Lecture Outline

Chapter 16

The Molecular Basis of Inheritance

Overview: Lifes Operating Instructions In April 1953, James Watson and Francis Crick shook the scientific world with an elegant dou le!helical model for the structure of deo"#ri onucleic acid, or $%A& 'our genetic endowment is the $%A #ou inherited from #our parents& %ucleic acids are uni(ue in their a ilit# to direct their own replication& )he resem lance of offspring to their parents depends on the precise replication of $%A and its transmission from one generation to the ne"t& It is this $%A program that directs the de*elopment of #our iochemical, anatomical, ph#siological, and +to some e"tent, eha*ioral traits& Concept 16.1 D ! is the genetic "aterial The search for genetic "aterial le# to D !. -nce )& .& /organ0s group showed that genes are located on chromosomes, the two constituents of chromosomes1proteins and $%A1were the candidates for the genetic material& 2ntil the 1934s, the great heterogeneit# and specificit# of function of proteins seemed to indicate that proteins were the genetic material& .owe*er, this was not consistent with e"periments with microorganisms, such as acteria and *iruses& )he disco*er# of the genetic role of $%A egan with research # Frederick 5riffith in 1967& .e studied 8treptococcus pneumoniae, a acterium that causes pneumonia in mammals& -ne strain, the 9 strain, was harmless& )he other strain, the 8 strain, was pathogenic& 5riffith mi"ed heat!killed 8 strain with li*e 9 strain acteria and in:ected this into a mouse& )he mouse died, and he reco*ered the pathogenic strain from the mouse0s lood& 5riffith called this phenomenon transfor"ation$ a phenomenon now defined as a change in genot#pe and phenot#pe due to the assimilation of foreign $%A # a cell&
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 For the ne"t 13 #ears, scientists tried to identif# the transforming su stance& Finall# in 1933, -swald A*er#, /acl#n /cCart#, and Colin /ac;eod announced that the transforming su stance was $%A& 8till, man# iologists were skeptical& <roteins were considered etter candidates for the genetic material& )here was also a elief that the genes of acteria could not e similar in composition and function to those of more comple" organisms& Further e*idence that $%A was the genetic material was deri*ed from studies that tracked the infection of acteria # *iruses& =iruses consist of $%A +or sometimes 9%A, enclosed # a protecti*e coat of protein& )o replicate, a *irus infects a host cell and takes o*er the cell0s meta olic machiner#& =iruses that specificall# attack acteria are called %acteriophages or :ust phages. In 1956, Alfred .ershe# and /artha Chase showed that $%A was the genetic material of the phage )6& )he )6 phage, consisting almost entirel# of $%A and protein, attacks >scherichia coli +>& coli,, a common intestinal acteria of mammals& )his phage can (uickl# turn an >& coli cell into a )6! producing factor# that releases phages when the cell ruptures& )o determine the source of genetic material in the phage, .ershe# and Chase designed an e"periment in which the# could la el protein or $%A and then track which entered the >& coli cell during infection& )he# grew one atch of )6 phage in the presence of radioacti*e sulfur, marking the proteins ut not $%A& )he# grew another atch in the presence of radioacti*e phosphorus, marking the $%A ut not proteins& )he# allowed each atch to infect separate >& coli cultures& 8hortl# after the onset of infection, the# spun the cultured infected cells in a lender, shaking loose an# parts of the phage that remained outside the acteria& )he mi"tures were spun in a centrifuge, which separated the hea*ier acterial cells in the pellet from lighter free phages and parts of phage in the li(uid supernatant& )he# then tested the pellet and supernatant of the separate treatments for the presence of radioacti*it#& .ershe# and Chase found that when the acteria had een infected with )6 phages that contained radiola eled proteins, most of the radioacti*it# was in the supernatant that
