1. Chapter 12: Molecular Genetics Discovery of DNA as genetic material Mid-1900s - scientists knew the following about chromosomes: They contained genetic information They were made up of DNA (deoxyribonucleic acid) and proteins They did NOT know whether the DNA or the protein was the actual genetic material. Several experiments were done to show that DNA was the genetic material.

2. Frederick Griffith’s experiment – 1928 Worked with 2 strains of bacteria S (smooth) strain caused pneumonia (coat protects it from host’s immune system, and host dies – top picture) R (rough) strain did not cause pneumonia (no coat, is killed by host’s immune system – bottom picture)

4. Results: Live S strain – mouse died Live R strain – mouse lived Heat-killed S strain – mouse lived Mixture of heat-killed S strain and live R strain – mouse died

5. When Griffith isolated live bacteria from the dead mice who has been injected with the mixture of heat-killed S strain and live R strain, he found the smooth trait. This suggested that the disease-causing trait had been passed to the live R bacteria. So live R bacteria were transformed into live S bacteria – wondered what the transforming substance was?

6. Today, we know why this happened. The transforming substance was DNA. The heat killed the S strain bacteria, but not its DNA. S strain DNA was taken up by live R strain bacteria, allowing it to grow the protective coat of the S strain. Transformed bacteria caused the mice to die. 

7. Oswald Avery’s experiment – 1944 Set out to identify transforming substance from Griffith’s experiment Isolated different molecules (DNA, proteins, lipids) from killed S cells Exposed live R cells to each molecule separately When live cells exposed to DNA, they transformed into S cells.

8. Avery concluded that when S cells were killed, their DNA was released R bacteria took that DNA in, and they transformed into S cells Avery’s conclusions were not widely accepted (although he was right!!)

9. Hershey and Chase experiment – 1952 Alfred Hershey and Martha Chase showed that DNA was the genetic material through two experiments with a T 2 (type 2) virus. They knew the following to be true: Viruses are made of DNA and protein Viruses inject genetic material into bacterium to reproduce

10. In first experiment, viral DNA was labeled with radioactive phosphorous. Virus was allowed to inject genetic material into bacterium.

11. In second experiment, viral protein was labeled with radioactive sulfur. Virus was allowed to inject genetic material into bacterium.

12. Because radioactive phosphorous was found inside the bacteria, and radioactive sulfur was not, they showed that DNA and not protein was the genetic material found in chromosomes. Animation

13. Discovery of the structure of DNA The following was known about DNA by the early 1950’s: DNA is made of nucleotides Phosphate Sugar – deoxyribose 1 of 4 bases – adenine, cytosine, thymine, and guanine Chargaff’s rules – Amount of adenine and thymine always equal Amount of cytosine and guanine always equal

14. DNA is in the shape of a double helix – discovered by Franklin & Wilkins through X-ray diffraction of DNA (a) 1953 - Watson & Crick used above information to construct 1 st model of DNA (b)

15. Structure of DNA DNA is a polynucleotide; nucleotides are composed of: Phosphate Sugar (deoxyribose) 1 of 4 nitrogen- containing bases - adenine (A), thymine (T), guanine (G), and cytosine (C)

16. There are 2 strands of nucleotides 2 strands are held together by hydrogen bonds Two strands twist around each other to form a double helix A & T, C & G are complementary base pairs (purine to a pyrimidine) Purines – A & G Pyrimidines – T & C

17. DNA strands are anti-parallel – run in opposite directions – orientation of sugars 5’ – pronounced 5 prime 3’ – pronounced 3 prime

18. When double helix is unwound, it resembles a ladder A & T pair with 2 hydrogen bonds C & G pair with 3 hydrogen bonds

20. DNA Replication Purpose: DNA makes an exact copy of itself prior to cell division; ensures that each new cell gets a complete copy of the DNA DNA Replication DNA Cell Division DNA

21. Steps: (enzymes in red) 1.Helicase attaches to DNA and breaks H 2 bonds between bases – DNA chain unwinds and unzips (Special proteins keep it unzipped) 2.RNA primase adds an RNA primer (short segment of RNA) to each strand of DNA 3.DNA polymerase attaches to separated strand, helping add complementary nucleotides to the new DNA strand

