Although the vast majority of DNA in most eukaryotes is found in the nucleus, some DNA is present within the mitochondria of animals, plants, and fungi and within the chloroplasts of plants. These organelles are the main cellular sites for ATP formation, during oxidative phosphorylation in mitochondria and photosynthesis in chloroplasts (Chapter 16). Many lines of evidence indicate that mitochondria and chloroplasts evolved from bacteria that were endocytosed into ancestral cells containing a eukaryotic nucleus, forming endosymbionts. Over evolutionary time, most of the bacte-rial genes encoding components of the present-day organelles were transferred to the nucleus. However, mitochondria and chloroplasts in today’s eukaryotes retain circular DNAs encoding proteins essential for organellar function as well as the ribosomal and transfer RNAs required for their translation. Thus eukaryotic cells have multiple genetic systems: a predominant nuclear system and secondary systems with their own DNA in the mitochondria and chloroplasts.
Cells were treated with a mixture of two dyes: ethidium bromide, which binds to DNA and emits a vermilion fluorescence, and DiOC6, which is incorporated specifically into mitochondria and emits a green fluorescence. Thus the nucleus emits a vermilion fluorescence, and areas rich in mitochondrial DNA fluoresce yellow — a combination of vermilion DNA and green mitochondrial fluorescence. [From Y. Huyashi and K. Veda, 1989, J. Cell Sci. 93:565.]
).
Since the dyes used to visualize nuclear and mitochondrial DNA do not affect cell growth or division, replication of mtDNA and division of the mitochondrial network can be followed in living cells using time-lapse microscopy. Such studies show that mtDNA replicates throughout interphase. At mitosis each daughter cell receives approximately the same number of mitochondria, but since there is no mechanism for apportioning exactly equal numbers of mitochondria to the daughter cells, some cells contain more mtDNA than others. All the mitochondria in eukaryotic cells contain multiple mtDNA molecules. Thus the total amount of mtDNA in a cell depends on the number of mitochondria, the size of the mtDNA, and the number of mtDNA molecules per mitochondrion.
Petite-strain mitochondria are defective in oxidative phosphorylation due to a deletion in mtDNA. (a) 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. (b) 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.
). For instance, petite yeast mutants exhibit
structurally abnormal mitochondria and are incapable of oxidative
phosphorylation. As a result, petite cells grow more slowly than wild-type
yeasts and form smaller colonies (hence the name “petite”).
Genetic crosses between different (haploid) yeast strains showed that the
petite mutation does not segregate with any known nuclear
gene or chromosome. In later studies, most petite mutants were found to contain
deletions of mtDNA.
Mitochondrial inheritance in yeasts is biparental: during the fusion of haploid cells, both parents contribute equally to the cytoplasm of the diploid. In mammals and most other animals, however, the sperm contributes little (if any) cytoplasm to the zygote, and virtually all of the mitochondria in the embryo are derived from those in the egg, not the sperm. Studies in mice have shown that 99.99 percent of mtDNA is maternally inherited, but a small part (0.01 percent) is inherited from the male parent. In higher plants, mtDNA is inherited exclusively in a uniparental fashion through the female parent (egg), not the male (pollen).
The entire mitochondrial genome from a number of different organisms has now been cloned and sequenced, and mtDNAs from all these sources have been found to encode rRNAs, tRNAs, and essential mitochondrial proteins. All proteins encoded by mtDNA are synthesized on mitochondrial ribosomes. 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 by processes discussed in Chapter 17.
Proteins and RNAs encoded by each of the two strands are shown separately. Transcription of the outer (H) strand occurs in the clockwise direction and of the inner (L) strand in the counterclockwise direction. The abbreviations for amino acids denote the corresponding tRNA genes. ND1, ND2, etc., denote genes encoding subunits of the NADH-CoQ reductase complex. The 207-bp gene encoding F0 ATPase subunit 8 overlaps, out of frame, with the N-terminal portion of the segment encoding F0 ATPase subunit 6. No mammalian mtDNA genes contain introns, although intervening DNA lies between some genes. [See D. A. Clayton, 1991, Ann. Rev. Cell Biol. 7:453.]
). It encodes the two
rRNAs found in mitochondrial ribosomes and the 22 tRNAs used to translate
mitochondrial mRNAs. Human mtDNA has 13 sequences that begin with an ATG
(methionine) codon, end with a stop codon, and are long enough to encode a
polypeptide of more than 50 amino acids; all of the possible proteins encoded by
these open reading frames have been identified. Mammalian mtDNA, in contrast to
nuclear DNA, lacks introns and contains no long noncoding sequences.
