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Ch. 11: Gene regulations How is cloning possible? Every cell has the same chromosomes Then….. Why does a heart muscle cell look different from a skin cell? Organisms respond to their environment by altering gene expression Central question: what regulates gene expression? Differentiation Differentiation is controlled by turning specific sets of genes on or off DNA Packing eukaryotic chromosomes condense during prophase of Mitosis helps regulate gene expression by preventing transcription – Nucleosomes – Tight helical fiber = – Supercoil = coiling of the tight helical fiber Metaphase chromosome Tight helical fiber (30-nm diameter) DNA double helix (2-nm diameter) Linker “Beads on a string” Nucleosome (10-nm diameter) Histones Supercoil (300-nm diameter) Animation: DNA Packing 700 nm X-chromosome inactivation – female mammals – one of the two X chromosomes is highly compacted and transcriptionally inactive (Barr body) – Occurs early in embryonic development, thus all cellular descendants have the same inactivated chromosome – Tortoiseshell fur coloration is due to inactivation of X chromosomes in heterozygous female cats Early embryo Two cell populations in adult Cell division and random X chromosome inactivation X chromosomes Allele for orange fur Allele for black fur Active X Inactive X Orange fur Inactive X Active X Black fur Eukaryotic gene expression – Each gene has its own promoter and terminator – Are controlled by interactions between numerous regulatory proteins and control sequences Regulatory proteins • Transcription factors - help RNA polymerase bind to the promoter • Activators – • Silencers Control sequences – Promoter – Enhancer – Related genes located on different chromosomes can be controlled by similar enhancer sequences Animation: Initiation of Transcription Enhancers Promoter Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Bending of DNA Transcription Alternative RNA splicing – Can involve removal of an exon with the introns on either side Animation: RNA Processing Exons 1 DNA RNA transcript 1 1 2 3 5 4 3 2 RNA splicing mRNA 4 3 2 5 or 5 1 2 4 5 Small RNAs control gene expression RNA interference (RNAi) – small, complementary RNAs bind to mRNA transcripts, blocking translation MicroRNA (miRNA) – MicroRNA + protein complex binds to complementary mRNA transcripts, blocking translation Animation: Blocking Translation Animation: mRNA Degradation Protein miRNA 1 miRNAprotein complex 2 Target mRNA 3 mRNA degraded 4 OR Translation blocked Control of gene expression also occurs with – Breakdown of mRNA – Initiation of translation – Protein activation – Protein breakdown Ex. Insulin formation Folding of polypeptide and formation of S—S linkages Initial polypeptide (inactive) Cleavage Folded polypeptide (inactive) Active form of insulin Epigenetic Inheritance This can be accomplished by acetylation or methylation of histones Regulation of Chromatin Structure Chemical modification of histone tails can affect the configuration of chromatin and thus gene expression Histone tails DNA double helix (a) Histone tails protrude outward from a nucleosome Addition of methyl groups to certain bases in DNA is associated with reduced transcription in some species Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription NUCLEUS Chromosome DNA unpacking Other changes to DNA Gene Gene Transcription Exon RNA transcript Intron Addition of cap and tail Splicing Tail mRNA in nucleus Cap Flow through nuclear envelope mRNA in cytoplasm CYTOPLASM Breakdown of mRNA Translation Brokendown mRNA Polypeptide Cleavage / modification / activation Active protein Breakdown of protein Brokendown protein Why so much control over gene expression? It allows cells to respond appropriately to their environment Signal transduction pathways convert messages received at the cell surface to responses within the cell via gene expression Three steps: 1. Reception – 2. Amplification/transduction – 3. Response - transcription factor is activated, enters nucleus, transcribes specific genes Signaling cell Signaling molecule Plasma Receptor membrane protein 1 2 3 Target cell Relay proteins Transcription factor (activated) 4 Nucleus DNA 5 mRNA Transcription New protein 6 Translation Cloning: How? Nuclear transplantation – Replacing the nucleus of an egg cell with a nucleus from an adult somatic cell. Allow embryo to form. Embryo can be used in: – Reproductive cloning – Therapeutic cloning – Grow embryonic stem cells in culture – Induce stem cells to differentiate and grow into organs, tissues, etc. Donor cell Nucleus from donor cell Reproductive cloning Implant blastocyst in surrogate mother Remove nucleus from egg cell Add somatic cell from adult donor Grow in culture to produce an Therapeutic early embryo cloning (blastocyst) Remove embryonic stem cells from blastocyst and grow in culture Clone of donor is born Induce stem cells to form specialized cells To clone or not to clone…. Benefits of reproductive cloning? Disadvantages of cloning? Human stem cell research Ethical concerns with reproductive cloning Ethical concerns with therapeutic cloning? Benefits: Human embryos – have the greatest potential to give rise to all cell types – Adult stem cells (bone marrow) or cord blood cells – can give rise to many but not all types of cells Ch 12: DNA Technology 1. DNA profiling 2. Genetically modified organisms/recombinant DNA technology 3. Gene therapy 4. Genomics 1. DNA profiling = analysis of DNA fragments to determine whether they come from a particular individual 3 steps: 1.. 