genetics of organelles - wordpress.com · 2019-10-05 · molecular genetics and genomics department...

Molecular Genetics and Genomics Department of Biology/Faculty of Sciences/LU

Dr. Fahd Nasr-All rights reserved

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Genetics of organelles I. Preamble1 One of the major concerns in a general theory for evolution is related to the transition from prokaryote-to-eukaryote system. The symbiotic origin of organelles found in eukaryotic cells was suggested more than ninety years ago; according to this hypothesis mitochondria and chloroplasts, the energy-converting organelles, descended from originally free- living bacteria via endosymbiotic events. In the mid-1980s, it was widely accepted, based on the ultra structural characteristics of eukaryotic cells and small subunit rDNA-based phylogeny that the first eukaryotic cell arose directly from an archaebacterial-like ancestor by evolving a nucleus, endomembrane and cytoskeleton. Since the beginning of the 1990s, a new hypothesis, termed chimeric, was raised stating that the eukaryotic nucleus has been formed from the cellular fusion between an archaebacterium and a Gram-negative eubacterium, explaining the chimeric nature of eukaryotic genomes, as revealed by various universal protein gene phylogenies. Summing up these different scenarios, the evolution of complex cells could be divided into two major phases. First, the emergence of the eukaryotic cell possessing a nucleus, endomembrane, and cytoskeleton. Second, the symbiotic origin of mitochondria resulting in the appearance of the first energetically efficient aerobic protozoan. The first living organisms are believed to have arisen more than 3.85 billion years ago (Fig. 1). Presumably, the metabolic and genetic system of the earliest cellular entities were more simple and rudimentary than that of any modern cell. Later, in parallel with the increase in genetic and cellular complexity, some genetic subsystems became non-compatible with exogenous parts. As cell designs gained greater complexity their flexibility continue to diminish until the organization of the cell could not change fundamentally any longer. When lateral gene transfer was restricted significantly, the first prokaryotes emerged, thereby initiating the second major stage of cellular evolution. This was the time of the origin of individuality and speciation, presumably through partial reproductive isolation. Despite the limitation in formerly general genetic mixing, the horizontal gene transfer has continued to be essential in the speciation dynamics of prokaryotes. Ancestral prokaryotes, the first modern cells, developed novel biochemical pathways for ATP production, such as the present –day forms of fermentation, and then oxidative phosphorylation. A considerable step was the development of photosynthesis, probably more than three billion years ago. The oxygen-producing photosynthesis resulted in, at least in parts, the appearance of molecular O2 in the atmosphere.

1 This chapter is based on a research project that had been prepared, under my supervision, by Misses Sara Al-Ghadban and Sara Banat during the academic year 2002-2003. Here, I would like to acknowledge the commitment and the honorable efforts of Misses Al-Ghadban and Banat that helped bring about this chapter to term. Their contribution is highly appreciated.



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Figure 1. The first eukaryotes appeared two billions years ago as a result of endosymbiotic events. The figure shows in a stepwise manner a possible scenario for the evolution of cells. Note that the rapid accumulation of oxygen, resulting from photosynthetic organisms, coincides with the first eukaryotes. The dotted lines indicate a dubious origin or divergence time (taken from Vellai and Vida, 19992).

II. Endosymbiotic theory and the origin of eukaryotes Evolutionists have long had a difficult time trying to account for the development of cells with nuclei. The first step, of course, is to agree on a suitable ancestor. The popular choice is bacteria (prokaryotes) which typically have only a single circular chromosome lying free in the cell. Unfortunately there are numerous structural and metabolic differences between the assumed ancestors and presumed descendants. The Endosymbiotic theory maintains that ancestors of eukaryotic cells were "symbiotic consortiums" of prokaryote cells with at least one and possibly more species (endosymbionts) involved. In other words, perhaps oxygen-breathing bacteria invaded an anareobic amoebalike bacteria, and each performed mutually benefiting functions. The bacteria would breathe for the anareobic amoebalike bacteria, and the amoebalike bacteria would navigate through new oxygen-rich waters in search of food. This way, each of the organisms would be benefiting from their symbiotic relationship as the waters and atmosphere changed over time. In support of this, notice that oxygen begins to accumulate between the first fossil records of prokaryotes and eukaryotes.

2 Vellai, T. and Vida, G. (1999) The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. Proc. R. Soc. Lond. B., 266: 1571-1577.



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Table 1. Differences between eukaryotic cells, prokaryotic cells, and organelles. The endosymbiosis theory postulates that mitochondria evolved from aerobic bacteria living within their host cell and chloroplasts evolved from endosymbiotic cyanobacteria (autotrophic prokaryotes). Several lines of evidence argue in favor of this theory: (i) Both mitochondria and chloroplasts can arise only from preexisting mitochondria and chloroplasts. They cannot be formed de novo i.e. in a cell that lacks them, because nuclear genes encode only some of the proteins of which they are made. (ii) Both mitochondria and chloroplasts have their own genome and it resembles that of prokaryotes not that of the nuclear genome. (iii) Both mitochondrial and chloroplast genomes consist of a single circular molecule of DNA (note that some species have linear genomes). (iv) Both mitochondria and chloroplasts have their own protein-synthesizing machinery, and it resembles that of prokaryotes not that found in the cytoplasm of eukaryotes. The first amino acid of their polypeptides is always formyl-Methionine as it is in bacteria (not methionine as in eukaryotic proteins). (v) A number of antibiotics (e.g., streptomycin) that act by blocking protein synthesis in bacteria also block protein synthesis within mitochondria and chloroplasts. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes. Conversely, inhibitors (e.g., diphtheria toxin) of protein synthesis by eukaryotic ribosomes do not have any effect on bacterial protein synthesis nor on protein synthesis within mitochondria and chloroplasts.

Prokaryotes Eukaryotes Mitochondria Chloroplasts

DNA 1 single, circular chromosome

Multiple linear chromosomes

1 single, circular chromosome

1 single, circular chromosome

Replication Binary fission Mitosis Binary fission Binary fission

Ribosomes 70 S 80 S 70 S 70 S

Electron transport chain

Found in plasma membrane around cell

Found in the mitochondria and

chloroplast

Found in plasma membrane

around mitochondria

Found in plasma membrane

around chloroplasts

Size ~ 1-10 microns ~ 50-500 microns ~ 1-10 microns ~ 1-10 microns

First amino acid formyl-Met Methionine formyl-Met formyl-Met

Antibiotic effect Streptomycin Diphtheria toxin

Yes No

No Yes

Yes No

Yes No

The original prokaryotic host cell ate or otherwise ingested aereobic bacteria (which may also have been a parasite), which reproduced so that subsequent generations of this new cell would also contain the newly ingested bacteria. These aereobic bacteria survived via the nutrients from the host prokaryotic cell, while multiple invaginations of the cell membrane helped prepare the aerobic bacteria for their new roles. Over time, both the prokaryotic host as well as the bacterial endosymbionts developed a mutually satisfying or beneficial existence and both entities lost their ability to function without the other. The ingested aerobic bacteria, which by definition are pro-oxygen, controlled and made possible the oxidative metabolism of what was the prokaryotic host cell. As the external world changed during the Precambrian times, the aerobic bacteria began to utilize and adapt their former roles to very similar functions with the prokaryotic cell. As a consequence, this former aerobic bacterium is



