The Molecular Basis of Inheritance

Transcription

1 hapter 6 he Molecular Basis of Inheritance Life s perating Instructions In 953, James Watson and Francis rick shook the world With an elegant double-helical model for the structure of deoxyribonucleic acid, or D oweroint Lectures for Biology, Seventh Edition eil ampbell and Jane Reece Lectures by hris Romero Figure 6. D, the substance of inheritance ereditary information Is encoded in the chemical language of D and reproduced in all the cells of your body D odes for the development of and the traits of all organisms. What are some traits you inherited from your parents? Your grandparents? ow did they figure it out? 6.: D is the genetic material Early in the 0th century he identification of the molecules of inheritance loomed as a major challenge to biologists Who was the guy who worked out heredity? he Search for the enetic Material: Scientific Inquiry he role of D in heredity Was first worked out by studying bacteria and the viruses that infect them Evidence hat D an ransform Bacteria Frederick riffith was studying Streptococcus pneumoniae bacterium that causes pneumonia in mammals e worked with two strains of the bacterium pathogenic strain and a nonpathogenic strain

2 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 EXERIME 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 riffith injected mice with the two strains as shown below: riffith called the phenomenon transformation ow defined as a change in genotype and phenotype due to the assimilation of external D by a cell Living S (control) cells Living R eat-killed Mixture of heat-killed S cells (control) cells (control) S cells and living R cells RESULS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. LUSI Figure 6. riffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. Evidence hat Viral D an rogram ells dditional evidence for D as the genetic material ame from studies of a virus that infects bacteria Viruses that infect bacteria, bacteriophages re widely used as tools by researchers in molecular genetics hage head ail ail fiber D Figure 6.3 Bacterial cell 00 nm lfred ershey and Martha hase erformed experiments showing that D is the genetic material of a phage known as he ershey and hase experiment EXERIME In their famous 95 experiment, lfred ershey and Martha hase used radioactive sulfur and phosphorus to trace the fates of the protein and D, respectively, of phages that infected bacterial cells. Mixed radioactively gitated in a blender to 3 entrifuged the mixture 4 Measured the labeled phages with separate phages outside so that bacteria formed radioactivity in bacteria. he phages the bacteria from the a pellet at the bottom of the pellet and infected the bacterial cells. bacterial cells. the test tube. the liquid Radioactive Empty Radioactivity hage protein protein shell (phage protein) in liquid Bacterial cell Batch : hages were grown with radioactive sulfur ( 35 S), which was incorporated into phage protein (pink). D Radioactive D hage D entrifuge ellet (bacterial cells and contents) Batch : hages were grown with radioactive phosphorus ( 3 ), which was incorporated into phage D (blue). entrifuge Radioactivity (phage D) ellet in pellet RESULS hage proteins remained outside the bacterial cells during infection, while phage D entered the cells. When cultured, bacterial cells with radioactive phage D released new phages with some radioactive phosphorus. Figure 6.4 LUSI ershey and hase concluded that D, not protein, functions as the phage s genetic material.

3 dditional Evidence hat D Is the enetic Materia rior to the 950s, it was already known that D Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a sugar, and a phosphate group Figure 6.5 Sugar-phosphate backbone end 4 4 hosphate Sugar (deoxyribose) end itrogenous bases 3 hymine () denine () ytosine () uanine () D nucleotide Erwin hargaff analyzed the base composition of D From a number of different organisms e found that D composition varies from one species to the next nd that the four bases (,,,) had corresponding frequencies in every organism e.g., nimal X denine-hymine (30% to 30%), uanine-ytosine (0% to 0%) Building a Structural Model of D: Scientific Inquiry nce most biologists were convinced that D was the genetic material RMSME Y he challenge was to determine how the structure of D could account for its role in inheritance f particular curiosity was the Y chromosome, which later was found to contain the following genes encoding for male traits Maurice Wilkins and Rosalind Franklin Were using a technique called X-ray crystallography to study molecular structure Rosalind Franklin roduced a picture of the D molecule using this technique Watson and rick deduced that D was a double helix hrough observations of the X-ray crystallographic images of D nm 3.4 nm Figure 6.6 a, b (a) Rosalind Franklin (b) Franklin s X-ray diffraction hotograph of D Figure 6.7a, c 0.34 nm (a) Key features of D structure (c) Space-filling model 3

