MOLECULAR AND CELL BIOLOGY

(Copyright 2003 by The McGraw-Hill Companies, Inc.)

Chapter 3: Chromosomes
In this chapter:
Chromosome tr!ct!re
"#$ %eplication
CH%&M&&M' T%(CT(%'
$ll essential bacterial genes are )o!n* in a single, circ!lar, *o!ble-stran*e* "#$ chromosome locate* in
the n!cleoi* region o) the cytoplasm. The bacterial chromosome is belie+e* to be attache* to the plasma
membrane an* speci)ies between ,,000 an* -,000 proteins. It is highly con*ense* an* consists o) "#$,
%#$, an* protein. In a**ition, there may be one or more plasmids. .lasmi*s are small circ!lar pieces o)
e/trachromosomal "#$ which may enco*e 200,00 proteins.
The genes o) e!1aryotes are *istrib!te* among a n!mber o) linear chromosomes that +ary in si2e an*
n!mber. '!1aryotic chromosomes are con*ense* by pac1ing the "#$ to *i))erent *egrees. Nucleosomes
consist o) "#$ wo!n* twice aro!n* an octet o) proteins calle* histones 3two each o) H2a, H2b, H3, an*
H44. $ppro/imately 200 base pairs 3bp4 o) the "#$ are wo!n* aro!n* the spherical bo*ies )orme* by the
histones, an* abo!t -0 bp o) "#$ connect the n!cleosomes. 5!rther compaction may be accomplishe* by
histone H1 bin*ing, which in*!ces the n!cleosomes to associate into a ring o) si/ n!cleosomes an* the
rings to associate into a cylin*er calle* a solenoi*. .hosphorylation o) histone H, res!lts in the *issociation
o) the solenoi* into an e/ten*e* n!cleosome )orm. The solenoi* is the )orm in which most o) the cell6s "#$
e/ists *!ring interphase. Howe+er, )!rther pac1ing can occ!r by certain proteins bin*ing the solenoi* an*
stim!lating it to loop bac1 an* )orth )rom a central core o) proteins calle* a scaffold. "ephosphorylation o)
topoisomerase II an* other proteins ca!ses *issociation o) the sca))ol* an* res!lts in the *econ*ensation
o) the chromosomes to the solenoi* )orm. In some e!1aryotes, ,7 loops o) the solenoi* )orm a *is1-li1e
str!ct!re an* the chromosome con*enses as h!n*re*s o) *is1s stac1 together. This is the )orm that is
pre*ominant *!ring n!clear *i+ision.
Heterochromatin is highly con*ense* "#$ that remains in the solenoi* )orm thro!gho!t the cell cycle
e/cept *!ring "#$ replication, when it *econ*enses. Most o) the genes associate* with heterochromatin
are not e/presse* beca!se o) the "#$6s con*ense* state. In contrast, euchromatin is *econ*ense* "#$
that e/ists in the solenoi* )orm or in an e/ten*e* n!cleosome )orm.
$ centromere is a highly constricte* region o) a mitotic or meiotic chromosome where the spin*le )ibers
attach. Comple/ se8!ences o) "#$ constit!te centromeres. I) the centromere is in the mi**le o) the
chromosome, the chromosome is sai* to be metacentric. I) the centromere is near the tip, it is calle*
telocentric. The short an* long arms o) the chromosome with respect to the centromere are *esignate* as
p an* q, respecti+ely. pecial staining techni8!es re+eal that each chromosome has a speci)ic pattern o)
*ar1 an* light regions calle* ban*s. Homologo!s chromosomes ha+e the same ban*ing pattern.
.rotein comple/es associate* with the centromeric regions are calle* kinetochores. 9inetochores bin*
microt!b!les o) the spin*le b!n*le an* )!nction to *istrib!te chromosomes as cells proli)erate. .ropagation
an* maintenance o) any piece o) "#$ re8!ires the presence o) one or more origin of replication sites
3OriR4 an* special en*s calle* telomeres. &rigin o) replication sites are special se8!ences where "#$
replication initiates. Telomeres protect the en*s o) linear chromosomes )rom cell!lar en2ymes that *egra*e
n!cleic aci*s )rom their en*s.
"#$ %'.:IC$TI&#
"#$ replication o) the E. coli chromosome begins at a single origin o) replication 3 oriC4 an* procee*s
bi*irectionally to a termination site locate* appro/imately hal)way aro!n* the circ!lar chromosome. "!ring
replication, the "#$ stran*s o) the *o!ble heli/ m!st be both !nwo!n* an* separate*.
"#$ replication is initiate* when a protein enco*e* by the gene dnaA bin*s repetiti+e ;-mer se8!ences at
the origin. !bse8!ently, helicases speci)ie* by dnaB an* inhibitory proteins enco*e* by dnaC bin*
repetiti+e ,3-mer se8!ences. $s helicase progresses -< to 3<, *issociation o) protein "naC allows the
helicase to !nwin* the "#$. The !nwin*ing pro*!ces positi+e s!perhelical t!rns in the rest o) the "#$,
ma1ing it energetically )a+orable to contin!e !nwin*ing the stran*s. To !nwin* the "#$, positi+e
s!perhelical t!rns ha+e to be remo+e* by c!tting the "#$ an* allowing it to rela/ or by intro*!cing negati+e
s!perhelical t!rns to compensate )or the positi+e ones. The intro*!ction o) negati+e s!perhelical t!rns
re8!ires energy an* an en2yme calle* DNA gyrase 3a topoisomerase4. "#$ gyrase is an en2yme that can
both remo+e positi+e s!percoils or intro*!ce negati+e s!percoils into the "#$an* thereby ma1e stran*
separation energetically more )a+orable. .res!mably the "#$ gyrase bin*s ahea* o) the !nwo!n* "#$
*!ring replication. ingle!stranded binding proteins 3=.s4 act to temporarily stabili2e the !nwo!n*
state.
"#$ replication begins with the synthesis o) a 30 n!cleoti*e long %#$ primer by an %#$ polymerase
calle* primase 3speci)ie* by dnaG4. The helicase an* primase s!bse8!ently )orm a comple/ en2yme
system 1nown as the primosome, which synthesi2es primers a)ter "#$ synthesis begins. Two catalytic
s!b!nits o) DNA polymerase III 3.olC4 associate with the templates an* the 3< en*s o) the primers an*
begin to polymeri2e *eo/yribon!cleoti*es into "#$. "#$ gyrase contin!es to remo+e positi+e s!percoils
an*>or intro*!ces negati+e s!percoils ahea* o) the primosome that is opening the two stran*s o) "#$. $t
+ario!s inter+als, the template signals the primase portion o) the primosome to polymeri2e primer %#$s
abo!t 30 n!cleoti*es long on only one template at the replication )or1. "#$ polymerase III polymeri2es
"#$ -6 to 36 )rom each o) the primers at the replication )or1. &ne stran*s o) "#$ is polymeri2e* towar* the
replication )or1 an* contin!es to be elongate* as the "#$ !nwin*s )!rther. The secon* stran* o) "#$ is
polymeri2e* away )rom the replication )or1. $s the "#$ !nwin*s )!rther, a new primer is synthesi2e* away
)rom the replication )or1 an* the "#$ polymerase synthesi2es "#$ )rom the last primer towar* the
pre+io!s %#$ primer. $s the "#$ polymerase rea*s the template stran*, it selects complementary
n!cleoti*es )or the nascent stran* base* on hy*rogen bon*ing capability.
