CHAPTER 11 MOLECULAR GENETICS

1. CHAPTER 11 MOLECULAR GENETICS ADVANCED BIOLOGY

2. At first, people believed that proteins served as the genetic material, even though DNA had already been discovered (1869). It wasn't until the 1940s that this theory was disproved. People believed that proteins were responsible for storing genetic information because of its complexity. They are made of 20 different amino acids, but DNA has only 4 bases. It was concluded that more complexity would account for diversity in organisms. But as more research was done, it was discovered that DNA was actually the genetic material. Introduction Studies of bacteria and viruses ushered in the field of molecular biology, the study of heredity at the molecular level, and revealed the role of DNA in heredity.

3. He postulated that information could somehow be transferred between different strains of bacteria. This was long before the discovery of DNA and was an inspired piece of scientific detective work. The Griffith Experiment Frederick Griffith From: https://en.wikipedia.org/wiki/Frederick_Griffith#/media/File:Griffithm.jpg Frederick Griffith, established that there was a transforming principle in bacterial genetics in a ground-breaking experiment, performed in 1928.

4. The Experiment Griffith used two strains of Pneumococcus bacteria, type III-S and type II-R. The III-S strain has a smooth polysaccharide coat which makes it resistant to the immune system of mice, whereas the II-R strain lacks this coat and so will be destroyed by the immune system of the host. For the first stage, Griffith showed that mice injected with III-S died but when injected with II-R lived and showed few symptoms. The next stage showed that if the mice were injected with type III-S that had been killed by heat, the mice all lived, indicating that the bacteria had been rendered ineffective. The interesting results came with the third part of the experiment, where mice were injected with a mixture of heat killed III-S and live II-R.The mice all died, indicating that some sort on information had been passed from the dead type III-S to the live type II-R. Blood sampling showed that the blood of the dead mice contained both live type III-S and live type II-R bacteria. Somehow the type III-S had been transformed into the type III-R strain, a process he named the transforming principle.

5. Griffith concluded that the type II-R had been "transformed" into the lethal III-S strain by a "transforming principle" that was somehow part of the dead III-S strain bacteria. Today, we know that the "transforming principle" Griffith observed was the DNA of the III-S strain bacteria. While the bacteria had been killed, the DNA had survived the heating process and was taken up by the II-R strain bacteria. The III-S strain DNA contains the genes that form the protective polysaccharide capsule. Equipped with this gene, the former II-R strain bacteria were now protected from the host's immune system and could kill the host. The exact nature of the transforming principle (DNA) was verified in the experiments done by Avery, McLeod and McCarty and by Hershey and Chase. Conclusion From https://en.wikipedia.org/wiki/Griffith%27s_experiment

6. In 1944, Oswald Avery and colleagues expanded upon the findings of Frederick Griffith to demonstrate that DNA is the genetic material. The Avery-McLeod-McCarty Experiment From http://vle.du.ac.in/mod/book/view.php?id=12964&chapterid=27920 Three scientists, Oswald Avery, Colin MacLeod, and Maclyn McCarty, managed to show that Frederick Griffith’s transforming factor was in fact DNA, i.e. DNA is the heritable substance.

7. The Experiment They prepared cultures containing the heat-killed III-S strain and then removed lipids and carbohydrates from the solution. Next they treated the solutions with different digestive enzymes (DNase, RNase or protease) to destroy the targeted compound. Finally, they introduced living II-R cells to the culture to see which cultures would develop transformed III-S bacteria. Only in the culture treated with DNase did the III-S strain bacteria fail to grow (i.e. no DNA = no transformation) From http://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/dna-experiments.html

8. The results indicated that DNA was the genetic component that was being transferred between cells. It was the first experimental evidence that showed that DNA was the genetic material. Despite this finding, the scientific community was reluctant to accept the role of DNA as a genetic material. It was only 8 years later, when Hershey and Chase conducted their experiment, that the concept was accepted. Conclusion