Invertebrate mtDNA is about the same size as human mtDNA, but yeast mtDNA is almost five times as large (≈78,000 bp). The mtDNAs from yeast and other lower eukaryotes encode many of the same gene products as mammalian mtDNA, as well as others whose genes are found in the nuclei of mammalian cells.
In contrast to other eukaryotes, which contain a single type of mtDNA, plants contain several types of mtDNA that appear to recombine with each other. Plant mtDNAs are much larger and more variable in size than the mtDNAs of other organisms. Even in a single family of plants, mtDNAs can vary as much as eightfold in size (watermelon = 330,000 bp; muskmelon = 2,500,000 bp). Unlike animal, yeast, and fungal mtDNAs, plant mtDNAs contain genes encoding a 5S mitochondrial rRNA, which is present only in the mitochondrial ribosomes of plants, and the α subunit of the F1 ATPase. The mitochondrial rRNAs of plants are also considerably larger than those of other eukaryotes. The recent sequencing of one of the smallest plant mtDNAs has revealed that long, noncoding regions and duplicated sequences are largely responsible for the greater length of plant mtDNAs.
Differences in the size and coding capacity of mtDNA from various organisms most likely reflect the movement of DNA between mitochondria and the nucleus during evolution. Direct evidence for this movement comes from the observation that several proteins encoded by mtDNA in some species are encoded by nuclear DNA in others. It thus appears that entire genes moved from the mitochondrion to the nucleus, or vice versa, during evolution.
The most striking example of this phenomenon involves the gene cox II, which encodes subunit 2 of cytochrome c oxidase. This gene is found in mtDNA in all organisms studied except for one species of legume, the mung bean: in this organism only, the cox II gene is nuclear. Many RNA transcripts of plant mitochondrial genes are edited, mainly by the enzyme-catalyzed conversion of selected C residues to U, and occasionally U to C. (RNA editing is discussed in Chapter 11.) The nuclear cox II gene of mung bean corresponds more closely to the edited cox II RNA transcripts than to the mitochondrial cox II genes found in other legumes. These facts are strong evidence that the cox II gene moved from mitochondrion to the nucleus during mung bean evolution by a process that involved an RNA intermediate. Presumably this movement involved a reversetranscription mechanism similar to that by which processed pseudogenes are generated in the nuclear genome from nuclear-encoded mRNAs.
As far as is known, all RNA transcripts of mtDNA and their translation products remain in the mitochondrion, and 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. In most eukaryotes, all of the tRNAs used for protein synthesis in mitochondria are encoded by mtDNAs. However, in wheat, in the parasitic protozoan Trypanosoma brucei (the cause of African sleeping sickness), and in ciliated protozoa, all mitochondrial tRNAs are encoded by the nuclear DNA and imported into the mitochondrion.
Reflecting the bacterial ancestry of mitochondria, mitochondrial ribosomes resemble bacterial ribosomes and differ from cytoplasmic ribosomes in their RNA and protein compositions, their size, and their sensitivity to certain antibiotics (see Figure 4-32). For instance, chloramphenicol blocks protein synthesis by bacterial and most mitochondrial ribosomes, but not by cytoplasmic ribosomes. Conversely, cycloheximide inhibits protein synthesis by eukaryotic cytoplasmic ribosomes but does not affect protein synthesis by mitochondrial ribosomes or bacterial ribosomes. In cultured mammalian cells the only proteins synthesized in the presence of cycloheximide are encoded by mtDNA and produced by mitochondrial ribosomes.
| 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 |
SOURCE: S. Anderson et al., 1981, Nature 290:457; P. Borst, in International Cell Biology 1980 – 1981, H. G. Schweiger, ed., Springer-Verlag, p. 239; C. Breitenberger and U. L. Raj Bhandary, 1985, Trends Biochem. Sci. 10:478 – 483; V. K. Eckenrode and C. S. Levings, 1986, In Vitro Cell Dev. Biol. 22:169 –176; J. M. Gualber et al., 1989, Nature 341:660 – 662; and P. S. Covello and M. W. Gray, 1989, Nature 341:662 – 666.
The severity of disease caused by a mutation in mtDNA depends on the nature of
the mutation and on the proportion of mutant and wild-type DNAs present in a
particular cell type. Generally, when mutations in mtDNA are found, cells
contain mixtures of wild-type and mutant
mtDNAs — a condition known as
heteroplasmy. Each time a mammalian somatic or germ-line
cell divides, the mutant and wild-type mtDNAs will segregate randomly into the
daughter cells, as occurs in yeast cells (see Figure 9-43). Thus, the mtDNA genotype fluctuates from one
generation and from one cell division to the next, and can drift toward
predominantly wild-type or predominantly mutant mtDNAs. Since all enzymes for
the replication and growth of mitochondria, such as DNA and RNA polymerases, are
imported from the cytosol, a mutant mtDNA should not be at a
“replication disadvantage”; mutants that involve large
deletions of mtDNA might even be at a selective advantage in replication.
