Laboratory Encounters in Plant Genomics Dr. Jan Stephens Colorado State University Summer workshop June 27th & 28th, 2006 2/17/2019

What is genetic engineering? What is genomics? What is biotechnology? What is genetic engineering? What can genetic engineering do? What are the basic procedures for producing a genetically modified plant product? 2/17/2019

Why is genetic engineering becoming such a popular science? 1. Trait Identification 2. Gene Discovery 3. Gene Cloning 4. Gene Verification 5. Gene Implantation 6. Cell Regeneration 7. Testing New Plant 8. Seed Production Why is genetic engineering becoming such a popular science? 2/17/2019

DNA STRUCTURE AND FUNCTION We can learn a lot about plants and the pathogens that make them sick by studying their DNA. DNA is located within every cell, and it contains the genetic “code” necessary for making a living thing http://www.biopatent.com/DNA3.gif 2/17/2019

DNA STRUCTURE AND FUNCTION The DNA double stranded helix was described by Watson and Crick in 1953 The DNA helix has a dual backbone of deoxyribose sugars and negatively charged phosphate molecules From: www.accessexcellence.org/RC/VL/GG/dna_molecule.html 2/17/2019

DNA STRUCTURE AND FUNCTION There are pairs of nucleotides or bases held together with hydrogen bonds between each sugar-phosphate strand The phosphate molecules have a negative charge that can be utilized to separate small pieces of DNA during electrophoresis 2/17/2019 http://users.rcn.com/jkimball.ma.ultranet/ BiologyPages/D/DoubleHelix.html

DNA STRUCTURE AND FUNCTION There are four different bases – adenine (A), thiamine (T), guanine (G) and cytosine (C) These are arranged in a special order that tells the cell what proteins to make http://www.web-books.com/MoBio/Free/Ch3B.htm 2/17/2019

DNA STRUCTURE AND FUNCTION The strands are “complementary” Both strands thus contain the same genetic information and can each serve as a model or template for the other strand www.accessexcellence.org/RC/VL/GG/dna_molccule.html 2/17/2019

DNA STRUCTURE AND FUNCTION DNA is found in the cell nucleus which is surrounded by a membrane DNA is packaged into chromosomes The nucleus is inside the cell which is also surrounded by a membrane and for plants by a cell wall http://web.jjay.cuny.edu/~acarpi/NSC/13-cells.htm 2/17/2019

A chromosome contains five types of histones: H1 (or H5), H2A, H2B, H3 and H4.  H1 and its homologous protein H5 are involved in higher-order structures.  The other four types of histones associate with DNA to form nucleosomes.  Each nucleosome consists of 146 bp DNA and 8 histones: two copies for each of H2A, H2B, H3 and H4.  The DNA is wrapped around the histone core, making nearly two turns per nucleosome. From http://www.web-books.com/MoBio/Free/Ch3D1.htm 2/17/2019

Histones are basic proteins, bristling with positively charged arginine and lysine residues. Histones are some of the most conserved molecules during the course of evolution. Histone H4 in the calf differs from H4 in the pea plant at only 2 amino acids residues in the chain of 102. The arrows point to the nucleosomes. You can see why the arrangement of nucleosomes has been likened to "beads on a string". From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleus.html 2/17/2019

DNA REPLICATION During cell division the DNA molecule replicates, so that each new cell receives an exact copy of genetic information. This occurs by the hydrogen bonds between the nucleotides breaking, allowing for the DNA ladder to unzip. Then, the separated strands unwind, and each strand becomes a template for a new complementary strand. 2/17/2019

DNA STRUCTURE AND FUNCTION The result is that in both offspring of a divided cell, each DNA molecule has one original strand and one newly synthesized strand. This mechanism, called DNA replication, ensures precise copying of the nucleotide base sequences in DNA. On average, one mistake may exist in every billion base pairs - the same as typing out the entire Encyclopedia Britannica five times and typing in a wrong letter only once! http://www.uvm.edu/~cgep/Pic/Replication.gif 2/17/2019

DNA replication must be precise DNA replication must be precise. Errors in a particular codon could lead to the wrong amino acid being specified at some point in a protein molecule. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/ReplicationFork.gif This could result in a shape change large enough to change the function of that protein. Errors in replication, and those induced by prolonged exposure to UV, X-rays, and radioisotopes, are known as mutations 2/17/2019

Transcription of DNA Protein synthesis uses the genetic code contained in the nucleotides as a blueprint The region of DNA that contains genes is “transcribed” or copied into another polymer called RNA Many of these RNA molecules undergo major changes before leaving the nucleus to act as messenger molecules (mRNA), that direct the synthesis of proteins. 2/17/2019

Transcription of DNA RNA is similar to DNA but it has ribose instead of deoxyribose sugar in the backbone, uracil (U) instead of thymine and is single stranded One of the two strands of DNA acts as a template for the synthesis of RNA. Thousands of RNA copies may be run off from the same DNA segment 2/17/2019 http://www.accessexcellence.org/RC/VL/GG/RNA_trans.html      