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recognized to have assumed the role of mitochondria. The evolution of eukaryotic plants and some of the other protists incorporated photosynthetic bacterial endosymbionts whereby a similar process occurred as with aerobic bacteria and mitochondria. The photosynthetic bacteria utilized their ability to perform photosynthesis for the former prokaryotic host cell, rather than just for itself. This is recognized to be the chloroplast. III. Mitochondria III.1. Location and structure Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. The number of mitochondria per cell appears to be relatively constant and characteristic for any given cell type. Mitochondria are often located near structures that requires ATP, the major product of their biochemical activity, or near a source of fuel, on which they depend. The mitochondria may make up a relatively large fraction of the total cytoplasmic volume; in the liver cell this is about 20 % and in a heart-muscle cell over 50%. Mitochondria are spherical, or nearly so, in brown fat cells, football-shaped in liver cells, cylindrical in the kidney, and threadlike in fibroblasts. Sometimes they have a very complex irregular structure, with extended processes, as in yeast cells. The most intensively studied mitochondria are those of rat liver, which electron microscopy shows to be about 2 µm long and somewhat less than 1µm wide in the intact cell. They are thus about the same size as bacteria (Table 1). Mitochondria have two membranes, an outer membrane that is smooth and somewhat elastic and an inner membrane that has folds, or invaginations, called cristae. The cristae appear to be devices for increasing the surface area of the inner membrane in relation to the mitochondrial volume. Inside the inner compartment is the matrix. This amorphous gel-like phase contains about 50% protein, some of which is organized into a reticular network apparently attached to the inner surface of the inner membrane. The matrix undergoes dramatic changes in volume and organization during changes in respiratory activity. The matrix also contains DNA and ribosomes. Mitochondria are energy-producing compartments found in nearly all eukaryotic cells (absent in some protozoa). The energy that mitochondria produce is in the form of ATP, an energy rich molecule that powers much of the work cells do. The process of producing ATP from glucose is often called the universal currency of cellular energy. It is a convenient way for cells to store the energy they need for such processes as protein manufacture, DNA replication, and the construction of new organelles. ATP is also required for such mechanical work as muscle contraction and cell movement. Following the first stages of sugar breakdown, the complicated process of energy transfer from sugar to ATP takes place within the animal cell's mitochondria. Besides supplying energy, mitochondria help to control the concentration of calcium and other electrically charged particles in the cytoplasm. They also break down and recycle the energy contained in fatty acids and amino acids.



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III.2. Growth and division Mitochondria are large enough to be observed by light microscopy in living cells. From several studies, the mitochondria in living cells are seen to be very dynamic – frequently dividing, fusing, and changing shape. Division and fusion of these organelles are topologically complex processes, because the organelles are enclosed by double membrane and the integrity of the separate mitochondrial compartments must be maintained. The copy number and shape of mitochondria vary dramatically in different cell types and can change in the same cell type under different physiological conditions, ranging form multiple spherical organelles to a single organelle with a branched structure (or reticulum). The arrangement is controlled by the relative rates of mitochondrial division and function, which is regulated by dedicated GTPases that reside on the mitochondrial membranes. The regulation of mitochondrial morphology and distribution is important for cell differentiation and fusion. As an example, mutations in Drosophila that impair mitochondrial fusion, and hence cause extensive mitochondrial fragmentation, block sperm development and produce infertility.

Table 2. Relative amounts of mitochondrial DNA in some cells and tissues (the large variation in the number and size of the mitochondria per cell in yeasts is due to mitochondrial fusion and fission.

Organism Tissue or Cell type

DNA molecules per organelle

Organelles per cell

Organelle DNA as % of total cellular DNA

Number of DNA molecule per cell

Rat Liver 5-10 1000 1 5.103-104

Yeast Vegetative 2-50 1-50 15 2-2500

Frog Egg 5-10 107 99 5.107-108

There can be many copies of the mitochondrial genomes in the space enclosed by each organelle’s inner membrane (Table 2). Mitochondrial DNA (mt DNA) is typically in the form of covalently closed circular double-stranded molecules (cccdsDNA). However, mtDNA is linear in certain ciliates e.g. Paramecium. How many of these genomes are present in a single organelle depends on the degree of organelle fragmentation; frequently, many genomes are housed in the same compartment. In most cells, the replication of the organelle DNA is not limited to the S phase of the cell cycle, when the nuclear DNA replicates, but occurs throughout the cell cycle – out of phase with cell division. Individual organelle DNA molecules seem to be selected at random for replication, so that in a given cell cycle, some may replicate more than once and others not at all. Nonetheless, under constant conditions, the process is regulated to ensure that the total number of organelle DNA molecules doubles in every cell cycle, as required if each cell type is to maintain a constant amount of organelle DNA. When conditions change, the total organelle mass per cell can be regulated according to needs. A large increase in mitochondria, for example, is observed if a resting skeletal muscle is repeatedly stimulated to contract for a prolonged period.



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Mitochondria cannot be synthesized de novo from scratch and can only make new ones by dividing mitochondria already present in the cell. When mitochondria divide they simply pinch into two, though not necessarily into equal halves. Bacteria divide also this way, which makes sense since they are the ancestors of mitochondria. It follows then that mitchondria may still use the same set of proteins to divide as bacteria. Many proteins are involved in bacterial division and the most widely occurring and conserved division protein is FtsZ. Just prior to division FtsZ molecules assemble to form a ring inside of the cell called the Z-ring. This ring appears to constrict the cell until it splits into two. In the cells of the most well studied eukaryotic organisms, such as animals and yeast, FtsZ is not present and their mitochondria appear to employ dynamin-like proteins to carry out mitochondrial division. Recently however, mitochondrial forms of FtsZ were discovered in other organisms such as algae and slime molds. Evidence for the involvement of FtsZ in mitochondrial division first came from the alga, Mallomonas splendens. Recently, we have discovered that the amoeba, Dictyostelium discoideum also uses FtsZ to divide its mitochondria.

Box 1- Functions of mitochondria Glycolysis or Embden-Meyerhof-Parnas pathway Glycolysis (from glyco, meaning “ sugar,” and lysis, meaning “splitting”) occurs in a series of nine steps, each catalyzed by a specific enzyme. This series of reactions is carried out by virtually all-living cells, from bacteria to the eukaryotic cells of plants and animals. Glycolysis is an anaerobic process that occurs in the ground substance of the cytoplasm. Glycolysis (from glucose the pyruvate) can be summarized by the overall equation: C6H12O6 +2NAD +2ADP+ 2Pi 2C3H4O3 + 2NADH2 + 2ATP Glucose pyruvate Thus, one glucose molecule is converted to two molecules of pyruvate. The net harvest- the energy recovered – is two molecules of ATP and the two molecules of NADH2. The two molecules of pyruvate have a total energy content of about 546 Kcal, which is a large portion of the 686 Kcal stored in the original glucose molecule. Krebs cycle The krebs cycle is named in honor of Sir Hans Krebs, who was largely responsible for its elucidation. Krebs postulated this metabolic pathway in 1973 and later received a Nobel Prize in recognition of his brilliant work. The krebs cycle is also called the tricarboxylic acid (TCA) cycle because it is initiated with the formation of an organic acid (citrate) that has three carboxylic acid groups. Before entering the krebs cycle, pyruvate is both oxidized and decarboxylated. In the course of this exergonic3 reaction, a molecule of NADH2 is produced from NAD. The original glucose molecule has now been oxidized to two acetyl (CH3CO) groups; two molecules of CO2 have been liberated; and two molecules of NADH2. Each acetyl group is then temporarily attached to coenzyme A (CoA)- a large molecule, a portion of which is panthotenic acid, one of the B-complex vitamins. The combination of the acetyl group and CoA is known as acetyl- CoA4.

3 Exergonic reaction refers to any reaction accompanied by the liberation of energy. 4 Fats and amino acids can also be converted to acetyl-CoA and enter the respiratory chain.



