DNA: The Molecule of Life

1. DNA: The Molecule of Life Molecular Genetics

2. DNA and RNA • Genes are segments of DNA on a chromosome that code for specific traits • DNA – nucleic acid called deoxyribonucleic acid that contains the instructions necessary for a cell to build proteins from amino acids. • RNA – ribonucleic acid plays a role in gene expression and protein synthesis

3. Isolating the Material of Heredity • 1869 Friedrich Miescher isolated a weakly acidic phosphorus-containing substance from the nuclei of white blood cells • Called it “nucleic acid” • Early 1900’s Pheobus Levene isolated two types of nucleic acid • Called them “ribose nucleic acid” (RNA) and “deoxyribose nucleic acid” (DNA) • Soon after, Thomas Hunt Morgan provided the first experimental evidence that genes are located on chromosomes • Working with fruit flies

4. Isolating the Material of Heredity • In 1928 Fredrick Griffith designed an experiment to study the bacteria that were responsible for a pneumonia epidemic in London • He discovered that the dead pathogenic bacteria passed on their disease-causing properties to live, non-pathogenic bacteria • He called this the transforming principle • Griffith died during world war II but several scientists built on his work

5. http://www.juliantrubin.com/bigten/dnaexperiments.html

6. Isolating the Material of Heredity • In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered: • When they treated heat-killed pathogenic bacteria with a protein-destroying enzyme, transformation still occurred • When they treated heat-killed pathogenic bacteria with a DNA-destroying enzyme, transformation did not occur • The results provided evidence that DNA has a role in transformation

7. http://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch8-1.htmlhttp://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch8-1.html

8. Isolating the Material of Heredity • In 1952, Alfred Hershey and Martha Chase used radioactive labeling to show that genes are made of DNA • They used a virus that contains a protein coat surrounding a length of DNA • This virus attaches to a bacteria cell and injects genetic information into the cell • The infected cell produces new viruses and bursts which releases the new viruses to infect other cells

9. Isolating the Material of Heredity • Hershey and Chase created two batches of the virus • In one they labeled the protein coat with radioactive sulfur • In the other, they labeled the DNA with radioactive phosphorus • They found that the radioactive phosphorus was found in the bacterial cells • They concluded that DNA must direct the cell to produce new viruses • animation

10. http://www.accessexcellence.org/RC/VL/GG/hershey.html

11. Structure of DNA • After isolating DNA and RNA, Levene determined that both molecules are made up of nucleotides (in long chains) • Nucleotide is composed of: • 5-carbon sugar (deoxyribose in DNA, ribose in RNA) • Phosphate • Nitrogen base (4 different types) • The 4 nitrogen bases belong to two chemical groups called purines and prymidines • Purines = Adenine (A) and Guanine (G) • Prymidines = Thymine (T) and Cytosine (C)

13. Structure of DNA • In the late 1940’s Erwin Chargaff found that nucleotides are not present in equal amounts in DNA and RNA • Nucleotides are present in varying proportions • He found that the number of adenine in DNA is equal to the number of thymine in a sample • The amount of cytosine is approximately equal to the amount of guanine • This constant relationship is called Chargaff’s rule • Video on Chargaff’s rule

14. Structure of DNA • In the early 1950’s, a British scientist Rosaslind Franklin used x-ray photography to analyze the structure of DNA • DNA has a helical structure with two regularly repeating patterns • Nitrogenous bases are located on the inside of the structure, and the sugar-phosphate backbone is located on the outside (near the watery nucleus) • Many argue that Franklin should have shared in the Nobel Prize for discovering the structure of DNA, but she died before it was handed out NOVA program on photo 51.

15. Structure of DNA • In 1953, James Watson and Francis Crick were the first to produce a structural model for DNA • Watson and Crick’s model of DNA closely resembles a twisted ladder • Deoxyribose sugar and phosphate molecules make up backbone (handrails of the ladder) • Paired nitrogen bases held together by weak hydrogen bonds make up the rungs (steps) of the ladder • The ladder twists to form a double helix • From Franklin’s images, Watson and Crick knew the distance between the sugar-phosphate handrails remained constant

16. Structure of DNA • The two strands that make up DNA are not identical, they are complementary to eachother • This is due to the complementary base pairs of A-T and C-G • The two strands are also antiparallel • The phosphate bridges run in opposite directions in the two strands • Each end of a double stranded DNA molecule contains the 5’ end of one strand and the 3’ end of the complementary strand

