Biochemical Identification of the Genetic Material. Griffith s bacterial transformation Key Concepts:

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1 Chapter ucleic Acid Structure, DA Replication, and Chromosome Structure Biochemical Identification of the Genetic Material Key Concepts: n Biochemical Identification of the Genetic Material n ucleic Acid Structure n An verview of DA Replication n Molecular Mechanism of DA Replication n Molecular Structure of Eukaryotic Chromosomes n n n n n What is the genetic material? Four criteria necessary for genetic material:. Information. Replication. Transmission 4. Variation Late 800s biochemical basis of heredity postulated Researchers became convinced that chromosomes carry the genetic information 90s to 940s scientists expected the protein portion of chromosomes would turn out to be the genetic material Griffith s bacterial transformation n Late 90s Frederick Griffith was working with Streptococcus pneumoniae bacteria n Two strains of S. pneumoniae: q Strains that secrete capsules look smooth (S) and infections are fatal in mice q Strains that do not secrete capsules look rough (R) and infections are not fatal in mice n The capsule shields the bacteria from the immune system, so they survive in the blood n Smooth strains (S) with capsule are fatal; rough strains (R) without capsule are not n If mice are injected with heat-killed type S, they survive (because bacteria are dead) n owever, mixing live R with heat-killed S kills the mouse q Blood is found to contain living type S bacteria q Known as transformation Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Control: Type S cells Injected living are virulent. type S bacteria into mouse. Control: Type R cells Injected living are benign. type R bacteria into mouse. 4

2 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Treatment Result Conclusion Control: Injected living type S bacteria into mouse. Type S cells are virulent. n ow is this possible? Control: Injected living type R bacteria into mouse. Control: Injected heatkilled type S bacteria into mouse. Type R cells are benign. eat-killed type S cells are benign. n Genetic material had been transferred from the heat-killed type S bacteria to the living type R bacteria n This gave them the capsule-secreting trait and was passed on to their offspring 4 Injected living type R and heat-killed type S bacteria into mouse. Virulent type S strain in dead mouse s blood Living type R cells have been transformed into virulent type S cells by a substance from the heat-killed type S cells. 5 n What was the biochemical basis of this transforming principle? At the time there was no way to know 6 Avery, MacLeod, and McCarty Used Purification Methods to Reveal That DA is the Genetic Material n 940s interest in finding biochemical basis of bacterial transformation n nly purified DA from type S could transform type R n But, purified DA might still contain traces of contamination that may be the transforming principle n Added Dase, Rase and proteases n Rase and protease had no effect n When Dase was added, no transformation took place n Surprising conclusion: DA is the genetic material Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. YPTESIS A purified macromolecule from type S bacteria, which functions as the genetic material, will be able to convert type R bacteria into type S. KEY MATERIALS Type R and type S strains of Streptococcus pneumoniae. Experimental level Conceptual level Purify DA from a type S strain. This involves breaking open cells and separating the DA away from other components by ± Dase centrifugation. ± Rase ± Protease + Type R cells A B C D E Mix the DA extract with type R bacteria. Allow time for the DA to be taken up by the type R cells, converting a few of them to type S. Also, carry out the same steps but add the enzymes Dase, Rase, or protease to the DA extract, which digest DA, RA, and proteins, Add respectively. As a control, don t add A B C D E antibody any DA extract to some type R cells. Control + DA + DA + DA + DA + Dase + Rase + Protease A B C D E Add an antibody, a protein made by the immune system of mammals, that specifically recognizes type R cells that haven t been transformed. The binding of the antibody causes the type R cells to aggregate. A B C D E