The "#$ synthesi2e* towar* the replication )or1 is synthesi2e* in a contin!o!s manner an* is calle* the
leading stran*. The opposite "#$ stran* is synthesi2e* in a *iscontin!o!s manner away )rom the
replication )or1 an* is re)erre* to as the lagging stran*. The lea*ing an* lagging stran*s are synthesi2e*
hal)way aro!n* the bacterial chromosome !ntil they enco!nter the lagging an* lea*ing stran*s synthesi2e*
at the other replication )or1. The %#$-"#$ )ragments that initially constit!te the lagging stran* are 1nown
as "ka#aki )ragments, name* a)ter the scientist who *isco+ere* them.
The %#$ primers are remo+e* by a "#$ repair en2yme calle* DNA polymerase I speci)ie* by polA. It
!ses neighboring "#$ as a primer an* polymeri2es "#$ )rom it, *isplacing the %#$ primer. $ "#$ ligase
remo+es nic1s in the "#$ by connecting the )ragments together. $opoisomerase I% is re8!ire* to
separate the two *a!ghter chromosomes.
"#$ replication in e!1aryotic chromosomes generally is initiate* )rom many origin o) replication sites.
%eplication )or1s procee* in both *irections )rom these sites. The sites that comprise yeast origins o)
replication are calle* autonomously replicating sequences 3$%s4 an* consist o) two regions that bin* a
*istinct set o) proteins that *estabili2e the *o!ble heli/. In one region, conser+e*, repeating ,,-mers bin* a
m!ltiprotein comple/ calle* the origin recognition comple& 3&%C4. ?hen proteins also bin* the other
region, the "#$ ben*s by interaction o) the proteins in the two regions. This *istortion o) the "#$ promotes
the separation o) paire* "#$ stran*s at the origin an* initiation o) %#$ primer synthesis.
'n2ymes similar to those in+ol+e* in bacterial "#$ replication are )o!n* in e!1aryotes. #!mero!s
topoisomerases, helicases, an* %#$ polymerases ha+e been )o!n* in e!1aryotes. DNA topoisomerase II
is in+ol+e* in relie+ing positi+e s!percoils in the "#$, whereas a helicase acti+ity separates the two stran*s
$t least )i+e *i))erent "#$ polymerases ha+e been )o!n* in e!1aryotic cells. The primase 3 DNA pola4
synthesi2es lagging stran* "#$. DNA pold cataly2es lea*ing stran* synthesis. DNA pole an* DNA polb
are responsible )or replacing the n!cleoti*e gaps create* when %#$ primers are remo+e* by
en*on!cleases. $ "#$ ligase repairs single stran*e* nic1s 3!nconnecte* a*@acent n!cleoti*es4 le)t in the
"#$. DNA polg per)orms "#$ replication in the mitochon*ria.
To complete replication o) a linear chromosome, %#$ primers at each en* o) the chromosome ha+e to be
remo+e* an* replace* by "#$. $ltho!gh %#$ primers can be remo+e* by e/on!cleases, none o) the
!s!al "#$ polymerases are able to replace the %#$ witho!t a "#$ primer. $n !n!s!al type o) "#$
polymerase 1nown as telomerase consists o) protein an* an %#$ template that the protein portion copies
repetiti+ely into "#$ in or*er to e/ten* one stran* o) the telomere. Th!s, telomerase is responsible )or
maintaining the length o) the chromosomes.
Chapter 4: Transcription and Gene Regulation
In this chapter:
Intro*!ction
.ro1aryotic Genes
Transcription Initiation an* Termination
The :ac &peron
'!1aryotic Gene %eg!lation
%#$ .rocessing
I#T%&"(CTI&#
The central *ogma o) molec!lar genetics proposes that the in)ormation in "#$ is !se* to ma1e %#$
molec!les thro!gh a process 1nown as transcription an* that the in)ormation in some %#$ is !se* to
ma1e proteins by a process calle* translation. Transcription is carrie* o!t by %#$ polymerases, whereas
translation is cataly2e* by en2ymes associate* with ribosomes. The %#$ molec!les an* proteins
synthesi2e* *!ring the *e+elopment an*>or maintenance o) an organism are responsible )or an organism6s
characteristics.
The in)ormation )or synthesi2ing a partic!lar %#$ is locate* in only one o) the two "#$ stran*s. The stran*
that contains the in)ormation )or ma1ing an %#$ molec!le an* that is Area*B by an %#$ polymerase is
calle* the template stran* or the sense stran*. The stran* that is complementary to the template is
sometimes calle* the nonsense stran* since it pro+i*es no in)ormation )or the ma1ing o) %#$ or protein.
#ot all templates )or co*ing %#$s occ!r on the same stran* o) "#$, howe+er. Messenger %#$ that
speci)ies the synthesis o) a protein is calle* sense 'NA, whereas %#$ complementary to sense %#$ is
calle* antisense 'NA.
Most genes, especially those enco*ing proteins, are reg!late* so they are e/presse* at the appropriate
time an* le+el nee*e* to maintain the cell or to promote its growth an* proli)eration.
.%&9$%C&TIC G'#'
tructural genes are the n!cleoti*e se8!ences o) "#$ that ser+e as templates )or the synthesis o) %#$s.
The a+erage length o) str!ct!ral genes speci)ying proteins in pro1aryotes is abo!t ,,000 bp, compare* to
abo!t ,0,000 bp in e!1aryotes. tr!ct!ral genes an* the controlling sites that reg!late the rate o)
transcription o) these genes are calle* operons.
'egulatory proteins are proteins that a))ect the e/pression o) str!ct!ral genes by bin*ing to controlling
sites near the str!ct!ral genes an* either acti+ating or repressing transcription. %eg!latory proteins that
stim!late gene transcription are terme* transcription factors or acti(ators.
'epressors are proteins that inhibit the initiation o) transcription when bo!n* to controlling se8!ences
calle* operators. .roteins that terminate transcription are re)erre* to as terminator proteins. Howe+er,
the en* o) transcription is most o)ten signale* by a speci)ic terminator se8!ence )o!n* in the "#$ or
newly transcribe* %#$. In general, acti+ators stim!late %#$ polymerase bin*ing to promoter sites on "#$
at the beginning o) str!ct!ral genes, whereas repressors inhibit %#$ polymerase bin*ing. )ontrolling
sites are short n!cleoti*e se8!ences o) "#$, !s!ally ,-030 bp long, that control the e/pression o)
str!ct!ral genes ne/t to them.
