19. Molecular Biology Copy

8/21/2019 19. Molecular Biology Copy

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MOLECULAR BIOLOGY

NUCLEOTIDES

Questions:

1. Give the soures o! "r#on "n$ nitro%en o! &urine "n$ &'ri(i$ine rin%. )on

M"' *++,*. N"(e - $ierent t'&es o! neu/eoti$es %ivin% their #io/o%i"/ i(&ort"ne.

)on M"' *++-0. h"t is the &"th2"' #re"3$o2n o! &urine nu/eoti$e4 )on M"' *++--. S"/v"%e &"th2"'.,. rite " note on &urine s"/v"%e &"th2"'. )on M"' *+1+5. h"t is %out4 Enu(er"te the /ini"/ 6n$in%s in %out. h"t is the (o$e o!

"tion o! the $ru% "//o&urino/ in this on$ition47. h"t is Lesh 8 N'h"n s'n$ro(e49. Lesh 8 N'h"n s'n$ro(e. )on M"' *++1. N"(e the in #orn error o! (et"#o/is( "ssoi"te$ 2ith h'&erurie(i" "n$

se/!8(uti/"tion.1+.Oroti "i$uri"

C;EMISTRY O< NUCLEOTIDES

N"(e - $ierent t'&es o! neu/eoti$es %ivin% their #io/o%i"/ i(&ort"ne. )on M"'

*++-

1. Nucleotides are precursors of deoxy-ribonucleic acid (DNA) and ribonucleic acid (RNA).

Nucleotides are also components of important co-enzymes like NAD+ and AD! and

metabolic re"ulators suc# as cA$% and c&$%.'. A nucleotide is made up of components

1. Nitro"enous base! (a purine or a pyrimidine)'. %entose su"ar! eit#er ribose or deoxyribose*. %#osp#ate "roups esteried to t#e su"ar.

. NUCLEOSIDE = nitro"enous base + pentose su"ar,. Nu/eoti$e >Nu/eosi$e (ono&hos&h"te? Nucleoside + %#osp#ate. Nu/ei "i$s >DNA @ RNA) polymers of nucleotide

/. The &urine #"ses adenine and "uanine* present in bot# RNA and DNA .0. The &'ri(i$ine #"ses cytosine! t#ymine and uracil. ytosine is present in bot# DNA

and RNA. 2#ymine is present in DNA and uracil in RNA.3. DNA >Deo' ri#onu/eoti$es? 2#ere are four di4erent types of nucleotides found in

DNA! di4erin" only in t#e nitro"enous base.1. d-adenosine + %i 5d-A$% (d-adenylic acid)'. d-"uanosine + %i 5d-&$% (d-"uanylic acid). d-cytidine + %i 5d-$% (d-cytidylic acid),. d-t#ymidine + %i 5d-2$% (d-t#ymidylic acid)

. RNA >Ri#onu/eoti$es?1. Adenosine + %i 5Adenosine monop#osp#ate (A$%) (Adenylic acid)'. &uanosine + %i 5&uanosine monop#osp#ate (&$%) (&uanylic acid). ytidine + %i 5ytidine monop#osp#ate ($%) (ytidylic acid)

,. 6ridine + %i 56ridine monop#osp#ate (6$%) (6ridylic acid). 7nosine + %i 57nosine monop#osp#ate (7$%) (7nosinic acid)

1+.Nu/eoti$es 8 #io/o%i"/ i(&ort"ne:1. Nucleotides are essential for RNA and DNA production and! t#erefore! proteins

cannot be synt#esized or cells proliferate. 2#ey precursors of A2%! &2%! 62%! 2%

and t#eir deri8ati8es.'. Nucleotides also ser8e as carriers of acti8ated intermediates in t#e synt#esis of

6D%-"lucose and D%-c#oline.. Nucleotides play an important role as 9ener"y currency: in t#e cell e". A2%,. ;ulfate donor Adenosine


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. 2#e met#yl "roup donor ;- adenosylmet#ionine./. 2#ey are structural components of se8eral essential coenzymes! suc# as coenzyme

A! AD! NAD+! and NAD%+.0. ;econd messen"ers cyclic adenosine mono p#osp#ate (cA$%) cyclic "uanosine

monop#osp#ate (c&$%)3. Allosteric re"ulator &2%=. c&$% ser8es as a second messen"er in response to nitric oxide (N>) durin"

relaxation of smoot# muscle.1?. 6D%-su"ar deri8ati8es participate in biosynt#esis of "lyco"en and proteo"lycans.11. 6D%-"lucuronic acid forms t#e urinary "lucuronide con@u"ates of bilirubin1'. 2% participates in biosynt#esis of sp#in"omyelin1. ;ynt#etic analo"s like -uorouracil! and /-mercaptopurine! for cancer treamnet.1,. 2#e purine analo" allopurinol! used in treatment of #yperuricemia and "out1. ytarabine is used in c#emot#erapy of cancer! and azat#ioprine is employed

durin" or"an transplantation to suppress immunolo"ic re@ection

BIOSYNT;ESIS O< )URINE NUCLEOTIDES

1. 2#e contribution of $ierent "to(s "re !ro( $ierent soures for t#e formation of

t#e purine rin"

1. N1 of purine is deri8ed from amino "roup of aspartate.'. ' and s arise from formate of N1?- formyl 2B.. N and N= are obtained from amide "roup of "lutamine,. ,! and N0 are contributed by "lycine.. /directly comes from >'.

C@N soures o! &urines'. 2Co met#ods of synt#esis

1. The $e novo s'nthesis t#e purine rin" is synt#esized from di4erent small

components. 2#e pat#Cay is expensi8e in terms of t#e use of #i"# ener"y

p#osp#ate bonds.'. S"/v"%e &"th2"' %urine is synt#esized from intermediates in t#e de"radati8e

pat#Cay for nucleotides.

DE NOO SYNT;ESIS O< )URINE

2#ere are 11 steps in t#e de no8o synt#esis pat#Cay

;tep ? %reparatory ;tep %#osp#o ribosyl purop#osp#ate (%R%%) synt#esis

Ribose-5-phosphate + ATP →ADP + Phospho ribosyl pyrophosphate (PRPP) (enzyme:

PRPP synthetase)

;tep 1 &lutamine + %R%% -p#osp#oribosylamine.(enzyme- "lutamyl

amidotransferase)

'0


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;tep ' -%#osp#oribosylamine + "lycine + A2% "lycinamide ribotide (&AR).