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Upon entering the Krebs cycle, the two-carbon acetyl group is combined with a four-carbon compound (oxaloacetate) to produce a six -carbon compound (citrate). In the course of the cycle, two of the six carbons are oxidized to CO2, and oxaloactate is regenerated- thus literally making this series a cycle. Each turn around the cycle uses up one acetyl group and regenerates one molecule of oxaloacetate, which is then ready to begin the Krebs cycle again. Some of the energy released by the oxidation of the carbon atoms is used to convert ADP to ATP (one molecule per cycle), some is used to convert NAD to NADH2 (three molecules per cycle). In addition, some of the energy is used to reduce a second electron carrier- the coenzyme flavin adenine dinucleotide (FAD). One molecule of FADH2 is formed from FAD per cycle. Oxygen is not directly involved in the Krebs cycle; the electrons and protons removed in the oxidation of carbon are all accepted by NAD and FAD: Oxaloacetate + acetylCoA + ADP + 3NAD + FAD Oxaloacetate + 2CO2 + CoA + ATP + 3NADH2+ FADH2 The respiratory chain The respiratory chain consists of four complexes of integral membrane proteins: The NADH dehydrogenase or complex I, succinate dehydrogenase or complex II, the cytochrome c reductase or complex III, the cytochrome c oxidase or complex IV and two freely-diffusible molecules: ubiquinone, and cytochrome c that shuttle electrons from one complex to the next. The respiratory chain accomplishes: a- The stepwise transfer of electrons from NADH (and FADH2) to oxygen molecules to form (with the aid of protons) water molecules (H2O); Cytochrome c can only transfer one electron at a time, so cytochrome c oxidase must wait until it has accumulated 4 of them before it can react with oxygen. b- Harnessing the energy released by this transfer to the pumping of protons (H+) from the matrix to the intermembrane space. c- Approximately 20 protons are pumped into the intermembrane space as the 4 electrons needed to reduce oxygen to water pass through the respiratory chain. d- The gradient of protons formed across the inner membrane by this process of active transport forms a miniature battery. e- The protons can flow back down this gradient, reentering the matrix, only through another complex of integral proteins in the inner membrane, the ATP synthase complex or complex V. Chemiosmosis in mitochondria The energy released as electrons pass down the gradient from NADH to oxygen is harnessed by the enzyme complexes of the respiratory chain to pump protons (H+) against their concentration gradient from the matrix of the mitochondrion into the intermembrane space across the inner mitochondrial membrane (an example of active transport). As the concentration of protons increases there (as the pH decreases), a strong transmembrane gradient is set up whose inherent energy, termed “proton motive force” or “pmf ”, can be used for chemical work: production of ATP through complex V. As in chloroplasts, the energy released as these electrons flow down their gradient is harnessed to the synthesis of ATP. The process is called chemiosmosis.

III.3. Mitochondrial genomes5 Mitochondrial DNA molecules ranges in size from less than 6,000 bp in Plasmodium falciparum (the human malaria parasite) to more than 300,000 bp in some land plants. In mammals, the mitochondrial genome is a DNA circle of about 16,500 bp (less than 0.001% of the size of the nuclear genome). It is nearly the same size in animals as diverse as Drosophila

5 Alberts, B. et al. (2002). Molecular Biology of the cell. New York, pp. 808-821.



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and sea urchin. There is no correlation between the size of mitochondrial genomes and the number of proteins they encode. While the human mitochondrial DNA encode 13 proteins, the 22-fold larger mitochondrial DNA of Arabidopsis encodes only 32 proteins – that is about 2.5-fold as many as human mitochondrial DNA. The extra DNA that is found in Arabidopsis, Marchantia, and other plant mitochondria may be “junk DNA”. The mitochondrial DNA of the protozoan Reclinomonas americana has 97 genes, more than the mitochondrion of any other organism analyzed so far. III.3.1. The human mitochondrial genome The relatively small size of human mitochondrial genome made it a particularly attractive target for early DNA- sequencing projects, and in the 1981, the complete sequence of its 16,569 bp was published. Compared with nuclear, chloroplast, and bacterial genomes, the human mitochondrial genome has several surprising features: a. High gene density: Unlike other genomes, nearly every nucleotide seems to be a part of

a coding sequence, either for a protein or for one of the rRNAs or tRNAs. Since these coding sequence run directly into each other, there is very little room left for regulatory DNA sequences.

b. Relaxed codon usage: Whereas 30 or more tRNAs specify amino acids in the cytosol and in chloroplasts, only 22 tRNAs are required for the mitochondrial protein synthesis. The normal codon-anticodon pairing rules are relaxed in mitochondria, so that the third base in the codon is entirely irrelevant (superwobble). Such “ 2 out of 3” pairing allows one tRNA to pair with any one of four codons and permits protein synthesis with fewer tRNA molecules.

c. Variant genetic code: comparisons of mitochondrial gene sequences and the amino acid sequences of the corresponding proteins indicate that the genetic code employed by mitochondria differ in some respects from the universal code: 4 of the 64 codons have different “meanings” from those of the universal genetic code (Table 3).

Table 3. Some differences between the “Universal” genetic code and mitochondrial genetic code. Italics and bolding indicate that the code differs from the “Universal” code.

Mitochondrial Codes

Codon “Universal” Code Mammals Invertebrates Yeasts Plants UGA STOP Trp Trp Trp STOP AUA Ile Met Met Met Ile CUA Leu Leu Leu Thr Leu AGA AGG Arg STOP Ser Arg Arg



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The observation that the genetic code is nearly the same in all organisms provides strong evidence that all cells have evolved from a common ancestor. How, then, does one explain the few differences in the genetic code in many mitochondria? A hint comes from the finding that the mitochondrial genetic code is different in different organisms. In the mitochondrion with the largest number of genes, that of the protozoan Reclinomonas, the genetic code is unchanged from the standard genetic code of the cell nucleus. Yet UGA, which is a stop codon elsewhere, is read as tryptophan in mitochondria of mammals, fungi, and invertebrates. Similarly, the codon AGG normally codes for arginine, but it codes for stop in the mitochondria of mammals and codes for the serine in the mitochondria of Drosophila. Such variation suggests that a random drift can occur in the genetic code in mitochondria. Presumably, the unusually small number of proteins encoded by the mitochondrial genome makes an occasional change in the meaning of a rare codon tolerable, whereas such a change in a large genome would alter the function of many proteins and thereby destroy the cell.

Table 4. Comparison between the human nuclear and mitochondrial genomes. In addition to their differences in genetic capacity and different genetic codes, the mitochondrial and nuclear genomes differ in many aspects of their organization and expression.

Nuclear genome Mitochondrial genome

Size 3,300,000 Kb 16.6 Kb

No. of different DNA molecules 23 (in XX) Or 24 (in XY) cells, all linear

One circular DNA molecule

Total no. of DNA molecules per cell

23 in haploid cells; 46 in diploid cells

Several thousands

Associated proteins Several classes of histone and non histone proteins

Largely free of proteins

Number of genes 30,000 – 35,000 37

Gene density ~ 1/ 40 Kb 1/ 0.45 Kb

Repetitive DNA Large fraction ~ 48% Very little

Transcription The great bulk of genes are transcribed individually

Continuous transcription of multiple genes

Introns Found in most genes Absent

% of coding DNA ~ 2-3% ~ 93%

Recombination At least once for each pair of homologs at meiosis Not evident

Inheritance Mendelian for sequences on X and autosomes; paternal for sequences on Y

Exclusively maternal (non-mendelian, cytoplasmic inheritance)



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III.3.1.a. Mitochondrial genes6 The human mitochondrial genome contains 37 genes: 28 are encoded by the heavy strand, and nine by the light strand. Of the 37 genes, a total of 24 specify a mature RNA product: 22 mitochondrial tRNA molecules and two mitochondrial rRNA molecules, a 23S rRNA (a component of the large subunit of mitochondrial ribosomes) and a 16S rRNA (a component of the small subunit of mitochondrial ribosomes). The remaining 13 genes encode polypeptides which are synthesized on mitochondrial ribosomes. Each of the 13 polypeptides encoded by the mitochondrial genome is a subunit of one of the mitochondrial respiratory complexes, the multi-chain enzymes of oxidative phosphorylation that are engaged in the production of ATP. Note, however, that there are a total of about 100 different polypeptide subunits in the mitochondrial oxidation phosphorylation system, and so the vast majority are encoded by the nuclear genes. All other mitochondrial proteins are encoded by the nuclear genome and are translated on cytoplasmic ribosomes before being imported into the mitochondria. Unlike its nuclear counterpart, the human mitonchondrial genome is extremely compact: approximately 93% of the DNA sequence represents coding sequence. All 37 mitochondrial genes lack introns and they are tightly packed. The coding sequences of some genes (notably those encoding the sixth and the eighth subunits of mitochondrial ATPase) show some overlap and, in most other cases, the coding sequences of neighboring genes are contiguous or separated by one or two noncoding bases. Some genes even lack termination codons; to overcome this deficiency, UAA codons have to be introduced at the post-transcriptional level via a process termed RNA editing.