22. Each side is done differently, since new nucleotides can be added to the 3’ end of the new strand only Leading strand – built continuously Lagging strand – elongates away from elongation fork. Made in small sections called Okazaki fragments Okazaki fragments are later connected by the enzyme DNA ligase

23. Overview of DNA replication

24. Ladder configuration and DNA replication

25. Each old strand of nucleotides serves as a template for each new strand. The process is semiconservative because each new double helix is composed of an old strand of nucleotides from the parent strand and one newly-formed strand (daughter strand). Proofreading and repair limits error rate to less than 1 per billion nucleotides. http://www.stolaf.edu/people/gian nini/flashanimat/ molgenetics/dna- rna2.swf Replication fork DNA replication song

26. DNA (Deoxyribonucleic acid) RNA (Ribonucleic acid) Sugar DeoxyriboseRibose Bases Adenine, thymine, guanine, cytosine Adenine, uracil, guanine, cytosine Strands Double-stranded with base pairing Single-stranded Helix YesNo Location NucleusNucleus, cytoplasm Types XXXXXXXXXMessenger, transfer, ribosomal

27. RNA vs. DNA

28. Messenger RNA - carries genetic information to the ribosomes Ribosomes - part of the cell where proteins are made Ribosomal RNA - found in the ribosomes Transfer RNA - transfers amino acids to the ribosomes

29. Making Proteins A gene is a segment of DNA that specifies the amino acid sequence of a protein. DNA is found in the nucleus of a cell; proteins are made outside the nucleus at the ribosomes.

31. Overview of gene expression

32. Two processes are involved in the synthesis of proteins in the cell: Transcription – DNA is copied into mRNA, which will take a copy of the DNA code to the ribosome to direct the making of protein; occurs in nucleus Translation - the process of building proteins, the sequence of bases of mRNA is “translated” into a sequence of amino acids; occurs in ribosome These processes are the same in all organisms

33. The Genetic Code DNA holds instructions to make a protein Instructions are copied into mRNA, which will be used to make a protein Codon - each three-letter unit of an mRNA molecule Each codon represents 1 amino acid There are 64 possible codons, and only 20 amino acids, so most amino acids have more than one codon

34. Messenger RNA codons

35. Transcription Purpose – Makes a copy of the DNA code that can leave the nucleus and travel to the ribosome to direct protein synthesis – mRNA Occurs in the nucleus Occurs at only 1 gene at a time Adenine in DNA pairs with uracil in RNA, not thymine Thymine in DNA pairs with adenine in RNA

36. Steps: 1.Starting at promoter (signals the start of a gene), segment of DNA unwinds and unzips 2.½ of DNA will serve as a template (DNA template strand is in the 3’ to 5’ direction; RNA in 5’ to 3’) 3.RNA polymerase joins the RNA nucleotides so that the codons in mRNA are complementary to the code in DNA. 4.Termination signal (signals end of gene) is reached, process ends, and DNA closes back up From video shown in class: http://www.youtube.com/watch?v=41_Ne5mS2ls Animation

37. Transcription and mRNA synthesis

38. RNA Processing DNA contains exons (parts of a gene that are expressed) and introns (intragene segments – not expressed) Before mRNA leaves the nucleus, the introns are removed so that only the exons remain The splicing of mRNA is done by ribozymes, enzymes composed of RNA. Primary mRNA/pre-mRNA (with introns & exons) is processed into mature mRNA (without introns).

40. Translation Protein constructed during this process Occurs at the ribosomes Key players in translation: mRNA (messenger RNA) Made during transcription, has codons Travels from nucleus to ribosome Contains copy of DNA code to make protein tRNA (transfer RNA) rRNA (ribosomal RNA)

41. Transfer RNA (tRNA) tRNA molecules bring amino acids to the ribosomes Free-floating in the cytoplasm of the cell Each tRNA has a sequence of nucleotides called an anticodon – it is this sequence that determines which amino acid each tRNA has

42. Complementary base pairing occurs between anticodons of tRNA and codons of mRNA – determines the sequence of amino acids to construct the polypeptide. If mRNA codon is AUG, tRNA anticodon would be UAC

43. Ribosomal RNA (rRNA) rRNA is made in the nucleolus (a cell structure found inside the nucleus) Ribosome made of a large subunit and small subunit that join just prior to protein synthesis

44. Ribosome has a binding site for mRNA and binding sites for two tRNA molecules at a time. Several ribosomes may attach and translate the same mRNA, therefore the name polyribosome (letter c below).