(;ynt#etae)

;tep &lycinamide ribotide + N1?-ormyl tetra#ydrofolate formyl "lycinamide

ribosyl - p#osp#ate.(formyl transferase)

;tep , ormyl"lycinamide ribosyl - p#osp#ate+ &lutamine formyl "lycinamidine

ribonucleotide (&A$) (synt#etase);tep ormyl "lycinamidine ribonucleotide + A2% -aminoimidazole ribonucletide

(A7R) (synt#etase)

;tep / -aminoimidazole ribonucletide + >' -amino- ,-carboxy-amino-

imidazole ribonucleotide (AA7R). (carboxylase)

;tep 0 -amino- ,-carboxy-aminoimidazole ribonucleotide + aspartate + A2% N-

succinyl--amino-imidazole carboxamide ribonucleotide. (;A7AR)

(synt#etase)

;tep 3 N-succinyl--amino-imidazole carboxamide ribonucleotide -

aminoimidazole , carboxamide ribonucleotide (A7AR) + umarate

(Adenosuccinate lyase)

;tep = -amino imidazole , carboxamide ribonucleotide (A7AR) + N1>-ormyl

tetra#ydrofolate formamino imidazole ,-carboxamide ribonucleotide

(formyl transferase)

;tep 1? ormamino imidazole ,-carboxamide ribonucleotide inosine

monop#osp#ate( /M)) + B'> (cyclo#ydrolase)

;tep 11 7$%+ Aspartate+ &2% A$% + umarate

'3


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'=


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SALAGE )AT;AY

1. A s"/v"%e &"th2"' is a pat#Cay in C#ic# nucleotides (purine and pyrimidine) are

synt#esizedfrom intermediates in t#e de"radati8e pat#Cay for nucleotides. 2#e free purines

(adenine! "uanine and #ypoxant#ine) are formed in t#e normal turno8er of nucleic

acids (particularly RNA)! and also obtained from t#e dietary sources. 2#e purines can

be directly con8erted to t#e correspondin" nucleotides.'. >n t#e contrary! in De No8o pat#Cays! t#e bases of t#e nucleotides are made from

scratc# by usin" simpler startin" materials (includin" amino acids). or t#is process!

A2% #ydrolysis is reEuired.. ;al8a"e pat#Cays recycle already used bases by reattac#in" t#em to a ribose. 2#e

sal8a"e pat#Cay economizes intracellular ener"y expenditure.,. ;al8a"e pat#Cay is important in eryt#rocytes and brain C#ere de no8o synt#esis is nor

operatin".. %R%% is startin" material in t#is pat#Cay. %#osp#oribosyl pyrop#osp#ate (%R%%) is t#e

donor of ribose -p#osp#ate in t#e sal8a"e pat#Cay./. 2#e free purines are sal8a"ed by tCo di4erent enzymes* adenine p#osp#o ribosyl

transferase (A%R2ase) and #ypoxant#ine "uanine p#osp#oribosyl transferase

(B&%R2ase). Adenine p#osp#or ribosyl transferase

i. A$enine)R)) F A$enosine (ono&hos&h"te ))i

Bypoxant#ine "uanine p#osp#o ribosyl transferase

ii. Gunine )R)) 8F Gu"nosine (ono &hos&h"te ))i


iii. ;'&o"nthine8%u"nine )R)) F Inosine

(ono&hos&h"te ))i0. Absence of enzymes of sal8a"e pat#Cay produces specic clinical syndromes. A defect

in t#e enzyme Bypoxant#ine "uanine p#osp#o ribosyl transferase(B&%R2) causes

Lesh8N'h"n s'n$ro(e .

Re%u/"tion o! )urine S'nthesis:

1. 2#e intracellular concentration of %R%% re"ulates purine synt#esis to a lar"e extent'. 7f A$% and $% are a8ailable in adeEuate amounts to meet t#e cellular reEuirements!

t#eir synt#esis is turned o4 at t#e amidotransferase reaction.. A$% in#ibits adenylsuccinate synt#etase C#ile $% in#ibits 7$% de#ydro"enase,. 2#e formation of A$% from 7$% reEuires &2%* similarly formation of &$% reEuires A2%.

Bence bot# &2% and A2% are made a8ailable in suFcient Euantities.

)urine S'nthesis Inhi#itors:

1. olic acid (2B) is essential for t#e synt#esis of purine nucleotides (reactions , and 1?).

;ulfonamides are t#e structural analo"s of paraaminobenzoic acid (%AGA). 2#ese sulfa

dru"s can be used to in#ibit t#e synt#esis of folic acid by microor"anisms. 2#is

indirectly reduces t#e synt#esis of purines and! t#erefore! t#e nucleic acids (DNA and

RNA). ;ulfonamides #a8e no inuence on #umans! since folic acid is not synt#esized

and is supplied t#rou"# diet.'. 2#e structural analo"s of folic acid (e.". met#otrexate) are Cidely used to control

cancer. 2#ey in#ibit t#e synt#esis of purine nucleotides (reaction , and 1?) and! t#us!

nucleic acids. Got# t#ese reactions are concerned Cit# t#e transfer of one-carbon

moiety (formyl "roup). $ercaptopurine in#ibits t#e con8ersion of 7$% to &$% and

A$%.2#ese in#ibitors also a4ect t#e proliferation of normally "roCin" cells. 2#is causes

?

http://en.wikipedia.org/wiki/Metabolic_pathwayhttp://en.wikipedia.org/wiki/Nucleotidehttp://en.wikipedia.org/wiki/Purinehttp://en.wikipedia.org/wiki/Pyrimidinehttp://en.wikipedia.org/wiki/Nucleotidehttp://en.wikipedia.org/wiki/Purinehttp://en.wikipedia.org/wiki/Pyrimidinehttp://en.wikipedia.org/wiki/Metabolic_pathway


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many side-e4ects includin" anemia! baldness! scaly skin etc.. Azaserine (diazo acetyl-H-;erine) is a "lutamine anta"onist and t#erefore in#ibits

reactions in8ol8in" "lutamine.,. >t#er synt#etic nucleotide analo"ues used as anticancer a"ents are /-t#io "uanine

and 3-aza "uanine.

DEGRADATION O< )URINE NUCLEOTIDES

Ste&s o! )urine #re"3$o2n1. A$% is rst deaminated to produce 7$% by A$% deaminase.'. 7$% is con8erted to inosine and &$% to "uanosine by t#e action of


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Disor$ers o! )urine Met"#o/is(:

;Y)ERURICEMIA I GOUT

1. 6ric acid is t#e end product of purine metabolism in #umans. 2#e normal concentration of

uric acid in t#e serum of adults is of -0 mKdl. 7n Comen! it is about 1 m" loCer t#an in

men. 2#e daily excretion of uric acid is ??-0?? m".

'. 7t is dened as serum uric acid concentration exceedin" 0 m"Kdl in male and / m"Kdl infemale. 2#e manifestations are due to t#e loC solubility of uric acid in Cater.