The simplest operon consists o) one str!ct!ral gene an* one promoter to ser+e as a bin*ing site )or %#$
polymerase. These operons are constituti(e, that is, they are e/presse* at all times. ome simple
operons may be reg!late* by an attenuator, which co*es )or %#$ str!ct!re that ca!ses the %#$
polymerase to premat!rely cease transcribing.
Most operons in bacteria consist o) n!mero!s str!ct!ral genes an* controlling sites. In bacteria, many
operons contain more than one str!ct!ral gene, or cistron. These polycistronic operons are transcribe*
into a single m%#$. 'ach protein-co*ing region in the m%#$ is *e)ine* by its own start codon, where
protein synthesis is initiate*, an* a nonsense codon, where protein synthesis is terminate*. &perons in
e!1aryotes are !s!ally monocistronic, that is, they contain a single gene. $ regulon is a gro!p o) operons
!n*er the control o) a reg!latory protein. The operons in a reg!lon are generally not contig!o!s.
T%$#C%I.TI&# I#ITI$TI&# $#" T'%MI#$TI&#
Transcription o) a str!ct!ral gene can only occ!r i) there is a promoter )or %#$ polymerase bin*ing near the
beginning o) the gene. In bacteria, promoters are !s!ally ,-030 bp long. The base pair se8!ence o) the
promoter *etermines the e))iciency with which %#$ polymerase bin*s to it an* th!s the e))iciency o)
transcription. In many cases, %#$ polymerase bin*ing to bacterial promoter sites is *epen*ent !pon a
)amily o) proteins calle* sigma 3s4 factors. 'ach type o) sigma )actor *etermines the 1in* o) promoter that
%#$ polymerase will recogni2e. =oth a sigma )actor an* %#$ polymerase are re8!ire* )or e))icient bin*ing
to a promoter. $)ter initiation, the sigma )actor will *issociate )rom the %#$ polymerase.
$ltho!gh pro1aryotic promoters +ary consi*erably, two short regions are share* in common by most
promoters. $ region on the nonsense stran* abo!t ,0 bp be)ore transcription initiates 3 D,0 se8!ence4 has
a consens!s-6-T$T$$T-36 an* another region abo!t 3- bp !p 3D3- se8!ence4 has a consens!s se8!ence
-6-TTG$C$-36. $ consensus sequence contains the n!cleoti*e se8!ence most commonly enco!ntere* in
a wi*e +ariety o) promoters.
Most terminators at the en* o) a m%#$ molec!le co*e )or a single stran*e* region that )ol*s on itsel) *!e
to hy*rogen bon*ing between complementary base pairs an* co*es )or a )inal region containing many
!racils. The hairpin str!ct!re that )orms interacts with the %#$ polymerase an* stim!lates its *etachment
)rom the "#$. ome terminators re8!ire terminator proteins s!ch as 'ho 3E4 to be acti+e. These %ho-
*epen*ent terminators lac1 a poly-( region in the 36 en* o) the terminator. The NusA protein is another
termination )actor that apparently bin*s *irectly to the %#$ polymerase an* cataly2es its release when it
comes to termination sites.
TH' :$C &.'%&#
The lactose operon pro+i*es a goo* mo*el system o) se+eral concepts o) pro1aryotic gene reg!lation. It
consists o) three str!ct!ral genes 3lacZ, lacY, an* lacA4 as well as three controlling sites 3lacCRP, lacP1,
an* lacO4. The str!ct!ral genes lacZ, lacY, an* lacA enco*e the en2ymes b!galactosidase, permease,
an* transacetylase, respecti+ely. Catabolism o) lactose is *epen*ent !pon these proteins. The controlling
sites lacCRP, lacP1, an* lacO are bin*ing sites )or the c$M. receptor protein, %#$ polymerase, an* the
lactose repressor, respecti+ely. The lactose reg!latory operon consists o) one str!ct!ral gene 3lacI4 an*
one controlling site 3lacP24. The str!ct!ral gene enco*es the lactose repressor, whereas the controlling site
is an %#$ polymerase bin*ing site 3a promoter4.
In the absence o) inducer, something that t!rns on transcription o) an operon, e/pression is inhibite* by
the bin*ing o) the lactose repressor at lacO. The repressor sterically hin*ers the bin*ing o) %#$
polymerase to lacP1 an* the initiation o) transcription. I) lactose is present, it can be con+erte* by the cell
into allolactose, which acts as an in*!cer )or this operon. ?hen in*!cer is present, it bin*s to the
repressor 3protein LacI4 an* inacti+ates it. Inacti+e LacI cannot bin* the operator, an* %#$ polymerase is
able to bin* to lacP1 an* initiate transcription o) the genes necessary )or lactose catabolism. '+en when an
operon is in*!ce* the cell *oes not rapi*ly )ill with m%#$ an* protein since m%#$ has an a+erage hal)-li)e
o) only 2.- min!tes. That means that 2.- min!tes a)ter m%#$ is synthesi2e*, hal) o) it will be *egra*e*.
.roteins are more stable than m%#$. $lso, as the cell synthesi2es m%#$ an* proteins, it *epletes its
stores o) certain energy molec!les. ?hen a cell is rapi*ly metaboli2ing, catabolite repression sh!ts *own
many catabolic operons, incl!*ing the lactose operon. This in+ol+es a small molec!le calle* cyclic
adenosine monophosphate 3c$M.4. The cell!lar le+el o) c$M. *ecreases when the synthesis o) lactose
m%#$ an* en2ymes increases, an* the le+el o) c$M. increases when these catabolic genes are no longer
e/presse*. The higher the c$M. le+el, the more c$M. bin*s to a protein calle* cyclic A*+ receptor
protein 3C%.4, which then !n*ergoes a con)ormational change that promotes bin*ing to an acti+ator
bin*ing site 3lacCRP4. This, in t!rn, !preg!lates transcription o) the lactose operon genes.
'(9$%C&TIC G'#' %'G(:$TI&#
In e!1aryotes, @!st as in bacteria, genes are reg!late* so that they are e/presse* at the right time an* at
the correct le+els to maintain the cell or promote growth an* proli)eration. Cells in m!lticell!lar organisms
m!st not only respon* to the chemicals in their en+ironment b!t m!st also wor1 together thro!gh comple/
signaling that o)ten a))ects gene acti+ity. The controlling sites in e!1aryotes are similar to those )o!n* in
bacteria, howe+er there are many more controlling sites an* proteins a))ecting each e!1aryotic gene.
Transcription )actors 3T5s4 attach to bin*ing sites in promoter regions an* stim!late %#$ polymerase
bin*ing to the promoter site. Transcription )actors are pro*!ce* constit!ti+ely since great n!mbers o) genes
*epen* on these )or e/pression.
,nhancers are bo!n* by transcription acti+ators 3T$s4 that are synthesi2e* in response to speci)ic signals.
Most enhancers, s!ch as the one that bin*s GalF, are locate* h!n*re*s or tho!san*s o) base pairs )rom
the promoter sites. Howe+er, some in*!ce* acti+ators, s!ch as 5os an* G!n, bin* +ery near promoter sites.
