Biotechnology
Biotechnology: What is it? Manipulation of organisms at the molecular level Recombinant DNA- DNA from two different sources (often different species) combined in vitro
Analyzing DNA Molecules Restriction Enzymes- used as defense in bacteria Cut DNA at specific restriction sites (4-6 bp long) though restriction digestion Hundreds of known restriction enzymes used as knives to chop up DNA fragments
Analyzing DNA: Gel Electrophoresis Uses a gel as a molecular sieve to separate nucleic acids or proteins by size A current is applied that causes charged molecules to move through the gel; larger molecules move more slowly Molecules are sorted into bands by their size
Analyzing DNA: Using RFLPs Restriction fragment analysis- DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis Short tandem repeats (STRs)- short repetitive sequences that are inherited between restriction sites Single nucleotide polymorphisms (SNPs)- single basepair sites that vary in a population When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphism (RFLP)
DNA Analysis: Southern Blotting Combines gel electrophoresis of DNA fragments with nucleic acid hybridization DNA probes hybridize with strands of DNA from the gel Used to find target sequences
Amplifying DNA: PCR Polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA Three-step cycle heating, cooling, and replication produces an exponentially growing population of identical DNA molecules
DNA Cloning Gene cloning- uses bacteria to make multiple copies of a gene To work directly with specific genes Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA
Creating Recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites Yields multiple restriction fragments Cut DNA in a staggered way, producing fragments with sticky ends that bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments
Choosing a Host Cell Prokaryote or Eukaryote? Bacteria used first Easily grown and manipulated in the lab Contain plasmids and many genetic markers Missing post-transcriptional mechanisms (removing introns) Use of saccharomyces (yeast) Rapid growth Easy to grow Small genome (12 million base pairs) Plant cells- totipotent
Choosing a Vector Vector- inserts the DNA into a cell DNA sequence must contain origin of replication (replicon) Insert after existing origin of replication Insert with a vector containing an origin of replication Four characteristics of vectors: Replicate independently inside the host cell Recognition sequence for restriction enzymes Reporter gene to signal presence in the new cell Small size compared to host genome
Plasmids as Vectors Fulfill all four necessary characteristics Also: antibiotic resistance is a good marker, and contain origin of replication
Viruses as Vectors Can accommodate larger genes (greater than 10,000bp) Infect cells naturally Requires eliminating lethal genes
Artificial Chromosomes Yeast Artificial Chromosome (YAC) Contains ori, telomeres, and centromere Basically a full chromosome Artificial restriction sites Marker genes for yeast metabolism
Vectors for Plants Agrobacterium Tumefaciens- soil bacterium that infects plants Inserts plasmid (Ti) that induces tumors in plants Useful genes can be inserted into this plasmid
How do we know the DNA is in the cell? Reporter genes Often antibiotic resistance
How do we know the DNA is in the cell? 3 types of DNA enter the cell: Foreign DNA only Plasmid only Recombinant plasmid (least likely)
Other Types of Reporter Genes Restriction inside lac operon Green fluorescent protein
Cloning Eukaryotic Genes Steps required to clone the hummingbird β-globin gene in a bacterial plasmid: 1. The hummingbird genomic DNA and a bacterial plasmid are isolated 2. Both are digested with the same restriction enzyme 3. The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends 4. Some recombinant plasmids now contain hummingbird DNA 5. DNA mixture is added to bacteria that have been genetically engineered to accept it 6. Bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids 7. This results in the cloning of many hummingbird DNA fragments, including the β-globin gene
Where does the DNA come from? Three main sources of DNA for cloning: Gene libraries Complementary DNA Artificial synthesis or mutation
Gene Libraries Entire human genome is like a library A really huge library (80 million base pairs per chromosome) Restriction enzymes break chromosomes into smaller pieces, which are inserted into vectors Plasmids- require 200,000 fragments to complete the human genome Phages- 50,000 fragments
Gene Libraries
cdna Libraries Only the genes transcribed in a particular tissue Enzyme reverse transcriptase produces DNA from RNA snapshot of cell activity
Artificial DNA Library and Mutations Automated process produces specific nucleotide sequences Important to also include the correct regulatory sequences Mutations can be created to test their effects on the cell Will taking off the signal sequence on a protein inhibit its ability to enter the endoplasmic reticulum