. &out is a disorder c#aracterized by #i"# le8els of uric acid. 2#e #yperuricemia can lead to

t#e deposition of mono -sodium urate crystals in t#e @oints! and an inammatory response

to t#e crystals! causin" rst acute and t#en pro"ressin" to c#ronic "outy art#ritis. uric acid

is deposited in cooler areas of t#e body to cause top#i. 2#us top#i are seen in distal @oints

of foot. 7ncreased excretion of uric acid may cause deposition of uric acid crystals in t#e

urinary tract leadin" to calculi or stone formation Cit# renal dama"e. &out may be eit#er

primary or secondary.-. )ri("r' %out lt is an inborn error of metabolism and is related to increased synt#esis of

purine nucleotides. 2#e folloCin" are t#e important causes of primary "outa. Abnormal %R%% synt#etase lack of allosteric control

b. Abnormal -p#osp#oribosyl amido transferase o8erproduction of purine nucleotidesdue to lack of feedback control

c. Deciency of enzymes of sal8a"e pat#Cay* increased a8ailability of %R%% and

decreased purines* so feedback in#ibition is lost.d. &lucose /-p#osp#atase deciency ln type 7 "lyco"en stora"e disease (8on &ierke


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b. 7ncrease renal excretion of urate by uricosuric dru"s! C#ic# decrease t#e

reabsorption of uric acid from kidney tubules! e.". probenecid.c. A//o&urino/

i. 7t #as similar structure like #ypoxant#ine. Allopurinol is an analo"ue of

#ypoxant#ine. Allopurinol is a competiti8e in#ibitor of xant#ine oxidase

t#ereby decreasin" t#e formation of uric acid.ii. 2#e dur" causes a rapid fall in serum uric acid le8el and an increase in

concentration of #ypoxant#ine and xant#ine in blood. Got# xant#ine and

#ypoxant#ine are more soluble and so are excreted easily in urine.iii. Jant#ine oxidase con8erts allopurinol to alloxant#ine. 7t is a more e4ecti8e

in#ibitor of xant#ine oxidase. 2#is is a "ood example of suii$e

inhi#itionH.i8. Dosa"e 7nitially 1?? to '?? m" daily. $aintenance '?? to /?? m" daily. Not

recommended in c#ildren.8. 7n addition to "out! t#e dru" can be used in secondary #yperuricaemia.

8i. Allopurinol also #as an in#ibitory action on t#e enzyme tryptop#an

pyrrolase.d. olc#icine! an anti-inammatory a"ent is 8ery useful to arrest t#e art#ritis in "out.e. 6rate oxidase 2#e dru" can be used in loCerin" uric acid le8el by oxidisin" uric

acid.)seu$o%out:

2#e clinical manifestations of pseudo"out are similar to "out. Gut t#is disorder is

caused by t#e deposition of calcium pyrop#osp#ate crystals in @oints. ;erum uric acid

concentration is normal in pseudo"out.

*. ;G)RT $e6ien' LESC;8NY;AN SYNDROMEa. 7t is an J-linked in#erited disorder of purine metabolism. 7ncidence is 11?!???

males.b. 7t is due to a defect of #ypoxant#ine-"uanine p#osp#o ribosyl (B&%R2ase). ;o! t#e

rate of sal8a"e pat#Cay is decreased resultin" in accumulation of %R%% and

decreased le8el of in#ibitory purine nucleotides.


i. Gunine )R)) 8F Gu"nosine (ono &hos&h"te

))i


ii. ;'&o"nthine8%u"nine )R)) F Inosine

(ono&hos&h"te ))ic. $utations t#at leads to t#e defect include deletions! frames#ift mutations! base

substitutions! and aberrant mRNA splicin".d. 2#e disease is c#aracterized by se/! (uti/"tion (includin" bitin" and #ead

ban"in") mental retardation! excessi8e uric acid and nep#ro-lit#iasis. &out

de8elops in later life. 2#e neurolo"ical manifestations su""est t#at t#e brain is

dependent on t#e sal8a"e pat#Cay for t#e reEuirements of 7$% and &$%.e. Allopurinol treatment t#at #elps to decrease uric acid production! #as no e4ect on

t#e neurolo"ical manifestations in t#ese patients

IMMUNODE;'&ourie(i"?

". A$enosine De"(in"se >ADA? De6ien'1. Adenosine deaminase plays a role in t#e breakdoCn of purines. Hymp#ocytes

#a8e t#e #i"#est acti8ity of t#is enzyme. A deciency of ADA results in an

accumulation of adenosine! C#ic# is con8erted to its ribonucleotide or

deoxyribonucleotide. As dA2% le8els rise! ribonucleotide reductase is in#ibited!

t#us pre8entin" t#e production of all deoxyribose-containin" nucleotides


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onseEuently! cells cannot make DNA and di8ide. 2#is Could lead decrease in 2

I G lymp#ocytes and a combined immunodeciency.'. 2#is autosomal recessi8e deciency causes recurrent infection and early deat#.

Antibiotics and periodic in@ections of immuno"lobulin Cill be life sa8in". Meekly

intramuscular in@ections of bo8ine ADA Cere found to be benecial.b. The $e6ien' o! &urine nu/eoti$e &hos&hor'/"se it is associated Cit# impairment

of 2-cell function but #as no e4ect on G-cell function. lt is belie8ed t#at d&2% in#ibits t#e

de8elopment of normal 2-cells.

DE NOO SYNT;ESIS O< )YRIMIDINE:

Ste& 1: C"r#"(o'/ )hos&h"te S'nthesis

&lutamine transfers its amido nitro"en to >' to produce carbamoyl p#osp#ate. 2#is

reaction is A2%-dependent and is catalysed by cytosomal enzyme carbamoyl

p#osp#ate synt#etase ll (%; 7l).

Ste& *: R"te Li(itin% Ste&:

arbamoyl p#osp#ate and aspartate combine to form carbamoyl aspartate. 2#e

enzyme is aspartyl transcarbamoylase (A2)! C#ic# is allosterically re"ulated. 2#e

atoms ' and N are deri8ed from carbamoyl p#osp#ate and t#e rest are from

aspartate.Ste& 0: ase)

Ste& -: Oi$"tion

Bydro"en atoms are remo8ed from and / positions! so t#at orotic acid is

produced. Lnzyme is di#ydro orotate de#ydro"enase (DB>DB). 7t reEuires NAD as co-

enzyme.

Ste& ,:


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. 6ridine p#osp#orylase adds ribose-1-p#ospate to t#e free base uracil! formin" uridine

monop#osp#ate. 6ridine kinase t#en p#osp#orylates t#is nucleoside into its

dip#osp#ate and trip#osp#ate forms.


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/


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-. Deoxyt#ymidine p#osp#orylase adds deoxyribose-1-p#osp#ate to t#ymine! formin"

deoxyt#ymidine monop#osp#ate. 2#ymidine kinase can t#en p#osp#orylate t#is

compound to deoxyt#ymidine dip#osp#ate and trip#osp#ate

Re%u/"tion o! )'ri(i$ine S'nthesis:

1. %; 77! A2 and DB>ase are present as a multienzyme complex and referred to as

AD%R2ase and >$% decarboxylase are also present as a sin"le functional

complex. Gecause of t#is clusterin" of enzymes! t#e synt#esis is Cell co-ordinated.'. Aspartate transcarbamoylase (A2) is allosterically in#ibited by 2%.. %; 77 (enzyme 1) is in#ibited by 62% and acti8ated by %R%%,. >$% decarboxylase is in#ibited by 6$%.

