In this article we will discuss about the genomes in chloroplast DNA and mitochondria DNA.
The phenomenon of extra-nuclear inheritance based on transmission of visible phenotypes through mitochondria and chloroplasts. Studies in the 70s revealed presence of DNA in these organelles. Both mitochondria and chloroplasts are present only in cells of lower and higher eukaryotic organisms. Detailed studies established that DNA in these organelles is similar to the DNA in prokaryotic bacteria.
The genomes of both mitochondria and chloroplasts code for all of their RNA species and some proteins that are involved in the function of the organelles. The DNA is in the form of a circular duplex molecule, except in some lower eukaryotes in which mitochondrial DNA is linear.
Each organelle contains several copies of the genome, and because there are-multiple organelles per cell, organelle DNA constitutes a repetitive sequence. Mitochondrial DNA (mtDNA) varies enormously in size, whereas chloroplast DNA (ctDNA) ranges in size between 120 and 200 kb.
Chloroplasts are present in green plants and photosynthetic protists. ctDNA sequence studied in a number of plants indicates uniformity in size and organisation. The differences in size are due mainly to the differences in lengths of introns and inter-genic regions as well as the number of genes. All cp DNAs contain a significant proportion of noncoding DNA sequences.
The ctDNA is double stranded circular, and devoid of histones and other proteins. In many cases, the GC content of cpDNA differs from that of nuclear DNA and mitochondrial DNA. Complete cpDNA sequences have been determined in tobacco (155, 844 bp) and rice (135, 42 bp).
Multiple copies of cpDNA are present in the nucleoid region of each chloroplast. In the green alga Chlamydomonas, one chloroplast contains 500 to 1500 cpDNA molecules. Chloroplasts divide by growing and then dividing into two daughter chloroplasts.
The proportion of introns in chloroplast DNA could be high, 38% in Euglena. Among the expressed genes in chloroplast genome, 70 to 90% of the genes encode proteins including those involved in photosynthesis, four genes code for rRNAs (one each for 16S, 23S, 4.5S and 5S), and about 30 genes encode tRNAs.
Chloroplast genome also contains genes for some of the proteins required for transcription and translation of the encoded genes, and most importantly, genes for photosynthesis. Most of the proteins in chloroplasts are encoded by the nuclear genes. The mRNA transcripts of the chloroplast genes are translated according to the standard genetic code.
However, the primary structures of several RNA transcripts are found to go through editing consisting of C to U transitions, that cause mRNA sequence to deviate from the sequence in the corresponding gene. Editing makes it difficult to convert chloroplast nucleotide sequences into amino acid sequences of the corresponding protein.
Most of the cpDNAs studied share a common feature, that is, a 10 to 24 kb segment present in two identical copies as an inverted repeat. The cpDNA also contains two copies of each of the rRNA genes which are located in these two identical repeat sequences in an inverted orientation.
Other genes that are found in the repeated sequence are therefore, also duplicated in the chloroplast genome. The location of these repeats defines a short single copy (SSC) region and a long single copy (LSC) region in chloroplast genome.
Chloroplast protein synthesis uses organelle-specific 70S ribosomes consisting of 50S and 30S subunits. The 50S subunit contains one copy each of 23S, 5S and 4.5S rRNAs, while the 30S subunit contains one copy of a 16S rRNA.
Among the ribosomal proteins, some are encoded by the nuclear DNA, some by the chloroplast genome. About 100 open reading frames (ORFs), putative protein coding genes, have been identified by computer analysis. Protein synthesis is similar to that in prokaryotes.
Each human cell contains hundreds of mitochondria each containing multiple copies of mitochondrial DNAs (mtDNA). Mitochondria generate cellular energy through the process of oxidative phosphorylation. As a by-product they produce most of the endogenous toxic reactive oxygen species Mitochondria are also the central regulators of apoptosis or programmed cell death.
These interrelated functional systems involve activities of about 1000 genes distributed in the nuclear genome and the mitochondrial genome. Due to their dependence on the nuclear genome, mitochondria are considered as semi-autonomous.
This has been shown by experiments in which mitochondria and mtDNA could be transferred from one cell to another. The donor cell was enucleated and its mitochondria-containing cytoplast fused with a recipient cell (technique of cybrid transfer).
The genomes of mitochondria show wide variation particularly among plants and protists. Most mitochondrial DNAs (mtDNA) consist of a closed circular double stranded supercoiled DNA molecules located in multiple nucleoid regions (similar to those in bacterial cells); some protists however, have varying lengths or multiple circular molecules of DNA as in the trypanosomes. mtDNA in the protist Amoebidium parasiticum consists of several distinct types of linear molecules with terminal and sub-terminal repeats.
Although most mtDNAs are in the size range of 15 to 60 kb, mtDNA in malarial parasite (Plasmodium spp) is only 6 kb long, while that of rice (Oryza sativa) is 490 kb, and cucurbits 2 Mb. There are about 40 to 50 coding genes in mitochondrial DNA, Plasmodium being an exception with 5 coding genes.
The large size of mitochondrial genomes in plants are due to noncoding inter-genic regions and their content of tandem repeats. Introns are present in many mtDNAs, and in some unusual cases, the genes are split into as many as 8 regions that are dispersed in the genome, and located on both strands of the DNA. Transcription takes place separately in portions of the split genes producing discrete pieces of RNA that are held together by base pairing of complementary sequences.
The mtDNA contains information for a number of mitochondrial compounds such as tRNAs, rRNA, and some of the polypeptide subunits of the proteins cytochrome oxidase, NADH- dehydrogenase and ATPase. Most of the other proteins found in mitochondria are encoded by the nuclear genome and transported into mitochondria.
These include DNA polymerase and other proteins for mtDNA replication, RNA polymerase and other proteins for transcription, ribosomal proteins for ribosome assembly, protein factors for translation, and the aminoacyl-tRNA synthetases.
The mitochondrial oxidative phosphorylation complexes are composed of multiple polypeptides, mostly encoded by the nuclear DNA (nDNA). However, 13 polypeptides are encoded by mtDNA. The mtDNA also codes for 12S and 16S rRNAs and 22 tRNAs required for mitochondrial protein synthesis. The mtDNA also contains a control region consisting of approximately 1000 base pairs constituting the promoter region and the origin of replication.
The mRNAs synthesised within the mitochondria remain in the organelle and are translated by mitochondrial ribosomes that are assembled within mitochondria. Mitochondrial ribosomes have two subunits. Mitochondria in human cells have 60S ribosomes consisting of a 45S and a 35S subunit.
There are only two rRNAs in mitochondrial ribosomes of most organisms, that is, 16S rRNA in large subunit and 12S rRNA in small subunit of most animal ribosomes. There is usually one gene for each rRNA in a mitochondrial genome. The proteins in mitochondrial ribosomes are encoded by the nuclear genome and transported into mitochondria from the cytoplasm.
Mitochondrial ribosomes are sensitive to most of the inhibitors of bacterial ribosome function such as streptomycin, neomycin and chloramphenicol. For protein synthesis, mitochondria of most organisms use a genetic code that shows differences from the universal genetic code. Only plant mitochondria use the universal nuclear genetic code.
Transcription of mammalian mtDNA is unusual in that each strand is transcribed into a single RNA molecule that is then cut into smaller pieces. In the large RNA transcripts that are produced, most of the genes encoding the rRNAs and the mRNAs are separated by tRNA gene.
The tRNAs in the transcript are recognised by specific enzymes and are cut out, leaving only the mRNAs and the rRNAs. A poly (A) tail is then added to the 3’end of each mRNA and CCA is added to the 3’end of each tRNA. There are no 5′ caps in mitochondrial mRNAs.
Mitochondrial DNA replication is semi-conservative and uses DNA polymerases that are specific to the mitochondria. The mtDNA replicates throughout the cell cycle, independently of nuclear DNA synthesis which takes place in S phase of cell cycle. Observations on mtDNA replication in animal mitochondria in vivo have resulted in a model referred to as the displacement loop (D loop) model as follows (Fig. 17.6).
The two strands of mtDNA in most animals have different densities because the bases are not equally distributed on both strands, called H (heavy) and L (light) strands. The synthesis of a new H strand starts at the replication origin for the H strand and forms a D-loop structure (Fig. 17.6).
As the new H strand extends to about halfway around the molecule, initiation of synthesis of a new L strand takes place at a second replication origin. Synthesis continues until both strands are completed. Finally, each circular DNA assumes a supercoiled form.
The mtDNA is maternally inherited and has a very high mutation rate. When a new mtDNA mutation occurs in a cell, a mixed intracellular population of mtDNAs is generated, known as heteroplasmy. During replication in a heteroplasmic cell, the mutant and normal molecules are randomly distributed into daughter cells.
When the percentage of mutant mtDNAs increases, the mitochondrial energy producing capacity declines, production of toxic reactive oxygen species increases, and cells become more prone for apoptosis. The result is mitochondrial dysfunction. Tissues most sensitive to mitochondrial dysfunction are brain, heart, kidney and skeletal muscle.
The mtDNA mutations are associated with a variety of neuromuscular disease symptoms, including various ophthalmological symptoms, muscle degeneration, cardiovascular diseases, diabetes mellitus, renal function and dementias.
The mtDNA diseases can be caused either by base substitutions or rearrangement mutation. Base substitution mutations can either alter protein (missense mutation) or rRNAs and tRNAs (protein synthesis mutations). Rearrangement mutations generally delete at least one tRNA and thus cause protein synthesis defects.
Missense mutations are associated with myopathy, optic atrophy, dystonia and Leigh’s syndrome. Base substitution mutations in protein synthesizing genes have been associated with a wide spectrum of neuromuscular diseases, and the more severe typically include mitochondrial myopathy.
Mitochondrial diseases are also associated with a number of different nuclear DNA mutations. Mutations in the RNA component of the mitochondrial RNAse have been implicated in metaphyseal chondrodysplasia or cartilage hair hypoplasia which is an autosomal recessive disorder resulting from mutation in nuclear chromosome 9 short arm position (9p13).