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9.0 Regulation and Recombinant DNA Related Sadava’s chapters: 1) Regulation of Gene expression 2) Genomes 3) Recombinant DNA Additional source: “A genetic switch” Mark Ptashne 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression The rate of a metabolic pathway can be regulated in two ways: • Allosteric regulation of enzyme-catalyzed reactions allows rapid fine-tuning. • Regulation of protein synthesis (regulation of the concentration of enzymes) is slower but conserves resources. 9.1 Regulation of Gene expression In case of lack of precursor whose metabolism involves a particular protein, prokaryotes can: • Inhibit the protein’s function • Hydrolyze the protein after it is made • Prevent mRNA translation at the ribosome • Hydrolyze mRNA, preventing translation • Down-regulate mRNA transcription Prokaryotes generally use the most efficient way—down regulating mRNA transcription. Less energy is wasted as it is early in protein synthesis. 9.1 Regulation of Gene expression E. coli must adapt quickly to food supply changes and can use glucose or lactose as a sole source of energy. Uptake and metabolism of lactose involve three proteins: • β-galactoside permease—a carrier protein that moves sugar into the cell • β-galactosidase—an enzyme that hydrolyses lactose • β-galactoside transacetylase—transfers acetyl groups to certain β-galactosides If E.coli is grown with glucose but no lactose present, no enzymes for lactose conversion are produced. 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression Besides the promoter, other sequences bind regulatory proteins that interact with RNA polymerase and regulate rate of transcription. Some bind positive regulators— enhancers; others repressors— silencers. The combination of factors present determines the rate of transcription. 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression Three criteria for DNA recognition by a protein motif: • Fits into major or minor groove • Has amino acids that can project into interior of double helix • Has amino acids that can bond with interior bases 9.1 Regulation of Gene expression • Genes to be regulated simultaneously may be far apart or on different chromosomes. • Gene expression is coordinated if they have the same regulatory sequences that bind same transcription factors. • Example: A regulatory sequence in plant genes called stress response element (SRE)—encodes for proteins needed to cope with drought. 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression • Epigenetics refers to changes in expression in a gene or set of genes, without a change in the DNA sequence. • Changes are sometimes heritable and stable, but are reversible. • Includes two processes: DNA methylation and chromosomal protein alterations. 9.1 Regulation of Gene expression • Some cytosine residues in DNA are modified by adding a methyl group covalently to the 5′ carbon—forms 5′ methylcytosine. • DNA methyltransferase catalyzes the reaction—usually in adjacent C and G residues. • Regions are called CpG islands—often in promoters. 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression Effects of DNA methylation: • Methylated DNA attracts proteins that are involved in repression of transcription and can inactivate DNA • Important in development—early demethylation allows many genes to become active • In cancer, misregulation can occur in oncogenes and tumor suppressors 9.1 Regulation of Gene expression Chromatin remodeling is the alteration of chromatin structure. • Nucleosomes contain DNA and histones in a tight complex, inaccessible to RNA polymerase. • Each histone has a positively charged “tail” at its N terminus with amino acids. • Histone acetyltransferases change the tail’s charge by adding acetyl groups to the amino acids. • This opens up nucleosomes and activates transcription. 9.1 Regulation of Gene expression 9.1 Regulation of Gene expression The “histone code”—histone modifications affect gene activation and repression: • Histone acetylation activates transcription • Histone methylation affects gene expression depending on which amino acid is involved (several lysines in particular) 9.2 Genomes 9.2 Genomes Genome sequence information is used to identify: • Open reading frames, or coding regions • Amino acid sequences of proteins • Regulatory sequences • RNA genes • Other noncoding sequences In comparative genomics, newly sequenced genomes are compared with sequences from other organisms. This can give information about the functions of sequences, and is used to trace evolutionary relationships. 9.2 Genomes Functional genomics assigns functions to the products of genes. H. influenzae chromosome has 1,738 open reading frames. When it was first sequenced, only 58 percent coded for proteins with known functions. Genome sequencing provides insights into microorganisms that cause human diseases and reveal new methods to combat them. Sequencing also reveals relationships between pathogenic organisms, suggesting that genes may be transferred between different strains. 