9. The Hershey-Chase Experiment Alfred Hershey and Martha Chase did their famous experiment in 1952 which helped confirm that DNA is the genetic material. From https://www.dnalc.org/view/16406-Gallery-18-Alfred-Hershey-and-Martha-Chase-1953.html

10. The Experiment Viruses (T2 bacteriophage) were grown in one of two isotopic mediums in order to radioactively label a specific viral component . Viruses grown in radioactive sulfur (35S) had radiolabelled proteins (sulfur is present in proteins but not DNA). Viruses grown in radioactive phosphorus (32P) had radiolabeled DNA (phosphorus is present in DNA but not proteins). The viruses were then allowed to infect a bacterium (E. coli) and then the virus and bacteria were separated via centrifugation. The larger bacteria formed a solid pellet while the smaller viruses remained in the supernatant. The bacterial pellet was found to be radioactive when infected by the 32P–viruses (DNA) but not the 35S–viruses (protein).

11. THE STRUCTURE OF NUCLEOTIDES DNA and RNA are nucleic acids. They are polymers of nucleotides One of the two strands of DNA is a DNA polynucleotide, a nucleotide polymer (chain). A nucleotide is composed of a a. nitrogenous base, b. five-carbon sugar, and c. phosphate group.

12. It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group. A nucleotide without a phosphate is called nucleoside Each type of DNA nucleotide has a different nitrogen-containing base: adenine (A) , cytosine (C) thymine (T) and guanine (G)

13. RNA (ribonucleic acid) is unlike DNA in that it uses the sugar ribose (instead of deoxyribose in DNA) and RNA has the nitrogenous base uracil (U) instead of thymine The nucleotides are joined to one another by a sugar-phosphate backbone. In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next. Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases

14. Scientific Discovery: DNA • DNA is a double-stranded helix • In 1952, after the Hershey-Chase experiment demonstrated that the genetic material was most likely DNA, a race was on to describe the structure of DNA and b. explain how the structure and properties of DNA can account for its role in heredity.

15. In 1953, James D. Watson and Francis Crick deduced the secondary structure of DNA, using X-ray crystallography data of DNA from the work of Rosalind Franklin and Maurice Wilkins and Chargaff’s observation that in DNA, • the amount of adenine was equal to the amount of thymine and ii. the amount of guanine was equal to that of cytosine

16. Watson and Crick reported that DNA consisted of two polynucleotide strands wrapped into a double helix. • The sugar-phosphate backbone is on the outside. b. The strands are anti-parallel to each other • The nitrogenous bases are perpendicular to the backbone in the interior. • Specific pairs of bases give the helix a uniform shape. • A pairs with T, forming two hydrogen bonds, and G pairs with C forming three hydrogen bonds.

17. In 1962, the Nobel Prize was awarded to James D. Watson, Francis Crick, and Maurice Wilkins. Rosalind Franklin probably would have received the prize as well but for her death from cancer in 1958. Nobel Prizes are never awarded posthumously. • The Watson-Crick model gave new meaning to the words genes and chromosomes. The genetic information in a chromosome is encoded in the nucleotide sequence of DNA

18. ORGANIZATION OF GENETIC MATERIAL • The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein • Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein  In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid • Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells • Histones are proteins that are responsible for the first level of DNA packing in chromatin. Histones have large amounts of positively charged amino acids (lysine and arginine) thus they can bond with negatively charged phosphate groups

19. Chromatin is organized into fibers 10-nm fiber. DNA winds around histones to form nucleosome “beads”. Nucleosomes are strung together like beads on a string by linker DNA 30-nm fiber. Interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber 300-nm fiber. The 30-nm fiber forms looped domains that attach to proteins • Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis. Loosely packed chromatin is called eu-chromatin, and is the one transcribe during transcription. During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin. Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions.