3 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. ershey and Chase 4 Remove type R cells by centrifugation. Plate the remaining bacteria (if any) that are in the supernatant onto petri plates. Incubate overnight. Centrifuge Type S cells in supernatant Type R cells in pellet n 95 studied a T virus that infects Escherichia coli q Bacterial virus is known as bacteriophage or phage n Phage coat made entirely of protein n DA found inside capsid 5 TE DATA A B C D E DA Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. DA Control DA extract DA extract + Dase DA extract + Rase DA extract + protease Protein Phage head (capsid) Sheath Tail fiber 6 CCLUSI DA is responsible for transforming type R cells into type S cells. Base plate 7 SURCE Avery,.T., MacLeod, C.M., and McCarty, M Studies on the Chemical ature of the Substance Inducing Transformation of Pneumococcal Types. Journal of Experimental Medicine 79:7 56. (a) Schematic drawing of T bacteriophage E. coli cell T genetic material being 50 nm injected into E. coli (b) An electron micrograph of T bacteriophage infecting E. coli Eye of Science/Photo Researchers, Inc. 0 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. n What does the phage inject into the bacteria DA or protein? n Shear force from a blender separates the phage coat from surface of the bacteria Experiment Experiment E. coli cells were E. coli cells were infected with infected with 5 S-labeled phage P-labeled phage and subjected to and subjected to blender treatment. blender treatment. Bacterial cell Bacterial cell Phage DA P-labeled phage DA 5 S-labeled sheared empty phage Sheared empty phage Using a Geiger counter, determine the amount of radioactivity in the supernatant. Geiger (radioisotope) counter 4 TE DATA n Tag each component with a radioactive label q 5 S labels proteins q P labels DA n Conclusion: DA is the genetic material Transfer to tube and centrifuge. Supernatant has 5 S-labeled empty phage. Pellet has E. coli cells infected with unlabeled phage DA. Transfer to tube and centrifuge. Supernatant has unlabeled empty phage. Pellet has E. coli cells infected with P-labeled phage DA. Total isotope in supernatant (%) Extracellular 5 S 00 Extracellular P 80 Blending removes 80% 60 of 5 S from cells Most of the P (65%) remains with intact cells Agitation time in blender (min)

4 ucleic Acid Structure Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. ucleotides Single strand Levels of DA Structure:. ucleotides the building blocks of DA and RA. Strand a linear polymer strand of DA or RA. Double helix the two strands of DA 4. Chromosomes DA associated with an array of different proteins into a complex structure 5. Genome the complete complement of genetic material in an organism Double helix DA associates with proteins to form a chromosome. 4 DA DA nucleotides n Formed from nucleotides (A, G, C, T) n ucleotides composed of three components q Phosphate group q Pentose sugar n Deoxyribose n DA = Deoxyribonucleic Acid q itrogenous base n Purines Adenine (A), Guanine (G) Phosphate n Pyrimidines Cytosine (C), Thymine (T) Deoxyribose Base P C Phosphate (a) DA nucleotide Deoxyribose Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Base Purines (double ring) Adenine (A) Guanine (G) Pyrimidines (single ring) C Thymine (T) Cytosine (C) 5 6 4

5 RA n Formed from nucleotides (A, G, C, U) RA nucleotides Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. n ucleotides composed of three components q Phosphate group q Pentose sugar n Ribose n RA = Ribonucleic Acid q itrogenous base n Purines Adenine (A), Guanine (G) n Pyrimidines Cytosine (C), Uracil (U) Phosphate Ribose Base P Base C Phosphate Ribose (b) RA nucleotide Adenine (A) Guanine (G) Uracil (U) Cytosine (C) 7 8 ucleotide numbering system n Sugar carbons are to 5 n Base attached to carbon on sugar n Phosphate attached to 5 carbon on sugar Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. 4 C 5 Thymine 6 P C 4 Phosphate Deoxyribose 9 Strands n ucleotides are covalently bonded n Phosphodiester bond phosphate group links two sugars n Backbone formed from phosphates and sugars n Bases project away from backbone n Written 5 to n ex: 5 TACG Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Backbone Bases C Thymine (T) P C 4 Adenine (A) P C 4 Cytosine (C) Phosphodiester linkage P C 4 Guanine (G) P C Single nucleotide 4 Phosphate Sugar (deoxyribose) 0 5

6 Solving the structure of DA n 95, James Watson and Francis Crick, proposed the structure of the DA double helix n Watson and Crick used Linus Pauling s method of working out protein structures using simple ball-and-stick models n Rosalind Franklin s X-ray diffraction results were crucial evidence, suggesting a helical structure with uniform diameter X-rays diffracted by DA onto photographic plate Pattern represents the atomic array in wet fibers Wet DA fibers X-ray beam Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Base-pairing n Erwin Chargoff analyzed base composition of DA from many different species n Results consistently showed amount of adenine (A) = amount of thymine (T) amount of cytosine (C) = amount of guanine (G) 4 6

7 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Watson and Crick n Put together these pieces of information n Found ball-and-stick model consistent with data q Double-stranded helix q Base-pairing: A with T and G with C Features of DA n Double stranded n Antiparallel strands n Right-handed helix n Sugar-phosphate backbone Complete turn of the helix.4 nm end end Bases ydrogen bond Sugar-phosphate backbone n James Watson, Francis Crick, and Maurice Wilkins awarded obel Prize in 96 n Bases on the inside n Stabilized by -bonding ne nucleotide 0.4 nm n Rosalind Franklin had died and the obel Prize is not awarded posthumously 5 n Specific base-pairing n ~0 nts per helical turn (a) Double helix end end nm 6 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. end end phosphate Adenine C P P C Guanine C Thymine C P P C Guanine Cytosine C P P C n Chargoff s rule q A pairs with T q G pairs with C q Keeps width consistent n Complementary DA strands q 5 GCGGATTT q CGCCTAAA 5 hydroxyl (b) Base pairing Cytosine ydrogen bond end Key Features Two strands of DA form a double helix. The bases in opposite strands hydrogenbond according to the AT/GC rule. The strands are antiparallel. There are ~0 nucleotides in each strand per complete turn of the helix. end n Antiparallel strands q ne strand 5 to q ther stand to 5 8 7