De%r"$"tion o! &'ri(i$ine nu/eoti$es:

1. ytosine and 6racil are de"raded in a similar Cay

1. irst pyrimidine nucletides are dep#osp#orylated to t#e nucleosides by O-

nucleotidases. %yrimidine nucleosides are t#en p#osp#orolysed into free pyrimidines

and pentose 1 p#osp#ate Cit# t#e #elp of nucleoside p#osp#orylases.'. ytosine Cill form uracil by deaminase.. 6racil! is t#en reduced to !/-di#ydrouracil by di#ydrouracil de#ydro"enase usin"

NAD%B.,. !/-di#ydrouracil is #ydrolyzed by #ydropyrimidine #ydrase to produce P-

ureidopropionic acid.. P-ureidopropionase con8ert P-ureidopropionic acid into >'! NB and P-alanine./. 2#e P-alanine can eit#er be used in t#e synt#esis of Anserine! carnosine or oA or can

be oxidised to acetate! NB and >'.

'. 2#ymine

1. 2#ymine released from t#ymidine or produced by t#e deamination of -met#ylcytosine

is reduced to di#ydrot#ymine by an NADB dependent de#ydro"enase'. Next is t#e #ydrase t#at brin"s about t#e #ydrolysis of di#ydrot#ymine to "i8e P-

ureidoisobutyric acid.. 2#e P-ureidoisobutyric acid is #ydrolysed by P-ureidoisobutyrase into >'! NB and P-

amino isobutyrate.

Disor$ers o! )'ri(i$ine Met"#o/is(:Oroti Ai$uri"

i. 2#e condition results from absence of eit#er or bot# of t#e enzymes! >%R2ase and >$%

decarboxylase. 7t is an autosomal recessi8e disease.ii. 2#ere is retarded "roCt# and me"aloblastic anemia. 2#e rapidly "roCin" cells are

more a4ected and #ence t#e anemia.iii. rystals are excreted in urine C#ic# may cause urinary tract obstruction. Due to lack of

feedback in#ibition orotic acid production is excessi8e.i8. 2#e condition can be successfully treated by feedin" cytidine or uridine. 2#ey may be

con8erted to 62% C#ic# can act as feedback in#ibitor.8. >rotic aciduria may also occur in ornit#ine transcarbamoylase deciency (urea cycle

enzyme) as carbamoyl p#osp#ate accumulates due to defecti8e con8ersion to

citrulline.8i. Allopurinol competes Cit# orotic acid for t#e enzyme orotate p#osp#oribosyl

transferase! leadin" to orotic aciduria and orotidinuria.

Anti"ner A%ents Atin% on )'ri(i$ines

i. $et#otrexate in#ibits di#ydrofolate reductase and t#ereby reduces t#e re"eneration of

2BA* it is a anticancer a"ent.ii. -uoro-uracil! -iodo uracil! -deoxy uridine! /-aza uridine! /-aza cytidine and -iodo-

'-deoxyuridine are anticancer dru"s! C#ic# competiti8ely in#ibit t#ymidylate synt#ase.iii. ytosine arabinoside C#ere ribose is replaced by arabinose is anot#er anticancer

a"ent.

0


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DEOJYRIBO NUCLEIC ACID

RE)LICATION O< DNA

1. Desri#e the (eh"nis( o! DNA re&/i"tion. A$$ " note on DNA re&"ir

(eh"nis(. Au% *++7*. N"(e the &ri(er reKuire$ !or DNA &o/'(er"se III. )on M"' *++0

0. De6ne the ter(s re&/i"tion tr"nsri&tion "n$ tr"ns/"tion. Desri#e theste&s invo/ve$ in &rotein #ios'nthesis. M"rh *++* Ot 19-. h"t is se(i8onserv"tive re&/i"tion4 E&/"in 2ith " si(&/e $i"%r"(. )on

De *++0,. h"t is O3""3i !r"%(ents4 )on M"' *++5

RE)LICATION

1. Durin" cell di8ision! eac# dau"#ter cell "ets an exact copy of t#e "enetic information

of t#e mot#er cell. 2#is process of copyin" t#e DNA is knoCn as DNA replication.'. 7n t#e dau"#ter cell! one strand is deri8ed from t#e mot#er cell* C#ile t#e ot#er strand

is neCly synt#esized. 2#is is called se(ionserv"tive type of DNA replication.

Ste&s:

1. >ri"in of replication (ori)

'. 6nCindin" of DNA. ormation of t#e replication fork,. 7nitiation and c#ain elon"ation. ormation of replication bubbles and li"ation of t#e neCly synt#esised DNA se"ments.

Ori%in o! Re&/i"tion >ori?

1. 2#ere are specic sites C#ere replication starts. 2#e ori"in of replication on t#e DNA

strand in bacteria is termed as ori. orrespondin" areas in #i"#er or"anisms are called

replicators C#ic# contain t#e base seEuences called t#e ori"in replication element

(>RL). 2#is area is reco"nized by specic proteins collecti8ely called t#e ori"in

reco"nition complex (>R).'. 2#e protein A or DnaA binds at specic sites of ori"in! and opens t#e duplex (double

strand)

Un2in$in% o! DNA:1. Relief of supercoil is done by a "roup of enzymes called topoisomerases.'. Belicase separates t#e strands of DNA! Cit#out a cut! but usin" ener"y from #ydrolysis

of A2%.0. DNA Re&/iso(e:

1. DNA replication needs more t#an '? enzymes and proteins! collecti8ely called

DNA replicase system or replisomes. L".i. Belicases mo8e on bot# directions! separatin" t#e strands in ad8ance of

t#e replication. 2#is forms a replication bubble Cit# tCo replication forks.ii. ;in"le stranded DNA bindin" proteins (;;G) stabilize t#e complex.

Re&/i"tion Bu##/e:

1. 2#e tCo complementary strands of DNA separate at t#e site of replication to form a

bubble. $ultiple replication bubbles are formed in DNA molecules for a rapidreplication process.'. 2#e enzyme primase in association Cit# sin"le-stranded bindin" proteins forms a

complex called primosome! and produces RNA primers.. DNA polymerase enzyme synt#esises a neC complementary strand of DNA! by

incorporatin" dN$% seEuentially! makin" use of sin"le stranded DNA as template.,. 2#e synt#esis of tCo neC DNA strands! simultaneously! takes place in t#e opposite

direction---one is in a direction (


14/54

A molecule of pyrop#osp#ate (%%i) is remo8ed Cit# t#e addition of eac# nucleotide.