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contained phage particles, not in the pellet with the acteria& When the# e"amined the acterial cultures with )6 phage that had radiola eled $%A, most of the radioacti*it# was in the pellet with the acteria& .ershe# and Chase concluded that the in:ected $%A of the phage pro*ides the genetic information that makes the infected cells produce new *iral $%A and proteins to assem le into new *iruses& )he fact that cells dou le the amount of $%A in a cell prior to mitosis and then distri ute the $%A e(uall# to each daughter cell pro*ided some circumstantial e*idence that $%A was the genetic material in eukar#otes& 8imilar circumstantial e*idence came from the o ser*ation that diploid sets of chromosomes ha*e twice as much $%A as the haploid sets in gametes of the same organism& ?# 193@, >rwin Chargaff had de*eloped a series of rules ased on a sur*e# of $%A composition in organisms& .e alread# knew that $%A was a pol#mer of nucleotides consisting of a nitrogenous ase, deo"#ri ose, and a phosphate group& )he ases could e adenine +A,, th#mine +),, guanine +5,, or c#tosine +C,& Chargaff noted that the $%A composition *aries from species to species& In an# one species, the four ases are found in characteristic, ut not necessaril# e(ual, ratios& .e also found a peculiar regularit# in the ratios of nucleotide ases that are known as Chargaff0s rules& In all organisms, the num er of adenines was appro"imatel# e(ual to the num er of th#mines +A) B AA,& )he num er of guanines was appro"imatel# e(ual to the num er of c#tosines +A5 B AC,& .uman $%A is 34&9A adenine, 69&3A th#mine, 19&9A guanine, and 19&7A c#tosine& )he asis for these rules remained une"plained until the disco*er# of the dou le heli"& &atson an# Cric' #iscovere# the #ou%le heli( %) %uil#ing "o#els to confor" to *+ra) #ata. ?# the eginnings of the 1954s, the race was on to mo*e from the structure of a single $%A strand to the three! dimensional structure of $%A& Among the scientists working on the pro lem were ;inus <auling in California and /aurice Wilkins and 9osalind Franklin in ;ondon& /aurice Wilkins and 9osalind Franklin used C!ra#
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cr#stallograph# to stud# the structure of $%A& In this techni(ue, C!ra#s are diffracted as the# passed through aligned fi ers of purified $%A& )he diffraction pattern can e used to deduce the three! dimensional shape of molecules& James Watson learned from their research that $%A was helical in shape, and he deduced the width of the heli" and the spacing of nitrogenous ases& )he width of the heli" suggested that it was made up of two strands, contrar# to a three!stranded model that ;inus <auling had recentl# proposed& Watson and his colleague Francis Crick egan to work on a model of $%A with two strands, the #ou%le heli(. 2sing molecular models made of wire, the# placed the sugar! phosphate chains on the outside and the nitrogenous ases on the inside of the dou le heli"& )his arrangement put the relati*el# h#dropho ic nitrogenous ases in the molecule0s interior& )he sugar!phosphate chains of each strand are like the side ropes of a rope ladder& <airs of nitrogenous ases, one from each strand, form rungs& )he ladder forms a twist e*er# ten ases& )he nitrogenous ases are paired in specific com inationsD adenine with th#mine and guanine with c#tosine& <airing like nucleotides did not fit the uniform diameter indicated # the C!ra# data& A purine!purine pair is too wide, and a p#rimidine!p#rimidine pairing is too short& -nl# a p#rimidine!purine pairing produces the 6!nm diameter indicated # the C!ra# data& In addition, Watson and Crick determined that chemical side groups of the nitrogenous ases would form h#drogen onds, connecting the two strands& ?ased on details of their structure, adenine would form two h#drogen onds onl# with th#mine, and guanine would form three h#drogen onds onl# with c#tosine& )his finding e"plained Chargaff0s rules& )he ase!pairing rules dictate the com inations of nitrogenous ases that form the ErungsF of $%A& .owe*er, this does not restrict the se(uence of nucleotides along each $%A strand& )he linear se(uence of the four ases can e *aried in countless wa#s& >ach gene has a uni(ue order of nitrogenous ases&