8 n Grooves are revealed in the space-filling model n Major groove q Proteins bind to affect gene expression n Minor groove q arrower Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Major groove Minor groove Major groove Minor groove An verview of DA Replication n Late 950s three different models were proposed for DA replication q Semiconservative Model q Conservative Model q Dispersive Model n ewly-made strands are daughter strands n riginal strands are parental strands 9 0 Semiconservative Mechanism riginal double helix Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. First round of replication Second round of replication Conservative Mechanism riginal double helix Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. First round of replication Second round of replication Parental strand Daughter strand (a) Semiconservative mechanism. DA replication produces DA molecules with parental strand and newly made daughter strand. (b) Conservative mechanism. DA replication produces double helix with both parental strands and the other with new daughter strands. 8

9 Dispersive Mechanism Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. riginal First round Second round double helix of replication of replication riginal double helix Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. First round of replication Second round of replication Parental strand Daughter strand (a) Semiconservative mechanism. DA replication produces DA molecules with parental strand and newly made daughter strand. (b) Conservative mechanism. DA replication produces double helix with both parental strands and the other with new daughter strands. (c) Dispersive mechanism. DA replication produces DA strands in which segments of new DA are interspersed with the parental DA. (c) Dispersive mechanism. DA replication produces DA strands in which segments of new DA are interspersed with the parental DA. 4 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Meselson and Stahl experiment Grow bacteria in 5 media. 5 medium (heavy) Transfer to 4 media and continue growth for <,.0,.0, or generations. 4 medium (light) 5 TE DATA n In 958, Matthew Meselson and Franklin Stahl devised an experiment to differentiate among the three proposed DA replication mechanisms Approximate generations after transfer to 4 medium. < Light alf-heavy n itrogen comes in a common light form ( 4 ) and a rare heavy form ( 5 ) Isolate DA after each generation. Transfer DA to CsCl gradient, and centrifuge. DA eavy n Grew E. coli in medium with 5 to label, then switched to medium with 4, collecting samples after each generation 4 CsCl gradient bserve DA under UV light. Centrifuge n riginal parental strands would be 5 while newly made strands would be 4 n Conclusion: Semiconservative DA replication 5 Meselson, M., Stahl, F., (958) The replication of DA in Escherichia coli, PAS, 44(7):67 8, Fig. 4a 6 9

10 Semiconservative replication n The two parental strands separate and serve as template strands n ew nucleotides must obey the AT/GC rule n End result: two new double helices with same base sequence as original G Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Incoming nucleotides C G A riginal ewly riginal (template) synthesized (template) 7 strand daughter strand strand (a) The mechanism of DA replication (b) The products of replication 8 G C A A T Replication fork G C T C Molecular Mechanism of DA Replication n rigin of replication provides an opening called a replication bubble that forms two replication forks n DA replication proceeds outward from forks n Bacteria have single origin of replication DA strands unwind. DA replication begins outward from two replication forks. DA replication continues in both directions. rigin of replication Replication forks Replication fork Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Circular bacterial chromosome DA strands unwind, and DA replication begins. DA replication is completed. rigin of replication Site where DA replication ends rigin of replication DA strands unwind, and DA replication begins at multiple origins of replication. DA replication is completed. n Eukaryotes have multiple origins of replication Replication fork Kinetochore proteins at the centromere (a) Bidirectional replication (b) Single origin of replication in bacteria (c) Multiple origins of replication in eukaryotes

11 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. n DA helicase q Binds to DA and travels 5 to using ATP to separate strand and move fork forward n DA topoisomerase q Relives additional coiling ahead of replication fork n Single-strand binding proteins q Keep parental strands open to act as templates DA topoisomerase travels slightly ahead of the replication fork and alleviates coiling caused by the action of helicase. Direction of replication fork Single-strand binding proteins coat the DA strands to prevent them from re-forming a double helix. DA helicase travels along one DA strand in the to direction and separates the DA strands. 4 4 n DA polymerase q Covalently links nucleotides q Deoxynucleoside triphosphates Deoxynucleoside triphosphates q Free nucleotides with three phosphate groups q Breaking covalent bond to release pyrophosphate (two phosphates) provides energy to connect nucleotides Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. (a) Action of DA polymerase Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. DA polymerase catalytic site Incoming deoxynucleoside triphosphates 4 end end G C Template G strand C C P C P end A A end P C P C T T C C C P C P G G P C P C C C C P C P G G end end end P C C C ew phosphoester bond + P P end Pyrophosphate An incoming nucleotide (a deoxynucleoside triphosphate) + (b) Chemistry of DA replication Phosphate