4 Franklin had concluded that D end 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 ydrogen bond end Figure 6.7b end (b) artial chemical structure end Watson and rick reasoned that there must be additional specificity of pairing 3 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 Sugar denine () Sugar Sugar hymine () Sugar Figure 6.8 uanine () ytosine () he Basic rinciple: Base airing to a emplate Strand Since the two s of D are complementary Each acts as a template for building a new in replication In D replication he parent molecule unwinds, and two new daughter s are built based on basepairing rules lay the D helix game: ine/dna_double_helix/ (a) he parent molecule has two complementary s of D. 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 D s. (c) Each parental now serves as a template that determines the order of nucleotides along a new, complementary. (d) he nucleotides are connected to form the sugar-phosphate backbones of the new s. Each daughter D molecule consists of one parental and one new. Figure 6.9 a d 4

5 D replication is semiconservative Each of the two new daughter molecules will have one old, derived from the parent molecule, and one newly made Figure 6.0 a c arent cell onservative model. he two parental s reassociate after acting as templates for new s, thus restoring the parental double helix. (b) Semiconservative model. he two s of the parental molecule separate, and each functions as a template for synthesis of a new, complementary. (c) Dispersive model. Each of both daughter molecules contains a mixture of old and newly synthesized D. (a) First replication Second replication Experiments performed by Meselson and Stahl Supported the semiconservative model of D replication EXERIME 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, 5. he bacteria incorporated the heavy nitrogen into their D. he scientists then transferred the bacteria to a medium with only 4, the lighter, more common isotope of nitrogen. ny new D that the bacteria synthesized would be lighter than the parental D made in the 5 medium. Meselson and Stahl could distinguish D of different densities by centrifuging D extracted from the bacteria. Bacteria Bacteria cultured in transferred to medium medium containing containing 5 4 RESULS Less 3 D sample 4 D sample dense centrifuged centrifuged after 0 min after 40 min (after first (after second More replication) replication) dense he bands in these two centrifuge tubes represent the results of centrifuging two D samples from the flask Figure 6. in step, one sample taken after 0 minutes and one after 40 minutes. LUSI Meselson and Stahl concluded that D replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 6.0. he first replication in the 4 medium produced a band of hybrid ( 5 4 ) D. his result eliminated the conservative model. second replication produced both light and hybrid D, a result that eliminated the dispersive model and supported the semiconservative model. First replication Second replication onservative model Semiconservative model D Replication: loser Look he copying of D Is remarkable in its speed and accuracy More than a dozen enzymes and other proteins articipate in D replication ecularbiology/dreplication.swf Dispersive model etting Started: rigins of Replication he replication of a D molecule Begins at special sites called origins of replication, where the two s are separated eukaryotic chromosome May have hundreds or even thousands of replication origins rigin of replication arental (template) Daughter (new) 0.5 µm Replication begins at specific sites where the two parental s separate and form replication bubbles. he bubbles expand laterally, as D replication proceeds in both directions. Bubble Replication fork 3 Eventually, the replication bubbles fuse, and synthesis of the daughter s is complete. wo daughter D molecules (a) In eukaryotes, D replication begins at many sites along the giant D molecule of each chromosome. Figure 6. a, b (b) In this micrograph, three replication bubbles are visible along the D of a cultured hinese hamster cell (EM). 5