Lac# ribonucleoside monop#osp#ate is added t#rou"# formation of a n t#e /e"$in% >!or2"r$? str"n$ t#e DNA is synt#esized continuously. >n t#e

/"%%in% >retro%r"$e? str"n$ t#e DNA is synt#esized in s#ort fra"ments! called

O3""3i !r"%(ents. 2#ese pieces are made in t#e normal <

< direction! and later @oined to"et#er.0. An enzyme capable of polymerisin" DNA in O 5 O direction does not exist in any

or"anism! so t#at bot# of t#e neCly replicated DNA strands cannot "roC in t#e same

direction simultaneously3. O3""3i &iees

a. 2#e small fra"ments of t#e discontinuously synt#esized DNA are called >kazaki

pieces. 2#ese are produced on t#e la""in" strand of t#e parent DNA. >kazaki

pieces are later @oined to form a continuous strand of DNA. DNA polymerase 7

and DNA li"ase are responsible for t#is process.b. ;e8eral >kazaki fra"ments (up to a t#ousand) must be seEuentially synt#esized

for eac# replication fork. 2o ensure t#at t#is #appens! t#e #elicase acts on t#e

la""in" strand to unCind dsDNA in a < to < direction.c. 2#e #elicase associates Cit# t#e primase to a4ord t#e latter proper access to

t#e template. 2#is alloCs t#e RNA primer to be made and! in turn! t#e

polymerase to be"in replicatin" t#e DNA. 2#is is an important reaction

seEuence since DNA polymerases cannot initiate DNA synt#esis de no8o. 2#e

mobile complex betCeen #elicase and primase #as been called a &ri(oso(e. d. As t#e synt#esis of an >kazaki fra"ment is completed and t#e polymerase is

released! a neC primer #as been synt#esized. 2#e same polymerase molecule

remains associated Cit# t#e replication fork and proceeds to synt#esize t#e

next >kazaki fra"ment.

=. 2#e template DNA strand (t#e parent) determines t#e base seEuence of t#e neCly

synt#esized complementary DNA.

=


15/54

0 "n$ , en$s o! DNA

The DNA )o/'(er"se Co(&/e

1. A number of di4erent DNA polymerase molecules en"a"e in DNA replication. 2#ese

s#are t#ree important properties (1) c#ain elon"ation! (') processi8ity! and ()

proofreadin".*. Ch"in e/on%"tion:

a. 6nder t#e inuence of DNA polymerase! t#e < #ydroxyl "roup of t#e end

nucleotide combines Cit# t#e < p#osp#ate "roup of t#e neC deoxynucleotide. 2#e pyrop#osp#ate is released from t#e deoxynucleoside tri p#osp#ate.

b. 2#is neCly added nucleotide Could noC polymerise Cit# anot#er one! formin"

t#e next p#osp#odiester bond. 2#e base pairin" rule is alCays obser8ed.

. )roeesivit' DNA polymerase 777 is a #i"#ly 9processi8e: enzymeQt#at is! it remains

bound to t#e template strand as it mo8es alon"! and does not di4use aCay-. )roo! re"$in%:

a. 2#e nucleotide seEuence of DNA Cill be replicated Cit#out errors. $isreadin" of

t#e template seEuence could result in mutations.a. DNA polymerase 777 #as a 9proofreadin": acti8ity t#rou"#


16/54

'. ompounds t#at in#ibit #uman topoisomerases are used as anticancer a"ents e.".

adriamycin! etoposide! doxorubicin. 2#e nucleotide analo"s t#at in#ibit DNA replication

are also used as anticancer dru"s e.". /-mercaptopurnie! -u orour acil.. $et#otrexate (in#ibits di#ydrofolate reductase) and ;-uorouracil (in#ibits t#ymidylate

synt#ase) block nucleotide synt#esis.,. 7n recent years! topoisomerase in#ibitors are bein" used. 2#ey block t#e unCindin" of

parental DNA strands and pre8ent replication.

, ste&s o! re&/i"tion:

DNA RE)AIR

1. List v"rious DNA re&"ir (eh"nis(s "n$ %ive their #io/o%i"/ i(&ort"ne. Ot *++0

C"uses o! errors in DNA:

1. Lndo"enousa. Replication errors

i. 7ncorrect base-pairin"ii. 7nsertion of one to a feC extra nucleotides

b. Reacti8e oxy"en species produced from normal metabolic byproducts'. Eo%enous

a. #emicals - nitrous acid* i"arette smoke contains carcino"ens suc# as t#e

aromatic polycyclic #ydrocarbonsb. Radiation-

i. 6ltra8iolet li"#t! can fuse tCo pyrimidines

ii. Bi"# ener"y ionizin" radiation! can cause double-strand breaksiii. Radiot#erapy c. #emot#erapyd. Siruses

Eets o! errors:

i. 7t is fortunate t#at a "reat ma@ority of t#e mutations probably occur in t#e DNA t#at

does not encode proteins!a nd conseEuentlyC ill not #a8e any serious impact on t#e

or"anism.ii. &ene $utation #an"e in a sin"le base pair in t#e #uman "enome can cause a

serious disease e.". sicklecell anemia.

,1

http://en.wikipedia.org/wiki/Reactive_oxygen_specieshttp://en.wikipedia.org/wiki/Radiotherapyhttp://en.wikipedia.org/wiki/Chemotherapyhttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Reactive_oxygen_specieshttp://en.wikipedia.org/wiki/Radiotherapyhttp://en.wikipedia.org/wiki/Chemotherapyhttp://en.wikipedia.org/wiki/Virus


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iii. 6ncontrolled cell di8isions anceri8. ell deat#

T'&es o! DNA $"("%es:

7. ;in"le-base alteration

A. Depurination

G. Deamination of cytosine to uracil

. Deamination of adenine to #ypoxant#ineD. Alkylation of base

L. 7nsertion or deletion of nucleotide

. Gase-analo" incorporation

77. 2Co-base alteration

A. 6S li"#tinduced t#ymine-t#ymine (pyrimidine) dimer

G. Gifunctional alkylatin" a"ent cross-linka"e

777. #ain breaks

A. 7onizin" radiation

G. Radioacti8e disinte"ration of backbone element

. >xidati8e free radical formation

7S. ross-linka"e

A. GetCeen bases in same or opposite strandsG. GetCeen DNA and protein molecules (e"! #istones)

T'&es o! re&"ir:

1. $ismatc# repair*. Gase excision- repair0. Nucleotide excisionrepair-. Double-strand break repair

Mis("th re&"ir:

1. 2#is mec#anism corrects a sin"le mismatc# base pair (e"! to A rat#er t#an 2 to

A) or a s#ort re"ion of unpaired DNA.'. 2#e ori"inal template DNA contains met#ylated residues. 2#e neCly synt#esised

strand Cill not #a8e met#ylated bases. ;o enzymes can reco"nize t#e ori"inal DNA

strand. 2#e mismatc#ed base is idendied by an endonuclease t#at makes

defecti8e strand remo8ed. A small se"ment of DNA Cit# correct seEuences of

bases is synt#esised by polymerase beta. 2#e "ap is lled by DNA li"ase.. aulty mismatc# repair #as been linked to #ereditary nonpolyposis colon cancer.

