WEEK 13

1. WEEK 13 Molecular Biology of the Gene Chapter 12 Pages 211-232 EXAM 4 is MAY 24th at 6pm!!

2. Last Week Mendelian Patterns of Inheritance Chromosome level Genotypes and Phenotypes This Week Understanding inheritance of genes at the molecular level How does DNA become expressed to form the over 1.5 million different species that have been discovered?

3. DNA=Genetic Material How did early research scientists find out that DNA, and not for ex. Protein, is the genetic material that gets passed down from generation to generation? During the 20th Century, researchers knew that Genetic material must be: • able to store information • stable in order to be replicated and passed from generation to generation • able to withstand changes/mutations  genetic variability (important for evolution)

4. Late 1920s Frederick Griffith Conducted series of experiments to find vaccine for Streptococcus pneumoniae (pneumococcus) = causes pneumonia in mammals Experiment: • Grew the bacteria on culture plates • Two strains ‘S’ (smooth colonies) and ‘R’ (rough colonies)

5. Griffith’s Transformation Experiment a. S strain is encapsulated and KILLS mice (virulent) • R strain is nonencapsulated and is nonvirulent • Heat-killed S strain do not kill mice • Heat-killed S and R strain together kill mice b/c R strain is transformed into the S strain

6. Griffith’s Results and Conclusions -Some kind of transfer between the S strain and the R strain occurred for the live S strain to be able to be removed from the dead mouse. -What was the transforming substance? How did the S strain become “alive” again in the presence of the R strain even when the S strain was heat killed?

7. DNA: The Transforming Substance • Researcher Oswald Avery and others were trying to determine if Protein or DNA was the genetic material responsible for transformation of one type of bacteria to another. • Proteins = 20 amino acids • DNA = 4 nucleotides • It was believed that protein was the genetic material because it had more variability to more genetic information Conducted his research at Rockefeller U. in NYC!!

8. Avery’s Experimentw/collaborators MacLeod and McCarty In vitro experiments Rnase= Enzyme that degrades RNA Protease =Enzyme that degrades Protein Dnase = Enzyme that degrades DNA

9. Avery’s Conclusions • DNA from the S strain causes R strain bacteria to be transformed which enables the R strain to form a capsule = virulent (this DNA codes for the capsule!) • Adding DNase prevents transformation! Therefore, DNA must be the material that transforms! • The molecular weight of the transforming DNA is equivalent to 1,600 nucleotides, enough for genetic variation • Addition of enzymes that degrade protein and RNA had no effect on ability of the R strain to transform to the S strain  neither protein nor RNA is the genetic material • DNA IS THE GENETIC MATERIAL!!

10. Transformation and Biology Today • Transformation = transfer of one gene from one type of organism to another - These modified organisms are called ‘Genetically modified organisms’ (GMOs) or transgenics • Foods like corn, tomatoes, cotton and rice are GMOs and are altered for pest resistance or longer shelf lives • Green fluorescent protein (GFP) sequences can be isolated and cloned from Jelly Fish and transferred to other organisms which causes these organisms to glow

11. GFP Trangenics Ex. Fish, pigs, mice (gene for GFP is inserted before a promoter that ‘turns the gene on’ and transcribes and translates the gene to produce the GF protein) We use GFP transgenics to understand development!

12. The Structure of DNA The nucleotide content of DNA is not fixed and varies greatly between species Ex. E. coli and Zeamays(corn) have approx. 25% of each type of nucleotide However, within a species, for example, humans only 0.1% of nucleotide bases differ 3 billion base pairs x .001 = 3 million base pair differences

13. The Structure of DNA The sugar in DNA is ‘deoxy’ b/c it lacks an oxygen at the 2’ Carbon

14. Chargaff’s Rules The percentage of A always equals the percentage of T The percentage of C always equals the percentage of G • Chargaff’s rules: • The amount of A, T, G and C in DNA varies from species to species • 2. In each species, the amount of A=T and the amount of C=G Complementary Base Pairing ALWAYS!!!