17. http://www.synapses.co.uk/genetics/tsg19.html

19. In a segment of DNA, the number of purines equals the number of pyrimidines; this is because of the base pairing rule • RULE: nitrogen bases always pair in complementary pairs • Adenine = Thymine • Guanine = Cytosine

20. Ex) if 15% of the bases were thymine, what percentage would be a) adenine b) guanine c) cytosine

21. Example • Determine the complementary strand of DNA: A T G C A G C I I I I I I I

22. Ribonucleic Acid (RNA) compared to DNA • The sugar component of RNA is ribose • RNA does not have the nucleotide thymine (T), in its place is uracil (U) • RNA remains single stranded • There are several types of RNA • mRNA, rRNA, tRNA DNA, RNA animation

23. DNA Replication (Synthesis) • Replication – DNA has the ability to replicate (or duplicate) itself. • This is why one cell is able to divide into two cells and each cell has identical genetic information • Replication takes place during S phase of interphase

24. DNA Replication (Synthesis) • Replication is semi-conservative • Each new molecule of DNA contains one strand of the original complementary DNA and one new parent strand • Each new strand conserves half of the original molecule • Meselson-Stahl Experiment • Replication takes place at several locations along the DNA molecule simultaneously • The steps are described in sequence to better understand the concept • BioFlix Replication

25. Replication starts at a specific nucleotide sequence called the replication origin • During replication, weak hydrogen bonds that hold complementary nitrogen bases together are broken (This causes the two edges to “unzip”) with a special group of enzymes called helicases (gyrase breaks the hydrogen bonds) • This creates two y-shaped areas (replication forks) at the end of the unwound area, the unwound area is called a replication bubble • The parent (original) strands are conserved while two new strands created from nucleotides are formed with them (they act as a template)

26. Free floating nucleotides (from diet) are attached to the exposed nitrogen bases according to the base pair rule with an enzyme called DNA polymerase • This process is called elongation • DNA polymerase attaches new nucleotides to the free 3’ end of a preexisting chain of nucleotides • Elongation can only take place in a 5’ to 3’ direction • This means that replication occurs in opposite directions along each strand of the parent DNA • One strand is replicated continuously, it is called the leading strand • The other strand is replicated in short segments, it is called the lagging strand • The short segments are called Okazaki fragments • These fragments are spliced together by an enzyme called DNA ligase

27. Since DNA polymerase cannot synthesize new fragments of DNA, and RNA primer serves as a starting point for the elongation of each new DNA strand • An enzyme called primase is required to construct a primer • When finished the strand, DNA polymerase removes the primer by eliminating the nucleotides in a 5’ to 3’ direction • Hydrogen bonds form between the nitrogen bases • Special proofreading enzymes (DNA polymerase) check the new strand of DNA for mistakes. Errors are removed by cutting the mistake out and using an endonuclease and replacing it with the correct nitrogen base

28. As soon as the newly formed strands are complete, they rewind automatically into the helix structure • Replication continues until the new strands are complete and the two DNA molecules separate from eachother • This is called termination • This replication produces two strands of DNA from one where each strand is composed of “half-old and half-new” http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2005/Durnbaugh/yfp.html Replication Fork Adding Nucleotides Replication Animation Replication Review

29. http://www.biosci.ohio-state.edu/~mgonzalez/Micro521/04.html http://distancelearning.ksi.edu/demo/bio378/lecture.htm

30. Genetic Engineering and Recombinant DNA • Human Genome Project - animation • Genetic Engineering – refers to the alteration of an organism’s genome (complete set of genes) by selectively removing, adding, or modifying DNA • Recombinant DNA – the process of cutting out DNA from one genome and placing the DNA into another genome resulting in a transgenic organism

31. Examples of transgenic organisms: • Genetically modified bacteria for use in medicine and bioremediation (environmental clean-up) • Transgenic plants to improve crop yield and resistance to environmental effects • Cloned animals (livestock) and organs for human use

32. Recombinant DNA – How do they do it? • Use restriction enzymes (endonucleases) to cut strands of DNA within their interior (at specific sequences) • animation • Then ligase (enzyme that fuses segments of DNA) is used as a biological glue