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 In April 1953, Watson and Crick pu lished a succinct, one! page paper in %ature reporting their dou le heli" model of $%A& Concept 16., Man) proteins wor' together in D ! replication an# repair )he specific pairing of nitrogenous ases in $%A was the flash of inspiration that led Watson and Crick to the correct dou le heli"& )he possi le mechanism for the ne"t step, the accurate replication of $%A, was clear to Watson and Crick from their dou le heli" model& During D ! replication$ %ase pairing ena%les e(isting D ! stran#s to serve as te"plates for new co"ple"entar) stran#s. In a second paper, Watson and Crick pu lished their h#pothesis for how $%A replicates& >ssentiall#, ecause each strand is complementar# to the other, each can form a template when separated& )he order of ases on one strand can e used to add complementar# ases and therefore duplicate the pairs of ases e"actl#& When a cell copies a $%A molecule, each strand ser*es as a template for ordering nucleotides into a new complementar# strand& -ne at a time, nucleotides line up along the template strand according to the ase!pairing rules& )he nucleotides are linked to form new strands& Watson and Crick0s model, semiconser*ati*e replication, predicts that when a dou le heli" replicates, each of the daughter molecules will ha*e one old strand and one newl# made strand& -ther competing models, the conser*ati*e model and the dispersi*e model, were also proposed& >"periments in the late 1954s # /atthew /eselson and Franklin 8tahl supported the se"iconservative "o#el proposed # Watson and Crick o*er the other two models& In their e"periments, the# la eled the nucleotides of the old strands with a hea*# isotope of nitrogen +15%,, while an# new nucleotides were indicated # a lighter isotope +13%,& 9eplicated strands could e separated # densit# in a centrifuge& >ach model1the semiconser*ati*e model, the conser*ati*e model, and the dispersi*e model1made specific predictions a out the densit# of replicated $%A strands&
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 )he first replication in the 13% medium produced a and of 15 13 h# rid + %! %, $%A, eliminating the conser*ati*e model& A second replication produced oth light and h# rid $%A, eliminating the dispersi*e model and supporting the semiconser*ati*e model& ! large tea" of en-)"es an# other proteins carries out D ! replication. It takes >& coli 65 minutes to cop# each of the 5 million ase pairs in its single chromosome and di*ide to form two identical daughter cells& A human cell can cop# its G illion ase pairs and di*ide into daughter cells in onl# a few hours& )his process is remarka l# accurate, with onl# one error per ten illion nucleotides& /ore than a doHen enH#mes and other proteins participate in $%A replication& /uch more is known a out replication in acteria than in eukar#otes& )he process appears to e fundamentall# similar for prokar#otes and eukar#otes& )he replication of a $%A molecule egins at special sites, origins of replication. In acteria, this is a specific se(uence of nucleotides that is recogniHed # the replication enH#mes& )hese enH#mes separate the strands, forming a replication E u le&F 9eplication proceeds in oth directions until the entire molecule is copied& In eukar#otes, there ma# e hundreds or thousands of origin sites per chromosome& At the origin sites, the $%A strands separate, forming a replication E u leF with replication for's at each end& )he replication u les elongate as the $%A is replicated, and e*entuall# fuse& D ! pol)"erases catal#He the elongation of new $%A at a replication fork& As nucleotides align with complementar# ases along the template strand, the# are added to the growing end of the new strand # the pol#merase& )he rate of elongation is a out 544 nucleotides per second in acteria and 54 per second in human cells& In >& coli, two different $%A pol#merases are in*ol*ed in replicationD $%A pol#merase III and $%A pol#merase I& In eukar#otes, at least 11 different $%A pol#merases ha*e een identified so far&
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 >ach nucleotide that is added to a growing $%A strand is a nucleoside triphosphate& >ach has a nitrogenous ase, deo"#ri ose, and a triphosphate tail& A)< is a nucleoside triphosphate with ri ose instead of deo"#ri ose& ;ike A)<, the triphosphate monomers used for $%A s#nthesis are chemicall# reacti*e, partl# ecause their triphosphate tails ha*e an unsta le cluster of negati*e charge& As each nucleotide is added to the growing end of a $%A strand, the last two phosphate groups are h#drol#Hed to form p#rophosphate& )he e"ergonic h#drol#sis of p#rophosphate to two inorganic phosphate molecules dri*es the pol#meriHation