The Molecular Basis of Inheritance

Transcription

1 hapter 16 he Molecular Basis of Inheritance Edited by Shawn Lester PowerPoint Lectures for Biology, Seventh Edition Neil ampbell and Jane Reece Lectures by hris Romero

2 verview: Life s perating Instructions In 1953, James Watson and Francis rick shook the world With an elegant double-helical model for the structure of deoxyribonucleic acid, or DN Figure 16.1

3 DN, the substance of inheritance Is the most celebrated molecule of our time Hereditary information Is encoded in the chemical language of DN and reproduced in all the cells of your body It is the DN program hat directs the development of many different types of traits

4 oncept 16.1: DN is the genetic material Early in the 20th century he identification of the molecules of inheritance loomed as a major challenge to biologists

5 he Search for the Genetic Material: Scientific Inquiry he role of DN in heredity Was first worked out by studying bacteria and the viruses that infect them

6 Evidence hat DN an ransform Bacteria Frederick Griffith was studying Streptococcus pneumoniae bacterium that causes pneumonia in mammals He worked with two strains of the bacterium pathogenic strain and a nonpathogenic strain

7 Griffith found that when he mixed heat-killed remains of the pathogenic strain With living cells of the nonpathogenic strain, some of these living cells became pathogenic EXPERIMEN Bacteria of the S (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal s defense system. Bacteria of the R (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Living S (control) cells Living R Heat-killed (control) cells (control) S cells Mixture of heat-killed S cells and living R cells RESULS Mouse dies Mouse healthy Mouse healthy Mouse dies Figure 16.2 Living S cells are found in blood sample. NLUSIN Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells.

8 Griffith called the phenomenon transformation Now defined as a change in genotype and phenotype due to the assimilation of external DN by a cell

9 Evidence hat Viral DN an Program ells dditional evidence for DN as the genetic material ame from studies of a virus that infects bacteria

10 Viruses that infect bacteria, bacteriophages re widely used as tools by researchers in molecular genetics Phage head ail ail fiber DN Figure 16.3 Bacterial cell 100 nm

11 lfred Hershey and Martha hase Performed experiments showing that DN is the genetic material of a phage known as 2

12 he Hershey and hase experiment EXPERIMEN In their famous 1952 experiment, lfred Hershey and Martha hase used radioactive sulfur and phosphorus to trace the fates of the protein and DN, respectively, of 2 phages that infected bacterial cells. 1 Mixed radioactively 2 gitated in a blender to 3 entrifuged the mixture 4 Measured the labeled phages with bacteria. he phages infected the bacterial cells. separate phages outside the bacteria from the bacterial cells. so that bacteria formed a pellet at the bottom of the test tube. radioactivity in the pellet and the liquid Phage Bacterial cell Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Batch 1: Phages were grown with radioactive sulfur ( 35 S), which was incorporated into phage protein (pink). DN Radioactive DN Phage DN entrifuge Pellet (bacterial cells and contents) Figure 16.4 Batch 2: Phages were grown with radioactive phosphorus ( 32 P), which was incorporated into phage DN (blue). entrifuge RESULS Phage proteins remained outside the bacterial cells during infection, while phage DN entered the cells. When cultured, bacterial cells with radioactive phage DN released new phages with some radioactive phosphorus. NLUSIN Pellet Radioactivity (phage DN) in pellet Hershey and hase concluded that DN, not protein, functions as the 2 phage s genetic material.

13 dditional Evidence hat DN Is the Genetic Materia Prior to the 1950s, it was already known that DN Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a sugar, and a phosphate group Figure 16.5 Sugar-phosphate backbone 5 end 5 P H 2 4 H H 1 H H 3 2 H P H 2 H H H H H P H 2 H H H H H 5 P H H Phosphate H H 3 2 H H H Sugar (deoxyribose) 3 end H Nitrogenous bases H 3 N N H hymine () H N N N H N H N H denine () H H H N H N N ytosine () H N N N N H N H H Guanine (G) DN nucleotide

14 Erwin hargaff analyzed the base composition of DN From a number of different organisms In 1947, hargaff reported hat DN composition varies from one species to the next his evidence of molecular diversity among species Made DN a more credible candidate for the genetic material

15 Building a Structural Model of DN: Scientific Inquiry nce most biologists were convinced that DN was the genetic material he challenge was to determine how the structure of DN could account for its role in inheritance

16 Maurice Wilkins and Rosalind Franklin Were using a technique called X-ray crystallography to study molecular structure Rosalind Franklin Produced a picture of the DN molecule using this technique Her research was instrumental in determining DN structure. - She did not receive a Nobel prize! (a) Rosalind Franklin Figure 16.6 a, b (b) Franklin s X-ray diffraction Photograph of DN

17 Watson and rick deduced that DN was a double helix hrough observations of the X-ray crystallographic images of DN G 1 nm G G 3.4 nm G Figure 16.7a, c G 0.34 nm (a) Key features of DN structure (c) Space-filling model

18 Franklin had concluded that DN Was composed of two antiparallel sugarphosphate backbones, with the nitrogenous bases paired in the molecule s interior he nitrogenous bases re paired in specific combinations: adenine with thymine, and cytosine with guanine

19 5 end H P Hydrogen bond 3 end H P H 2 G H 2 P P H 2 G H 2 P P H 2 H 2 P Figure 16.7b H 3 end (b) Partial chemical structure H 2 P 5 end

20 Watson and rick reasoned that there must be additional specificity of pairing Dictated by the structure of the bases Each base pair forms a different number of hydrogen bonds denine and thymine form two bonds, cytosine and guanine form three bonds

21 H N N H H 3 N N H N Sugar N N denine () Sugar hymine () H N H N N N H N Sugar N N N H Sugar Figure 16.8 H Guanine (G) ytosine ()

22 oncept 16.2: Many proteins work together in DN replication and repair he relationship between structure and function Is manifest (clear or obvious) in the double helix

23 he Basic Principle: Base Pairing to a emplate Strand Since the two strands of DN are complementary Each strand acts as a template for building a new strand in replication

24 In DN replication he parent molecule unwinds, and two new daughter strands are built based on basepairing rules G G G G G G G G G G G G (a) he parent molecule has two complementary strands of DN. Each base is paired by hydrogen bonding with its specific partner, with and G with. (b) he first step in replication is separation of the two DN strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) he nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each daughter DN molecule consists of one parental strand and one new strand. Figure 16.9 a d

25 DN replication is semiconservative Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand Parent cell (a) onservative model. he two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. First replication Second replication (b) Semiconservative model. he two strands of the parental molecule separate, and each functions as a template for synthesis of a new, complementary strand. Figure a c (c) Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DN.

26 Experiments performed by Meselson and Stahl Supported the semiconservative model of DN replication Figure EXPERIMEN Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15 N. he bacteria incorporated the heavy nitrogen into their DN. he scientists then transferred the bacteria to a medium with only 14 N, the lighter, more common isotope of nitrogen. ny new DN that the bacteria synthesized would be lighter than the parental DN made in the 15 N medium. Meselson and Stahl could distinguish DN of different densities by centrifuging DN extracted from the bacteria. 1 RESULS Bacteria cultured in medium containing 15 N 3 DN sample 4 DN sample centrifuged centrifuged after 20 min after 40 min (after first (after second replication) replication) he bands in these two centrifuge tubes represent the results of centrifuging two DN samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes. 2 Bacteria transferred to medium containing 14 N Less dense More dense

27 NLUSIN Meselson and Stahl concluded that DN replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure he first replication in the 14 N medium produced a band of hybrid ( 15 N 14 N) DN. his result eliminated the conservative model. second replication produced both light and hybrid DN, a result that eliminated the dispersive model and supported the semiconservative model. First replication Second replication onservative model Semiconservative model Dispersive model

28 DN Replication: loser Look he copying of DN Is remarkable in its speed and accuracy More than a dozen enzymes and other proteins Participate in DN replication DN Helicase DN Polymerase DN clamp Single-Strand Binding (SSB) Proteins opoisomerase DN Gyrase DN Ligase Primase elomerase

29 Getting Started: rigins of Replication he replication of a DN molecule Begins at special sites called origins of replication, where the two strands are separated

30 eukaryotic chromosome May have hundreds or even thousands of replication origins rigin of replication Parental (template) strand Daughter (new) strand 0.25 µm 1 Replication begins at specific sites where the two parental strands separate and form replication bubbles. Bubble Replication fork 2 he bubbles expand laterally, as DN replication proceeds in both directions. 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. wo daughter DN molecules (a) In eukaryotes, DN replication begins at many sites along the giant DN molecule of each chromosome. Figure a, b (b) In this micrograph, three replication bubbles are visible along the DN of a cultured hinese hamster cell (EM).

31 Elongating a New DN Strand Elongation of new DN at a replication fork Is catalyzed by enzymes called DN polymerases, which add nucleotides to the 3 end of a growing strand New strand emplate strand 5 end 3 end 5 end 3 end Sugar Phosphate Base G G G G Figure H Nucleoside triphosphate P P H Pyrophosphate3 end 2 P 5 end 5 end

32 ntiparallel Elongation How does the antiparallel structure of the double helix affect replication?

33 DN polymerases add nucleotides nly to the free 3 end of a growing strand long one template strand of DN, the leading strand DN polymerase III can synthesize a complementary strand continuously, moving toward the replication fork

34 o elongate the other new strand of DN, the lagging strand DN polymerase III must work in the direction away from the replication fork he lagging strand Is synthesized as a series of segments called kazaki fragments, which are then joined together by DN ligase

35 Priming DN Synthesis DN polymerases cannot initiate the synthesis of a polynucleotide hey can only add nucleotides to the 3 end he initial nucleotide strand Is an RN or DN primer

36 nly one primer is needed for synthesis of the leading strand But for synthesis of the lagging strand, each kazaki fragment must be primed separately

37 1 Primase joins RN nucleotides into a primer emplate strand 2 DN pol III adds DN nucleotides to the primer, forming an kazaki fragment. 3 RN primer fter reaching the next RN primer (not shown), DN pol III falls off. fter the second fragment is primed. DN pol III adds DN nucleotides until it reaches the first primer and falls off kazaki fragment DN pol 1 replaces the RN with DN, adding to the 3 end of fragment DN ligase forms a bond between the newest DN and the adjacent DN of fragment 1. 7 he lagging strand in this region is now complete Figure verall direction of replication

38 ther Proteins hat ssist DN Replication Helicase, topoisomerase, single-strand binding protein re all proteins that assist DN replication able 16.1

39 summary of DN replication 1 Helicase unwinds the parental double helix. verall direction of replication Leading strand rigin of replication Lagging strand 2 Molecules of singlestrand binding protein stabilize the unwound template strands. 3 he leading strand is synthesized continuously in the 5 3 direction by DN pol III. DN pol III Lagging strand VERVIEW Leading strand Leading strand 5 3 Parental DN 4 Primase begins synthesis of RN primer for fifth kazaki fragment. Replication fork Primase DN pol III Lagging Primer 4 strand 3 DN pol I 2 DN ligase DN pol III is completing synthesis of 6 DN pol I removes the primer from the 5 end 7 DN ligase bonds the fourth fragment, when it reaches the RN primer on the third fragment, it will dissociate, move to the replication fork, and add DN nucleotides to the 3 end of the fifth fragment primer. of the second fragment, replacing it with DN nucleotides that it adds one by one to the 3 end of the third fragment. he replacement of the last RN nucleotide with DN leaves the sugarphosphate backbone with a free 3 end. the 3 end of the second fragment to the 5 end of the first fragment. Figure 16.16

40 he DN Replication Machine as a Stationary omplex he various proteins that participate in DN replication Form a single large complex, a DN replication machine (replisome) he DN replication machine Is probably stationary during the replication process

41 Proofreading and Repairing DN DN polymerases proofread newly made DN Replacing any incorrect nucleotides In mismatch repair of DN Repair enzymes correct errors in base pairing

42 In nucleotide excision repair Enzymes cut out and replace damaged stretches of DN 1 thymine dimer distorts the DN molecule. 2 nuclease enzyme cuts the damaged DN strand at two points and the damaged section is removed. Nuclease DN polymerase 3 Repair synthesis by a DN polymerase fills in the missing nucleotides. Figure DN ligase 4 DN ligase seals the Free end of the new DN o the old DN, making the strand complete.

43 Replicating the Ends of DN Molecules he ends of eukaryotic chromosomal DN Get shorter with each round of replication End of parental DN strands 5 3 Leading strand Lagging strand 5 Lagging strand 3 Primer removed but cannot be replaced with DN because no 3 end available for DN polymerase 3 Last fragment RN primer 5 Previous fragment Removal of primers and replacement with DN where a 3 end is available Second round of replication Figure New leading strand 3 New lagging strand Shorter and shorter daughter molecules Further rounds of replication

44 Eukaryotic chromosomal DN molecules Have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DN molecules Figure µm

45 If the chromosomes of germ cells became shorter in every cell cycle Essential genes would eventually be missing from the gametes they produce n enzyme called telomerase atalyzes the lengthening of telomeres in germ cells, fetal cells, and cancer cells type of reverse transcriptase!

46 Somatic cells have finite number of cell divisions (40-60?) Hayflick limit elomeres shorten with each cell division When these cells reach their limit, they enter senescence and stop dividing, then they will die elomere length limits human lifespan to maximum of ~125 years ould telomerase lead to immortality?