Gene Expression and DNA Technology Set 3b Test on 7/28

Gene Expression What causes DNA (genes) to be transcribed (mRNA) and then translated (amino acids/ polypeptides/proteins)? What makes one gene expressed, and another not? Would really want to make ALL your proteins?

Operons Operons turn genes on and off in prokaryotes. A simple switch, based on the cell environment. Ex.) lac operon turns on the lactase, which breaks down lactose when lactose is present.

Parts of an Operon Regulatory gene (DNA) – where repressor protein is coded for Not part of the operon, technically. Promoter (DNA) – where RNA polymerase binds Operator (DNA) – where the repressor protein binds Structural gene/loci – the functional gene that is controlled.

Repressors Repressors stop RNA polymerase from coding the operon. Can be on “active” in the presence of a chemical, or off in it’s presence. Ex) lac repressor is active when there is no lactose. Inactive when there is lactose. Ex) trp repressor is active when there is tryptophan. Inactive when there is no tryptophan.

Differentiation Different cells become different because they make different proteins. Different types of cells make different proteins because different combinations of genes are active in each type Muscle cell Pancreas cells Blood cells Fiure 11.2

Cell division in culture Differentiated cells can retain all of their genetic potential Especially in plants. Most differentiated cells retain a complete set of genes Your brain cells have all the DNA for your white blood cells, and visa versa Root of carrot plant Root cells cultured in nutrient medium Cell division in culture Plantlet Adult Plant Single cell Figure 11.3

DNA packing in eukaryotic chromosomes helps regulate gene expression A chromosome contains DNA wound around clusters of histone proteins This beaded fiber is further wound and folded DNA packing blocks gene expression by preventing access of transcription proteins to the DNA Presumably… DNA double helix (2-nm diameter) Histones Linker “Beads on a string” Nucleosome (10-nm diameter) Tight helical fiber (30-nm diameter) Supercoil (300-nm diameter) Metaphase chromosome 700 nm TEM

Epigenetic Inheritance Inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence A new field of genetics… Allows traits to be passed differently than just in the DNA. This can be accomplished by acetylation or methylation of histones Basically, changing the proteins that the DNA coils around, which can be passed in the gametes on to the later generation.

Histone acetylation seems to loosen chromatin structure and thereby enhance transcription Addition of methyl groups to certain bases in DNA is associated with reduced transcription in some species Epigenetics NOVA movie Figure 19.4 b (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones

Two cell populations in adult In female mammals, one X chromosome is inactive in each somatic (body) cell Called a “Barr Body” Early embryo X chromosomes Allele for orange fur Allele for black fur Cell division and random X chromosome inactivation Two cell populations in adult Active X Inactive X Orange fur Black fur Figure 11.5

Complex assemblies of proteins control eukaryotic transcription A variety of regulatory proteins interact with DNA and with each other to turn the transcription of genes on or off Enhancers Promoter Gene DNA Activator proteins Other proteins Transcription factors RNA polymerase Bending of DNA Transcription Figure 11.6 More than just a single repressor protein. Multiple activators, many proteins involved. Changes the shape of DNA, often from “far” away.

Eukaryotic RNA may be spliced in more than one way After transcription, alternative splicing may generate two or more types of mRNA from the same transcript DNA RNA transcript mRNA Exons or RNA splicing Figure 11.7

Regulation at the translation stage and later Breakdown of mRNA The lifetime of an mRNA molecule helps determine how much protein is made Initiation of Translation Among the many molecules involved in translation are a great many proteins that control the start of polypeptide synthesis Protein Activation After translation is complete polypeptides may require alteration to become functional Protein Breakdown Some of the proteins that trigger metabolic changes in cells are broken down within a few minutes or hours

Blockage of translation RNA interference by single-stranded RNAi can lead to degradation of an mRNA or block its translation RNAi NOVA movie Recent data shows that RNAi can also work as a decoy for promoters, binding transcription factors so that they don’t initiate transcription. The micro- RNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double- stranded RNA, cutting it into shorter segments. One strand of each short double- stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 4 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation. 5 5 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Degradation of mRNA OR Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond Figure 19.9

Page 217, right column Very important image. Figure 11.9 NUCLEUS Chromosome Gene RNA transcript mRNA in nucleus mRNA in cytoplasm Polypeptide Active protein Breakdown of protein Cleavage / modification / activation Translation Breakdown of mRNA CYTOPLASM Flow through nuclear envelope Splicing Addition of cap and tail Transcription DNA unpacking Other changes to DNA Exon Intron Cap Tail Broken- down mRNA Broken- down protein Figure 11.9 Page 217, right column Very important image.