33. Production of human insulin • Gene in the human genome that codes for insulin is cut out using restriction enzymes • The plasmid of an E-coli bacteria is cut using restriction enzymes so that the gene for insulin can be inserted using a ligase • Bacteria can read the DNA and produce insulin for us to later harvest and use

34. Another example is the insertion of the gene that codes for growth hormone into animals so that they grow faster Note: Biotechnology refers to the use of organisms or biological products for commercial and/or industrial processes - video

35. What are the Issues? • Costs/where money is spent • Motivation for the product • Biological characteristics of the product • Heath effects • Environmental effects • Freedom of Information/Privacy • Who Owns the technology/patents • Issues Animation

36. Gel Electrophoresis • Technique used to separate DNA fragments by size for the purpose of identification in paternal or criminal suits (animation) • Sample of DNA is cut using restriction enzymes from hair, blood, skin, etc. This produces a number of DNA segments of different lengths. • The different pieces of DNA (referred to as restriction-fragment-length-polymorphisms or RFLP) are tagged with a radioactive isotope

37. 3) Using an agarose gel that contains holes or wells along one side, the samples of DNA are inserted into the wells. A known sample is loaded with it as a comparison

38. 4) Electric current is run through the gel, causing the movement of the negatively charged DNA fragments. The shortest strands move the farthest (lowest weight) and the longer strands (heavier) will not move as far.

39. 5) This causes separation of the DNA into bands. The gel is left to set 6) When combined with staining or X-ray film, the patterns are used to determine the presence or absence of particular DNA or proteins

41. DNA Fingerprinting • Developed in 1985 – used to identify whether or not a sample of DNA comes from a specific person • People have similar DNA, however every human (with the exception of identical twins, triplets, etc.) have some unique noncoding segments of DNA called introns; exons are segments of DNA that actually code for proteins

42. Sample of DNA is placed through gel electrophoresis as well as samples from individuals who are suspected as “owners” of the sample • Because of introns, each individual will have a different number of sites where the restriction enzyme will cut • This results in a unique number and length of fragments which produces a unique banding pattern (fingerprint) when x-rayed • Fingerprints are used to identify criminals, paternity or kinship • Animation

43. Lane A: DNA from crime scene cut with Enzyme 1 • Lane B: DNA from crime scene cut with Enzyme 2 • Lane C: DNA from Suspect 1 cut with Enzyme 1 • Lane D: DNA from Suspect 1 cut with Enzyme 2 • Lane E: DNA from Suspect 2 cut with Enzyme 1 • Lane F: DNA from Suspect 2 cut with Enzyme 2

44. Protein Synthesis • Genetic code is determined by the arrangement of nitrogen bases within the strands of DNA • Each gene codes for the production of a specific protein • DNA RNA protein translation transcription

45. Proteins • Proteins are composed of 20 different amino acids that are strung together in endless combinations • Compose cell membranes, cell organelles, muscle filaments, hair, hair color, enzymes (regulate speed of chemical reactions in cells), antibodies (disease-control agents), hormones

46. Genetic Code • It takes the code of 3 nucleotides (a codon) to code for one amino acid • Humans can make 12 of the 20 amino acids, we must consume the other 8 essential amino acids • Simple protein = 8 amino acids • Complex protein = 50 000 amino acids • Sequencing amino acids is determined by DNA • Replacement of a single amino acid can change a protein

47. Genetic Code • The genetic code has three important characteristics • The genetic code is redundant (more than one codon can code for the same amino acid) • The genetic code is continuous (reads as a series of three letter codons without spaces, punctuation or overlap) • The genetic code is nearly universal (almost all organisms use the same code – this is good for genetic engineering and biotechnology)

48. Role of DNA in protein synthesis • DNA is in nucleus, but protein synthesis occurs on the ribosomes in the cytoplasm • Carrier molecule (mRNA – messenger RNA) is responsible for reading the information from the DNA (transcription) and carry it to the ribosomal RNA (rRNA) in the cytoplasm where it will be translated into an amino acid sequence by transfer RNA (tRNA)

49. RNA (Ribonucleic Acid) • Different from DNA in that: • It’s single stranded • It contains the sugar ribose • It is located throughout the cell (DNA is only in the nucleus with some also in the mitochondria) • It contains the base uracil (U) instead of thymine (T) • There are three types: mRNA, rRNA, tRNA • It’s shorter (no introns)

50. Transcription • Protein synthesis begins with transcription (RNA synthesis) of DNA • DNA never leaves the nucleus (protected)