20. Histones can undergo chemical modifications that result in changes in chromatin organization. • Acetylation : Adding an acetyl group neutralizes the positive charge and activates genes by opening up the chromatin structure. In formation of the Barr body the H4 histone is greatly under acetylated • Methylation : Addition of methyl groups to arginine and lysine correlates with inactivation of genes

21. Phosphorylation • Phosphate groups can be added to hydroxyl groups of amin acids, serine and histidine. This introduces a negative charge to the protein. Increased phosphorylation is associated with specific times during the cell cycle and has been linked to gene activation

22. DNA REPLICATION • DNA replication depends on specific base pairing • In their description of the structure of DNA, Watson and Crick noted that the structure of DNA suggests a possible copying mechanism. • DNA replication follows a semiconservative model. • The two DNA strands separate. • Each strand is used as a pattern to produce a complementary strand, using specific base pairing. • Each new DNA helix has one old strand with one new strand.

23. Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope. • The first replication produced a band of hybrid DNA, eliminating the conservative model • A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model (see figure on the next page).

24. DNA Replication: A Closer Look • DNA replication proceeds in two directions at many sites simultaneously • DNA replication begins at special sites called the “origins of replication” where • DNA unwinds at the origin to produce a “bubble,” • Replication proceeds in both directions from the origin, and

25. replication ends when products from the bubbles merge with each other. • At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating • DNA replication occurs in the 5´ to 3´ direction. • a. Replication is continuous on the 3´ to 5´ template. • b. Replication is discontinuous on the 5´ to 3´ template, forming short segments

26. Two key proteins are involved in DNA replication. • DNA ligase joins small fragments into a continuous chain. • DNA polymerase adds nucleotides to a growing chain and proofreads and corrects improper base pairings. • Helicases are enzymes that untwist the double helix at the replication forks • Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template • Topoisomerase corrects “over winding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands. It cuts and rejoins the helix

27. The sequence of DNA replication is as follows • Enzyme DNA Helicase unwinds our double helix into two strands. Single-strand binding proteins bind to the unwound DNA strands to keep them separate. • An enzyme called primase start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template to form an initial nucleotide strand that is a short RNA primer.

28. A primer is needed because DNA polymerases do not intitiate synthesis of a polynucleotide. They only add nucleotides to the 3` end. The primer is short (5–10 nucleotides long), and the 3 end on the template serves as the starting point for the new DNA strand. • Enzymes DNA polymerases catalyze the elongation of new DNA at a replication fork. The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells. The incoporation of nucleotides by DNA polymerase is shown in the figure on the right

29. DNA polymerases add nucleotides only to the free 3 end of a growing strand; therefore, a new DNA strand can elongate only in the 5 to 3 direction • Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork

30. To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork. The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase (see figure on left). The proteins that participate in DNA replication form a large complex, a “DNA replication machine

31. Proofreading and Repairing DNA • DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides. DNA polymerases and DNA ligase also repair DNA damaged by harmful radiation and toxic chemicals. • In mismatch repair of DNA, repair enzymes correct errors in base pairing. • DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) • In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA

32. Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes • The usual replication machinery provides no way to complete the 5 • ends, so repeated rounds of replication produce shorter DNA molecules • Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres

33. Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules • It has been proposed that the shortening of telomeres is connected to aging • DNA replication ensures that all the somatic cells in a multicellular organism carry the same genetic information.

34. DNA TRANSCRIPTION & RNA TRANSLATION • The Flow of Genetic Information from DNA to RNA to Protein. • DNA Genotype Expression The DNA genotype is expressed as proteins, which provide the molecular basis for phenotypic traits. • Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation • DNA specifies traits by dictating protein synthesis. • The molecular chain of command is from DNA in the nucleus to RNA and RNA in the cytoplasm to protein.

35. Transcription is the synthesis of RNA under the direction of DNA. • Translation is the synthesis of proteins under the direction of RNA. • The connections between genes and proteins • The initial one gene–one enzyme hypothesis was based on studies of inherited metabolic diseases. • The one gene–one enzyme hypothesis was expanded to include all proteins. • Most recently, the one gene–one polypeptide hypothesis recognizes that some proteins are composed of multiple polypeptides (see figure below).