Viruses Viruses inject their DNA code into the cells of the infected cells. “DNA viruses” use DNA to put into the cells Retroviruses use RNA, which is converted to DNA, which is then put into the cells Then the virus uses the cell’s processes to make new virus proteins and DNA/RNA to create new virus particles

Cloning and DNA technology

To Clone or Not to Clone? A clone is an individual created by asexual reproduction and thus is genetically identical to a single parent

ANIMAL CLONING Nuclear transplantation can be used to clone animals Nuclear transplantation can be used to clone animals Remove nucleus from egg cell Add somatic cell from adult donor Grow in culture to produce an early embryo (blastocyst) Implant blastocyst in surrogate mother Remove embryonic stem cells from blastocyst and grow in culture Induce stem cells to form specialized cells (therapeutic cloning) Clone of donor is born (reproductive cloning) Donor cell Nucleus from donor cell Figure 11.10

Therapeutic cloning can produce stem cells with great medical potential Like embryonic stem cells, adult stem cells can perpetuate themselves in culture and give rise to differentiated cells Adult stem cells in bone marrow Cultured embryonic stem cells Different culture conditions Heart muscle cells Different types of differentiated cells Nerve cells Blood cells Figure 11.12

Unlike embryonic stem cells adult stem cells normally give rise to only a very limited range of cell types

Stem Cells Initial cells that can become many different types of cells Important for development, because all cells start as one There are genes, such as the Hox gene which control segmentation Which then controls development

Signal transduction pathways convert molecular messages to cell responses This is an important part of development. How it gets “going”, tells which cell to do what… Signaling cell Signal molecule Receptor protein Plasma membrane Target cell Relay proteins Transcription factor (activated) Transcription Nucleus DNA mRNA New protein Translation 1 2 3 4 5 6 Figure 11.14

THE GENETIC BASIS OF CANCER Cancer cells, which divide uncontrollably result from mutations in genes whose protein products affect the cell cycle

Proto-Oncogenes A mutation can change a proto-oncogene (a normal gene that promotes cell division) into an oncogene, which causes cells to divide excessively Proto-oncogene DNA Mutation within the gene Multiple copies of the gene Gene moved to new DNA locus, under new controls Oncogene New promoter Hyperactive growth- stimulating protein in normal amount Normal growth- stimulating protein in excess Figure 11.16A

Tumor-Suppressor Genes Mutations that inactivate tumor suppressor genes have similar effects as oncogenes Tumor-suppressor gene Mutated tumor-suppressor gene Normal growth- inhibiting protein Cell division under control Defective, nonfunctioning protein Cell division not under control Figure 11.16B

Oncogene proteins and faulty tumor-suppressor proteins can interfere with normal signal transduction pathways Growth-inhibiting factor Receptor Nonfunctional transcription factor (product of faulty p53 tumor-suppressor gene) Relay proteins Transcription factor (activated) Transcription Translation Protein that inhibits cell division cannot trigger transcription Protein absent (cell division not inhibited) Normal product of p53 gene Figure 11.17B Growth factor Target cell Receptor Hyperactive relay protein (product of ras oncogene) issues signals on its own Normal product of ras gene Relay proteins Transcription factor (activated) DNA Nucleus Transcription Translation Protein that stimulates cell division Figure 11.17A