15. Rosalind Franklin: The Dark Lady of DNA A book by Brenda Maddox Chronicles the life and research of Rosalind Franklin Chemist 1920-1958 Studied DNA structure using X-ray crystallography DNA structure was determined by her work with X-ray diffraction patterns Diffraction patterns (in the shape of an X) showed that DNA is a double helix Her research was used without her knowledge or permission, died before nobel prize was awarded to Watson, Crick and Wilkins, little recognition

16. X-Ray Crystallography/Diffraction Crystallized DNA

17. Watson and Crick • Used Franklin’s X-rays to build a double helix structure that fit the mathematical measurements provided by the x-rays • Deduced the spacing between base pairs in order to obtain a complete turn of the double helix • Determined that the DNA strands in the double helix are ANTIPARALLEL

18. ANTIPARALLEL DNA STRANDS The 5’ end of one strand is always parallel to the 3’ end of the other strand!! Structure allows for complementary base pairing

19. 5’3’ DNA is made in a 5’ to a 3’ direction ‘ = prime ‘ Refers to the Carbon position number in the nucleotide

20. Notice the antiparallel structure of DNA The sugars on one side all point up The sugars on the other strand all point down One strand ends 5’ (up) and the other strand ends 3’ (down)

21. CHROMOSOME STRUCTURE • Eukaryotic chromosomes contain a single double helix DNA molecule • Made up of 50% protein • Protein is called Histones • 5 types of histones- H1, H2A, H2B, H3 and H4 • H3 and H4 have little variation between species– highly conserved sequences! • Histones package DNA • Humans have about 2 m of DNA in a 5 micron nucleus

22. NUCELOSOMES: The “beads” of protein (histones) and DNA Resembles a string of pearls

23. EUCHROMATIN HETERCHROMATIN

24. Major Features of the DNA Structure • The DNA strands are antiparallel • The antiparallel nature of the DNA strands allows for the formation of a double helix • DNA is comprised of 4 nucleotide bases • Complimentary base pairing allows for the helical structure of DNA

25. DNA REPLICATION DNA replicates during the S phase of Interphase during mitosis and meiosis Each DNA strand of the double helix acts as a template for a new strand to be formed DNA replication is semi-conservative because each new strand is paired with the old template strand

26. DNA REPLICATION DNA replication requires: • Unwinding- DNA is “unzipped” by helicase via the breaking of the weak H bonds between the nucleotides • Complementary Base Pairing- New nucleotides are always paired in a complementary manner (A=T, C=G), done by DNA Polymerase • Joining- Nucleotides must join together to form new DNA strands, done by DNA Polymerase

27. SEMI-CONSERVATIVE DNA REPLICATION DNA replication must occur before a cell can divide! HOW? Helicase unwinds DNA Each strand acts as a template for the creation of a new strand Complementary nucleotides are added in order of the template sequence from available nucleotides in the cell From one helix, two new ‘daughter’ helices are formed Each helix has 1 new and 1 old strand w/ same sequence as the ‘parent’ strand

28. A Closer Look Carbon atoms are numbered, note the 3’ and 5’ locations 1 Strands run in opposite directions, DNA polymerase attaches a new nucleotide to the 3’ Carbons of the previous nucleotide ONLY! 2 Replication begins when an RNA primer with a sequence that Is complementary to the DNAlays down in the area to be replicated Helicase unwinds the DNA helix at the replication fork 3 The leading strand can immediately begin replication in the 5’ to 3’ direction off of the 3’ end 4

29. Leading and Lagging Strands

30. A Closer Look at DNA Replication The lagging strand forms discontinuously 5 The discontinuous fragments are called Okazaki fragments 6 DNA ligase joins the Okazaki fragments together 7

31. PROKARYOTIC DNA REPLICATION 1 Replication begins at the Origin of replication 4 3 DNA polymerase binds to each side of the circular DNA and replication begins 2 and terminates at the termination region Total replication time = 20-40 minutes!

32. Prokaryotic v Eukaryotic DNA Replication PROKARYOTES Replication can occur in two directions at once because the DNA is circular EUKARYOTES Replication can occur at numerous replication Forks/bubbles and later join together DNA is replicated within hours!

33. Telomeres Telomeres are not copied by DNA polymerase Telomeres are added to chromosomes by an enzyme called TELOMERASE TELOMERASE Adds repeated sequences to the ends of chromosomes Ex. TTAGGG

34. DNA Polymerase DNA polymerase makes a mistake about once per 100,000 base pairs  several errors! Therefore, DNA polymerase is able to proofread the strand that it is creating DNA pol can recognize a mismatched nucleotide and remove it When corrections are accounted for, DNA pol actually only has an error rate of 1 in 1,000,000 base pairs, very accurate!

