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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 171-172, 304-312 Gene structure pp 405 -408 Molecular Genetic Techniques and Cloning Chapter 9 Journal of Experimental Biology Volume 201, pp 1177-1195 (1998) Kwast et al. Regulation of Gene expression in Eukaryotes Gene regulation in prokaryotes and/or single cellular organisms is different in that gene regulation is primarily concerned with responding to external stimuli. These include nutrients, temperature. Although multicellular organsims also respond to such stimuli, eg plants, another level of gene regulation is involved. For multicellular organisms the right gene must be activated at the right time in the right cell. Examples of gene regulation at all levels have been documented, transcriptional Eukaryotic messages have 3’ untranslated region can also vary in size from tens to hundreds of bases Also have a poly A tail A series of A residues that are added in a non template dependant manner after transcription The amount of information in Eukaryotic DNA is 1 X 109 bp Estimated to be 35, 000 genes The function of non-coding DNA is not known, obviously some of the non-coding DNA acts as “promoter” regions etc. However still lots of DNA that appears to have no apparent function. Gene - Is a piece of DNA that encodes a functional unit - remember have ribosomal and tRNA genes so genes not restricted to encoding proteins Central Dogma of molecular biology is that DNA is the genetic material that is replicated and passed onto succeeding generations. It is transcribed into RNA, which is subsequently translated into protein. RNA - Three types Ribosomal RNA (rRNA) - as name suggests found in ribosomes which function to synthesise proteins Messenger RNA (mRNA) - This type of RNA specifies the sequence of amino acids in a protein by triplet codon bases. The mRNA sequence is translated into a protein sequence. Transfer RNA (tRNA) - This RNA acts as an intermediate between the mRNA and protein. Through complementary base pairing to the mRNA it delivers the amino acid coded in the mRNA to the ribosome. Proteins - all proteins are encoded for by mRNA and synthesised on ribosomes. These have various functions including structural (collagen, cell membrane proteins), enzymatic (digestive, intracellular metabolism), signals (insulin) and defence (antibodies). Obviously get exception to this flow of information 1) RNA viruses 2) RNA editing 3) Ribozymes 4) Prions If RNA is reversed transcribed to produce DNA, the DNA is called cDNA, as it is complementary to the RNA copy. No process of reverse translation known to date Relationship between the structure of protein, mRNA and Genes This is not the structural relationship between Protein, mRNA and genomic DNA in Eukaryotic cells The relationship is far more complicated and it is important to get the terminology correct as you will come across it in texts etc. Eukaryotic messages have a 5’ Cap Also have a 5 ‘ untranslated region can Vary from tens to hundreds of bases Again this is not the relationship between mRNA and genomic DNA Genomic genes in Eukaryotes contain intervening sequences known as Introns. Therefore the initial RNA transcript contains both exons and introns, the introns are removed from the primary RNA transcript in a process call splicing How do we know where translation starts in the mRNA First in frame AUG Not necessarily the first AUG but the first in frame AUG Find this from: I) Protein sequence - care as lots of eukaryotic proteins are processed ii) Sequence analysis on a computer How do we know where transcriptional begins Transcriptional start site Need to know before we can start about promoter elements etc Two methods 1) S1 nuclease mapping 2) Primer extension 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 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 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. cerevisiae C. elegans # Cells Size Chromosomes Predicted ORFs %Coding A. thaliana H. sapiens ~1000 >1x106 >1x106 12Mbp 97Mbp 125Mbp 3.2 Gbp 16 6 5 23 ~6,000 ~19,000 ~28,000 ~35,000 72% 27% 50% 1.5% 1 yeast carbon metabolism 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) to… Oxidative Metabolism (Ethanol TCA cycle) Mitochondrial structure • 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 Overview of aerobic respiration Four large, multi-subunit protein complexes - complex I is a NADHubiquinone 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 Outline of Tricarboxylic Acid Cycle 3-Carbons One pyruvate molecule is completely oxidised to CO2 CO2 4-Carbons 6-Carbons NADH NADH + CO2 FADH NADH + CO2 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 Organisation of the mitochondrial chromosome 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. Yeast mitochondrial chromosome yeast mtDNA human mtDNA maize mitochondrial genome Synthesis of mitochondrial proteins 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. 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 104 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. Human Evolution and mtDNA • Mitochondria divide by fission and are not made de novo • they are inherited mainly from the mother: >99% of our mitochondria are derived from those (1000 or so) present in our mother’s ovum Human Evolution and mtDNA 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 400-800 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. 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, 477-483 Human Evolution and mtDNA 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. However, when this was done with polymorphisms in the Y chromosome, ‘Adam’ was also traced back to Africa, at about the same period. Human Evolution and mtDNA 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. This suggests that modern Homo sapiens came out of Africa at about that time and migrated through Europe and Asia, replacing other early humans