B"se Eision8Re&"ir:

1. 2#e bases cytosine! adenine and "uanine can under"o spontaneous depurination to

form uracil! #ypoxant#ine and xant#ine. 2#ese altered bases do not exist in t#e

normal DNA! and t#erefore need to be remo8ed. 2#is is carried out by base excision

repair.'. A defecti8e DNA in C#ic# cytosine is deaminated to uracil is acted upon by t#e

enzyme uracil DNA "lycosylase. 2#is results in t#e remo8al of t#e defecti8e base

uracil.. An endonuclease cuts t#e backbone of DNA strand near t#e defect and remo8es a feC

bases. 2#e "ap so created is lled up by t#e action of repair DNA polymerase and DNA

li"ase.

Nu/eoti$e eisionre&"ir:

1. 2#e DNA dama"e due to ultra8iolet li"#t! ionizin" radiation and ot#er en8ironmental

factors often results in t#e modication of certain bases! strand breaks! cross-linka"s

etc.'. A n excision nuclease (exinuclease) cuts t#e DNA on eit#er side of t#e dama"ed DNA.

2#is defecti8e piece is de"raded. 2#e "ap created by t#e nucleotide excision is lled

,'


18/54

up by DNA polymerase C#ic# "ets li"ated by DNA li"ase. Jeroderma pi"mentosum (J%) is a rare autosomal recessi8e disease. 2#e a4ected

patients are p#otosensiti8e a nd susceptible to skin cancers. lt is noC reco"nized t#at

J % is due to a defect in t#e nucleotide excision repair.

Dou#/e8str"n$ #re"3 re&"ir:

1. 2#e defect occurs as a result of ionizin" radiation or oxidati8e free radical "eneration.

2#is defect may lead to c#romosomal translocation! broken c#romosomes! and nallycel7 deat#.'. D;Gs can be repaired by #omolo"ous recombination or non-#omolo"ous end @oinin".. 7n non#omolo"ous system! t#e ends of tCo DNA fra"ments are brou"#t to"et#er by a

"roup of proteins t#at e4ect t#eir reli"ation. BoCe8er! some DNA is lost in t#e process.

7n #omolo"ous recombination repair! uses t#e enzymes t#at normally perform "enetic

recombination betCeen #omolo"ous c#romosomes durin" meiosis.

DE


19/54

;tructure

1. 2#e mRNA is a complementary copy of t#e template strand of t#e DNA.'. 2#e "ene present in DNA is transcribed into mRNA* 2#e m-RNA carries a specic

seEuence of nucleotides in 9triplets: called codons! responsible for t#e synt#esis of a

specic protein molecule.. '-T of total RNA in t#e cell* de"raded Euickly* t#ymine is not present in RNA* instead

uracil Cill be incorporated.,. 2#e < terminal of mRNA is UcappedU by a 0-met#yl"uanosine trip#osp#ate. 2#e cap is

in8ol8ed in t#e reco"nition of mRNA by t#e translation mac#inery! and also #elps

stabilize t#e mRNA by pre8entin" t#e attack of tRNA?.1. 2#ere are about /? di4erent species and constitute about 1T of t#e total RNA in t#e

cell. 2#ey are 8ery stable.'. 2#ey transfer amino acids from cytoplasm to t#e ribosomal protein synt#esizin"

mac#inery.. 2#e transfer RNAs contain extensi8e internal base pairin" and acEuire clo8er leaf like

structure. 2#ey also contain a si"nicant proportion of unusual bases. 2#ese include

di#ydrouracil (DB6)! pseudouridine! and #ypoxant#ine. $oreo8er many bases are

met#ylated.,. All tRNA molecules contain four main arms.

,,


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a. 2#e "e&tor "r(: 7t is at t#e < Lnd. 7t carries t#e amino acid. 2#is area #as 0

base pairs. 2#e end seEuence is A-snRNAs?: snRNAs! a subset of t#e small RNAs! are

si"nicantly in8ol8ed in rRNA and mRNA processin" and "ene re"ulation. 2#ey are

in8ol8ed in mRNA splicin" and take part in t#e formation of spliceosomes. All of t#em

are located in t#e nucleus. 2#ey complex Cit# specic proteins! to form small nuclear

ribonucleoprotein particles (;nRN%s). 7t is pronounced as U;nurpsU. %roduction of

autoantibodies a"ainst 9;nurps: cause systemic lupus eryt#ematosis (;HL)! a fatal

autoimmune disease.0. Miro8RNA >(iRNA). 2#ey alter t#e function of mRNA. 2#ey are moderately stable.

miRNAs cause in#ibition of "ene expression by decreasin" specic protein production.-. S("// Inter!erin% RNAs >siRNAs?: duplexes betCeen siRNA and mRNA results

inreduced specic protein production. siRNAs are freEuently used to decrease or

Uknock-doCnU specic protein le8els in experimental contexts in t#e laboratory.

TRANSCRI)TION

1. De6ne the ter(s re&/i"tion tr"nsri&tion "n$ tr"ns/"tion. Desri#e the ste&s

invo/ve$ in &rotein #ios'nthesis. )on M"rh *++* Ot 19*. rite in $et"i/s "#out the initi"tion e/on%"tion "n$ ter(in"tion o! tr"nsri&tion.

Give "n "ount o! &ost tr"nsri&tion"/ &roessin%.>MGR U?

,


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Intro$ution:

1. Centr"/ Do%(" o! Mo/eu/"r Bio/o%' 7t states t#at! in a cell! information oCs

from t#e nuclear DNA to RNA to protein synt#esis i.e. UDNA makes RNA makes proteinU'. Geno(e: 2#e total DNA ("enetic information) contained in an or"anism or a cell is

re"arded as t#e "enome. 2#e "enome is t#e store#ouse of biolo"ical information. lt

includes t#e c#romosomes in t#e nucleus and t#e DNA in mitoc#ondria! and

c#loroplasts. 2#e study of t#e structure and function of "enome is "enomics.. 2#e Cord %ene refers to t#e functional unit of t#e DNA t#at can be transcribed.,. Co$ons: 2#e letters A! &! 2! and correspond to t#e nucleotides found in DNA. Mit#in

t#e protein codin" "enes t#ese nucleotides are or"anized into t#ree-letter code Cords

called codons! and t#e collection of t#ese codons makes up t#e "enetic code. odon

consists of a seEuence of t#ree nucleotides* i.e. it is a triplet code.

RNA )OLYMERASES

2#ere are se8eral distinct RNA polymerases in eukaryotic cells.

1. RNA polymerase 7 synt#esizes rRNAs in8ol8ed in facilitatin" protein synt#esis by

t#e ribosome.'. RNA polymerase 77 is responsible for t#e synt#esis of mRNA and miRNAs.. RNA polymerase 777 catalyzes t#e synt#esis of tRNAs.

,. RNA polymerase Cit# t#e subunit structure W'PPϖσ

is called t#e #oloenzyme.TRANSCRI)TION:

1. 2ranscription is a process in C#ic# ribonucleic acid (RNA) is synt#esized from DNA. 2#e

"enetic master plan of an or"anism is contained in the seKuene o!