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, and tissues that require most or all of the mtDNA in the cell to synthesize functional mitochondrial proteins. Leber’s hereditary optic neuropathy (degeneration of the optic nerve, accompanied by increasing blindness), for instance, is caused by a missense mutation in the mtDNA gene encoding subunit 4 of the NADH-CoQ reductase. Any of several large deletions in mtDNA cause another set of diseases including chronic progressive external ophthalmoplegia and Kearns-Sayre syndrome, which are characterized by eye defects and, in Kearns-Sayre syndrome, also by abnormal heartbeat and central nervous system degeneration. A third condition, causing “ragged” muscle fibers (with improperly assembled mitochondria) and associated uncontrolled jerky movements, is due to a single mutation in the TYCG loop of the mitochondrial lysine tRNA. As a result of this mutation, the translation of several mitochondrial proteins apparently is blocked.
As we discuss in Chapter 16, the structure of chloroplasts is similar in many respects to that of mitochondria. Like mitochondria, chloroplasts contain multiple copies of the organellar DNA and ribosomes, which synthesize some chloroplast-encoded proteins using the “standard” genetic code. Other chloroplast proteins are fabricated on cytosolic ribosomes and are incorporated into the organelle after translation.
Chloroplast DNAs are circular molecules of 120,000 – 160,000 bp, depending on the species. The complete sequences of several chloroplast DNAs have been determined, including those from liverwort (121,024 bp) and tobacco (155,844 bp). The liverwort chloroplast genome has two inverted repeats, each consisting of 10,058 bp, that contain the rRNA genes and a few other duplicated genes. Despite the difference in size, the overall organization and gene composition of the liverwort and tobacco DNAs are very similar; the size differential is due primarily to the length of the inverted repeat in which some genes are duplicated.
Of the ≈120 genes in chloroplast DNA, about 60 are involved in RNA transcription and translation, including genes for rRNAs, tRNAs, RNA polymerase subunits, and ribosomal proteins. About 20 genes encode subunits of the chloroplast photosynthetic electron transport complexes and the F0F1 ATPase complex. Also encoded in the chloroplast genome is the larger of the two subunits of ribulose 1,5-bisphosphate carboxylase, which is involved in the fixation of carbon dioxide during photosynthesis.
Reflecting the endosymbiotic origin of chloroplasts, some regions of chloroplast DNA are strikingly similar to those of the DNA of present-day bacteria. For instance, chloroplast DNA encodes four subunits of RNA polymerase that are highly homologous to the subunits of E. coli RNA polymerase. One segment of chloroplast DNA encodes eight proteins that are homologous to eight E. coli ribosomal proteins; the order of these genes is the same in the two DNAs.
Liverwort chloroplast DNA has some genes that are not detected in the larger tobacco chloroplast DNA, and vice versa. Since the two types of chloroplasts contain virtually the same set of proteins, these data suggest that some genes are present in the chloroplast DNA of one species and in the nuclear DNA of the other, indicating that some exchange of genes between chloroplast and nucleus has occurred during evolution.
Mitochondria and chloroplasts are believed to have evolved from bacteria tha formed a symbiotic relationship with ancestral cells containing a eukaryotic nucleus. Most of the genes originally within these organelles have been transferred to the nuclear genome over evolutionary time, leaving different genes in the organelle DNAs of different organisms.
Mammalian mtDNAs are only ≈16 kb in length; they contain no introns and very little noncoding DNA. Yeast and plant mtDNAs are much longer. All mtDNAs encode rRNAs, tRNAs, and some of the proteins involved in mitochondrial electron transport and ATP synthesis.
Most mtDNA is inherited from egg cells rather than sperm, and mutations in mtDNA result in a maternal cytoplasmic pattern of inheritance.
Mitochondrial ribosomes resemble bacterial ribosomes in their structure, sensitivity to chloramphenicol, and resistance to cycloheximide.
The genetic code of animal and fungal mtDNAs differs from that of bacteria and the nuclear genome in that several codons encode alternative amino acids or stop signals. The mitochondrial code differs between different animals and fungi. Plant mitochondria appear to use the standard nuclear and bacterial genetic code.
Mutations in mtDNA can cause human neuromuscular disorders, probably because of the high demand for ATP in these tissues. Patients generally have a mixture of wild-type and mutant mtDNA in their cells (heteroplasmy). The severity of the phenotype is greater, the higher the fraction of mutant mtDNA.
Chloroplast DNA is circular and contains ≈120 – 160 kb, depending on the plant species. It encodes ≈120 proteins and uses the standard genetic code.