T$s ca!se the "#$ to loop bac1 on itsel) when they interact with the T5s near the promoter. This
interaction between enhancer sites an* initiator sites is !s!ally necessary )or transcription abo+e a basal
le+el. )oacti(ators are acti+ator proteins that o)ten connect T5s an*>or T$s with the %#$ polymerase an*
may be essential )or gene e/pression.
?hereas only one %#$ polymerase )!nctions in bacterial cells, three *i))erent %#$ polymerases are
in+ol+e* in e!1aryotic n!clear transcription. The three polymerases initiate transcription only with speci)ic
combinations o) T5s an* T$s. 'NA polymerase I transcribes the genes that speci)y -.7, 27, an* ,7
r%#$. This polymerase is o)ten )o!n* associate* with chromosomes in the n!cleoli. 'NA polymerase II
transcribes )rom promoters that control the synthesis o) pre!m'NA, which consists o) co*ing 3e&ons4 an*
nonco*ing 3introns4 regions. 'NA polymerase III recogni2es promoters that control the synthesis o)
relati+ely short %#$s s!ch as t%#$s, - r%#$, an* others.
The bin*ing o) %#$ polymerase to promoter sites is *epen*ent on a n!mber o) T5s s!ch as the $-IID
comple&, which is )!nctionally comparable to sigma )actors in bacteria. T5II" is the )irst to bin* close to the
promoter at a site calle* a $A$A bo/ or Hogness bo/ abo!t D20 to DF0 bp be)ore the transcription start
site. &nce bo!n*, T5II" helps organi2e other T5s re8!ire* )or initiation o) %#$ synthesis. The comple/ o)
transcription )actors an* %#$ polymerase comprise the pre!initiation comple&, which yiel*s basal le+els
o) transcription. In*!ction to higher le+els re8!ires the presence o) other acti+ators bin*ing to enhancer
elements. $cti+ator proteins li1e )tf, p1, an* "ct!1 ca!se the "#$ to loop bac1 !pon itsel) so that the
acti+ator proteins interact with the pre-initiation comple/, signaling the %#$ polymerase to begin
synthesi2ing high le+els o) %#$.
%#$ .%&C'I#G
$)ter transcription, %#$ in e!1aryotes !n*ergoes signi)icant processing. Transcripts that speci)y proteins
are mo*i)ie* in the n!cle!s by the a**ition o) H-methylg!anine caps at their -6 en*s an* poly!A tails
appro/imately ,0002-0 n!cleoti*es long at their 36 termini. The pre-m%#$ is con+erte* into m%#$ by the
e/cision o) introns an* splicing o) e/ons.
Most splicing is carrie* o!t by en2yme comple/es, calle* spliceosomes, in the n!cle!s. pliceosomes
consist o) F *i))erent small nuclear ribonucleoprotein particles 3sn%#.s4 that wor1 together to bring the
en*s o) e/ons in a primary transcript near each other. The sn%#.s are constr!cte* )rom si/ to ten proteins
an* one or two o) the )i+e small nuclear 'NAs 3sn%#$s4 *esignate* .1, .2, .4, ./, an* .0. The
sn%#.s are generally *esignate* by the sn%#$s they contain. (, sn%#. bin*s to the -6 e/on-intron
@!nction, (- sn%#. attaches near the 36 intron-e/on @!nction, whereas (F-(I sn%#. bin*s near (-, an*
(2 associates where a lariat branch point will )orm. The spliceosome, in partic!lar (, sn%#., c!ts at the
36 en* o) an e/. (2 sn%#$ cataly2es the )ormation o) the lariat, whereas (- cataly2es the c!t at the - 6 en*
o) e/on I an* the splicing o) e/on - to e/on I.
In the simplest case, a spliceosome promotes the e/cision o) an intron between two e/ons an* the splicing
together o) the two e/ons. In more complicate* cases, a spliceosome may promote alternati(e splicing,
the splicing o) a pre-m%#$ into *i))erent combinations o) targete* e/ons. The m%#$ is s!bse8!ently
transporte* to the cytoplasm where it is translate* into proteins.
Chapter 5: Translation
In this chapter:
The Genetic Co*e
Translation in .ro1aryotes
Translation in '!1aryotes
TH' G'#'TIC C&"'
$ gro!p o) three a*@acent n!cleoti*es in "#$ is transcribe* into three complementary %#$ n!cleoti*es,
which in t!rn are translate* into a single amino aci* within a polypepti*e chain. =eing a triplet co*e, F3 J IF
*i))erent combinations e/ist, which is many more than are necessary to enco*e 20 *i))erent amino aci*s.
'ach co*ing triplet is re)erre* to as a codon. 'ach m%#$ co*on is con+entionally written with the -6
n!cleoti*e at the le)t an* the 36 n!cleoti*e at the right, beca!se protein synthesis begins at the -6 en*s o)
m%#$ molec!les an* procee*s towar* their 36 en*s. The co*e is highly degenerate in that more than one
co*on can speci)y the same amino aci*. =eca!se o) co*e *egeneracy, many changes 3mutations4 can
occ!r in a gene that will ha+e no e))ect on the amino aci* composition o) the gene pro*!ct. !ch changes
are re)erre* to as silent m!tations. The complementary base pairing between an m%#$ co*on an* its
anticodon in a t%#$ is !s!ally m!ch less restraine* at the thir* position than in the other two positions o)
the triplet. This phenomenon, calle* 1obble, allows the same t%#$ to recogni2e more than one m%#$
co*on in many cases.
The co*on -6-$(G-36 near the en* o) an m%#$ molec!le is the !s!al start 3initiation4 codon that places
methionine at the beginning 3amino en*4 o) all nascent e!1aryotic polypepti*e chains. i/ty-one co*ons are
sense codons that speci)y amino aci*s. There are three co*ons that are not recogni2e* by any t%#$:
($$, ($G, an* (G$. These are terme* nonsense codons or stop codons, beca!se they pro+i*e part o)
the signal that protein synthesis sho!l* stop at that point. The complete* polypepti*e can be release* )rom
its cognate t%#$ an* )rom the ribosome.
T%$#:$TI&# I# .%&9$%C&T'
%ibosomes contain two sites )or bin*ing t%#$ molec!les: the aminoacyl site 3$ site4, where each t%#$
molec!le )irst attaches, an* the peptidyl site 3. site4, where a t%#$ hol*s the growing polypepti*e chain.
The bacterial ribosome has two ma@or s!b!nits: a 30 s!b!nit to which the m%#$ an* t%#$s become
bo!n*, an* a -0 s!b!nit to which t%#$s also bin*. 'ach t%#$ becomes charged by a t'NA aminoacyl
synthetase that attaches a speci)ic amino aci* to each species o) t%#$. 'ach t%#$ molec!le has a loop
containing a triplet o) ribon!cleoti*es, calle* the anticodon, that can base pair with a complementary triplet
co*on in m%#$.