2.Amplify (copy) markers for analysis – 3.Compare sizes of amplified fragments by gel electrophoresis 1. Select genetic marker to analyze Short tandem repeats (STRs) are genetic markers used in DNA profiling – STRs = – STR analysis compares the lengths of STR sequences at specific regions of the genome – Current standard for DNA profiling is to analyze 13 different STR sites STR site 1 STR site 2 Crime scene DNA Number of short tandem Number of short tandem repeats match repeats do not match Suspect’s DNA 2. Amplify the DNA sample Polymerase chain reaction (PCR) = method of amplifying a specific segment of a DNA molecule Relies upon a pair of primers = Repeated cycle of steps for PCR: 1. Sample is heated to separate DNA strands 2. Sample is cooled and primer binds to specific target sequence 3. Target sequence is copied with DNA polymerase Cycle 1 yields 2 molecules Genomic DNA 3 1 3 5 3 Target sequence 5 5 5 3 Cycle 2 yields 4 molecules 5 5 2 Cool to allow 3 Heat to primers to form separate DNA strands hydrogen bonds with ends of target sequences 5 3 5 3 Primer 3 5 DNA polymerase adds nucleotides to the 3 end of each primer 5 3 New DNA Cycle 3 yields 8 molecules 3. Gel electrophoresis separates DNA molecules based on size – DNA samples placed at one end of a porous gel – Current is applied and DNA molecules move from the negative electrode toward the positive electrode – DNA fragments appear as bands, visualized through staining or radioactivity or fluorescence Video: Biotechnology Lab Mixture of DNA fragments of different sizes Power source Longer (slower) molecules Gel Shorter (faster) molecules Completed gel Crime scene 1 DNA isolated 2 DNA of selected markers amplified 3 Amplified DNA compared Suspect 1 Suspect 2 Mixture of DNA fragments A “band” is a collection of DNA fragments of one particular length Longer fragments move slower Shorter fragments move faster DNA attracted to + pole due to PO4– groups Power source Applications of DNA profiling Forensics - to show guilt or innocence Establishing paternity Identification of human remains Species identification – Evidence for sale of products from endangered species 2. Recombinant DNA technology/ Genetically Modified organisms – Recombinant DNA is formed by joining DNA sequences from two different sources: 1. . 2. . – Bacterial Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors Recombinant cells and organisms can mass-produce gene products – Common prokaryotic host: E. coli bacterium – Has many advantages in gene transfer, cell growth, and quantity of protein production – Common eukaryotic hosts: – Yeast: S. cerevisiae – “Pharm” animals – Will secrete gene product in milk Advantages of recombinant DNA products Genetically modified (GM) Transgenic organisms contain at least one gene from another species Agrobacterium tumefaciens Plant cell DNA containing gene for desired trait 1 Ti plasmid Insertion of gene into plasmid 3 2 Recombinant Ti plasmid Introduction into plant cells Regeneration of plant DNA carrying new gene Restriction site Plant with new trait Pros? GM plants GM animals Cons? 3. Gene therapy One possible procedure: – insert functional gene into a virus – virus delivers the gene to an affected cell – Viral DNA & gene insert into the patient’s chromosome – Return the cells to the patient for growth and division Cloned gene (normal allele) 1 Insert normal gene into virus Viral nucleic acid Retrovirus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow 4 Inject cells into patient 4. Genomics Genomics = Applications: – Evolutionary relationships: Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans – Medical advancement: Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast Human Genome Project Goals: – To determine the nucleotide sequence all DNA in the human genome – To identify the location and sequence of every human gene Results of the Human Genome Project – 21,000 genes in 3.2 billion nucleotide pairs – Only 1.5% of the DNA codes for proteins – The remaining 88.5% of the DNA contains – Control regions (promoters, enhancers) – Unique noncoding DNA – Repetitive DNA Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (15%)