Cancers result from a series of genetic changes in a cell lineage Colon cancer develops in a stepwise fashion Colon wall 1 Increased cell division Oncogene activated 2 Growth of polyp Tumor-suppressor gene inactivated 3 Growth of malignant tumor (carcinoma) Second tumor- suppressor gene inactivated Cellular changes: DNA changes: Figure 11.18A

Accumulation of mutations can also lead to cancer UV radiaton Smoking/Tobacco use Chromosomes mutation 1 2 3 4 mutations Normal cell Malignant cell Figure 11.18B

Researchers can insert desired genes into plasmids, creating recombinant DNA and insert those plasmids into bacteria Bacterium Bacterial chromosome Plasmid 1 isolated 3 Gene inserted into plasmid 2 DNA Cell containing gene of interest Gene of interest Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Figure 12.1

Restriction enzymes cut DNA. DNA ligase puts it back together. Creating recombinant DNA using restriction enzymes and DNA ligase Restriction enzyme recognition sequence G A A T T C C T T A A G DNA 1 2 3 4 C T T A A A AT TC G Addition of a DNA fragment from another source Two (or more) fragments stick together by base-pairing G A AT T C C T TA A G 5 DNA ligase pastes the strand Restriction enzyme cuts the DNA into fragments Recombinant DNA molecule Sticky end Restriction enzymes cut DNA. DNA ligase puts it back together. Figure 12.2

Cloning a gene in a bacterial plasmid E.coli 1 Isolate DNA from two sources Human cell 2 Cut both DNAs with the same restriction enzyme Plasmid DNA Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V 5 Put plasmid into bacterium by transformation Recombinant bacterium 6 Clone the bacterium Figure 12.3 Bacterial clone carrying many copies of the human gene

Recombinant cells and organisms can mass-produce gene products Table 12.6

Therapeutic hormones In 1982, humulin, human insulin produced by bacteria Became the first recombinant drug approved by the Food and Drug Administration Figure 12.7A

Gene therapy (or the alteration of an afflicted individual’s genes) may someday help treat a variety of diseases Cloned gene (normal allele) 1 Insert normal gene into virus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome 4 Inject cells into patient Bone marrow Bone marrow cell from patient Viral nucleic acid Retrovirus Figure 12.13

Gel electrophoresis sorts DNA molecules by size + – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter Completed gel Figure 12.10

How Restriction Fragments Reflect DNA Sequence Restriction fragment length polymorphisms (RFLPs) reflect differences in the sequences of DNA samples Crime scene Suspect w x y z Cut DNA from chromosomes C G A T Figure 12.11A

After digestion by restriction enzymes the fragments are run through a gel – + Longer fragments Shorter x w y z 1 2 Figure 12.11B

Detecting a harmful allele using restriction fragment analysis 1 2 3 4 5 Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe Detection of radioactivity (autoradiography) I II III Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe) Film Figure 12.11C

DNA profiling can help solve crimes Figure 12.12B Defendant’s blood Blood from defendant’s clothes Victim’s Figure 12.12A

The polymerase chain reaction (PCR) method is used to amplify DNA sequences. It can be used to clone a small sample of DNA quickly, producing enough copies for analysis 1 2 4 8 Initial DNA segment Number of DNA molecules Figure 12.14

gene within plant chromosome Recombinant DNA technology can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens DNA containing gene for desired trait Plant cell Ti plasmid 1 Insertion of gene into plasmid using restriction enzyme and DNA ligase 2 Introduction into plant cells in culture 3 Regeneration of plant Recombinant Ti plasmid T DNA carrying new gene within plant chromosome T DNA Plant with new trait Restriction site Figure 12.18A

Sowing A Gene Revolution, Scientific American, (September 2007), 297, 104-111

Sowing A Gene Revolution, Scientific American, (September 2007), 297, 104-111

Transgenic organisms are those that have had genes from other organisms inserted into their genomes Chimera: Organism that has cells or genes from another individual of the same or a different species. Figure 12.18B

Examples of Transgenics Cows, sheep or goats that produce proteins in their milk e.g. tPA (tissue plasminogen activator) Mice to study what a gene does or as a disease model. Cows that produce milk with less cholesterol or lactose Influenza resistant pigs