Translation in pro1aryotes begins at a start co*on. The bacterial m%#$ translation initiation co*on 3$(G4
enco*es N-)ormylmethionine, whereas internal $(G co*ons speci)y methionine. The 36-($C--6 antico*on
o) the t%#$ pairs 3in antiparallel )ashion4 with the complementary -6-$(G-36 co*on in the m%#$.
In the )irst step o) translation initiation, three protein initiation factors 3I5,, I52, I534 an* GT. bin* to the
30 ribosomal s!b!nit. The 30 s!b!nit bin*s to a hine!Dalgarno 3-"4 recognition se8!ence on the
m%#$ *!e to base pairing interactions with the ,I r%#$ component o) the ribosome. Then, a t%#$
loa*e* with the amino aci* N-)ormylmethionine 3t%#$
)Met
4 bin*s to the 30-m%#$ comple/. I53 is release*
an* then GT. hy*rolysis releases I5,, I52, G"., an* phosphate, allowing the -0 s!b!nit to @oin the
30m%#$- t%#$
)Met
comple/ to )orm a complete H0 initiation comple&. The t%#$
)Met
en*s !p in the .
site o) the ribosome. This completes the initiation phase.
The elongation phase procee*s with the help o) a gro!p o) protein elongation factors when a secon*
acti+ate* t%#$ enters the $ site 3again by speci)ic co*on-antico*on base pairing4. This places N-
)ormylmethionine an* the incoming amino aci* ne/t to one another so that a pepti*e bon* can be )orme*
between them by action o) an en2ymatic, ribosomal component calle* peptidyl transferase. The amino-
acyl bon* that hel* the N-)ormylmethionine to the 36-en* o) its t%#$ is bro1en sim!ltaneo!sly with the
)ormation o) the pepti*e bon*. The m%#$ translocates three n!cleoti*es thro!gh the ribosome an* a new
co*on is now positione* in the $ site. This process is repeate* !ntil a stop co*on is enco!ntere* at the $
site. $t termination, the polypepti*e is release* )rom the t%#$ with the help o) release factors. The
m%#$ an* the last t%#$ are release* )rom the ribosome as the ribosome *issociates into its 30 an* -0
s!b!nits.
T%$#:$TI&# I# '(9$%C&T'
The process o) translation in e!1aryotes is essentially the same as that in bacteria, b!t *i))ers in se+eral
important ways. tr!ct!rally, the ribosomal s!b!nits o) e!1aryotes consist o) F0 an* I0 s!b!nits that
together )orm an 70 comple/. $ltho!gh most bacterial m%#$s speci)y m!ltiple proteins, e!1aryotic
m%#$s co*e )or a single nascent polypepti*e chain. Howe+er, some newly synthesi2e* polypeti*e chains
may s!bse8!ently be en2ymatically clea+e* into two or more )!nctional protein components.
&nly three well-*e)ine* initiation )actors are re8!ire* )or translation o) E. coli m%#$s, b!t many more are
nee*e* in e!1aryotes. "!ring initiation, a special initiator t%#$ 3t%#$
iMet
4 brings an !n)ormylate*
methionine into the )irst position on the ribosome. In e!1aryotes the F0 ribosomal s!b!nit is tho!ght to
attach at the cappe* -< termin!s rather than at a hine-"algarno se8!ence as in pro1aryotes. It then sli*es
along !ntil it reaches the )irst $(G start co*on. Three *i))erent elongation )actors in e!1aryotes replace
those )o!n* in bacteria. Howe+er, a single release )actors acts in e!1aryotes.
Chapter 6: Mutations
In this chapter:
Types o) M!tations
M!tagens
Chromosomal $berrations
TC.' &5 M(T$TI&#
*utations are heritable changes in the genetic material that gi+e rise to alternati+e )orms o) any gene.
These alternate )orms are calle* alleles. There are two broa* types o) m!tations, those that a))ect the gene
an* those that a))ect whole chromosomes 3chromosomal aberrations4. Gene m!tations at the n!cleoti*e
le+el are calle* point mutations. $ny errors in the replication o) a gene within the "#$ molec!le res!lting
in the insertion, *eletion, or s!bstit!tion o) one or more bases will gi+e rise to a m!tation. '+en tho!gh the
cell has mechanisms to impro+e the )i*elity o) "#$ replication, e+ery once in a while a spontaneo!s
mista1e is ma*e lea*ing to a heritable change in the "#$ se8!ence. In the laboratory, m!tation rates can
be greatly increase* by e/posing cells to chemicals or physical agents calle* mutagens.
Many m!tations are *!e to the instability o) the n!cleoti*e bases in the "#$. #!cleoti*e bases may
!n*ergo str!ct!ral changes calle* tautomeric shifts, which res!lt in the re*istrib!tion o) electrons an*
protons so that the bases no longer pair normally. G may pair with T or $ with C, res!lting in heritable
changes in the n!cleoti*e se8!ence.
$ransitions occ!r when the mispairing res!lts in the replacement o) one p!rine )or another or one
pyrimi*ine )or another. $rans(ersions res!lt when a p!rine is replace* by a pyrimi*ine or +ice +ersa.
=eca!se the str!ct!ral changes lea*ing to transitions are relati+ely small, they occ!r more )re8!ently than
trans+ersions, which re8!ire more s!bstantial mo*i)ications o) a molec!le.
.oint m!tations res!lting )rom base s!bstit!tions in a gene that co*e )or a polypepti*e may res!lt in
missense2 nonsense, or silent m!tations. Missense m!tations res!lt in the replacement o) one sense
co*on )or another, altering the amino aci* enco*e* in that position. $ nonsense m!tation creates one o) the
three stop co*ons 3(G$, ($$, ($G4 an* res!lts in polypepti*es that are shorter than the normal ones.
ilent m!tations are changes in the se8!ence o) the co*on that *o not alter the enco*e* amino aci*.
-rameshift mutations are the res!lt o) n!cleoti*e or base insertions or *eletions within the co*ing region
o) a gene. The genetic co*e is translate* by the protein synthesis apparat!s by rea*ing se8!ential gro!ps
o) three bases that ma1e !p a co*on, beginning )rom the start co*on. I) a single base is a**e* or remo+e*,
all o) the co*ons )rom that point on will be change*. $ tr!ncate* protein may res!lt i) a co*on is m!tate* to
one o) the three stop co*ons.
M(T$G'#
Chemical an* physical m!tagens can ca!se m!tations by replacing one base with another in the "#$
molec!le, ca!sing str!ct!ral changes in a base so that it ca!ses it to mispair, ca!sing insertions or
*eletions, or *amaging a base so m!ch that it is !nable to pair with any other normal base.
3ase analogs are s!))iciently similar to the normal nitrogeno!s bases in "#$ that they can be incorporate*
into a replicating "#$ molec!le by "#$ polymerases. &nce incorporate*, howe+er, base analogs ha+e
abnormal base-pairing properties so that they pro*!ce m!tations *!ring s!bse8!ent "#$ replication
cycles. 5or e/ample, --bromo!racil an* 2-amino-p!rine are two common base analogs.
