Organelle DNAs

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 phosphoryla-tion in mitochondria and photosynthesis in chloroplasts (Chapter 8). Many lines of evidence indicate that mitochondria and chloroplasts evolved from bacteria that were endo-cytosed into ancestral cells containing a eukaryotic nucleus, forming endosymbionts. Over evolutionary time, most of the bacterial genes encoding components of the present-day organelles were transferred to the nucleus. However, mitochondria and chloroplasts in today's eukaryotes retain 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.

Mitochondria Contain Multiple mtDNA Molecules

Individual mitochondria are large enough to be seen under the light microscope, and even the mitochondrial DNA

▲ EXPERIMENTAL FIGURE 10-35 Dual staining reveals the multiple mitochondrial DNA molecules in a growing Euglena gracilis cell. Cells were treated with a mixture of two dyes: ethidium bromide, which binds to DNA and emits a red fluorescence, and DiOC6, which is incorporated specifically into mitochondria and emits a green fluorescence. Thus the nucleus emits a red fluorescence, and areas rich in mitochondrial DNA fluoresce yellow—a combination of red DNA and green mitochondrial fluorescence. [From Y Hayashi and K. Ueda, 1989, J. Cell Sci. 93:565.]

(mtDNA) can be detected by fluorescence microscopy. The mtDNA is located in the interior of the mitochondrion, the region known as the matrix (see Figure 5-26). As judged by the number of yellow fluorescent "dots" of mtDNA, a Euglena gracilis cell contains at least 30 mtDNA molecules (Figure 10-35).

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 in most organisms 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. By isolating mitochondria from cells and analyzing the DNA extracted from them, it can be seen that each mitochondrion contains 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. Each of these parameters varies greatly between different cell types.

mtDNA Is Inherited Cytoplasmically and Encodes 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 (Figure 10-36). For instance, petite yeast mutants exhibit structurally abnormal mitochondria and are incapable of ox-idative 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.

In the mating by fusion of haploid yeast cells, both parents contribute equally to the cytoplasm of the resulting diploid; thus inheritance of mitochondria is biparental (see Figure 10-36a). In mammals and most other multi-cellular organisms, however, the sperm contributes little (if any) cytoplasm to the zygote, and virtually all 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 uni-parental 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

Mating by cell fusion
Meiosis: random distribution of mitochondria to daughter cells

All haploid cells respiratory-proficient

All haploid cells respiratory-proficient

Respiratory-proficient Petite Respiratory-


Respiratory-proficient Petite Respiratory-

proficient 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 cytosolic ribo-somes and are imported into the organelle by processes discussed in Chapter 16.

The Size and Coding Capacity of mtDNA Vary Considerably in Different Organisms

Surprisingly, the size of the mtDNA, the number and nature of the proteins it encodes, and even the mitochondrial genetic code itself vary greatly between different organisms. Human mtDNA, a circular molecule that has been completely sequenced, is among the smallest known mtDNAs, containing 16,569 base pairs (Figure 10-37). 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 (methio-nine) codon, end with a stop codon, and are long enough to encode a polypeptide of more than 50 amino acids; all 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 noncod-ing sequences.

The mtDNA from most multicellular animals (meta-zoans) is about the same size as human mtDNA and encodes similar gene products. In contrast, yeast mtDNA is almost five times as large («78,000 bp). The mtDNAs from yeast and other fungi encode many of the same gene products as mammalian mtDNA, as well as others whose genes are found in the nuclei of metazoan cells.

^ FIGURE 10-36 Cytoplasmic inheritance of the petite mutation in yeast. Petite-strain mitochondria are defective in oxidative phosphorylation owing 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 haploid 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.

► FIGURE 10-37 The coding capacity of human mitochondrial DNA (mtDNA). 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. Mammalian mtDNA genes do not contain introns, although intervening DNA lies between some genes. [See D. A. Clayton, 1991, Ann. Rev. Cell Biol. 7:453.]



In contrast to other eukaryotes, which contain a single type of mtDNA, plants contain several types of mtDNA that appear to recombine with one another. 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 kb; muskmelon = 2500 kb). 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 a subunit of the F1 ATPase. The mitochondrial rRNAs of plants are also considerably larger than those of other eukaryotes. Long, noncoding regions and duplicated sequences are largely responsible for the greater length of plant mtDNAs. I

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 or ganism 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 12.) The nuclear cox II gene of mung bean corresponds more closely to the edited cox II RNA transcripts than to the mitochon-drial cox II genes found in other legumes. These observations are strong evidence that the cox II gene moved from the mitochondrion to the nucleus during mung bean evolution by a process that involved an RNA intermediate. Presumably this movement involved a reverse-transcription mechanism similar to that by which processed pseudogenes are generated in the nuclear genome from nuclear-encoded mRNAs.

Products of Mitochondrial Genes Are Not Exported

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 mito-chondrial ribosomes, although all but one or two of the ri-bosomal proteins (depending on the species) are imported from the cytosol. In most eukaryotes, all 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 protozoans, most mitochondrial tRNAs are encoded by the nuclear DNA and imported into the mitochondrion.

Reflecting the bacterial ancestry of mitochondria, mitochondrial ribosomes resemble bacterial ribo-somes and differ from eukaryotic cytosolic ribo-somes in their RNA and protein compositions, their size, and their sensitivity to certain antibiotics (see Figure 4-24). For instance, chloramphenicol blocks protein synthesis by bacterial and mitochondrial ribosomes from most organisms, but cycloheximide does not. This sensitivity of mitochondrial ri-bosomes to the important aminoglycoside class of antibiotics is the main cause of the toxicity that these antibiotics can cause. Conversely, cytosolic ribosomes are sensitive to cyclo-heximide and resistant to chloramphenicol. In cultured mammalian cells the only proteins synthesized in the presence of cycloheximide are encoded by mtDNA and produced by mi-tochondrial ribosomes. I

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; remarkably, the code even differs in mitochondria from different species (Table 10-3). Why and how these differences arose during evolution is mysterious. UGA, for example, is normally a stop codon, but is read as tryptophan by human and fungal mitochondrial translation systems; however, in plant mitochondria, UGA is still recognized as a stop codon. AGA and AGG, the standard nuclear codons for arginine, also code for arginine in fungal and plant mtDNA, but they are stop codons in mammalian mtDNA and serine codons in Drosophila mtDNA.

As shown in Table 10-3, plant mitochondria appear to utilize the standard genetic code. However, comparisons of the amino acid sequences of plant mitochondrial proteins with the nucleotide sequences of plant mtDNAs suggested that CGG could code for either arginine (the "standard" amino acid) or tryptophan. This apparent nonspecificity of the plant mitochondrial code is explained by editing of mitochondrial RNA transcripts, which can convert cytosine residues to uracil residues. If a CGG sequence is edited to UGG, the codon specifies tryptophan, the standard amino acid for UGG, whereas unedited CGG codons encode the standard arginine. Thus the translation system in plant mitochondria does utilize the standard genetic code. I

Mutations in Mitochondrial DNA Cause Several Genetic Diseases in Humans

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 mtDNAs 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 10-36). Thus, the mtDNA genotype, which fluctuates from one generation and from one cell division to the next, 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

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