12 Features of DA polymerase. DA polymerase cannot begin synthesis on a bare template strand q q q Requires a primer to get started DA primase makes the primer from RA The RA primer is removed and replaced with DA later. DA polymerase only works 5 to DA polymerase is able to covalently link nucleotides together from a primer, which is made by DA primase. Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. RA primer DA polymerase can link nucleotides only in the to direction. (a) eed for a primer (b) to direction of DA synthesis Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. n Leading strand q DA synthesized in as one long molecule q DA primase makes a single RA primer q DA polymerase adds nucleotides in a 5 to direction as it slides forward n Lagging strand q DA synthesized 5 to but as kazaki fragments q kazaki fragments consist of RA primers plus DA n In both strands q RA primers are removed by DA polymerase and replaced with DA q DA ligase joins adjacent DA fragments DA strands separate at an origin of replication, creating replication forks. Primers are needed to initiate DA synthesis. The synthesis of the leading strand begins in the direction of the replication fork. In the lagging strand, the first kazaki fragment is made in the opposite direction. The leading strand elongates, and a second kazaki fragment is made. Replication forks Leading strand Primer First kazaki fragment of the lagging strand Second kazaki fragment RA primer Direction of replication fork First kazaki fragment

13 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. rigin of replication 4 The leading strand continues to elongate. A third kazaki fragment is made, and the first and second are connected together. Third kazaki fragment First and second kazaki fragments have been connected to each other. Leading strand Replication Replication fork fork Lagging strand Lagging strand Leading strand (b) Replication from an origin Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. DA DA primase makes RA primers to begin primase DA polymerase III continues to elongate the replication process. the leading strand. In the lagging strand, DA polymerase III synthesizes DA DA from the second primer. DA polymerase I removes the first primer and replaces it with DA. RA primer Missing Second covalent bond primer DA polymerase III makes DA from the Third RA primers. DA primase hops back to primer the opening of the fork and makes a second RA primer for the lagging strand. Clamp protein Direction of replication fork 4 In the lagging strand, DA ligase forms a DA DA covalent bond between the first and second polymerase I polymerase III kazaki fragments. A third kazaki Leading fragment is made. The leading strand strand continues to elongate. Second DA primer polymerase III DA primase First RA primer DA ligase Lagging strand (kazaki fragment) Third primer replication is very accurate n Three mechanisms for accuracy. ydrogen bonding between A and T, and between G and C is more stable than mismatched combinations. Active site of DA polymerase is unlikely to form bonds if pairs mismatched. DA polymerase can proofread to remove mismatched pairs n n DA polymerase backs up and digests linkages ther DA repair enzymes as well 5

14 DA Polymerases Are a Family of Enzymes With Specialized Functions n Important issues for DA polymerase are speed, fidelity, and completeness n early all living species have more than one type of DA polymerase n Genomes of most species have several DA polymerase genes due to gene duplication n Independent genetic changes produce enzymes with specialized functions n E. coli has 5 DA polymerases q DA polymerase III multiple subunits, responsible for majority of replication q DA polymerase I a single subunit, rapidly removes RA primers and fills in DA q DA polymerases II, IV and V DA repair and can replicate damaged DA n DA polymerases I and III stall at DA damage n DA polymerases II, IV and V don t stall but go slower and make sure replication is complete n umans have or more DA polymerases q Designated with Greek letters q DA polymerase α its own built in primase subunit q DA polymerase δ and ε extend DA at a faster rate q DA polymerase γ replicates mitochondrial DA q When DA polymerases α, δ or ε encounter abnormalities they may be unable to replicate q Lesion-replicating polymerases may be able to synthesize complementary strands to the damaged area 4

15 Telomeres n Series of short nucleotide sequences repeated at the ends of chromosomes in eukaryotes n Specialized form of DA replication only in eukaryotes in the telomeres n Telomere at does not have a complementary strand and is called a overhang Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Telomere repeat sequences G G G T G G G T G G G T G G G C C C A C C C A C C C A T G G G T overhang n DA polymerase cannot copy the tip of the strand with a end q o place for upstream primer to be made n If this replication problem were not solved, linear chromosomes would become progressively shorter n Telomerase enzyme attaches many copies of DA repeat sequence to the ends of chromosomes n Shortening of telomeres is correlated with cellular senescence n Telomerase function is reduced as an organism ages n 99% of all types of human cancers have high levels of telomerase