46. Three Steps of Translation 1)Chain initiation 2)Chain elongation 3)Chain termination. Enzymes are required for each step, and the first two steps require energy. http://www.youtube.com/watch?v=D5vH4Q_tAkY http://www.youtube.com/watch?v=41_Ne5mS2ls From video shown in class: Animation

47. Chain Initiation Small ribosomal subunit attaches to the mRNA near the start codon. The anticodon of tRNA, called the initiator RNA, pairs with the start codon at the P site on ribosome. Large ribosomal subunit joins.

48. Chain Elongation The initiator tRNA passes its amino acid to a tRNA-amino acid complex that has come to the second binding site, the A site. The ribosome moves forward and the tRNA at the second binding site is now at the first site, a sequence called translocation. The previous tRNA leaves the ribosome at the E site of the ribosome

50. Chain Termination A stop-codon is reached. A release factor (an enzyme) breaks the polypeptide from the last tRNA The ribosome falls away from the mRNA molecule and separates into its two subunits A newly synthesized polypeptide may function alone or become part of a protein.

52. Review of Gene Expression DNA in the nucleus contains a triplet code; each group of three bases stands for one amino acid. During transcription, an mRNA copy of the DNA template is made. The mRNA is processed before leaving the nucleus. The mRNA joins with a ribosome, where tRNA carries the amino acids into position during translation.

55. Control of Gene Expression in Prokaryotes The lac operon Regulator gene codes for active repressor, which automatically attaches to the operator. RNA polymerase cannot attach to promoter, and transcription does not occur

56. When lactose attaches to repressor, it becomes inactive and cannot attach to the operator. Now RNA polymerase can attach to the promoter, transcription occurs, and the genes are expressed

57. Control of Gene Expression in Eukaryotes In eukaryotes, cells differ in which genes are being expressed – based on cell function – ex. nerve vs. muscle. One way that eukaryotes can control gene expression is through proteins called transcription factors Two types Those that guide and stabilize the binding of RNA polymerase to a promotor Those that control the rate of transcription (by controlling how DNA is folded or preventing activators from binding)

58. During development, cells become specialized. This differentiation is controlled by a set of genes called Homeobox (Hox) genes. They code for transcription factors and are active in specific parts of the DNA corresponding to specific parts of the body that is developing. They control what body part will develop in a specific location.

60. RNA interference: Some viruses have double-stranded RNA An enzyme called dicer can cut this RNA into small segments When they attach to protein complexes in the cell, one of the strands breaks down. The remaining section attaches to molecules of mRNA, causing them to break and preventing translation

62. Gene Mutations Definition - a change in the sequence of bases within a gene Causes – Mutations can be spontaneous or caused by environmental influences called mutagens. Cancer causing mutagens are called carcinogens Mutagens include radiation (X-rays, UV radiation), and organic chemicals (in cigarette smoke and pesticides).

63. Types – Frameshift mutations – one or more bases are inserted or deleted from a sequence of DNA can result in nonfunctional proteins can result in no protein at all – stop codon where there shouldn’t be one Point mutations – One base is substituted for another May result in change of amino acid sequence May not affect protein at all

64. Types of point mutations: Silent mutation - the change in the codon results in the same amino acid Ex: UAU  UAC both code for tyrosine Nonsense mutation - a codon is changed to a stop codon; resulting protein may be too short to function Ex: UAC  UAG (a stop codon)

65. Missense mutation - involves the substitution of a different amino acid, the result may be a protein that cannot reach its final shape Ex: Hb s which causes sickle-cell disease

67. In general, mutations can have any of the following effects: No change in proteins made or appearance Wrong protein is made No protein in made New appearance may result

68. Repair of Mutations DNA polymerase proofreads the new strand against the old strand and detects mismatched pairs, reducing mistakes to one in a billion nucleotide pairs replicated. If errors occur in sex cells – mutation may be passed onto offspring If errors occur in body cells - cancer may result

69. Transposons: Jumping Genes Transposons are specific DNA sequences that move from place to place within and between chromosomes. These “jumping genes” can cause a mutation to occur by altering gene expression. It is likely all organisms, including humans, have transposons.