9.2 Genomes DNA can also be analyzed directly from environmental samples. Metagenomics—genetic diversity is explored without isolating intact organisms. Shotgun sequencing is used to detect presence of known microbes, as well as heretofore unidentified organisms. It is estimated that 90 percent of the microbial world has been invisible to biologists and is only now being revealed by metagenomics. 9.2 Genomes Mycoplasma genitalium can survive in the laboratory with only 382 functional genes. In 2010 a synthetic bacterial genome was synthesized and transplanted into a M. capricolum cell This could have many useful benefits, but also raises many ethical concerns. 9.2 Genomes 9.2 Genomes 9.2 Genomes There are major differences between eukaryotic and prokaryotic genomes: • Eukaryotic genomes are larger and have more protein-coding genes • Eukaryotic genomes have more regulatory sequences. Greater complexity requires more regulation • Much of eukaryotic DNA is noncoding, including introns, gene control sequences, and repeated sequences • Eukaryotes have multiple chromosomes; each must have an origin of replication (ori), a centromere, and a telomeric sequence at each end 9.2 Genomes 9.2 Genomes 9.2 Genomes Eukaryotes have closely related genes called gene families. These arose over evolutionary time when different copies of genes underwent separate mutations. For example: Genes encoding the globin proteins all arose from a single common ancestral gene. 9.2 Genomes 9.2 Genomes 9.2 Genomes • Moderately repetitive sequences are repeated 10–1,000 times • They include the genes for tRNAs and rRNAs • Single copies of the tRNA and rRNAgenes would be inadequate to supply the large amounts of these molecules needed by cells 9.2 Genomes Some interesting facts about the human genome: • Protein-coding regions make up less than 2%, about 24,000 genes • Each gene codes for several proteins due to posttranscriptional mechanisms (e.g., alternative splicing) • An average gene has 27,000 base pairs • Over 50 percent of the genome is transposons and other repetitive sequences • 97 percent of the genome is the same in all people • The chimpanzee shares 95% of the human genome and the rhesus macaque 91%. 9.2 Genomes 9.2 Genomes Genetic variation can affect an individual’s response to a particular drug. Pharmacogenomics makes it possible to predict whether a drug will be effective. The aim is to personalize drug treatments. 9.3 Recombinant DNA Restriction endonucleases are bacterial enzymes used to cut DNA. DNA ligase catalyzes the joining of DNA fragments. Restriction enzymes and DNA ligase are used to cut DNA into fragments and then splice them together in new combinations. 9.3 Recombinant DNA 9.3 Recombinant DNA 9.3 Recombinant DNA 9.3 Recombinant DNA 9.3 Recombinant DNA 9.3 Recombinant DNA Biotechnology is the use of living cells or organisms to produce materials useful to people. Examples: • Using yeasts to brew beer and wine • Using bacteria to make cheese, yogurt, etc. • Using microbes to produce antibiotics, alcohol, and other products 9.3 Recombinant DNA After wounds heal, blood clots are dissolved by plasmin. Plasmin is stored as an inactive form called plasminogen. Conversion of plasminogen is activated by TPA. TPA can be used to treat strokes and heart attacks, but large quantities are needed. Can be made using recombinant DNA technology. 9.3 Recombinant DNA Pharming: Production of pharmaceuticals in farm animals or plants. Example: Transgenes are inserted next to the promoter for lactoglobulin (a protein in milk). The transgenic animal then produces large quantities of the protein in its milk. Human growth hormone (for children suffering deficiencies) can now be produced by transgenic cows. Only 15 such cows are needed to supply all the children in the world suffering from this type of dwarfism. 9.3 Recombinant DNA Crops with improved nutritional characteristics: • Rice does not have β-carotene, but does have a precursor molecule • Genes for enzymes that synthesize β-carotene from the precursor are taken from daffodils and inserted into rice by the Ti plasmid 9.3 Recombinant DNA Recombinant DNA is also used to adapt a crop plant to an environment. Example: Plants that are salt-tolerant. Genes from a protein that moves sodium ions into the central vacuole were isolated from Arabidopsis thaliana and inserted into tomato plants. 9.3 Recombinant DNA Concerns over biotechnology: • Genetic manipulation is an unnatural interference in nature • Genetically altered foods are unsafe to eat • Genetically altered crop plants are dangerous to the environment • The complexity of the biological world makes it impossible to predict all potential environmental effects of transgenic organisms. • In fact, some spreading of transgenes has been detected.