Alkylating agents ca!se m!tations by chemically altering bases so that they pair !p with a speci)ic base
other than the normally complementary base. Intercalating agents are planar molec!les that can insert
themsel+es between the stac1e* bases within the *o!ble heli/. These agents alter the molec!le o) "#$ in
s!ch a way that "#$ polymerases may insert or s1ip one or more bases *!ring replication, o)ten res!lting
in )rameshi)t m!tations. 5or e/ample, pro)la+in, acri*ine orange, an* ethi*i!m bromi*e are intercalators.
?hen !ltra+iolet light is absorbe* by a*@acent pyrimi*ines in one stran* o) a "#$ molec!le, a *imer )orms.
These *imers inter)ere with proper base pairing *!ring "#$ replication. The impact is so e/tensi+e that the
normal replicati+e process is stoppe* !ntil these *imers are repaire*.
CH%&M&&M$: $='%%$TI&#
Two types o) chromosomal aberrations can occ!r in cells: changes in chromosome str!ct!re an* changes
in chromosome n!mber. tr!ct!ral changes incl!*e *eletions, *!plications, in+ersions, an* translocations.
Deletions are chromosomal changes in which one or more genes or chromosomal segments are lost.
Duplications occ!r when one or more copies o) a chromosomal segment are present on the same or
*i))erent chromosomes. "eletions an* *!plications can occ!r in the same m!tational e+ent when two
homologo!s "#$ stran*s o+erlap, brea1 at the same time at two *i))erent 3nonhomologo!s4 points, an*
then re@oin with the wrong stran*. &ne o) the stran*s will be missing one or more genes, an* the reciprocal
stran* will ha+e an e/tra copy o) one or more genes. In(ersions occ!r when a brea1age in one o) the
chromosomes occ!rs an* the segment rotates ,70K be)ore it re@oins. $ranslocations ta1e place when
nonhomologo!s chromosomes brea1 an* e/change segments.
In *iploi* 32n4 organisms, there are two ma@or types o) chromosomal aberrations that are the res!lt o)
changes in chromosome n!mber. These are polyploidy an* aneuploidy. .olyploi*y res!lts when cells
ac8!ire one or more sets o) chromosomes beyon* the AnormalB n!mber o) sets. 5or e/ample, triploids 33n4
contain one e/tra set o) chromosomes, an* wo!l* there)ore be sterile, since they cannot pro*!ce balance*
gametes by meiosis.
Aneuploids are the res!lt o) changes in the in*i+i*!al n!mber o) homologo!s chromosomes in a set. This
!s!ally res!lts )rom nondis4unction *!ring meiosis 3see 5ig!re I-34. The ane!ploi* con*ition that res!lts
in three copies o) a gi+en chromosome is 1nown as trisomy 32nL,4.
Chapter 8: Recombinant DNA Technology
In this chapter:
Cloning
%estriction 'n*on!cleases
Mectors
Host Cells
C:&#I#G
"isco+eries in molec!lar biology ha+e allowe* scientists to *!plicate nat!ral genetic trans)er phenomena in
the laboratory an* to *e+elop metho*s to intro*!ce almost any type o) genetic in)ormation into an
organism. 5enetic engineering is the creation o) new "#$, !s!ally by lin1ing "#$ )rom *i))erent
organisms together by arti)icial means !sing en2ymes 1nown as restriction en#ymes.
)loning is the pro*!ction o) many copies o) the newly engineere* "#$. The ampli)ication o) a speci)ic
clone* gene or genes, co!ple* with a mar1e* increase in pro*!ction o) their protein pro*!cts, ma1es it
relati+ely easy to e/tract an* p!ri)y these proteins in the laboratory. $ s!itable plasmi* 3+ector4 is selecte*
in which to insert a *esire* gene 3*onor "#$4. =oth *onor "#$ an* +ector are *igeste* with the same
restriction en2yme, an* then inc!bate* together with ligase to @oin the *onor "#$ )ragments with the
plasmi*. The res!lt is a recombinant plasmi* that contains the *esire* "#$ )ragment. The recombinant
plasmi* is then !se* to trans)orm a host bacterial cell, creating a new genetic strain o) bacteria that stably
maintains the recombinant plasmi*.
The goal o) cloning is to isolate a *esire* gene or segment o) "#$ )rom an organism an* intro*!ce it into a
s!itable host cell to obtain large 8!antities o) the "#$. &)ten, this *onor "#$ is !se* )or the large-scale
pro*!ction o) important proteins, b!t the "#$ may also be !se* in the *etection o) in)ectio!s agents or
abnormal cells. #ormally, the *onor "#$ is a small portion o) the genome o) a cell, an* it is present as one
or two copies in each cell. There)ore, be)ore *onor "#$ can be e/tracte*, a s!))icient n!mber o) cells
containing the *esire* "#$ m!st be obtaine*, either )rom a small segment o) tiss!e or by c!lt!ring the
cells. The cells m!st then be *isr!pte* an* the genetic material 3either in chromosomes or in plasmi*s4
e/tracte*.
%'T%ICTI&# '#"&#(C:'$'
'estriction endonucleases are bacterial en2ymes that recogni2e speci)ic n!cleoti*e se8!ences within a
*o!ble-stran*e* "#$ molec!le an* clea+e at those locations. These en2ymes c!t "#$ into )ragments o)
+ario!s lengths, *epen*ing on the n!mber o) times the en2yme6s recognition site is repeate* within the
molec!le. Most restriction en*on!cleases !se* in cloning recogni2e base pair se8!ences )o!r to eight
n!cleoti*es long an* c!t within these se8!ences. Many restriction en2ymes ha+e recognition se8!ences
1nown as palindromes. .alin*romes are se8!ences that are i*entical when rea* in the -6N36 *irection on
both stran*s o) the "#$ molec!le. %estriction en*on!cleases may c!t the "#$ to pro*!ce )ragments with
cohesi(e 3Astic1yB4 ends or blunt ends.
The res!lting )ragments o) a restriction en2yme *igestion can be +is!ali2e* by a proce*!re 1nown as
electrophoresis. 'lectrophoresis in+ol+es the mo+ement o) charge* molec!les or ions thro!gh a semisoli*
s!pport me*i!m !n*er the in)l!ence o) an electrical )iel*. $garose gels are common me*ia )or the
electrophoresis o) "#$. The agarose gel, cast as a thin slab in a mol* with sample wells at one en*, is
s!bmerge* in a b!))er sol!tion with the sample well si*e towar* the negati+e pole 3 cathode4. The samples
are *ispense* into the wells an* a c!rrent )rom the power s!pply is applie* to the system. ince n!cleic
aci*s ha+e a negati+e charge at pHJ7.0, they migrate within the gel matri/ )rom the negati+e to the positi+e
pole 3anode4 at a rate *epen*ent !pon their si2e, shape, an* total charge. "#$ molec!les are in+isible to
the na1e* eye, b!t can be seen in gels by staining them with a sol!tion o) a *ye calle* ethidium bromide,
which intercalates between the stac1e* bases o) the "#$ molec!le an* )l!oresces.