Table 5. The limited autonomy of the mitochondrial genome

Encoded by the mitochondrial genome

Encoded by the nuclear genome

Components of oxidative phosphorylation system NADH CoQ reductase, complex I 7 subunits >41 subunits Succinate reductase, complex II 0 subunits 4 subunits CoQ-Cytochrome c reductase, complex III 1 subunits 10 subunits Cytochrome c oxidase, complex IV 3 subunits 10 subunits ATP synthase, complex V 2 subunits 14 subunits Total 13 subunits >80 subunits Components of protein synthesis apparatus tRNA components 22 tRNA None rRNA components 2 rRNA None Ribosomal proteins None ~80 Total 24 ~80 Other mitochonrial proteins None All7

6 Strachan, T. and Read, A.P. (2000). Human Molecular genetics 2. Bath Press, UK., pp: 141-142. 7 e.g. mtDNA and mtRNA polymerases plus numerous other enzymes, structural and transport proteins, etc.



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III.3.1.b. Mitochondrial DNA replication and transcription The only significant region lacking any known coding DNA is the displacement (D) loop region. This is the region in which a triple-stranded DNA structure is generated by synthesizing an additional short piece of the H-strand DNA, known as 7S DNA. Replication of mtDNA proceeds via a displacement mechanism The replication of both H and L strands is unidirectional and starts at specific origins. As to the H strand, the origin is in the D loop and only after about two – thirds of the daughter H strand has been synthesized (by using the L strand as a template and displacing the old H strand) does the origin for L strand duplication become exposed. Thereafter, replication of the L strand proceeds in the opposite direction, using the H strand as a template. The D loop contains the predominant promoter of transcription of both L and H strands. Unlike transcription of nuclear genes, in which individual genes are almost always transcribed separately using individual promoters, transcription of the mitochondrial DNA starts at the promoters in the D loop region and continues, in opposite directions for the two strands, round the circle to generate large multigenic transcripts. The mature RNAs are subsequently generated by cleavage of the multigenic transcripts. III.3.2. The rice mitochondrial genome8 Rice is an important cereal crop worldwide and it could play a major role as a model for cereal genomics. The entire mitochondrial genome of rice (Oryza stavia L.), a monocot plant, has been sequenced. It was found to comprise 490,520 bp, with an average G + C content of 43.8 %. A total of 35 genes for known proteins, 3 rRNAs, 2 pseudoribosomal proteins, 17 kinds of tRNAs, and 5 pseudo tRNAs were identified. No apparent strand bias was observed for the presence of genes. ORFs capable of encoding more than 150 amino acids were sought based on the universal codon usage. In addition to the 35 protein-coding genes mentioned, 19 other ORFs were deduced; however, only 10 of these were found to be transcribed. A greater degree of variation in terms of presence /absence and integrity of genes was observed among the ribosomal protein genes and tRNA genes of rice, Arabidopsis, and sugar beet. Transcription and post-transcriptional modification (RNA editing) in rice mitochondrial sequence were also examined. In all, 491 Cs in the genomic DNA were converted to Ts in cDNA. The frequency of RNA editing differed markedly depending upon the ORF considered. Sequences derived from plastid and nuclear genomes make up 6.3 % and 13.4 % of the mitochondrial genome, respectively. The degree of conservation of plastid sequences in the mitochondrial genome ranged from 61 % to 100 %, suggesting that gene transfer has occurred very recently. All of the protein–coding genes of the plastid origin in rice mitochondria seems to be non-functional, as a result of many sequence alterations and the absence of RNA editing. In contrast, 7 tRNAs of plastid origin are likely to be functional. Three plastid DNA fragments that were incorporated into the mitochondrial genome were subsequently transferred to the

8 Notsu, Y. et al. (2002). The complete sequence of the rice (Oryza stavia L.) mitochondrial genome: frequent DNA sequences acquisition and loss during evolution of the flowering plants. Mol Genet Genomics, 268: 434-445.



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nuclear genome. Nineteen fragments having homology with known retrotransposon and transposon sequences, but different from those of dicots, were identified. Nuclear sequences might have been transferred to the mitochondrial genome. Extensive reshuffling of the fragmented DNA is indicated in either case, although the mechanism of such events and the orientation of sequence flow is not clearly understood. The results indicate frequent and independent flow of DNA sequences to and from the mitochondrial genome during flowering plant evolution and this may account for the genetic fluidity and plasticity of mitochondrial genome in higher plants

Box 2- Gene transfer Mitochondria are the direct descendants of a bacterium that was engulfed by a primitive eukaryotic host cell. Over time the mitochondrion has lost or transferred the majority of its genetic information to the nucleus. Much of this transfer occurred soon after the establishment of the mitochondrial endosymbiosis. Gene transfer and functional activation appear to have ceased in animals, as evidenced by their nearly constant mitochondrial gene content, however, a number of evolutionary recent gene transfer events from the mitochondrion to the nucleus have been identified in plants, indicating that gene transfer is still taking place. These gene transfer events provide a unique window of opportunity to study gene transfer, a process of fundamental importance, to the establishment and evolution of organelles in eukaryotic cells9. Activation of nuclear gene after the transfer from an organelle requires the acquisition of a number of elements for regulation and targeting, including a promoter, polyadenylation signal, and a mitochondrial targeting presequence. On transfer to the nucleus, it is important that the gene acquires regulatory and trafficking sequences relatively quickly, before it becomes inactivated by random mutations. In addition, the gene product must be retargeted to the mitochondrion, sorted to its correct intramitochondrial location, and assembled into appropriate holoenzyme. Gene transfer to the nucleus is an ongoing process, in that a number of recent gene transfer events have been detected that have resulted in some variability in the coding capacities of mitochondrial genomes even among relatively closely related organisms. In addition, plant nuclear genomes can harbor large pieces of mitochondrial DNA, which serve no apparent function and hence are commonly referred to as “ promiscuous DNA”. Occasionally, DNA transfer in the reverse direction can also occur, as revealed in the detection of copia-, gypsy-, and LINE-like retrotransposon fragments in the mitochondrial genome of Arabidopsis thaliana10.

III.3.3. Mitochondrial plasmids In addition to a large and complex main mitochondrial genome, the mitochondria of many species of higher plants contain a variety of smaller DNA molecules. These molecules can be regarded as extrachromosomal replicons or plasmids, which can replicate autonomously

9 Daley, D.O. et al. (2002). Gene transfer from mitochondria to nucleus: novel mechanisms for gene activation from Cox2. The Plant Journal, 30: 11-21. 10 Kulda, J. et al. (2002). Loss of the mitochondrial cox2 intron 1 in a family of monocotyledonous plants and utilization of mitochondrial intron sequences for the construction of nuclear intron. Mol Genet Genomics, 267: 223-230.