35. The Central Dogma of Molecular Biology Viruses may or may not follow the central dogma!

37. DNA RNA • 3 CLASSES OF RNA • Messenger RNA (mRNA) • Transfer RNA (tRNA) • Ribsomal RNA (rRNA) RNA carries the information! RNA is single stranded In RNA, U (uracil) replaces T (thymine) found in DNA

38. 3 Classes of RNA mRNA- Messenger RNA -takes message from DNA in the nucleus to the ribosomes in the cytoplasm tRNA- Transfer RNA - pairs with mRNA during translation - transfers amino acids to the ribosomes rRNA- Ribosomal RNA - makes up the ribosomes, along with other proteins, where polypeptides/proteins are made

39. DNA is TRANSCRIBED to RNA is TRANSLATED to PROTEIN DNA segments (nucleotides) code for Protein (polypeptides) 2 steps involved in synthesizing protein 1- Transcription DNA serves as a template for formation of RNA transcripts 2- Translation mRNA transcript directs the sequence of amino acids in a polypeptide

40. Genes Genes are specific segments of DNA with a particular arrangement of nucleotides Genes are found on chromosomes Genes code for proteins The nucleotide sequence of a gene specifies the order of amino acids in a polypeptide Each gene follows the genetic code for the sequence of an amino acid

41. The Genetic Code The genetic code is a triplet code known as a CODON A codon consists of 3 bases of RNA An amino acid is assigned to each codon Some codons code for the same amino acid (degenerate-amino acids have more than one codon) *protects against mutations!!! Some codonscode for a ‘start’ signal to begin translation and some codons code for ‘stop’end translation. There is 1 start codon (AUG) and 3 stop codons (UAA, UGA, UAG)

44. The Genetic Code The genetic code is Universal to all living organisms (mitochondria and chloroplasts differ slightly from this code) Universality of the code provides strong evidence that we all share a common evolutionary heritage! Because organisms share the same code, transfer of genes is possible between species

45. Transcription DNA  RNA Translation RNA  Protein Occurs in the nucleus Occurs in the cytoplasm

46. 1. mRNA Transcription Complementary RNA is made from a DNA template Strand The gene sequence is identified on the DNA helix DNA unzips (Helicase) RNA polymerase attaches to a region of DNA called a Promoter = INITIATION The promoter sequence indicates where to begin trans- cription RNA polymerase joins nucleotides together in the 5’ to 3’ direction = ELONGATION Elongation occurs until a stop sequence causes RNA polymerase to stop transcribing and to fall away from the DNA template strand The new mRNA transcript is released

47. mRNA Processing After transcription, mRNA is further processed The mRNA transcript is called, “Pre-mRNA” The pre-mRNA receives a 5’ Cap and a 3’ poly-A tail (The cap is a modified guanine (G) that tells the ribosome where to attach when translation begins The poly-A tail is 150-200 Adenines (A) which facilitates the transport of the mRNA out of the nucleus) Introns and exons. EXONS are EXPRESSED, INTRONS are removed and are NOT expressed. Intronss are spliced from the pre-mRNA by SPLICEOSOMES (spliceosomes contain small, nuclear RNAs (snRNAs)) snRNAs recognize which introns to remove snoRNAs (small nucleolar RNAs help process rRNA and tRNA)

48. Alternative mRNA Splicing Not all exons stay in a pre-mRNA Some mRNAs contain only some of the possible exons that are available from a DNA sequence Therefore, exons are ‘alternatively spliced’ b/c what is an exon in one mRNA could be an intron in another mRNA

49. Why have introns anyway? Introns were originally thought of as ‘junk DNA’ because it was spliced out Some introns give rise to microRNAs(miRNAs) which regulate the translation of mRNAs miRNAs can bond with mRNAs via complementary base pairing and prevent translation from occurring Introns are thought to encourage crossing-over during meiosis  exon shuffling  evolution of new genes

50. Transcription DNA  RNA Translation RNA  Protein Occurs in the nucleus Occurs in the cytoplasm