The graphical representation o) recognition sites )or two or more restriction en*on!cleases is 1nown as a
restriction map )or that molec!le. 9nowing the restriction maps )or commonly !se* plasmi*s an*
bacteriophage "#$ genomes allows scientists to plan cloning strategies )or isolating an* mo+ing aro!n*
pieces o) "#$ that contain genes o) interest.
M'CT&%
$)ter a *esire* segment o) "#$ is c!t away )rom the *onor6s genome with restriction en*on!cleases, it is
ligated into a (ector "#$ molec!le, !s!ally a plasmi* or a bacteriophage genome. 6igases are en2ymes
that cataly2e the )ormation o) a phospho*iester bon* between the 36-hy*ro/yl gro!p o) a segment o) *onor
"#$ an* the -6-phosphate gro!p o) the +ector "#$.
$ +ector is a "#$ molec!le into which )oreign "#$ molec!les are ligate* an* inserte* into cells so that the
recombinant "#$ can be replicate*. $ goo* +ector sho!l* be stable, sel)-replicating, small, easily isolate*,
easily *etecte* an* co!l* ha+e a +ariety o) single c!ts. .lasmi* +ectors m!st also contain a mar1er, s!ch
as an antibiotic resistance gene, to )acilitate the selection o) bacterial cells that contain the plasmi*.
Mectors can be intro*!ce* into host cells by trans)ormation or trans*!ction. $ransformation is a
mechanism o) genetic trans)er between bacteria in which the *onor "#$ e/ists cell-)ree in the recipient
bacteri!m6s imme*iate en+ironment. "#$ can be nat!rally release* into the en+ironment when cells *ie
an* s!bse8!ently lyse. '/perimentally, "#$ containing genes o) interest, !s!ally within a plasmi*, can be
intro*!ce* into the en+ironment in or*er to trans)orm bacterial cells. The ability o) a recipient bacteri!m to
ta1e !p )ree "#$ an* become trans)orme* is 1nown as competence. ome strains o) bacteria are
nat!rally competent. In others, competence is a brie) physiological state *!ring the e/ponential growth
phaseO in these bacteria, Ca
2L
ions enhance the le+el o) competence. $ransduction, on the other han*, is
a mechanism o) "#$ !pta1e by bacteria in which the *onor "#$, consisting o) )ragments o) the bacterial
chromosome, is intro*!ce* into a bacterial cell +ia a phage +ector.
$n e&pression (ector is a +ector that carries a gene that can be e))iciently transcribe* an* translate* by
the host cell.
H&T C'::
$ n!mber o) bacterial an* yeast strains ha+e been *e+elope* )or recombinant "#$ e/periments. In or*er
)or a gi+en plasmi* to be replicate* by a host cell, the cell m!st recogni2e its origin o) replication site 3 oriC4.
%ecombinant plasmi* +ectors are normally intro*!ce* into competent cells by trans)ormation an* then
selecte* !sing appropriate cell c!lt!re me*ia. 5or e/ample, i) the +ector contains an ampR gene that
enco*es resistance to ampicillin, the c!lt!re me*ia wo!l* incl!*e that antibiotic to ens!re that only
trans)orme* cells will grow.
$nother metho* )or intro*!cing recombinant "#$ molec!les into host cells is electroporation. In this
metho*, a s!spension o) e/ponentially growing host cells is mi/e* with a sol!tion o) recombinant "#$
molec!les an* e/pose* to a high electric )iel* )or a )ew millisecon*s. The high +oltage alters the str!ct!re
o) the membrane so that pores are temporarily )orme*, allowing plasmi* "#$ to enter the cell. This metho*
is )ast an* e))icient.
I) bacteria are !se* as the host to clone e!1aryotic genes, certain steps m!st be ta1en to ma1e it possible
)or the bacteria to ma1e sensible m%#$ an* )!nctional proteins, since bacteria *o not possess
mechanisms )or processing e!1aryotic pre-m%#$ molec!les. To *o this, it is necessary to isolate alrea*y
processe* m%#$ )rom the *onor e!1aryotic cells an* con+ert the single-stran*e* %#$ to *o!ble-stran*e*
"#$. 'e(erse transcriptase )rom retro+ir!ses !ses %#$ templates )or synthesi2ing "#$. The res!lting
"#$ molec!les, 1nown as c"#$ 3)or complementary "#$4, can then be !se* )or cloning in bacteria since
they posses only intron-)ree protein-co*ing genetic in)ormation.
Chapter 9: Nucleic Acid Manipulations
In this chapter:
#!cleic $ci* Hybri*i2ation
The .olymerase Chain %eaction
#!cleic $ci* e8!encing
#(C:'IC $CI" HC=%I"IP$TI&#
5rom *e+elopments in the area o) genetic engineering an* molec!lar biology, a power)!l tool 1nown as
DNA hybridi#ation has emerge*. This techni8!e is !se* to *etect the presence o) "#$ )rom pathogens in
clinical specimens an* to locate speci)ic genes in cells. "#$ hybri*i2ation ta1es a*+antage o) the ability o)
n!cleic aci*s to )orm stable, *o!ble-stran*e* molec!les when two single stran*s with complementary
bases are bro!ght together !n*er )a+orable con*itions.
In "#$ hybri*i2ation assays, "#$ )rom a +ir!s or cell is *enat!re* with al1ali to separate the stran*s. The
single stran*s o) "#$ are then attache* to a soli* s!pport s!ch as a nitrocell!lose or nylon membrane so
that the stran*s *o not reanneal. The "#$ is attache* to the membrane by its s!gar-phosphate bac1bone
with the nitrogeno!s bases pro@ecting o!twar*. To characteri2e or i*enti)y the target "#$, a single-stran*e*
"#$ or %#$ molec!le o) 1nown origin, calle* a probe, is a**e* to the membrane in a b!))ere* sol!tion.
This allows the )ormation o) hy*rogen bon*s between complementary bases. The probe, so calle* beca!se
it is !se* to see1 or probe )or "#$ se8!ences, is labele* with a reporter gro!p, which may be a
ra*ioacti+e atom or an en2yme whose presence can be easily *etecte*.
The probe is allowe* to react with the target "#$O then any !nreacte* probe is remo+e* by washing in
b!))ere* sol!tions. $)ter the washes, all that remains on the nitrocell!lose is the target "#$ an* any probe
molec!les that ha+e attache* to complementary se8!ences in the target "#$, )orming stable hybri*s.
Hybri*i2ation o) target an* probe "#$s is *etecte* by assaying )or the probe6s reporter gro!p. I) the
reporter gro!p is *etecte*, hybri*i2ation has ta1en place. I) no reporter gro!p is *etecte*, it can be
ass!me* that the target molec!le *oes not ha+e se8!ences that are complementary to those o) the probe,
an* hence, the gene or "#$ segment so!ght is not present in the sample.