Translation of the code Three nucleotides encode a single amino acid As mRNA moves through the ribosome the appropriate amino acids are added to the growing protein molecule http://web.mit.edu/esgbio/www/dogma/trl.html 2/17/2019

Table of Standard Genetic Code TTT Phe (F) TTC Phe (F) TTA Leu (L) TTG Leu (L) TCT Ser (S) TCC Ser (S) TCA Ser (S) TCG Ser (S) TAT Tyr (Y) TAC TAA STOP TAG STOP TGT Cys (C) TGC TGA STOP TGG Trp (W) CTT Leu (L) CTC Leu (L) CTA Leu (L) CTG Leu (L) CCT Pro (P) CCC Pro (P) CCA Pro (P) CCG Pro (P) CAT His (H) CAC His (H) CAA Gln (Q) CAG Gln (Q) CGT Arg (R) CGC Arg (R) CGA Arg (R) CGG Arg (R) ATT Ile (I) ATC Ile (I) ATA Ile (I) ATG Met (M) START ACT Thr (T) ACC Thr (T) ACA Thr (T) ACG Thr (T) AAT Asn (N) AAC Asn (N) AAA Lys (K) AAG Lys (K) AGT Ser (S) AGC Ser (S) AGA Arg (R) AGG Arg (R) GTT Val (V) GTC Val (V) GTA Val (V) GTG Val (V) GCT Ala (A) GCC Ala (A) GCA Ala (A) GCG Ala (A) GAT Asp (D) GAC Asp (D) GAA Glu (E) GAG Glu (E) GGT Gly (G) GGC Gly (G) GGA Gly (G) GGG Gly (G) 2/17/2019

Mutations Missense mutations Nonsense mutations EXAMPLE: sickle-cell disease http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.html#insertions Nonsense mutations With a nonsense mutation, the new nucleotide changes a codon that specified an amino acid to one of the STOP codons (TAA, TAG, or TGA). 2/17/2019

Mutations e.g. Cystic fibrosis Patient B, the substitution of a T for a C at nucleotide 1609 converted a glutamine codon (CAG) to a STOP codon (TAG). The protein produced by this patient had only the first 493 amino acids of the normal chain of 1480 and could not function. 2/17/2019 http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.html#insertions

STRAWBERRY EXTRACTION PROCEDURE Extraction of DNA STRAWBERRY EXTRACTION PROCEDURE Add extraction buffer to the zipper bag with strawberry. Close bag, let in as little air as possible. Mush the strawberry thoroughly for 5 minutes, without breaking the bag. Place the zipper bags with fruit and extraction solution into the hot water bath for about 10-15 minutes. Occasionally shake the bag to distribute heat. 2/17/2019

Extraction of DNA Put the mashed bags of strawberry and solution into the ice bath for 1 minute. Remove and mush the strawberry more. Repeat this procedure 5 times. Filter this mixture through the cheesecloth filter. All students can combine their solutions at this point. Let the solution drain for 5 minutes. Aliquot approximately 2 ml of the strawberry solution into each test tube. 2/17/2019

Extraction of DNA Carefully, without disruption of the test tube contents add approximately 2 ml of ice-cold ethanol to each tube. Do this by letting the drops run slowly down the side of the test tube and rest on top of the strawberry mixture. Let the solution sit for 2 minutes without disturbing it. The DNA will appear as transparent, slimy, white mucus that you can “hook” up with the bent paperclip. 2/17/2019

Extraction of DNA WHAT IS THE PURPOSE OF EACH STEP? Why do we “mush” the strawberry fruit? Crushing the strawberry fruit physically breaks apart the cell walls. Why do we use shampoo? After the cell walls have been disrupted during mechanical mashing of the fruit, the detergent in the shampoo disrupts the cell and nuclear membranes of each cell to release the DNA. It does this by dissolving lipids and proteins that hold the membranes together. 2/17/2019

Why do we need to cool the mixture Why do we need to cool the mixture? DNases or enzymes that destroy DNA are present in the cell’s cytoplasm. They are there to protect the cell from invasion by viruses. Once the nuclear membrane is destroyed by the soap the DNA is now susceptible to the DNases and will quickly be degraded. However, these enzymes are temperature sensitive and cooling the solution slows down the process of degradation. What does the cold ethanol do? Everything except the DNA will dissolve in ethanol. The ethanol pulls water from the DNA molecule so that it then collapses in on itself and precipitates. The DNA will become visible as white mucous strands that can be spooled with the wooden applicator stick. . 2/17/2019

What does the salt do? The salt neutralizes the negative charges on the DNA and thus enables the DNA strands to stick together. It also causes proteins and carbohydrates to precipitate. Why can’t we use room temperature ethanol? The colder the ethanol is the greater the amount of DNA that is precipitated. (You could try having some of the students use room temperature ethanol and see if the amount of DNA they can spool is the same or less than that for the groups using the ice-cold ethanol.) 2/17/2019