Control of Gene Expression

Cloning S0matic cell nuclear transfer

 Researchers clone animals by nuclear transplantation  SOMATIC CELL NUCLEAR TRANSFER: A nucleus of an egg cell is replaced with the nucleus of a somatic cell from an adult  Thus far, attempts at human cloning have not succeeded in producing an embryo of more than six cells  Embryonic development depends on the control of gene expression (aka protein synthesis) Somatic Cell Nuclear Transfer

 In reproductive cloning, the embryo is implanted in a surrogate mother. The goal is to produce a cloned offspring.  In therapeutic cloning, the idea is to produce a source of embryonic stem cells.  Stem cells can help patients with damaged tissues.  Stem cells are NOT specialized in structure and function, therefore they can take on the role of damaged cells in damaged tissues. Reproductive vs. Therapeutic Cloning

Remove nucleus from egg cell Donor cell Add somatic cell from adult donor Grow in culture to produce an early embryo (blastocyst) Nucleus from donor cell Implant blastocyst in surrogate mother Remove embryonic stem cells from blastocyst and grow in culture Clone of donor is born (REPRODUCTIVE cloning) Induce stem cells to form specialized cells for THERAPEUTIC use Dolly the Sheep Reproductive vs. Therapeutic Cloning

Genetic Regulation in Prokaryotes

 Proteins interacting with DNA turn prokaryotic genes on or off in response to environmental changes.  The process by which genetic information flows from genes to proteins is called gene expression.  Our earliest understanding of gene control came from the bacterium E. coli (Reminder: Bacteria are prokaryotes.) Proteins turn genes on or off

Cellular Differentiation & the Cloning of Eukaryotes

 Differentiation yields a variety of cell types, each expressing a different combination of genes  In multicellular eukaryotes, cells become specialized as a zygote develops into a mature organism  Different types of cells make different kinds of proteins.  Different combinations of genes are active in each type. Cellular Differentiation

 Differentiated cells may retain all of their genetic potential. (Even if turned off, the gene is still present.)  Most differentiated cells retain a complete set of genes  In general, all somatic cells of a multicellular organism have the same genes.  So a carrot plant can be grown from a single carrot cell. Retain Genetic Potential

 The cloning of tadpoles showed that the nuclei of differentiated animal cells retain their full genetic potential Early Nuclear Transfer Tadpole (frog larva) Intestinal cell Frog egg cell Nucleus UV Nucleus destroyed Transplantation of nucleus Eight-cell embryo Tadpole

 The first mammalian clone, a sheep named Dolly, was produced in 1997  Dolly provided further evidence for the developmental potential of cell nuclei. Dolly again

 Connection: Reproductive cloning of nonhuman mammals has applications in basic research, agriculture, and medicine.  Scientists clone farm animals with specific sets of desirable traits.  Piglet clones might someday provide a source of organs for human transplant. Applications of Nonhuman Mammalian Cloning

 Connection: Because stem cells can both perpetuate themselves and give rise to differentiated cells, they have great therapeutic potential  Adult stem cells can also perpetuate themselves in culture and give rise to differentiated cells  But they are harder to culture than embryonic stem cells.  They generally give rise to only a limited range of cell types, in contrast with embryonic stem cells. Therapeutic Cloning

 Differentiation of embryonic stem cells in culture Cultured embryonic stem cells Different culture conditions Different types of differentiated cells Heart muscle cells Nerve cells Liver cells

 DNA packing in eukaryotic chromosomes helps regulate gene expression.  A chromosome contains a DNA double helix wound around clusters of histone proteins.  DNA packing tends to block gene expression. Gene Regulation in Eukaryotes

DNA double helix (2-nm diameter) Metaphase chromosome 700 nm Tight helical fiber (30-nm diameter) Nucleosome (10-nm diameter) Histones “Beads on a string” Supercoil (200-nm diameter)

 In female mammals, one X chromosome is inactive in each cell.  An extreme example of DNA packing in interphase cells is X chromosome inactivation Female Mammals EARLY EMBRYO Cell division and X chromosome inactivation X chromosomes Allele for orange fur Allele for black fur TWO CELL POPULATIONS IN ADULT Active X Inactive X Orange fur Inactive X Active X Black fur

Genetic Biotechnology

 Since ancient times, humans have bred animals and plants to increase the likelihood of certain desirable traits.  Example: Hunting dogs or larger fruits  Two methods are used:  Hybridization: crossing different parent organisms with different forms of a trait to produce an offspring with a specific trait  Example: hybrid rice that produces greater yield and another hybrid rice that contains greater nutritional properties  Disadvantage: Time-consuming and expensive  Inbreeding: crossing closely related parent organisms who have been bred to have desired traits to ensure that the traits are passed on  Example: German shepherd dogs  Disadvantage: Undesirable recessive traits are often passed down with the desirable traits. Selective Breeding

 A process by which an organism’s DNA is manipulated in order to insert the DNA of another organism (creates recombinant DNA)  Purpose: Incorporate the desirable traits of one organism into another organism  Example: Bioluminescent trait – A type of jellyfish contains a protein (GFP: green fluorescent protein) that causes it to glow. Scientists insert the DNA that codes for GFP into the DNA of mosquito larvae so that they will glow. Mosquito populations can be controlled as the larvae are more easily located.  Produces “genetically modified organisms” Genetic Engineering

 Selective breeding and genetic engineering require scientists to use special tools or processes to manipulate DNA.  Restriction Enzymes: cut DNA into smaller fragments with “sticky ends” that allow it to connect to other fragments of DNA  Gel Electrophoresis: electrical currents separate DNA fragments based on size allowing fragments to be sorted and studied DNA Tools

Useful in genetic engineering where DNA of one organism is inserted into the DNA of another organism (recombinant DNA) Example: EcoRI restriction enzyme cuts a GAATTC sequence Restriction Enzymes Restriction enzymes are found naturally in bacteria cells. The bacteria developed the enzymes to fight against viruses. They chop up the viral DNA that gets inserted into their cells.

Electrical currents run through the DNA samples that have been cut into fragments. Smaller fragments travel more quickly from the – electrode to the + electrode. Gel Electrophoresis Separated fragments can be studied or combined with other fragments to create recombinant DNA.

 When a particular new DNA sequence has been developed in recombinant DNA, bacteria cells are used to make multiple copies.  The bacteria cells are heated which causes pores to open in their cell walls.  The recombinant DNA moves through the pores into the bacteria cell.  As the bacteria cell replicates, the recombinant DNA is replicated, too.  Purpose: to create many copies of the desirable sequence that can be used in genetically-modified organisms Gene Cloning

 Learning the sequence of DNA fragments can allow scientists to understand the function of certain sequences of bases.  Restriction enzymes are used to cut large DNA strands into shorter fragments.  Dyes are used to color known bases, and the known colored known bases bind to the unknown sequence. The unknown sequence can be read by following complementary base- pairing rules. DNA Sequencing

 Purpose: create copies of selected DNA segments when the sample size of DNA is too small to run all of the needed tests  Uses a thermal cycler to separate the DNA double helix and DNA polymerase enzymes to create copies of the selected segments.  Extremely useful in forensic science and medicine Polymerase Chain Reaction

 Purpose: Insert beneficial genes into a needy organism  How?  A mutated gene is located on a chromosome.  A “normal” gene is inserted into a chromosome to replace the dysfunctional one using a “viral vector.”  The virus infects a cell and injects its genetic material including the “normal” gene. The cell begins replicated the “normal” gene, and the mutation is corrected. Gene Therapy