of the nucleotide to the new strand& )he strands in the dou le heli" are antiparallel& )he sugar!phosphate ack ones run in opposite directions& >ach $%A strand has a 30 end with a free h#dro"#l group attached to deo"#ri ose and a 50 end with a free phosphate group attached to deo"#ri ose& )he 50 30 direction of one strand runs counter to the 30 50 direction of the other strand& $%A pol#merases can onl# add nucleotides to the free 30 end of a growing $%A strand& A new $%A strand can onl# elongate in the 50 30 direction& Along one template strand, $%A pol#merase III can s#nthesiHe a complementar# strand continuousl# # elongating the new $%A in the mandator# 50 30 direction& )he $%A strand made # this mechanism is called the lea#ing stran#. )he other parental strand +50 30 into the fork,, the lagging stran#$ is copied awa# from the fork& 2nlike the leading strand, which elongates continuousl#, the lagging stand is s#nthesiHed as a series of short segments called O'a-a'i frag"ents. -kaHaki fragments are a out 1,444I6,444 nucleotides long in >& coli and 144I644 nucleotides long in eukar#otes& Another enH#me, D ! ligase$ e*entuall# :oins the sugar! phosphate ack ones of the -kaHaki fragments to form a single $%A strand& $%A pol#merases cannot initiate s#nthesis of a pol#nucleotide& )he# can onl# add nucleotides to the 30 end of an e"isting chain that is ase!paired with the template strand& )he initial nucleotide chain is called a pri"er.
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 In the initiation of the replication of cellular $%A, the primer is a short stretch of 9%A with an a*aila le 30 end& )he primer is 5I14 nucleotides long in eukar#otes& .ri"ase$ an 9%A pol#merase, links ri onucleotides that are complementar# to the $%A template into the primer& 9%A pol#merases can start an 9%A chain from a single template strand& After formation of the primer, $%A pol III adds a deo"#ri onucleotide to the 30 end of the 9%A primer and continues adding $%A nucleotides to the growing $%A strand according to the ase!pairing rules& 9eturning to the original pro lem at the replication fork, the leading strand re(uires the formation of onl# a single primer as the replication fork continues to separate& For s#nthesis of the lagging strand, each -kaHaki fragment must e primed separatel#& Another $%A pol#merase, $%A pol#merase I, replaces the 9%A nucleotides of the primers with $%A *ersions, adding them one # one onto the 30 end of the ad:acent -kaHaki fragment& )he primers are con*erted to $%A efore $%A ligase :oins the fragments together& In addition to primase, $%A pol#merases, and $%A ligases, se*eral other proteins ha*e prominent roles in $%A s#nthesis& /elicase untwists the dou le heli" and separates the template $%A strands at the replication fork& )his untwisting causes tighter twisting ahead of the replication fork, and topoiso"erase helps relie*e this strain& 0ingle+stran# %in#ing proteins keep the unpaired template strands apart during replication& )o summariHe, at the replication fork, the leading strand is copied continuousl# into the fork from a single primer& )he lagging strand is copied awa# from the fork in short segments, each re(uiring a new primer& It is con*entional and con*enient to think of the $%A pol#merase molecules as mo*ing along a stationar# $%A template& In realit#, the *arious proteins in*ol*ed in $%A replication form a single large comple", a $%A replication Emachine&F /an# protein!protein interactions facilitate the efficienc# of this machine& For e"ample, helicase works much more rapidl# when it is in contact with primase& )he $%A replication machine is pro a l# stationar# during the replication process&
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 In eukar#otic cells, multiple copies of the machine ma# anchor to the nuclear matri", a framework of fi ers e"tending through the interior of the nucleus& )he $%A pol#merase molecules Ereel inF the parental $%A and Ee"trudeF newl# made daughter $%A molecules& 1n-)"es proofrea# D ! #uring its replication an# repair #a"age in e(isting D !. /istakes during the initial pairing of template nucleotides and complementar# nucleotides occur at a rate of one error per 144,444 ase pairs& $%A pol#merase proofreads each new nucleotide against the template nucleotide as soon as it is added& If there is an incorrect pairing, the enH#me