16 Telomere Eukaryotic chromosome Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Telomere Telomerase moves 6 nucleotides to the right and begins to make another repeat. Molecular Structure of Eukaryotic Chromosomes Telomerase binds to a DA repeat sequence. G G G T G G G T G G G C C C A Repeat sequence Telomerase synthesizes a 6-nucleotide repeat sequence. T A A U C C CAAU G G G T G G G T G G G T GG GT C C C A A A U C C C AAU RA in telomerase Telomerase G G G T G G G T GG G T G G G T G G G T C C C A A A U C C CAAU 4 Primase makes an RA primer near the end of the telomere, and DA polymerase synthesizes a complementary strand in the to direction. The RA primer is eventually removed. G G G T G G G T G G G T G G G T G G G T C C C A C C C A C C CA A U C C C A A U C C C RA primer that is eventually removed n Typical eukaryotic chromosome may be hundreds of millions of base pairs long q Length would be meter q But must fit in cell 0-00µm n Chromosome q Discrete unit of genetic material n Chromosomes composed of chromatin q DA-protein complex 6 Three levels of DA compaction. DA wrapping q DA wrapped around histones to form nucleosome q Shortens length of DA molecule 7-fold. 0-nm fiber q Current model suggests asymmetric, D zigzag of nucleosomes q Shortens length another 7-fold Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. ucleosome: 8 histone proteins + 46 or 47 nucleotide base pairs of DA B 4 B A 4 nm DA Linker region Amino terminal tail of histone protein Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. (a) Micrograph of a 0-nm fiber (b) Three-dimensional zigzag model a: Photo courtesy of Dr. Barbara amkaloz 0 nm

17 . Radial loop domains q q Interaction between 0-nm fibers and nuclear matrix Each chromosome located in discrete territory Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Protein fiber inside the nucleus 0-nm fiber Radial loop domain Cell division n When cells prepare to divide, chromosomes become even more compacted q Euchromatin not as compact q etrochromatin much more compact n Level of compaction is not uniform q q eterochromatin Euchromatin Protein that attaches the base of a DA loop to a protein fiber n Metaphase chromosomes highly compacted Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. nm DA double helix 00 nm Anchoring of radial loop domains to the nuclear matrix (d) Radial loop domains (a) DA double helix nm Wrapping of DA around histone proteins 4 Further compaction of radial loops to form heterochromatin istones 700 nm ucleosome (b) ucleosomes ( beads on a string ) istone Formation of a -dimensional zigzag structure via histone and other DA-binding proteins (e) eterochromatin 5 Metaphase chromosome with copies of the DA,400 nm (c) 0-nm fiber 0 nm a: Dr. Gopal Murti/Visuals Unlimited; b: Ada L. lins and Donald E. lins/biological Photo Service; c: Courtesy Dr. Jerome B. Rattner, Cell Biology and Anatomy, University of Calgary (f) Metaphase chromosome d: Courtesy of Paulson, J.R. & Laemmli, U.K. James R. Paulson, U.K. Laemmli, The structure of histonedepleted metaphase chromosomes, Cell, :87 8, Copyright Elsevier 977; e-f: Peter Engelhardt/ Department of Virology, aartman Institute

18 Copyright The McGraw-ill Companies, Inc. Permission required for reproduction or display. DA double helix nm (a) DA double helix nm Wrapping of DA around histone proteins istones ucleosome (b) ucleosomes ( beads on a string ) istone Formation of a -dimensional zigzag structure via histone and other DA-binding proteins (c) 0-nm fiber 0 nm 00 nm Anchoring of radial loop domains to the nuclear matrix (d) Radial loop domains 4 Further compaction of radial loops to form heterochromatin 700 nm (e) eterochromatin 5 Metaphase chromosome with copies of the DA,400 nm (f) Metaphase chromosome a: Dr. Gopal Murti/Visuals Unlimited; b: Ada L. lins and Donald E. lins/biological Photo Service; c: Courtesy Dr. Jerome B. Rattner, Cell Biology and Anatomy, University of Calgary; d: Courtesy of Paulson, J.R. & Laemmli, U.K. James R. Paulson, U.K. Laemmli, The structure of histonedepleted metaphase chromosomes, Cell, :87 8, Copyright Elsevier 977; e-f: Peter Engelhardt/Department of Virology, aartman Institute 69 8