6 Elongating a ew D Strand Elongation of new D at a replication fork Is catalyzed by enzymes called D polymerases, which add nucleotides to the end of a growing ew emplate end end end end ntiparallel Elongation ow does the antiparallel structure of the double helix affect replication? Sugar hosphate Base Figure 6.3 ucleoside triphosphate yrophosphate end end end D polymerases add nucleotides nly to the free end of a growing long one template of D, the leading D polymerase III can synthesize a complementary continuously, moving toward the replication fork o elongate the other new of D, the lagging D polymerase III must work in the direction away from the replication fork he lagging Is synthesized as a series of segments called kazaki fragments, which are then joined together by D ligase Synthesis of leading and lagging s during D replication D pol Ill elongates D s only in the direction. ne new, the leading, arental D can elongate continuously as the replication fork progresses. kazaki 3 he other new, the fragments lagging must grow in an overall direction by addition of short segments, kazaki fragments, that grow (numbered here in the order D pol III they were made). riming D Synthesis D polymerases cannot initiate the synthesis of a polynucleotide hey can only add nucleotides to the end he initial nucleotide Is an R or D primer emplate Leading Lagging 3 4 D ligase joins kazaki fragments by forming a bond between their free ends. his results in a continuous. emplate D ligase Figure 6.4 verall direction of replication 6

7 nly one primer is needed for synthesis of the leading rimase joins R nucleotides into a primer. emplate D pol III adds D nucleotides to the primer, forming an kazaki fragment. But for synthesis of the lagging, each kazaki fragment must be primed separately R primer 3 fter reaching the next R primer (not shown), D pol III falls off. 4 fter the second fragment is primed. D pol III adds D nucleotides until it reaches the first primer and falls off. kazaki fragment 5 D pol replaces the R with D, adding to the end of fragment. 6 D ligase forms a bond 7 he lagging between the newest D in this region is now and the adjacent D of complete. fragment. Figure 6.5 verall direction of replication ther roteins hat ssist D Replication elicase, topoisomerase, single- binding protein are all proteins that assist D replication ype II opoisomerases ass D Strands hrough ne nother able 6. Stabilization of the cleavage complex for (topo I or) topo II causes D damage that is difficult to repair due to the large protein molecule attached to the broken D. summary of D replication verall direction of replication Leading Lagging elicase unwinds the rigin of replication parental double helix. Molecules of single binding protein synthesized continuously in the 3 he leading is stabilize the unwound direction by D pol III. Lagging Leading template s. VERVIEW D pol III he D Replication Machine as a Stationary omplex he various proteins that participate in D replication Form a single large complex, a D replication machine arental D 4 rimase begins synthesis of R primer for fifth kazaki fragment. Replication fork rimase D pol III rimer Lagging 4 Leading D pol I 3 D ligase he D replication machine Is probably stationary during the replication process 5 D pol III is completing synthesis of 6 D pol I removes the primer from the end 7 D ligase bonds the fourth fragment, when it reaches the of the second fragment, replacing it with D the end of the R 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 D nucleotides to the end last R nucleotide with D leaves the sugarphosphate backbone with a free end. fragment. of the fifth fragment primer. Figure 6.6 7

8 roofreading and Repairing D D polymerases proofread newly made D Replacing any incorrect nucleotides In mismatch repair of D Repair enzymes correct errors in base pairing In nucleotide excision repair Enzymes cut out and replace damaged stretches of D thymine dimer distorts the D molecule. nuclease enzyme cuts the damaged D at two points and the damaged section is removed. uclease D polymerase 3 Repair synthesis by a D polymerase fills in the missing nucleotides. Figure 6.7 D ligase 4 D ligase seals the Free end of the new D o the old D, making the complete. Replicating the Ends of D Molecules he ends of eukaryotic chromosomal D et shorter with each round of replication End of parental Leading D s Lagging Last fragment revious fragment Eukaryotic chromosomal D molecules ave at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of D molecules Lagging rimer removed but cannot be replaced with D because no end available for D polymerase R primer Removal of primers and replacement with D where a end is available Second round of replication ew leading ew lagging Further rounds of replication Shorter and shorter Figure 6.8 daughter molecules Figure 6.9 µm 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 8