$eo'ri#onu/eoti$es in its DNA. Ribonucleic acid (RNA) are t#e 9Corkin" copies: of

t#e DNA.'. 2ranscription produces messen"er RNAs t#at are translated into seEuences of amino

acids to produce polypeptide c#ains or proteins! and ribosomal RNAs! transfer RNAs!

and additional small RNA molecules etc.. >ne of t#e tCo strands of DNA ser8es as a template called non-codin" strand or sense

strand and produces Corkin" copies of RNA molecules. 2#e ot#er DNA strand C#ic#

does not participate in transcription is referred to as codin" strand or antisense strand.

TRANSCRI)TION )ROCESS: 2ranscription in8ol8es t#ree di4erent sta"es-initiation!elon"ation and termination

Initi"tion

1. 2ranscription be"ins Cit# t#e bindin" of t#e RNA polymerase to a "ene in DNA. RNA

polymerases must be able toa. Reco"nize t#e start point for transcription of eac# "ene andb. #oose t#e appropriate strand of DNA to use as a template.c. ontrol t#e freEuency of transcription.

'. or abo8e purposes! t#e "ene contains a codin" re"ion and a re"ulatory re"ion.a. 2#e o$in% re%ion #as t#e DNA seEuence ("ene) t#at is transcribed into RNA.b. 2#e codin" re"ion of DNA includes not only t#e "ene for mRNA synt#esis! but

also t#e initiator! promoter! and terminator re"ions as Cell as t#e introns and is

called a tr"nsri&tion unit.c. 2#e re"ulatory re"ion consists of folloCin" tCo classes of seEuences in DNA.i. 2#e &ro(oter seKuenes controls t#e bindin" of RNA polymerase to

DNA and identies t#e start point of transcription in t#e DNA. %romoter

seEuences #a8e tCo components.1. 2#e proximal component! called t#e 2A2A box C#ic# directs RNA

polymerase 77 to t#e correct site (delity)'. Distal component called AA2 I& boxes C#ic# specify t#e

freEuency of initiation.a. 7n eukaryotes! proteins knoCn as %ener"/ tr"nsri&tion !"tors (or basal

,/


22/54

factors) bind to t#e 2A2A box and facilitate t#e bindin" of RNA polymerase 77.

2#is bindin" process in8ol8es at least six basal transcription factors.a. Anot#er class of seEuence elements can eit#er increase or decrease t#e rate of

transcription initiation of eukaryotic "enes. 2#ese elements are called eit#er

enh"ners or re&ressors! dependin" on #oC t#ey e4ect RNA synt#esis.

. After 277D binds to t#e 2A2A box and 2G%! 8e more transcription factors and RNA

polymerase combine around t#e 2A2A box in a series of sta"es to form C#at is knoCn ast#e &reiniti"tion o(&/e.

E/on%"tion )roess o! Tr"nsri&tion

1. 2ranscription! t#e synt#esis of RNA from a DNA template! is carried out by RNA

%olymerases. ;ynt#esis of t#e neC RNA molecule occurs in t#e O-to-O direction. 2#e

ribonucleoside trip#osp#atesO adenosine trip#osp#ate (A2%)! &2%! 2%! and 62% ser8e

as t#e precursors. Lac# nucleotide base pairs seEuentially Cit# t#e complementary

deoxyribonucleotide base on t#e DNA template. 2#e polymerase forms an ester bond

betCeen t#e α -p#osp#ate on t#e ribose O -#ydroxyl of t#e nucleotide precursor and

t#e ribose O-#ydroxyl at t#e end of t#e "roCin" RNA c#ain.'. 2#e RNA% mo8es alon" t#e DNA template. NeC nucleotides are incorporated in t#e

nascent mRNA! one by one! accordin" to t#e base pairin" rule.

. A tr"nsri&tion #u##/e containin"RNA%! DNA and nascent RNA is formed.

RNA% #as no nuclease acti8ity* so

t#ere is no proof readin". Anot#er

di4rence in RNA synt#esis is t#at RNA

polymerase does not reEuire a primer,. As t#e RNA% mo8es on t#e DNA

template! t#e DNA #elix unCinds

doCnstream and Cinds at t#e

upstream areas.

Tr"nsri&tion #u##/e

Ter(in"tion o! Tr"nsri&tion:

1. 2#e elon"ation of t#e sin"le-stranded RNA c#ain continues until a termination si"nal is

reac#ed.'. 2ermination includes remo8al of RNA polymerase from DNA and t#e release of t#e RNA

molecule. 2ermination is brou"#t about by tCo means

a. ;pecic seEuences on t#e DNA molecule function as t#e si"nal for termination

of t#e transcription process. 2#is alloCs t#e RNA to fold back on itself! formin"

,0

http://en.wikipedia.org/wiki/Preinitiation_complexhttp://en.wikipedia.org/wiki/Preinitiation_complex


23/54

a loop. 2#is structure is knoCn as a 9#airpin:. Additionally! @ust beyond t#e

#airpin! t#e RNA transcript contains a strin" of Us at t#e


24/54

b. %oly-A tailin"c. Lndonuclease clea8a"ed. $et#ylatione. Remo8al of intronsf. ;plicin" of exons (connect to"et#er).

. 7n bacteria! mRNA is not c#an"ed* and translation of mRNA starts e8en before completion of

transcription. %ost-transcriptional processin" is not only form RNA but for tRNA and rRNA

as Cell.,. The , "&&in% 2#is is t#e rst of t#e processin" reactions. 2#e ! end of mRNA is

transcription capped Cit# 0-met#yl"uanosine by an unusual


25/54

1. 2#e "enetic materials of some animal and plant 8iruses are made up of RNA instead of

DNA. Retro8irus is a sub"roup of RNA 8iruses. 2#e #uman immunodeciency 8irus

(B7S) causin" A7D; is a retro8irus.'. Bere t#e RNA acts as a template. Gased on t#is RNA! t#e enzyme! RNA dependent DNA

polymerase or re8erse transcriptase Cill make a neC DNA strand.. 2#e neC DNA acts as a template to produce double stranded DNA. 2#us "enetic

information is transferred from RNA to DNA a re8erse processZ,. ;ome of t#e tumor 8iruses Cere also s#oCn to possess re8erse transcriptase. 2#e

presence of t#e enzyme may be taken as an indication of a retro8irus infection.

Inhi#itors o! RNA S'nthesis

1. Atino('in D Actinomycin D binds Cit# DNA template strand and blocks t#e

mo8ement of RNA polymerase. 2#is Cas t#e 8ery rst antibiotic used for t#e treatment

of tumors.'. Ri!"(&in: lt is an antibiotic Cidely used for t#e treatment of tuberculosis and leprosy.