Three common )ormats are !se* in soli*-phase hybri*i2ation assaysO *ot blot, o!thern blotting, an* in situ
hybri*i2ation. In the dot blot assay, a speci)ie* +ol!me o) sample or specimen is spotte* onto a small area
o) nitrocell!lose membrane, which is then carrie* thro!gh the proce*!re *escribe* abo+e. outhern
hybridi#ation assays in+ol+e restriction en2yme *igestion an* agarose gel electrophoresis o) the target
"#$ prior to the hybri*i2ation assay. The *i))erent ban*s on the agarose gel are trans)erre* by capillary
action onto a nitrocell!lose or nylon membrane in a blotting apparat!s. "!ring the trans)er, each o) the
"#$ ban*s is trans)erre* onto the membrane in the same relati+e position that it ha* in the gel. $)ter the
trans)er, the target "#$ is probe* an* *etecte*, as in the *ot blot assay. In situ hybridi#ation assays
in+ol+e the probing o) intact cells or tiss!e sections a))i/e* to a microscope sli*e. This type o) soli*-phase
assay has the a*+antage that one cannot only *etect the presence o) target "#$ in intact cells b!t also
*etermine the location o) s!ch target "#$ within a tiss!e. $n important application o) in situ hybri*i2ation is
)or the *etection o) +ir!ses an* certain types o) bacteria within in)ecte* cells.
TH' .&:CM'%$' CH$I# %'$CTI&#
The replication o) genetic material is carrie* o!t by en2ymes calle* "#$ polymerases. These en2ymes
initiate the synthesis o) "#$ starting )rom a primer bo!n* to a template. The primers are generally ; to 2-
bases in length an* establish the site where "#$ replication begins. ?ith the polymerase chain reaction
3.C%4, any partic!lar stretch o) genetic material can be pinpointe* an* replicate* n!mero!s times simply
by selecting a pair o) primers that )lan1 the *esire* stretch o) "#$. The .C% is pre*icate* on the annealing
o) two oligon!cleoti*es 3primers4 o) 1nown composition to a target se8!ence o) interest an* the e/tension
o) the oligon!cleoti*es with a "#$ polymerase. 'ach reaction is repeate* s!bse8!ent to a *enat!ration
step, th!s allowing )or e/ponential ampli)ication.
The .C% in+ol+es three temperat!re inc!bations or steps that are repeate* 20--0 times. &ne repetition o)
three steps is calle* a cycle. In the )irst step, calle* denaturation, the two stran*s o) the target "#$
molec!le are separate* 3*enat!re*4 by heating the "#$ to ;FKC to brea1 the hy*rogen bon*s between
bases, yiel*ing two separate stran*s. In the secon* step, calle* annealing, two primers hybri*i2e to
complementary se8!ences in the single stran*s. The primers are short 320030 bases in length4, synthetic
stretches o) single-stran*e* "#$. They are selecte* so that one primer is complementary to one en* o) the
gene o) interest on one stran*, while the secon* primer is complementary to the opposite en* on the other
stran*. The primers )orm hy*rogen bon*s with 3anneal to4 their complementary se8!ences, )orming stable,
*o!ble stran*e* molec!les. $nnealing temperat!res range between 3H an* I0KC. "!ring the thir* step,
e&tension, or elongation, the primers are e/ten*e* by a thermostable "#$ polymerase at H2KC.
#(C:'IC $CI" 'Q('#CI#G
Nucleic acid sequencing re+eals the genetic co*e o) a "#$ molec!le. It may be carrie* o!t !sing one o)
two metho*s, each o) which res!lts in the pro*!ction o) "#$ )ragments o) +ario!s lengths, *i))ering )rom
each other by a single base an* )rom which one can in)er the n!cleic aci* se8!ence o) the molec!le. This
is accomplishe* !sing denaturing polyacrylamide gels. ?hereas agarose gels can separate "#$
molec!les *i))ering in length by 300-0 bases, polyacrylami*e gels can *iscriminate among "#$ molec!les
*i))ering in length by a single base. "enat!ring gels ca!se the "#$ molec!le to become single stran*e*
an* remain that way thro!gho!t the entire process o) electrophoresis. "enat!ring gels contain urea an*
are r!n at ele+ate* temperat!res, both o) which promote the separation o) the two stran*s o) the "#$
molec!le.
$gain, the "#$ m!st be labele* in or*er to be +is!ali2e*. The most common )orm o) labeling is with
ra*ioacti+e isotopes, in partic!lar, 32., 33., or 3-. $)ter electrophoresis, the gel is *rie* an* place* ne/t
to a sheet o) /-ray )ilm in a *ar1 place. "!ring this time the ra*ioacti+e particles emitte* )rom the isotope in
each "#$ molec!le Ae/poseB the )ilm, an* a)ter *e+elopment, a *ar1 ban* is seen on the )ilm at the
position where the "#$ ban* was locate* in the gel. This pict!re, calle* an autoradiograph, is a mirror
image o) the position o) the "#$ ban*s in the gel.
There are two metho*s that can be !se* to se8!ence "#$ molec!les. The *a&am!5ilbert metho* is
base* on clea+age o) "#$ at speci)ic sites by chemicals rather than en2ymes. Howe+er, this metho* is
sel*om !se* anymoreO the anger metho* is pre)erre*. In the anger metho*, the en2ymatic synthesis o)
"#$ ta1es place by the se8!ential )ormation o) a phospho*iester bon* between the )ree -6-phosphate
gro!p o) an incoming n!cleoti*e an* the 36-&H gro!p o) the growing chain. This process ta1es place
thro!gho!t the length o) the "#$ molec!le. Dideo&ynucleotides lac1 a 36-&H gro!p, an* ha+e a 36-H
gro!p instea*. In the presence o) a *i*eo/yn!cleoti*e, the synthesis o) "#$ stalls beca!se the
*iphosphate bon* cannot be )orme*. The chain growth terminates at that point, an* the last base on the 3 6-
en* o) the chain is a *i*eo/y terminator. This mo*i)ication o) anger6s metho* o) "#$ se8!encing is 1nown
as dideo&y termination sequencing.
In the anger se8!encing techni8!e, )o!r *i))erent reaction mi/t!res are !se* to se8!ence a "#$
)ragment. 'ach reaction mi/t!re contains the template "#$ molec!le to be se8!ence*, ra*ioacti+ely
labele* primers, all )o!r *eo/yn!cleoti*es, "#$ polymerase, an* a *i))erent *i*eo/y terminator 3**$T.,
**CT., **GT., or **TT.4. ?hen one o) these terminators is incorporate* in the newly synthesi2e* "#$
stran*, it will stop )!rther synthesis o) that stran*O the res!lt is that all the stran*s o) +ario!s lengths in the
reaction mi/t!re en* in the same base. The ra*ioacti+e pro*!cts are separate* by electrophoresis an*
+is!ali2e* by a!tora*iography. 'eading from the bottom of the gel 7shortest fragments terminated
closest to the /8!end9 up1ard re(eals the base sequence complementary to that of the template
strand: