The Molecular Basis of Inheritance

Transcription

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

2 Scientific History he march to understanding that DN is the genetic material.h. Morgan (1908) Frederick riffith (1928) very, Mcarty & MacLeod (1944) Erwin hargaff (1947) Hershey & hase (1952) Watson & rick (1953) Meselson & Stahl (1958)

3 hromosomes related to phenotype.h. Morgan s work with Drosophila associated phenotype with specific chromosome (white-eyed male had specific X chromosome)

4 enes are on chromosomes Morgan s conclusions: genes are on chromosomes But which carries the genetic material protein or the DN? Initially proteins were thought to be genetic material Why? What s so impressive about proteins?!

5 Evidence hat DN an ransform Bacteria Frederick riffith was studying Streptococcus pneumoniae (a bacterium that causes pneumonia in mammals) He worked with two strains of the bacterium: a pathogenic ( smooth ) strain and a nonpathogenic ( rough ) strain riffith 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

6 he ransforming Principle mix heat-killed pathogenic & live pathogenic live non-pathogenic heat-killed non-pathogenic strain of bacteria strain of bacteria pathogenic bacteria bacteria. B.. D. mice die mice live mice live mice die ransformation = change in genotype and phenotype due to the assimilation of external DN by a cell

7 Evidence for DN as the ransforming Principle very, Mcarty and MacLeod set out to determine which transform non-pathogenic bacteria purified both DN and proteins separately from Streptococcus pneumonia bacteria injected protein into bacteria no effect injected DN into bacteria transformed harmless bacteria into virulent bacteria

8 dditional Evidence Hershey and hase s classic blender experiment worked with bacteriophage (viruses that infect bacteria) grew phages in 2 media, radioactively labeled with either 35 S in their proteins or 32 P in their DN infected bacteria with labeled phages Why use Sulfur vs. Phosphorus?

9 Hershey & hase Blender 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.

10 Hershey & hase Protein coat labeled with 35 S 2 bacteriophages are labeled with radioactive isotopes S vs. P DN labeled with 32 P bacteriophages infect bacterial cells Which radioactive marker is found inside the cell? bacterial cells are agitated to remove viral protein coats Which molecule carries viral genetic info? 35 S radioactivity found in the medium 32 P radioactivity found in the bacterial cells

11 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 Sugar-phosphate backbone Nitrogenous bases end P H 2 4ʹ H H 1ʹ H H 2ʹ H H H 3 N N H hymine () P H 2 H H H H H P H 2 H H H H H H N N N H N H N H denine () H H H N H N N ytosine () Figure 16.5 P H 2 1ʹ 4ʹ H Phosphate H H 2ʹ H H H Sugar (deoxyribose) end H N N N N H N H H uanine () DN nucleotide

12 Erwin hargaff analyzed the base composition of DN from a number of different organisms In 1947, hargaff reported that: that DN composition varies from one species to the next pointing to molecular diversity among species the 4 bases are not in equal quantity (Ex: in humans, = 30.9%, = 29.4%, = 19.9%, = 19.8%) Rules = = made DN a more credible candidate for the genetic material

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

14 Maurice Wilkins and Rosalind Franklin used a technique called X-ray crystallography to study molecular structure Maurice Wilkins Watson and rick deduced that DN was a double helix through observations of the X-ray crystallographic images of DN (a) Rosalind Franklin (b) Franklin s X-ray diffraction Photograph of DN

15 Franklin had concluded that DN was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule s interior he nitrogenous bases are paired in specific combinations: adenine with thymine, and cytosine with guanine 1 nm 3.4 nm Figure 16.7a, c 0.34 nm (a) Key features of DN structure (c) Space-filling model

16 end H P Hydrogen bond end H P H 2 H 2 P P H 2 H 2 P P H 2 H 2 P Figure 16.7b H end (b) Partial chemical structure H 2 P end

17 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: adenine () and thymine () form two bonds, cytosine () and guanine () form three bonds N Sugar N Sugar Figure 16.8 H N N H H 3 N H N N N Sugar denine () hymine () H N H N N H N N N N H Sugar H uanine () ytosine ()

18 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 he parent molecule unwinds, and two new daughter strands are built based on base-pairing rules (a) he parent molecule has two complementary strands of DN. Each base is paired by hydrogen bonding with its specific partner, with and 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

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

20 Meselson and Stahl Experiments performed by Meselson and Stahl supported the semiconservative model of DN replication 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 2 Bacteria transferred to medium containing 14 N Figure DN sample 4 DN sample centrifuged centrifuged after 20 min after 40 min (after first (after second replication) replication) Less dense More dense 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.

21 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

22 Scientific History.H. Morgan (1908) genes are on chromosomes Frederick riffith (1928) a transforming factor can change phenotype very, Mcarty & MacLeod (1944) transforming factor is DN Erwin hargaff (1947) hargaff rules: =, = Hershey & hase (1952) confirmation that DN is genetic material Watson & rick (1953) determined double helix structure of DN Meselson & Stahl (1958) semi-conservative replication

23 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

24 he replication of a DN molecule begins at origins of replication, where the two strands are separated 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).

25 Elongating a New DN Strand Elongation of new DN at a replication fork is catalyzed by DN polymerases, which add nucleotides to the end of a growing strand New strand end emplate strand end end end Sugar Phosphate Base H P P Pyrophosphate H end Figure Nucleoside triphosphate end 2 P end

26 How does the antiparallel structure of the double helix affect replication? DN polymerases add nucleotides only to the free end of a growing strand long one template strand of DN, DN polymerase III can synthesize a complementary strand, the leading strand, continuously moving toward the replication fork

27 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 (kazaki fragments), which are then joined together by DN ligase

28 Synthesis of leading and lagging strands during DN replication 1 DN pol Ill elongates DN strands only in the direction. Parental DN 2 kazaki fragments DN pol III emplate strand 1 2 ne new strand, the leading strand, can elongate continuously as the replication fork progresses. 3 he other new strand, the lagging strand must grow in an overall direction by addition of short segments, kazaki fragments, that grow (numbered here in the order they were made). 3 Leading strand Lagging strand DN ligase joins kazaki fragments by forming a bond between their free ends. his results in a continuous strand. Figure emplate strand DN ligase verall direction of replication

29 Priming DN Synthesis DN polymerases can N initiate the synthesis of a polynucleotide hey can only add nucleotides to the end he initial nucleotide strand is an RN (or DN) primer nly one primer is needed for synthesis of the leading strand For synthesis of the lagging strand, each kazaki fragment must be primed separately

30 1 Primase joins RN nucleotides into a primer. emplate strand 2 DN pol III adds DN nucleotides to the primer, forming an kazaki fragment. 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. 2 kazaki fragment DN pol 1 replaces the RN with DN, adding to the end of fragment DN ligase forms a bond 7 he lagging strand between the newest DN in this region is now and the adjacent DN of complete. fragment Figure verall direction of replication

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

32 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 direction by DN pol III. DN pol III Lagging strand VERVIEW Leading strand Leading strand 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 1 5 DN pol III is completing synthesis of 6 DN pol I removes the primer from the end 7 DN ligase bonds the fourth fragment, when it reaches the of the second fragment, replacing it with DN the end of the RN primer on the third fragment, it will nucleotides that it adds one by one to the end second fragment to dissociate, move to the replication fork, of the third fragment. he replacement of the the end of the first and add DN nucleotides to the end last RN nucleotide with DN leaves the sugar- fragment. of the fifth fragment primer. phosphate backbone with a free end. Figure 16.16

33 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 he DN replication machine is probably stationary during the replication process

34 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

35 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.

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

37 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

38 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 catalyzes the lengthening of telomeres in germ cells