens have been shown to bind preferentially to mtDNA rather than to nuclear DNA. (Allen and Coombs, 1980) High frequencies of homoplasmic mutations in mtDNA of tumors rather than in mtDNA of normal tissues of the same patients. ( Fliss , 1978) Preferential accumulation of mutated mtDNAs in tumor cells. (Gallardo, 2006)

80. ANCESTRAL MARKERS What is an ancestral marker? Ancestral markers are “mutations”, little changes or “hiccups” that occur in the genetic code of the mtDNA .  Most commonly found mutations in mtDNA is called a “SNP” (single nucleotide polymorphism). 

81. DNA consists of two chains of nucleotides, designated A, C, T, and G.  The unique combination of nucleotides in the chain is called a “genetic code” and holds genetic information.  Short segment of mtDNA , namely locations 1 to 45. 

82. T” at location 40 is replaced by a “G”.  This mutation is documented as follows: Location :  40 Nucleotide Change:  T>g (also indicated as T40G)

83. Sample report

84.    Remember, only one of the two chains in the pair is shown when reporting the sequence!

85. Technology used to detect mutations  All mutations detected in the sequence are indicated in pink.   Benefit - Can accurately read entire lengths of your DNA.  Limitation - Only approximately 400 to 500 nucleotides can be read at a time. DNA Sequencing

86. A single sequencing test can detect all of the markers in the HVR1 region and another sequencing test can detect all of the markers in the HVR2 region. Most of the mutations found in the mtDNA are located in HVR1 and HVR2. The HVR1 region is the most widely studied region of the mtDNA for ancestral studies. However, DNA Sequencing is not the best method for examining mutations in the Coding Region of mtDNA .

87.  It targets specific nucleotides.  Only the nucleotides that provide useful information are tested. This is the best method for efficiently testing large regions of DNA.  “SNP” testing

88. applications Forensics Mitochondrial DNA analysis is an appropriate method for: Charred remains Degraded specimens Old skeletal and fingernail samples Hair shafts

89. “Ancient DNA” field has shown that highly degraded samples contain populations of intact DNA molecules that are severely restricted in size . Additionally, mtDNA is quite useful in forensic investigations of remains recovered of a missing person or mass disaster.   Biological material from known, maternal relatives, even quite distant, can be used as a reference for direct comparison to the recovered remains.   

90. The two variable regions, hypervariable region 1 (HV1) and hypervariable region 2 (HV2) are amplified, detected, and analyzed for forensic identification.

91. WHY IS mtDNA IMPORTANT?? 13 of its 37 genes are involved in oxidative phosphorylation . The remaining 24 genes are involved in the creation of transfer RNA ( tRNA ) and ribosomal RNA ( rRNA ) which help to turn amino acids into proteins.

92. summary Mitochondrial DNA is the genetic material from mitochondria, which is the cellular organic cell, responsible for generating the necessary energy. Maternally Inherited. mtDNA is useful for the purpose of tracing ancestry. mtDNA does not recombine. Mutations, HV1, HV2

93. THANK YOU!