remo*es the wrong nucleotide and then resumes s#nthesis& )he final error rate is onl# one per ten illion nucleotides& $%A molecules are constantl# su :ect to potentiall# harmful chemical and ph#sical agents& 9eacti*e chemicals, radioacti*e emissions, C!ra#s, and ultra*iolet light can change nucleotides in wa#s that can affect encoded genetic information& $%A ases ma# undergo spontaneous chemical changes under normal cellular conditions& /ismatched nucleotides that are missed # $%A pol#merase or mutations that occur after $%A s#nthesis is completed can often e repaired& >ach cell continuall# monitors and repairs its genetic material, with 144 repair enH#mes known in >& coli and more than 134 repair enH#mes identified in humans& In "is"atch repair$ special enH#mes fi" incorrectl# paired nucleotides& A hereditar# defect in one of these enH#mes is associated with a form of colon cancer& In nucleoti#e e(cision repair$ a nuclease cuts out a segment of a damaged strand& $%A pol#merase and ligase fill in the gap& )he importance of the proper functioning of repair enH#mes is clear from the inherited disorder "eroderma pigmentosum& )hese indi*iduals are h#persensiti*e to sunlight& 2ltra*iolet light can produce th#mine dimers etween ad:acent th#mine nucleotides& )his uckles the $%A dou le heli" and interferes with $%A replication& In indi*iduals with this disorder, mutations in their skin cells are left uncorrected and cause skin cancer& The en#s of D ! "olecules are replicate# %) a special
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"echanis". ;imitations of $%A pol#merase create pro lems for the linear $%A of eukar#otic chromosomes& )he usual replication machiner# pro*ides no wa# to complete the 50 ends of daughter $%A strands& 9epeated rounds of replication produce shorter and shorter $%A molecules& <rokar#otes do not ha*e this pro lem ecause the# ha*e circular $%A molecules without ends& )he ends of eukar#otic chromosomal $%A molecules, the telo"eres$ ha*e special nucleotide se(uences& )elomeres do not contain genes& Instead, the $%A t#picall# consists of multiple repetitions of one short nucleotide se(uence& In human telomeres, this se(uence is t#picall# ))A555, repeated etween 144 and 1,444 times& )elomeres protect genes from eing eroded through multiple rounds of $%A replication& )elomeric $%A tends to e shorter in di*iding somatic cells of older indi*iduals and in cultured cells that ha*e di*ided man# times& It is possi le that the shortening of telomeres is somehow connected with the aging process of certain tissues and perhaps to aging in general& )elomeric $%A and specific proteins associated with it also pre*ents the staggered ends of the daughter molecule from acti*ating the cell0s s#stem for monitoring $%A damage& >ukar#otic cells ha*e e*ol*ed a mechanism to restore shortened telomeres in germ cells, which gi*e rise to gametes& If the chromosomes of germ cells ecame shorter with e*er# cell c#cle, essential genes would e*entuall# e lost& An enH#me called telo"erase catal#Hes the lengthening of telomeres in eukar#otic germ cells, restoring their original length& )elomerase uses a short molecule of 9%A as a template to e"tend the 30 end of the telomere& )here is now room for primase and $%A pol#merase to e"tend the 50 end& It does not repair the 30!end Eo*erhang,F ut it does lengthen the telomere& )elomerase is not present in most cells of multicellular organisms& )herefore, the $%A of di*iding somatic cells and cultured cells tends to ecome shorter& )elomere length ma# e a limiting factor in the life span of
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certain tissues and of the organism& %ormal shortening of telomeres ma# protect organisms from cancer # limiting the num er of di*isions that somatic cells can undergo& Cells from large tumors often ha*e unusuall# short telomeres, ecause the# ha*e gone through man# cell di*isions& Acti*e telomerase has een found in some cancerous somatic cells& )his o*ercomes the progressi*e shortening that would e*entuall# lead to self!destruction of the cancer& Immortal strains of cultured cells are capa le of unlimited cell di*ision& )elomerase ma# pro*ide a useful target for cancer diagnosis and chemotherap#&

Lecture Outline for Campbell/Reece Biology, 7th Edition, Pearson Education, Inc.

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