Molecular Biology Largely Concerned with Gene Expression What Turns it On/Off? How that is Achieved? How Much?

Regulation of Gene Expression - 2 lectures In Eukaryotes Regulation of Gene Expression is Complex - Not just on and off -  Vary magnitude

Regulation of Gene Expression 1 - Revision of Eukaryotic Gene Structure 2- Need for Global regulation of genes

Molecular cell Biology 5th Edition Mitochondria structure, aerobic and anaerobic metabolism pp , Gene structure pp Molecular Genetic Techniques and Cloning Chapter 9 Journal of Experimental Biology Volume 201, pp (1998) Kwast et al.

Will use control of gene expression by oxygen as an example Take place in all organisms - fungi, plants and animals Critical for survival Evolved early so can use comparative approaches to Understand

Oxygen is Toxic Will Not deal with Reactive Oxygen Species - Rather will deal with Role of oxygen in respiration - Oxidative Phosphorylation

Aerobic respiration essential for survival of multi-cellular Organisms Some micro-organisms can survive anaerobically Anaerobic - yeast - Industrial applications Plants - Loss of Yield - results in large loss of life and/or commercial income Animals - Medical conditions - hear disease, cancer etc

Genome Information S. cerevisiaeC. elegansA. thalianaH. sapiens # Cells1 ~1000 >1x10 6 >1x10 6 Size 12Mbp 97Mbp 125Mbp 3.2 Gbp Chromosomes Predicted ORFs ~6,000 ~19,000 ~28,000 ~35,000 %Coding72% 27%50% 1.5%

Aerobic metabolism: (oxidative metabolism) Glycolysis TCA cycle oxidative phosphorylation Anaerobic metabolism: (fermentation) Glucose  Acetyl CoA  Ethanol Diauxic shift: Metabolic change as fermentable carbon source is used up from… Glucose Fermentative (Glycolysis  Ethanol) Oxidative Metabolism (Ethanol  TCA cycle) to… yeast carbon metabolism

Two membranes Inner membrane invaginated Numbers of mitochondria per cell vary but usually 100s/cell Matrix contains the TCA cycle (and other) soluble enzymes Inner membrane contains metabolite transporters and the electron transport chain Mitochondrial structure

Overview of aerobic respiration

Four large, multi-subunit protein complexes - complex I is a NADH- ubiquinone reductase - complex II is succinate dehydrogenase (part of the TCA cycle) - complex III is the ubiquinone -cytochrome c reductase - complex IV is cytochrome oxidase The respiratory electron transport chain

NADH + CO 2 One pyruvate molecule is completely oxidised to CO 2 4-Carbons 3-Carbons CO 2 6-Carbons NADH + CO 2 NADH FADH Outline of Tricarboxylic Acid Cycle The NADH and FADH produced are oxidised by the respiratory electron transport chain

Mitochondria have their own DNA and Ribosomes Mitochondria have some of their own DNA, ribosomes, and can make many of their own proteins. The DNA is circular and lies in the matrix in structures called "nucleoids". Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA). mitochondrial DNA

Human mtDNA small, double stranded circular chromosome 16,569 bp in total no non-coding DNA no introns polycistronic replication which is initiated from the D (displacement)- loop region followed by splicing of transcript to form messages. Organisation of the mitochondrial chromosome

human mtDNA yeast mtDNA Yeast mitochondrial chromosome

maize mitochondrial genome

In all organisms, only a few of the proteins of the mitochondrion are encoded by mtDNA, but the precise number varies between organisms Subunits 1, 2, and 3 of cytochrome oxidase Subunits 6, 8, 9 of the Fo ATPase Apocytochrome b subunit of complexIII Seven NADH-CoQ reductase subunits (except in yeast) The nucleus encodes the remaining proteins which are made in the cytosol and imported into the mitochondrion. Most of the lipid is imported. Synthesis of mitochondrial proteins

Mitochondria are largely maternally inherited in higher animals and plants In mammals, most of the mitochondrial DNA (mtDNA) is inherited from the mother. This is because the sperm carries most of its mitochondria its tail and has only about 100 mitochondria compared to 100,000 in the oocyte. Although sperm mitochondria penetrate the egg, most are degraded after a few hours. As the cells develop, more and more of the mtDNA from males is diluted out. Hence less than one part in 10 4 or 0.01% of the mtDNA is paternal.

Mitochondria are largely maternally inherited in higher animals and plants This means that mutations of mtDNA are passed from mother to child. It also has implications for the cloning of mammals with the use of somatic cells. The nuclear DNA would be from the donor cell, but the mtDNA would be from the host cell. This is how Dolly the sheep was cloned. In plants, the cytoplasm, including the mitochondria and the plastids, are contributed only by the female gamete and not by the pollen - again, mutations in organelle DNA are inherited maternally.

Mitochondria divide by fission and are not made de novo Human Evolution and mtDNA they are inherited mainly from the mother: >99% of our mitochondria are derived from those (1000 or so) present in our mother’s ovum

D-loop: origin of mtDNA replication Human evolution can be traced by analysis of the base sequence in a small part of the mitochondrial genome which does not encode a gene and which is quite variable. - the so-called D-loop. Human Evolution and mtDNA

The D-Loop of the mtDNA is the start of replication/transcription site and contains bp Unlike the rest of mtDNA in humans, which is highly conserved, this region is very variable between people It also has a very high frequency of change during evolution (about 2% per million years) Human Evolution and mtDNA

By comparing different groups, we can get a glimpse of human evolutionary lines. Eg, African individuals have more variability between each other than do Asians, indicating that the former have had more time to accumulate changes - ie, the Africans are a more ancient group. Human Evolution and mtDNA

This makes the D-loop a very powerful tool for the study of evolutionary relationships between organisms and for DNA typing of individuals. In addition, because of the large number of mitos in a cell, extracting mtDNA is easier from small amounts of tissue - and it can be readily separated form other DNA by centrifugation on CsCl gradients. Human Evolution and mtDNA

Extrapolating this in evolutionary terms, this means that all mitochondria came from a “single” ancestral female - the so-called “Mitochondrial Eve”. References: Proceedings of National Academy Sci (USA) 91:8739 (1994) Science 279: 28 (1998) However, this is based on the assumption that mitochondrial inheritance is strictly clonal. Recent evidence shows that mitos from sperm do enter the egg and last for several hours. If recombination occurs between mitos, then the Eve hypothesis may be incorrect - or at least the timing would be incorrect. Proc. R. Soc. Lond. B (1999) 266,

But we have to be careful: the rate of change in mtDNA may not be constant and heteroplasmy (due to recombination of mtDNA) may cause complications. Also, mtDNA represents a single lineage and other genetic changes need to be traced also. Human Evolution and mtDNA However, when this was done with polymorphisms in the Y chromosome, ‘Adam’ was also traced back to Africa, at about the same period.

Assuming that the rate of change in the D-loop is constant and due only to mutation, the number of difference s between Africans can be use to calculate when their common ancestor lived. This works out to be about 200,000 years ago. Human Evolution and mtDNA This suggests that modern Homo sapiens came out of Africa at about that time and migrated through Europe and Asia, replacing other early humans