Prokaryotic and Organelle Genetics How Genes Travel on Chromosomes Prokaryotic and Organelle Genetics LECTURE OUTLINE A General Overview of Bacteria Bacterial Genomes Gene Transfer in Bacteria The Genetics of Chloroplasts and Mitochondria Non-Mendelian Inheritance of Chloroplasts and Mitochondria mtDNA Mutations and Human Health Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14

The genetics of Chloroplast and Mitochondria

Mitochondria and chloroplasts in eukaryotes have characteristics of prokaryotic cells Endosymbiotic theory – mitochondria and chloroplasts are descended from bacteria that fused with nucleated cells Mitochondria – organelles that produce energy for metabolic processes, found in all eukaryotic cells Each cell has many mitochondria, highest number in cells with high energy requirements Similar in size and shape to modern aerobic bacteria Produces energy in the form of ATP Chloroplasts – organelles that capture energy from light and store it as carbohydrates, found in plant and algal cells Structural similarities to certain cyanobacteria, which are capable of photosynthesis. 40-50 per cells in corn In humans, nerve, muscle, and liver cells each carry more than a thousand mitochondria.

1) The genomes of mitochondria (mtDNA) Located within highly condensed structures (nucleoids) Number of nucleoids per mitochondria varies depending on growth conditions and energy needs of cell mtDNA replication occurs independent of cell cycle Random occurrence of replication – some mtDNAs replicate many times, and other mtDNAs don't replicate at all In most species, mtDNA is circular Some species (Tetrahymena, Paramecium, Chlamydomonas, Hansenula) have linear mtDNA Protozoan parasites have a single mitochondrion (kinetoplast) with large network of minicircles and maxicircles The replication of mtDNA molecules, as well as the division of the mitochondria, can occur throughout the cell cycle independent of the replication of genomic nuclear DNA (which occurs only during S phase) and of the cell division at the end of mitosis

Kinetoplast DNA network . In certain protozoan parasites, there is a single mitochondrion, or kinetoplast, that contains a large interlocking network of DNA molecules present in mini- and maxicircles.The Kinetoplast DNA network maxicircles contain most of the genes usually found on mtDNA, while the minicircles play a role in RNA editing,

The size and gene content of mitochondrial genomes varies from organism to organism Table 14.1 Table 14.2

Mitochondrial genome variation across species Mitochondrial genome in humans is very compact Adjacent genes either touch each other or overlap slightly Virtually no intergenic regions No introns Mitochondrial genome of S. cerevisiae is 4X larger than in humans and other animals Long intergenic regions Has introns Although both these organelles replicate and express all the genes in their own DNA, their genomes encode only some of the proteins they require for their activities.

Editing of RNA transcripts Mitochondrial evolution has also led to some remarkable variations on the basic mechanisms of gene expression. Editing of RNA transcripts

CONCLUSION OF THESE OBSERVATIONS???? Protozoan parasites have kinetoplast the DNA arranged as a series of interlocking maxi- and minicircles. minicircles carry no protein-encoding genes. maxicircle DNA do carry and express genes. Kinetoplast has both RNAs that looked like the strange fragments of kinetoplast genes and related RNAs that could encode recognizable mitochondrial proteins CONCLUSION OF THESE OBSERVATIONS???? kDNA encodes a precursor (the strange fragment observed) for each mRNA. After transcription, the cellular machinery turns these precursors into functional mRNAs through the insertion or deletion of nucleotides. protozoan parasites have a single, large mitochondrion—the kinetoplast—which contains much more DNA than the mitochondria of other organisms and which has the DNA arranged as a series of interlocking maxi- and minicircles. DNA sequencing shows that the minicircles carry no protein-encoding genes. The detection of transcripts from maxicircle DNA, however, confi rms that these larger circles do carry and express genes.

Editing of RNA transcripts RNA editing first discovered in mtDNA transcripts of trypanosomes (a protozoan parasite) Transcription of mtDNA produces pre-mRNA that is converted to mature mRNAs by RNA- editing RNA editing produces start and stop codons for translation as well as internal codons RNA editing also identified in some plants and fungi Fig. 14.31 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14

Without RNA editing, the pre-mRNAs do not encode polypeptides. The extent of RNA editing varies from mRNA to mRNA and from organism to organism. In trypanosomes, the RNA editing machinery adds or deletes uracils. In plants, the editing adds or deletes cytosines. Without RNA editing, the pre-mRNAs do not encode polypeptides. Some pre-mRNAs lack a fi rst codon suitable for translation initiation; others lack a stop codon for the termination of translation. RNA editing creates both types of sites, as well as many new codons within the genes.

Mitochondrial translation differs from translation of mRNAs from nuclear genes Similar aspects of translation in prokaryotes Initiation of translation by N-formyl methionine and tRNAfMet Translation in prokaryotes and mitochondria is inhibited by chemicals (e.g. chloramphenicol and erythromycin) that don't affect translation of nuclear mRNAs The genetic code for nuclear and mitochondrial genes is different Table 14.3

2) The genomes of chloroplasts (cpDNA) Includes genes for some photosynthetic enzymes and for gene expression Ranges in size from 120-217 kb, but most are 120-160 kb Much uniform than mtDNA Closely packed genes with little intergenic sequence (like human mtDNA) but has introns (like yeast mtDNA) Similarities to bacteria RNA polymerases of choloroplasts and bacteria are similar Translation in prokaryotes and chloroplasts is inhibited by chemicals (e.g. chloramphenicol and streptomycin) that don't affect translation of nuclear mRNAs

The cpDNA-encoded proteins include many of the molecules that carry out photosynthetic electron transport and other aspects of photosynthesis, AND ALSO: RNA polymerase, translation factors, ribosomal proteins, and other molecules active in chloroplast gene expression.

Nuclear and organellar genomes cooperate with each other Assembly and maintenance of functional organelles depend on both organelle and nuclear gene products e.g. the 7 subunits of cytochrome c oxidase in most organisms are encoded by 3 mitochondrial genes and 4 nuclear genes Organelles don't carry all the genes needed for translation (semiautonomous) Because mitochondria and chloroplasts do not carry all the genes for the proteins (and in some organisms, the tRNAs) they need to function and reproduce, these organelles are semiautonomous, requiring the constant provision of proteins (and tRNAs) encoded by nuclear genes. Fig. 14.32

Number and genomic location of oxidative phosphorylation genes Fig. 14.32 Although some organelles in some species have many more genes than others, all are dependent on RNA and protein products encoded by nuclear genes. The location of oxidative phosphorylation genes is shown.

Gene transfer between organelles and the nucleus Evidence for transfer via an RNA intermediate COXIII gene in plants – encodes component of mitochondrial electron transport chain Present in the nuclear genomes of some plants but in the mitochondrial genomes of other plants Some plant species have a non-functional mitochondrial gene that contains an intron and the functional nuclear gene doesn't have the intron Evidence for transfer at the DNA level Some plant mtDNAs contain large fragments of cpDNA Nonfunctional, partial copies of organelle genes are present in the nuclear genome

High rate of mutation in mtDNA mtDNA evolves ~10X more rapidly than does nuclear DNA More errors in replication and less efficient repair Provides valuable tool for studying evolutionary relationships of closely- related species But, has little value for studying evolutionary relationships of distantly- related species Sequence analyses of mtDNA have shown that the maternal lineage of all present-day humans, no matter what ethnic group they belong to, traces back to a few female ancestors who lived in Africa some 200,000 years ago.

https://www. sciencenews https://www.sciencenews.org/article/scientists- find-clue-why-mitochondrial-dna-comes-only- mom

Non-Mendelian inheritance of organelles Mendel performed reciprocal crosses in which either the male or female plant carried the wild-type or variant allele. no difference in inheritance based on which parent showed the variant. 1909 – green vs. variegated leaves in four-o'-clocks Variegated offspring produced when ovules of variegated plants were fertilized with pollen from green plants No variegated offspring produced when ovules of green plants were fertilized with pollen from variegated plants

The first example of non-Mendelian inheritance uncovered by geneticists was seen in the flowering plants known as four-o’clocks. The egg cell is many times larger than the pollen cells, and contain both mitochondria and chloroplasts. Pollen is small and is essentially devoid of organelles, and thus organelle DNA. So any trait that is encoded by the organelle DNA will be contributed by the female The results can be explained in the following manner.  All of the organelle DNA that is found in an embryo is from the female. 

Maternal inheritance of Xenopus mtDNA Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 Two closely-related species of frogs DNA probes from mtDNA were used to identify mtDNA present in F1 offspring Fig. 14.34

A maternally inherited neurodegenerative disorder in humans Leber's hereditary optic neuropathy (LHON) A disease in which flaws in the mitochondria’s electron transport chain lead to optic nerve degeneration and blindness G-to-A substitution in gene for an NADH dehydrogenase subunit causes Arg-to-His missense substitution The substitution alters an arginine-specifying codon in the NADH dehydrogenase subunit 4 gene to a histidine codon. NADH dehydrogenase is a flavoprotein that contains iron-sulfur centres. Fig. 14.35

Not all offspring show signs of the disease, and not all siblings manifesting the condition have symptoms of the same severity. The random allotment to daughter cells of a large number of mitochondria during mitosis helps explain these observations. The resulting protein product diminishes the effi ciency of electron fl ow down the respiratory transport chain, reducing the cell’s production of ATP and causing a gradual decline in cell function and ultimately cell death. Because optic nerve cells have a relatively high requirement for energy

Distribution of organelles during mitosis Heteroplasmic cells contain a mixture of organelle genomes Homoplasmic cells contain only one type of organelle genome Mitotic progeny of homoplasmic cells are also homoplasmic Mitotic progeny of heteroplasmic cells can be either heteroplasmic, homoplasmic wild-type, or homoplasmic mutant Uneven distribution of organellar genomes has distinct phenotypic consequences Homoplasmy causes earlier appearance of the disease as well as more severe symptoms in LHON patients. A diploid cell contains dozens to thousands of organelle DNAs. It is therefore not possible to use the terms “homozygous” and “heterozygous” to describe a cell’s complement of mtDNA or cpDNA.

How Distribution Affects Phenotype After fertilization, as a result of the mitotic divisions of embryonic development, the random segregation of mutation-carrying mitochondria from heteroplasmic cells can produce tissues with completely normal ATP production and tissues of low energy production.

Mechanisms that contribute to uniparental inheritance Gamet size In most higher eukaryotes, the male gamete is much smaller than the female gamete. the zygote receives a very large number of maternal organelles and, at most, a very small number of paternal organelles. In some organisms, cells degrade the organelles or the organellar DNA of male gametes In some plants, the early divisions of the zygote distribute most or all of the paternal organelle genomes to cells that are not destined to become part of the embry In certain animals, details of fertilization prevent a paternal cell from contributing its organelles to the zygote. Events of fertilization allow only the sperm nucleus to enter the egg, physically excluding the paternal mitochondria.

Some organisms exhibit biparental inheritance of organellar genomes 1909 – reciprocal crosses between green and variegated geraniums Both types of crosses produces green, white, and variegated offspring in varying proportions Chloroplast traits inherited from both parents Fig. 14.37

Principles of non-Mendelian inheritance: A summary 1) In the inheritance of organelle genomes from one generation to the next, there is a 4:0 segregation of parental alleles, instead of the 2:2 pattern seen for the alleles of nuclear genes. 2) In most organisms, transmission of organelle- encoded traits is uniparental, mainly maternal, although in a few organisms transmission is biparental. 3) With both uniparental and biparental inheritance, when the parents transmit organelles of more than one genotype, mitotic segregation of those genotypes occurs in the offspring. This segregation of genotypes during mitosis is a consequence of the random partitioning of organelles during cell division.

mtDNA mutations and human health Maternal pattern of inheritance Symptoms vary enormously among family members Myoclonic epilepsy and ragged red fiber disease (MERFFF) Range of symptoms: uncontrolled jerking, muscle weakness, deafness, heart problems, kidney problems, progressive dementia Mutations in mitochondrial tRNAs (e.g. tRNALys) Disruption of mitochondrial transport chain Individuals affected by MERFF are heteroplasmic Severity of phenotype depends on percentage of mutant mtDNA (see Fig. 14.39 and 14.40) http://hihg.med.miami.edu/code/http/modules/education/Design/Print.asp?CourseNum=2&LessonNum=4

Muscle cell of MERRF patient. Transmission electron micrograph of muscle mitochondria from patients expressing MERRF. Mutant mitochondria are highly abnormal, showing paracrystalline arrays and crista degeneration.

The proportion of mutant mitochondria determines the severity of the MERFF phenotype and the tissues affected Tissues with higher energy requirements are less tolerant of mutant mitochondria Tissues with low energy requirements are affected only when the proportion of wild-type mitochondria is greatly reduced Fig. 14.40

Mitochondrial mutations may have an impact on aging Oxidative phosphorylation system in the mitochondria generates free radicals, which can damage DNA Accumulation of mtDNA mutations over time may result in age-related decline in oxidative phosphorylation Evidence in support of role of mtDNA and aging: Percentage of heart tissue with a mitochondrial deletion increases with age Brain cells of people with Alzheimer’s disease (AD) have abnormally low energy metabolism 20% to 35% of mitochondria in brain cells of most AD patients have mutations in cytochrome c oxidase genes, which may explain the low energy metabolism

Mitochondrial DNA Tests as Evidence of Kinship in Argentine Courts Between 1976 and 1983, the military dictatorship of Argentina kidnapped, incarcerated, and killed more than 10,000 university students, teachers, social workers, union members, and others who did not support the regime. Many very young children disappeared along with the young adults, and close to 120 babies were born to women in detention centers. In 1977, the grandmothers of some of these infants and toddlers began to hold vigils in the main square of Buenos Aires to bear witness and inform others about the disappearance of their children and grandchildren. They soon formed a human rights group—the “Grandmothers of the Plaza de Mayo.”

CONTD The extremely polymorphic noncoding region makes it possible to identify grandchildren through a direct match with the mtDNA of only one person—their maternal grandmother, or mother’s sister or brother— rather than through statistical calculations assessing data from four people.

END OF THE 14th chapter!!!