Searching the Gene Library A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene This process is called nucleic acid hybridization A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is Then we would synthesize this probe
DNA Chip
Cloning Organisms Organismal cloning produces one or more organisms genetically identical to the parent that donated the single cell One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism
But what about animals? Nuclear transplantation- the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg Older the donor nucleus, the lower the percentage of normally developing tadpoles
Gene Expression and Development
Studying Gene Expression In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mrnas in place in the intact organism
Organism Development Determination- sets the fate of a cell Differentiation- specific structures and functions arise Morphogenesis- shaping of differentiated cells Growth- increase in body size through cell division Differential gene expression- leads to differences in cells throughout multicellular organism
Cytoplasmic Determinants Maternal substances in the egg that influence early development As the zygote divides by mitosis, cells contain different cytoplasmic determinants, which lead to different gene expression Egg s cytoplasm contains RNA, proteins, and other substances Distributed unevenly in the unfertilized egg
Induction of Cells Signals from nearby embryonic cells influence differentiation Induction- signal molecules from embryonic cells cause transcriptional changes in nearby target cells Interactions between cells induce differentiation of specialized cell types
Determination and Differentiation Determination commits a cell to its final fate, precedes differentiation Cell differentiation is marked by the production of tissue-specific proteins For example: Myoblasts produce muscle-specific proteins and form skeletal muscle cells MyoD is one of several master regulatory genes that produce proteins that commit the cell to becoming skeletal muscle The MyoD protein is a transcription factor that binds to enhancers of various target genes
Pattern Formation Pattern formation - development of a spatial organization of tissues and organs In animals, pattern formation begins with the establishment of the major axes Positional information- molecular cues that control pattern formation Tells a cell its location relative to the body axes and to neighboring cells
Pattern Formation in Fruit Flies Pattern formation in the fruit fly Drosophila melanogaster Combining anatomical, genetic, and biochemical approaches, researchers have discovered developmental principles Some of these patterns are common to many other species, including humans In Drosophila, cytoplasmic determinants in the unfertilized egg determine the axes before fertilization After fertilization, the embryo develops into a segmented larva with three larval stages
How is the development controlled? Edward B. Lewis, Christiane Nüsslein-Volhard, and Eric Wieschaus Nobel 1995 Prize for decoding pattern formation in Drosophila Lewis demonstrated that genes direct the developmental process
Fly Development Nüsslein-Volhard and Wieschaus studied segment formation Created mutants, conducted breeding experiments, and looked for corresponding genes Needle in a haystack- 13,700 genes in Drosophila Breeding experiments were complicated by embryonic lethals, embryos with lethal mutations Cytoplasmic determinants are part of maternal genome They found 120 genes essential for normal segmentation
Axis Establishment Maternal effect genes- encode for cytoplasmic determinants that initially establish the axes of the body of Drosophila Also called egg-polarity genes because they control orientation of the egg and consequently the fly One maternal effect gene, the bicoid gene, affects the front half of the body An embryo whose mother has a mutant bicoid gene lacks the front half of its body and has duplicate posterior structures at both ends
Bicoid Research Example of the gradient hypothesis, in which gradients of substances called morphogens establish an embryo s axes and other features The bicoid research is important for three reasons: It identified a specific protein required for some early steps in pattern formation It increased understanding of the mother s role in embryo development It demonstrated a key developmental principle that a gradient of molecules can determine polarity and position in the embryo
Segmentation Genes Establish number, boundaries, and polarity of fruit fly segments (3 hours after fertilization, 1 cell with 6,000 nuclei) Gap genes- organize broad areas along anterior posterior axis Pair rule genes- divide embryo into units of two segments each Segment polarity genes- determine boundaries and anterior posterior location of segments
Hox Genes Expressed in different combinations along the axis, determine cell fates within each section Mutations lead to legs from the head, or two pairs of wings Homeobox- region common to hox genes, contains homeodomain which is common to transcription factors that activate development in all animals Conservation through all animals suggests importance