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in mitochondria, because they are present in high copy number relative to the main genome. These smaller DNA molecules can be either circular or linear. Linear mitochondrial plasmids are quite frequently found in fungi and less often in higher plants, but they seem to be absent from most animal cells. They usually contain terminal inverted repeats and have proteins covalently attached to their 5' termini, thus suggesting a model for their complete replication (no end shortening occurs). They share this DNA structure with some DNA viruses, e.g. adenovirus or Bacillus subtillis bacteriophage 29 and various transposable elements such as Ac and Ds of maize and Tam from Antirrhinum majus. Although the knowledge of this linear DNA is very limited, its most striking known feature is that it can be transmitted to progeny plants through the pollen. In many higher plants mitochondria are transmitted uniparentally from the maternal plant to the progeny, and thus the main genomic DNAs and smaller DNAs in mitochondria are also transmitted maternally. III.4. Mitochondrial mutations Mitonchondria are inherited virtually exclusively from oocyte11. A number of diseases resulting from point mutations, deletions or rearrangements of mitonchondria genome, therefore display a characteristic maternal inheritance pattern. Mitonchondrially encoded disorders cannot be parentally transmitted. If an oocyte contains a mixture of mitonchondria with normal and mutated mitonchondrial DNA, both will be transmitted to daughter cells. Unlike the nuclear genome, there is no mechanism for ensuring equal segregation of the different species to progeny. Different daughter cells may contain different proportions of the mitochondrial variant. In the mature individual, correspondingly, some tissues may contain greater or lesser proportions of abnormal mitonchondria. This situation in which both normal and mutated mitonchondria are contained in the same cell, is known as heteroplasmy12 and complicates genetic and phenotypic predictions. On the other hand, homoplasmy refers to the condition in which only one mitochondrial genome type is present in the cell. The level of heteroplasmy may determine the likelihood of an individual being affected. Different individuals within a family may be affected to differing degrees of severity depending on the level of heteroplasmy, or may even have different clinical features because of different tissue distributions of the mutation.

11 In most cases, animals follow a model of standard maternal inheritance, in which mitochondrial DNA is passed only from the mother to progeny. The marine mussels of the genus Mytilus, and other Bivalves, however have an unusual system of mitochondrial DNA transmission termed doubly uniparental inheritance (DUI). Under this mode inheritance, males receive paternally derived mtDNA from their fathers (M type), which comes to dominate in the male gonad. Males also receive their mother’s mtDNA (F type), which comes to dominate in their somatic tissues. Females receive both their mother’s F type and their father’s M type, but the M type normally disappears by 24-48 h after fertilization. To summarize, male Mytilus mussels are heteroplasmic and females are normally homoplasic [Dalziel, A.C. and Stewart, D.T. (2002). Tissue-specific expression of male-transmit mtDNA and its implications for rates of molecular evolution in Mytilus mussels (Bivalvia: Mytilidea). Genome, 45: 348-355.] 12 The term heteroplasmy is also given to the cloned progeny that harbor mtDNAs from both the donor and the recipient cytoplasms.



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III.5. Mitochondrial protein import machinery Since the mitochondrial genome encodes a limited number of proteins, most mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and imported into the mitochondria. The mitochondrial genome (86Kb) of the yeast S. cerevisiae contains 54 genes, among which only nine code for proteins and thus, 400-500 of the approximately 6000 proteins encoded by the nuclear genome are probably targeted to the mitochondria13. Since the cell is compartmentalized (i.e. encloses several membrane-bounded subcompartments such as peroxisome, nucleus, Golgi apparatus, etc.) and proteins synthesized in the cytosol have to be correctly targeted to their final location, specific information for targeting to each compartment is required. Imported mitochondrial proteins are synthesized either as precursors consisting of the mature protein with additional N- or C- terminal presequence containing the targeting information or as preproteins carrying internal targeting information (e.g. the carrier protein family, the best known member of which is ADP/ATP translocase). Moreover, in the case of mitochondria, import involves various subcompartments, the outer membrane, intermembrane space, inner membrane and matrix. The proteins targeted to each of these subcompartments therefore require specific targeting information and an import pathway involving common or distinct proteins at certain steps. Cytosolic chaperones interact specifically with the newly-synthesized preproteins, which they sort, transport and maintain in an open conformation using an ATP-dependent mechanism. Since precursors are prone to misfolding, aggregation, or simply folding in the cytosol, the main roles of cytosolic chaperones (Hsp70) in import might be to prevent these events and to maintain the precursors in an import-competent form. The function of Hsp70 chaperones is not specific for mitochondrial preproteins, since the addition of purified Hsp70 is sufficient to stimulate the import of several precursor proteins in vitro.

Box 3- Ricekttsia prowazekii as potential ancestor of mitochondria14 The Ricekttsia are -proteobacteria that multiply in eukaryotic cells only. R. prowazekii is the agent of epidemic, louse-borne typhus in humans. The genome of Ricekttsia is a small one, containing only 1,111,523 bp with an average G + C content of 29.1%. The genome contains 834 complete ORFs with an average length of 1,005 bp. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either Ricekttsia or mitochondrial genomes, but a complete set of genes encoding components of the TCA and the respiratory-chain complex is found in Ricekttsia. ATP production in Ricekttsia is the same as that in mitochondria. Many genes involved in biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome.

13 Duby, G. and Boutry, M. (2002). Mitochondrial protein import machinery and targeting information. Plant Science, 162: 477-490. 14 Andersson, S. G. E. et al. (1998). The genome project of Rickettsia prowazekii and the origin of mitochondria. Nature, 396: 133-143



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Phylogenetic analysis based on sequences of rRNA and heat shock proteins indicate that mitochondria may be derived from the -proteobacteria. Indeed, the closet ancestor to mitochondria seems to be the Ricekttsia. The reduction in genome size in mitochondria and Ricekttsia is likely to have occurred independently in two lineages. Most of the genes supporting mitochondrial activities are nuclear. Many of the 300 proteins encoded in the nucleus of the yeast S. cerevisiea but destined for service within the mitochondria are close homologues of their counterparts in R. prowazekii. Nearly one-quarter of these proteins required for bioenergetic processes and another one-third of them are required for the expression of the genes encoded in the mitochondrial genome. In total, more than 150 nucleus-encoded mitochondrial proteins share significant sequence homology with R. prowazekii. Another group of 58 nucleus-encoded mitochondrial proteins represents components of the mitochonrial transport machinery and regulatory system. These include proteins found in the mitochondrial outer membrane and others involved in splicing reactions. Such proteins have probably been secondarily recruited to mitochondria from genomes not necessarily related to that of the -protobacterial ancestor. The mitochondrial genome of the early diverging, fresh water protozoan Reclinomonas americana is more like that of a bacterium than any other mitochondrial genome sequenced so far. This genome contains 67 protein coding genes, most of which provide components for the genetic processes and the bioenergetic system. Several gene clusters in this mitochondrial genome are reminiscent of those in bacteria. Most similarities represent retained, ancestor traits present in the common ancestor of bacteria and mitochondria. The Rickettsia ATP/ADP translocases are monomers with 12 transmembrane regions each, whereas the mitochondrial translocases are dimers with six transmembrane regions per dimer. Since no relationship between the primary structures of the mitochondrial and Rickettsia ATP/ADP translocases, these transport systems may then have originated independently. Nevertheless, phylogenetic reconstructions based on components of the NADH dehydrogenase complexes indicate that there is a close evolutionary relation between R. prowazekii and mitochondria. The study of R. prowazekii genome sequence supports the idea that aerobic respiration in eukaryotes originated from an ancestor of the Rickettsia, as indicated previously by phylogenetic reconstructions based on the rRNA gene sequences.

IV. Chloroplasts IV.1. Structure Chloroplasts are small compartments found in the cells of algae and plants that contain the molecular machinery for harvesting sunlight and converting its energy into food. This process is called photosynthesis, and the food it produces enables plants and algae to grow and ultimately feed the rest of us. The chloroplast is one of several types of organelles termed plastids15, all of which derived from the same precursor proplastid. The latter is the progenitor of various plastids found in the root and shoot meristem, embryos, endosperm, and in young developing leaves. Chloroplasts differentiated from proplastids undergo a secondary set of divisions that result in a large population of small chloroplasts in each mesophyll cells.

15 There are several types of plastids. The photosynthetic chlorophyll-containing chloroplasts are green. The conversion of photosynthetic chloroplasts into yellow carotenoid-rich chromoplasts is seen in the ripening of bananas; the conversion of chloroplasts to lycopene-containing red chromoplasts is seen in the ripening of tomatoes. Each compartment of the eukaryotic cell is unique. A particular biochemistry can be favored in one compartment (e.g. chloroplasts or chromoplasts) while the environment in another compartment (e.g. the cytoplasm) is unfavorable [Bogorad, L. (2000). Engineering Chloroplasts: an alternative site for foreign genes, proteins, reactions and products. TIBTECH, 18: 257-263.]



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As a green photosynthetic plastid, chloroplasts typically measures 5m in diameter, are 1 to 2 m thick, and occupy up to 70% of the surface area of a cell and approximately 20% of the total volume in mature leaf cells. Electron microscopy reveals that the chloroplast, like the mitochondrion, comprises three different membranes (the double membrane envelope and the thylakoid membrane), which enclose the three distinct soluble phases (Intermembrane space, stroma, and the thylakoid lumen). The stroma is the site of carbon fixation, amino acid synthesis and many other pathways. In the thylakoid membrane system light is captured and ATP synthesized, whereas in thylakoid lumen several extrinsic photosynthetic proteins as well as polypeptides operating in the folding and proteolysis of the thylakoid proteins are housed16. IV.2. How do chloroplasts divide? Chloroplasts divide by a process of binary fission in which constriction of the envelope membrane occurs. This process is morphologically and genetically similar to bacterial cell division. Recent genetic approaches for understanding chloroplast division and development using Arabidopsis clearly indicate a close similarity to the genetic control of prokaryotic cells. Although considerable progress has been made in elucidating the molecular mechanisms of plastid binary fission in higher plants, an understanding of how cells control their plastid number is completely lacking. A close correlation between the leaf mesophyll cell size and the number of chloroplasts within the cell clearly indicates that cell size is a primary determinant of the chloroplast number. Further, it appears that chloroplast division is initiated only after chloroplasts have attained a certain size. Some arc mutants of Arabidopsis with greatly enlarged chloroplasts show continued chloroplast development with normal internal structure. As a consequence, chloroplast division appears to be a process independent of chloroplast development. This implies that the chloroplast division event is an integral part of normal leaf cell development and that the evolution of higher plants led to each photosynthetic cell containing many small chloroplasts rather than a few large ones. Ultra structural evidence has shown that electron-dense plastid-diving rings (PD rings) form at the plastid division site. PD rings have an outer ring on the cytoplasmic face of the plastid outer membrane and an inner ring on the stromal face of the plastid inner membrane. The precise behavior of PD rings has been observed in the Cyanidiophyceae, the red algae. PD rings appear at the initiation of chloroplast division and continue to squeeze the membrane until the final division stage. The formation site and behavior during division of both the FtsZ ring and PD rings show-striking similarities. The FtsZ protein is assembled into an 80-nm-wide FtsZ ring at the future division sites in the stroma within the chloroplast envelope. When the chloroplast begins to constrict, the 40-nm-wide inner PD ring and the 20-nm-wide outer PD ring appear17. The model for chloroplast division was proposed based on the observation of these rings. The formation of the FtsZ ring is the earliest step in the assembly of the division

16 Leister, D. (2002). Chloroplast research in the genomic age. TRENDS in Genetics, 19: 47-56. 17 Kuroiwa, H. et al. (2002). Chloroplast division machinery as revealed by immunofluorescence and electron microscopy. Planta, 215: 185-190.



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apparatus at the programmed position on the dividing plane. With invagination of the chloroplast envelop, the inner and outer PD rings appear to be lining the FtsZ ring in the stromal and cytosolic sides, respectively. The complex division apparatus gradually constricts, accompanied by disassembly of the FtsZ and inner PD ring proteins. In the last stage of division, the FtsZ ring disappears, while the outer PD ring becomes a wide, thick, rigid structure that allows the completion of chloroplast division. IV.3. Function of the chloroplast Photosynthesis is the physico-chemical process by which plants, algae and photosynthetic bacteria use light energy to drive the synthesis of organic compounds. In plants, algae and certain types of bacteria, the photosynthetic process results in the release of molecular oxygen and the removal of carbon dioxide from the atmosphere that is used to synthesize carbohydrates (oxygenic photosynthesis). Other types of bacteria use light energy to create organic compounds but do not produce oxygen (anoxygenic photosynthesis18). Photosynthesis provides the energy and reduced carbon required for the survival of virtually all life on our planet, as well as the molecular oxygen necessary for the survival of oxygen consuming organisms. In addition, ancient photosynthetic organisms produced the fossil fuels currently being burned to provide energy for human activity. Although photosynthesis occurs in cells or organelles that are typically only a few microns across, the process has a profound impact on the earth's atmosphere and climate. The photosynthetic apparatus of the chloroplast captures light energy and converts it into biologically useful energy that is, for example, available for: (1) manufacturing sugars and amino acids; (2) reducing nitrate to ammonium; (3) making starch; (4) synthesizing complex organic compounds; and (5) assembling, chloroplast proteins that are required for the chloroplasts themselves to function.

Box 4- Photosynthesis consists of both “ Light” and “Dark” Reactions It is convenient to divide the processes of photosynthesis into four stages, each occurring in a defined area of the chloroplast: Absorption of light: The initial step in photosynthesis is the absorption of light by chlorophyll attached to proteins in the thylakoid membranes. Chlorophyll is a ringed compound similar in structure to heme, except that a magnesium atom, Mg2+ (rather than an iron atom Fe3+), is in the center and besides the four central 5-atom rings, there is an additional 5-atom ring. The energy of the absorbed light is used first to remove electrons from unwilling donor- water, in green plants, forming oxygen: 2H2O O2 + 4H+ + 4e- The electrons are then transferred to a primary acceptor. All these reactions occur in a complex of proteins termed a Photosystem, in the thylakoid membrane.

18 Occurs only under anaerobic conditions, and only one type of chlorophyll-containing reaction center appears to occur in a given species. Electrons ejected from the bacteriochlorophyll follow a cyclic path, thus causing the transmembrane pumping of protons and generating pmf. The pmf can be used for the synthesis of ATP.



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Electron transport: Electrons move from the primary electron acceptor through a chain of electron transport molecules in the thylakoid membrane until they reach the ultimate electron acceptor, usually NADP+, reducing it to NADPH. The transport of electrons is coupled to the movement of protons across the membrane from the stroma to the thylakoid lumen, in much the same way that a proton-motive force (pmf) is established across the mitochondrial inner membrane during electron transport. Thus the overall reaction of stages 1 and 2 can be summarized as: 2H2O + 2NADP+ 2H++ 2NADPH+ O2 Many bacteria do not use water as the donor electrons. Rather, they use molecules such as hydrogen gas (H2) or hydrogen sulfide (H2S) as the ultimate source of electrons to reduce NADP+. In these bacteria the overall reactions of the first stages of photosynthesis would therefore be: H2S + NAD+ H+ + NADH + S and H2 + NAD+ H+ + NADH Generation of ATP: protons move down their concentration gradient form the thylakoid lumen to the stroma through a set of transport proteins, which couples proton movement to the synthesis of ATP from ADP and Pi: H+ + ADP3- + Pi

2- ATP4 - + H2O This use of the pmf to synthesize ATP is very similar to the process occurring during oxidative phosphorylation in the mitochondria. CO2 Fixation- the conversion of CO2 into Carbohydrates: The ATP4- and the NADPH generated by the second and third stages of photosynthesis provide the energy and electrons to drive the synthesis of polymers of six – carbon sugars from CO2 and H2O: 6CO2 + 18ATP4- + 12 NADPH + 12H2O C6H12O6+ 18ADP3- +18Pi

2- + 12NADP+ + 6H+ All four stages of photosynthesis are tightly coupled and controlled so as to produce the amount of carbohydrates required by the plant.

IV.4. Chloroplast genomes The first phase of chloroplast genomics culminated with the completion of tobacco (Nicotana tabacum) and liverwort (Marchantia polymorpha) chloroplast genome sequences in 1986. The present literature has witnessed the discovery of new plastid-encoded traits, the use of plastids for foreign gene expression, and an appreciation of their diversity, particularly outside the vascular plants. Two recent major foci have emerged: (i) functional studies, ranging from details of photosynthesis to gene expression and cell biology; (ii) genomics whose major goal is to obtain functional data as well as evolutionary and comparative analysis of complete nuclear and organelle genomes of different species. These are usually selected because of their importance for sciences e.g. model organism, commercial crops, etc. At present, complete genome sequences have been obtained from virtually all the major algal lineages, including the C. reinhardtii19 sequence and the Synechocyctis20 sp. PCC 6803 genome. The latter represents

19 The unicellular, green alga Chlamydomonas reinhardtii has many characteristics that make it an ideal organism for elucidating the function, biosynthesis, and regulation of the photosynthetic apparatus. Photosynthetic mutants of C. reinhardtii are viable because this alga can be grown heterotrophically with acetate as a sole source of carbon, and because C. reinhardtii is haploid during vegetative growth mutation are almost immediately expressed and specific mutants phenotypes can be readily observed as colonies on solid medium. Furthermore, C. reinhardtii lends itself to in vivo procedures that are difficult or impossible to perform with more complex systems, and the “molecular toolkit” with which investigators can manipulate genes and gene expression in C.



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the presumed ancestor of chloroplast. With chloroplast post genomic era a new chapter of chloroplast molecular genetics, where evolutionary forces and intracellular mechanisms that shape genome architecture, gene expression, and ecological adaptation, are revealed21. Plastids are organelles characteristic of plants cells. As endosymbiotic remnants of a free-living cyanobacterial progenitor, plastids have, over evolutionary time, lost the vast majority of their genes in a similar fashion to that of the mitochondrial ancestor. Indeed, depending on the organism, contemporary plastomes22 , which are in average 150 Kb in size, contain only 60-200 open reading frames (ORFs). The plastomes of green algae and flowering plants are remarkably similar in the sequences of their genes. However, the organization of genes on the plastid chromosome differs drastically. Although identical plastome copies are contained in each cell plant, the organelles themselves can vary to a large extent in their morphology and function. The genome of the chloroplasts found in Marchantia polymorpha (a liverwort) contains 121,024 bp in a closed circle. These make up some 128 genes, which include: duplicated genes encoding each of the four subunits (23S, 16S, 4.5S, and 5S) of the ribosomal RNA (rRNA) used by the chloroplast, 37 genes encoding all the tRNA molecules used for translation within the chloroplast. Moreover, there are four genes encoding some of the subunits of the chloroplast RNA polymerase, a gene encoding the large subunit of the enzyme RUBISCO (ribulose biphosphate carboxylase oxygenase); nine genes for components of photosytems II and I, six genes encoding parts of the chloroplast ATP synthase, and genes for 19 of the ~60 proteins used to construct the chloroplast ribosome. All these gene products are used within the chloroplast, but all the chloroplast structures also depend on proteins encoded by nuclear genes, translated in the cytosol, and imported into the chloroplast. IV.5. Chloroplast import machinery During the course of evolution most of the endosymbiotic genes were transferred to the host nucleus. Today most of the chloroplastic proteins are synthesized in the cytosol and subsequently imported into the organelle. Protein import into chloroplasts has been analyzed in vitro and found to occur post-translationally. The precursor proteins are generally synthesized in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These translation systems are a complex mixture of many proteins several of which might actually bind to or interact with newly synthesized proteins and thereby

reinhardtii is extensive and has become increasingly sophisticated in recent years. Selectable markers are available for identification of nuclear and chloroplast transformants, as are relatively simple procedures to introduce DNA into cells. Reporter genes, such as GFP and arylsulfatase, and anti-sense and RNAi-based suppression of mRNA levels have been effectively used in C. reinhardtii. The many advantageous features of C. reinhardtii have earned in the epithet “green yeast”. Many types of physiological, genetic, and molecular manipulations of C. reinhardtii have become rountine and have made this organism ripe for extensive genetic studies [Shrager, J. et al. (2003). Chlamydomonas reinhardttii Genome Project. A guide to the generation and use of the cDNA Information. Plant Physiology, 131: 401-408. 20 A genus of unicellular cyanobacteria (blue-green algae) in which the cells are coccoid and divide by equal binary fission in two or three plans, giving rise to clusters of cells. 21 Simpson, C.L. and Stern, D.B. (2002). The Treasure Trove of Algal Chloroplast Genomes. Surprises in Architecture and Gene Content, and Their Functional Implications. Plant Physiology, 129: 957-966. 22 Chloroplast genome is termed plastome.



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influencing protein import reactions. Indeed it has been shown that newly synthesized proteins in a reticulocyte lysate system bind to chaperons of the HSP70 family either during or shortly after completion of their synthesis. The association with HSP70 is most likely independent of the presence of a presequence and occurs for many if not all proteins, which are targeted in a post-transcriptional event to a compartment in the eukaryotic cell. A presequence-specific association was found for ornithine carbamylase and a soluble factor from reticulocyte lysate, which was called presequence binding factor (PBF) and had a positive effect on import. Wheat germ lysate contains a soluble precursor guidance complex that binds to chloroplast transit peptide. The guidance complex consists of HSP70, 14-3-3 proteins and further unidentified components. However, wheat germ lysate seems to contain also components, which inhibit protein import into chloroplasts. The import inhibitor, which is a polypeptide, does not associate with the guidance complex or the subunits of the translocon, it acts in a concentration-dependent manner directly on the translation product and not by influencing a component of the translocon on the chloroplast surface. Hence, once the precursor has engaged the translocon the inhibitor can no longer bind to the prepotein. Furthermore, the inhibitor might be a component of a regulatory system that could enable the plant cell to respond rapidly to different physiological needs23. Passage through the chloroplast membranes is achieved in a manner similar to that of the mitochondria. Chloroplast proteins pass the outer and the inner membranes of the envelope into the stroma, a process involving the same types of passage as into the mitochondrial matrix. But some proteins are transported yet further, across the stacks of the thylakoid membrane into the lumen. Proteins destined for the thylakoid membrane or lumen must cross the stroma en route. Chloroplast targeting signal resemble that of mitochondrial targeting signal. The leader consists of ~50 amino acids, and the N-terminal half is needed to recognize the chloroplast envelope. A cleavage between positions 20-25 occurs during or following passage across the envelope, and proteins destined for the thylakoid membrane or lumen have a new N-terminal leader that guides recognition of the thylakoid membrane. There are several different systems in the chloroplast that catalyze import of proteins into the thylakoid membrane. V. End-notes V.1. A deeper look of endosymbiosis The origin of mitochondria and plastids form different bacterial endosymbionts has been a widely accepted hypothesis for several decades. However, the extent of additional gene transfer from bacterial to eukaroytic genomes is still being discovered. Phylogenies for many universal proteins, such as some metabolic enzymes, show the Archaea, rather then the Bacteria, as the outgroup in the universal tree of life, which indicates a clause relationship between bacteria and the eukaryotes. Further evidence for the early integration of bacterial genes into the eukaryotic genome comes from studies of proteins from simple protists such as

23 Schleiff, E. et al. (2002). Chloroplast protein import inhibition by a soluble factor from wheat germ lysate. Plant Molecular Biology, 50: 177-185.



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Giardia intastinalis and Trichomonas vaginalis (Parabasalidea1), which commonly lack mitochondria (that is, they are amitochondria); rRNA phylogenies indicate that these are the earliest evolved eukaryotic lineages. Early views on these protists, once collectively called the “Archezoa”, were that they evolved before mitochondrial biogenesis, which is the endosymbiosis –driven fixation of an -proteobacterium in a eukaryotic cell. However, molecular studies showed that these amitochondrial protists have genes for several proteins that are not only targeted to the mitochondria in higher eukaryotes but that are also of bacterial origin. Parabasalidea as well as some ciliates and fungi that lack mitochondria, have specialized organelles called hydrogenosomes24, which ferment pyruvate and produce hydrogen. The hydrogenosome lacks any genome that might help trace its origins; however, phylogenetic analysis of hydrogenosome-targeted proteins indicates a possible shared evolutionary path with mitochondria, and the hydrogenosome itself might be a derived from mitochondrion. Therefore, it is plausible that amitochondrial protists secondarily lost their organelles, but not before the successful fixation of several bacterial genes in the nuclear genome25. Recently some scientists have called for further consideration of the origins of the mitochondrial and chloroplast proteomes. The most striking aspect of mitochondrial evolution is the extent of the partnership between the host and the endosymbiont. In the yeast Saccharomyces cerevisiae, there are ~ 400 nuclear proteins that are involved in mitochondrial interactions, but half of these have no bacterial counterparts. Endosymbiosis seems to have been a two-way process. The bacterial endosymbiont directly contributed some of its genes to the genome of the host (either an archeon or an early eukaryote). Experiments indicate that gene transfer, at least in yeast, might be biased in the direction from the mitochondria to the nuclear genome (1 transfer event per 105 generations versus near negligible transfers from the nucleus to the mitochondria). However, the host must have also targeted or invented nuclear genes for communication and support to the new organelles. V.2. Why do mitochondria and chloroplasts have their own genetic systems? Why do mitochondria and chloroplasts have their own separate genetic systems, when other organelles that share the same cytoplasm, such as peroxisomes and lysosomes, do not? The question is not trivial, because maintaining a separate genetic system is costly: more than 90 proteins -including many ribosomal proteins, aminoacyl-tRNA synthases, DNA and RNA polymerases, and RNA-processing and RNA-modifying enzymes-must be encoded by nuclear genes specifically for this purpose. The amino acid sequences of most of these proteins in the mitochondria and chloroplasts differ from those of their counterparts in the nucleus and cytosol, and it appears that these organelles have relatively few proteins in common with the

1 Parabasalidea is a superorder of protozoa that consists of two orders: Hypermastigida and Trichomonadida. 24 Hydrogenosome is a membrane-limited intracellular organelle present in certain eukaryotic microorganisms such as Entamoeba hystolytica. It contains iron-sulphur proteins and flavoproteins as components of an electron transport chain used in the oxidation of pyruvate into acetate, CO2 and H2 (here protons are used as electron acceptors). 25 Brown, J. R. (2003). Ancient Horizontal Gene Transfer. Nature Reviews Genetics, 4: 121-132.



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rest of the cell. This means that the nucleus must provide at least 90 genes just to maintain each organelle’s genetic system. The reason for such a costly arrangement is not clear, and the hope that the nucleotide sequences of the mitochondrial and chloroplast genomes would provide the answer has proved to be unfounded. Scientists cannot think of compelling reasons why the proteins made in the mitochondria and chloroplasts should be made there rather then in the cytosol.

Box 5- What are the possible fates of genes in endosymbiosis? The endosymbiotic hypothesis seems to provide a partial explanation for the observed HGT between the bacteria and the eukaryotes. The diagram shows the five possible fates of a single orthologous gene found in both the bacterial endosymbiont and the host (either an archeabacterium or proto-eukaryote) after endosymbiosis (modified from Brown 2003).

The rarest scenario (panel a) is the retention of the gene in the genomes of both the organelle (formerly the endosymbiont) and the host cell. In the case of the mitochondria the overwhelming evolutionary trend is towards the reduction of mitochondrial genome size. For example, the sugar beet Beta vulgaris has the largest known mitochondrial genomes, at 368,799 bp, but it is far smaller than the genome of the bacterium Mycoplasma genitalium, which, at 580,074 bp, is the smallest known bacterial genome. Similarly, the number of protein-encoding genes in mitochondria range from 2 in Plasmodium falciparum to 67 in other protist, Reclinomonas americana, whereas Bartonella henselae, perhaps the closest known -proteobacterial relative of the mitochondrium, has ~1,600 genes. The second possibility (panel c) is that the organelle gene is lost from the organism and that the product encoded by a gene in the host genome now functions in two compartments: the organelle and the cytoplasm. Although it is no longer encoded by the organelle genome, this type of a gene would be expected to have homologues in other contemporary bacterial species. A third possibility (panel e) is that the organelle gene is transferred to the host genome, where it coexists with the host copy. For example, animals, plants, fungi and slime moulds have two separate genes, both of which code for methionyl-tRNA synthetases. One isoform functions in the

Recipient or host cell Endosymbiont

Endosymbiosis

Orthologousgenes

(a)

(b) (c) (d)

(e)



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cytoplasm and is more closely related to archaeal orthologues, whereas the second isoform, targeted to the mitochondria, is most similar to bacterial orthologues. The organelle gene could be transferred to the eukaryotic host genome, where it might substitute for an existing gene or add a new function (panel b). This phenomenon is called "endosynbiotic gene replacement". The more specialized instances of this are represented by the case of amitochondrial protists that have the organelle gene but not the organelle. This phenomenon-termed cryptic endosymbiosis-invokes the idea of a temporal state of endosymbiosis, followed by the loss of the bacterial endosymbiont. The final scenario (panel d) depicts the case in which an unrelated gene encodes a protein that has acquired a new role in maintaining the organelle. This nuclear protein might be a replacement for an existing gene product (which was lost from the organelle genome) or have an entirely new function in the endosymbiont. Recent genome analyses indicate that possibly more than half of the mitochondrial proteome originated from species other than the endosymbiont.

At one time, it was suggested that some proteins have to be made in the organelle because they are too hydrophobic to get to their site in the membrane from the cytosol. More recent studies, however, make this explanation implausible. In many cases, even highly hydrophobic subunits in the various mitochondrial enzyme complexes are highly conserved in evolution; their site of synthesis is not. The diversity in the location of genes coding for the subunits of the functionally equivalent proteins in different organisms is difficult to explain by any hypothesis that postulates a specific evolutionary advantage of present-day mitochondrial or chloroplast genetic systems. Perhaps the organelle genetic systems are an evolutionary dead-end. In terms of the endosymbiont hypothesis, this would mean that the process whereby the endosymbionts transferred most of their genes to the nucleus stopped before it was complete. Further transfers may have been ruled out, for mitochondria, by recent alterations in the mitochondrial genetic code that made the remaining mitochondrial genes nonfunctional if they were transferred to the nucleus.

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