36. Translation of Codons into Amino Acid Sequence Genetic information written in codons is translated into amino acid sequences Genetic information written in codons is translated into amino acid sequences Protein construction requires a conversion of a nucleotide sequence to an amino acid sequence Transcription rewrites the DNA code into RNA, using the same nucleotide “language

37. TRANSLATION

38. The flow of information from gene to protein is based on a triplet code: The genetic instructions for the amino acid sequence of a polypeptide chain are written in DNA and RNA as a series of non-overlapping three-base “words” called codons. Translation involves switching from the nucleotide “language” to the amino acid “language.” Each amino acid is specified by a codon 64 codons are possible. Some amino acids have more than one possible codon.

39. The Genetic Code • The genetic code dictates how codons are translated into amino acids • Characteristics of the genetic code • Three nucleotides specify one amino acid. • 61 codons correspond to amino acids. • AUG codes for methionine and signals the start of transcription. • 3 “stop” codons signal the end of translation (See the dictionary of the genetic code).

41. The genetic code is • redundant, with more than one codon for some amino acids, • unambiguous in that any codon for one amino acid does not code for any other amino acid, • nearly universal—the genetic code is shared by organisms from the simplest bacteria to the most complex plants and animals, and • without punctuation in that codons are adjacent to each other with no gaps in between

42. DNA Transcription • Transcription produces genetic messages in the form of RNA • An RNA molecule is transcribed from a DNA template by process that resembles the synthesis of a DNA strand during DNA replication. • RNA nucleotides are linked by the transcription enzyme RNA polymerase. • Specific sequences of nucleotides along the DNA mark where transcription begins and ends

43. The “start transcribe” signal is a nucleotide sequence called a promoter. • Transcription begins with initiation, as the RNA polymerase attaches to the promoter. • During the second phase, elongation, the RNA grows longer. As the RNA peels away, the DNA strands rejoin. • Finally, in the third phase, termination, the RNA polymerase reaches a sequence of bases in the DNA template called a terminator, which signals the end of the gene. • The polymerase molecule now detaches from the RNA molecule and the gene.

44. RNA Processing • Eukaryotic RNA is processed before leaving the nucleus as mRNA • Messenger RNA (mRNA) encodes amino acid sequences and conveys genetic messages from DNA to the translation machinery of the cell, which in • prokaryotes occurs in the same place that mRNA is made • but in eukaryote, mRNA must exit the nucleus via nuclear pores to enter the cytoplasm

45. Eukaryotic mRNA has introns, interrupting sequences that separate exons, the coding regions. • Eukaryotic mRNA undergoes processing before leaving the nucleus. • RNA splicing removes introns and joins exons to produce a continuous coding sequence. • A cap and tail of extra nucleotides are added to the ends of the mRNA to • facilitate the export of the mRNA from the nucleus, • protect the mRNA from attack by cellular enzymes, and iii. help ribosomes bind to the mRNA.

46. tRNA Molecules Transfer RNA molecules serve as interpreters during translation • A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long • Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf. Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule tRNA is roughly L-shaped as shown in the figure below • Transfer RNA (tRNA) molecules function as a language interpreter, converting the genetic message of mRNA into the language of proteins.

47. Transfer RNA molecules perform this interpreter task by picking up the appropriate amino acid and using a special triplet of bases, called an anticodon, to recognize the appropriate codons in the mRNA. • Accurate translation requires two steps: – First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNAsynthetase – Second: a correct match between the tRNA anticodon and an mRNA codon

48. VIRAL GENOME • The viral genome is the complete genetic complement contained in a DNA or RNA molecule in a virus. • Viral DNA may become part of the host chromosome • A virus is essentially “genes in a box ,” an infectious particle consisting of a bit of nucleic acid, wrapped in a protein coat called a capsid and in some cases, a membrane envelop.

49. Reproductive cycle of viruses • Viruses have two types of reproductive cycles. • In the lytic cycle, • viral particles are produced using host cell components, • the host cell lyses, and • viruses are released

50. Lysogenic cycle • Viral DNA is inserted into the host chromosome by recombination. • Viral DNA is duplicated along with the host chromosome during each cell division. • The inserted phage DNA is called a prophage. • Most prophage genes are inactive.