Rifampin binds Cit# t#e p-subunit of prokaryotic RNA polymerase and in#ibits its

acti8ity.. a-Amanitin lt is a toxin produced by mus#room! Amanita p#alloides. 2#is mus#room is

delicious in taste but poisonous due to t#e toxin o-amanitin C#ic# ti"#tly binds Cit#

RNA polymerase ll of eukaryotes and in#ibits transcription

GENETIC CODE @ CODONS

1. h"t is " o$on4 )on M"' *++-*. h"t is %eneti o$e4 Mention t2o i(&ort"nt !e"tures o! %eneti o$e. h"t

"re s'non'(ous o$ons4 )on De *++00. h"t is %eneti o$e4 E&/"in its !e"tures. )on "&r *+++

CODONS

1. Centr"/ Do%(" o! Mo/eu/"r Bio/o%': 2#e oC of "enetic information is dependent ont#e seEuence of bases in DNA! its mRNA transcript and t#e seEuence of amino acids in a

protein.

'. 2#e "enetic information for t#e synt#esis of proteins is contained in DNA ori"inally in t#e

codin"strand and as it is complementary to t#e template strand! and t#en t#e information

is transcribed to mRNA.. 2#e t#ree nucleotide (triplet) base seEuences in mRNA act as code Cords for amino acids

in protein is called codon. 2#e collection of codons is knoCn as &enetic code and it is

arran"ed in a table.

?


26/54

,. ;ince t#ere are four di4erent bases! t#ey can "enerate /, (,) di4erent codons. 2#eir

nucleotide seEuences are alCays Critten from t#e X end to t#e X end. 2#ere are 1 tRNA

species! carryin" '? amino acids! C#ic# translate /1 codons.. 2#ere are t#ree codons C#ic# do not code for any particular amino acids. 2#ey are

nonsense o$onsP! more correctly termed as punctuator codons or terminator codons.

2#ey put UfullstopU to t#e protein synt#esis. 2#ese t#ree codons are 6AA! 6A&! and 6&A./. L8ery amino acid except met#ionine is represented by se8eral codons. odons t#at

represent same amino acid are called as s'non'(ous o$ons. 7n synonymous

codons! t#e base of t#e t#ird position is insi"nicant! because t#e codons di4erin" only

in t#e t#ird base represent t#e same amino acid.0. ;e8eral codons ser8e special functions.a. 2#e initi"tion o$on (A6&) is t#e most common si"nal for t#e be"innin" of a

polypeptide in all cells.b. 2#e ter(in"tion o$ons (6AA! 6A&! and 6&A)! also called stop codons or nonsense

codons! normally si"nal t#e end of polypeptide synt#esis and do not code for any

knoCn amino acids.. #an"in" a sin"le nucleotide base on t#e mRNA c#ain is called a 9point mutation:.

GENETIC CODE

1. %rotein synt#esis is called translation because t#e 9lan"ua"e: of t#e nucleotide

seEuence on t#e mRNA is translated into t#e 9lan"ua"e: of an amino acid seEuence.

2#e process of translation reEuires a "enetic code! t#rou"# C#ic# t#e information

contained in t#e nucleic acid seEuence is expressed to produce a specic seEuence of

amino acids.'. 7t consists of a specic seEuence of nucleotides at a "i8en position on a "i8en

c#romosome t#at codes for a specic protein (or! in some cases! an RNA molecule). A

"ene #as


27/54

codon and t#e complementary nucleotide in t#e anticodon is called Cobblin". 2#e

pairin" of codon and anticodon can Cobble at t#e t#ird letter. or example! &&6! &&

and &&A are t#e codes for "lycine* all t#ree Cill pair Cit# t#e anticodon 7 (7

7nosinic acid) of "lycine tRNA. 2#e de"eneracy of "enetic code and Cobblin"

p#enomenon to"et#er Cill reduce t#e e4ect of mutations.3. Ter(in"tor Co$ons 2#ere are t#ree codons C#ic# do not code for any particular

amino acids. 2#ey are 9nonsense codons:! more correctly termed as punctuatorcodons or terminator codons. 2#ey put UfullstopU to t#e protein synt#esis. 2#ese t#ree

codons are 6AA! 6A&! and 6&A. 6&A is a stop codon* but in special circumstances! it

stands for seleno-cysteine (t#e U'1stU aminoacid).=. Initi"tor Co$on 7n most of t#e cases! A6& acts as t#e initiator codon. A6& also acts

as t#e codon for met#ionine. 7n a feC proteins! &6& may be t#e initiator codon.1?. Mitohon$ri" h"ve $ierent o$es. 2#e protein synt#esisin" mac#inery of

mitoc#ondria is distinct from t#at in t#e cytoplasm. 2#ere are only about '' tRNAs in

mitoc#ondria* but t#ere are 1 tRNA species in cytoplasm.

TRANSLATION OR )ROTEIN BIOSYNT;ESIS

1. De6ne the ter(s re&/i"tion tr"nsri&tion "n$ tr"ns/"tion. Desri#e the ste&s

invo/ve$ in &rotein #ios'nthesis. M"rh *++* Ot 19*. )ost tr"ns/"tion"/ (o$i6"tions0. E&/"in )rotein s'nthesis in $et"i/. A$$ " note on $ru%s th"t inhi#it &rotein

s'nthesis.

Tr"ns/"tion:

1. 2#e pat#Cay of protein synt#esis is called translation because t#e 9lan"ua"e: of t#e

nucleotide seEuence on t#e mRNA is translated into t#e 9lan"ua"e: of an amino acid

seEuence. 2#e process of translation reEuires a "enetic code! because t#e seEuence of

amino acids in t#e protein is determined by t#e nucleotide base seEuence in t#e DNA

C#ic# is transcribed to mRNA and translated into protein Cit# t#e #elp of ribosomes.'. 2#ere are Cide 8ariations in t#e cells C#ic# synt#esize proteins. Hi8er cells produce

albumin and blood clottin" factors for export into t#e blood for circulation. 2#e 7i8er

cells are ric# in t#e protein biosynt#etic mac#inery! and may be re"arded as t#e

protein factory in t#e #uman body. Lryt#rocytes lack t#e mac#inery for translation! and

t#erefore cannot synt#esize proteins.. 2#e tRNAs act as adapter molecules betCeen mRNA and t#e amino acids coded by it.

2#e nucleotides of codons #a8e no aFnity for amino acids. ;o t#e tRNA molecules act

as mediators betCeen t#e mRNA and amino acids.,. 2ranslation is a cytoplasmic process. 2#e mRNA is translated from < to < end. 7n t#e

polypeptide c#ain synt#esized! t#e rst amino acid is t#e amino terminal one. 2#e

c#ain "roCt# is from amino terminal to carboxyl terminal.

'


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Ste&s o! &rotein s'nthesis:

1. ReKuire(ent o! Co(&onentsa. All t#e amino acids present in a protein must be present at t#e time of synt#esis. 7f

one amino acid is missin" translation stops.b. >ne specic type of tRNA is reEuired for eac# amino acid. ;ome amino acids may #a8e

more t#an one specic tRNA molecule.c. Aminoacyl-tRNA synt#etases catalyze a tCo-step reaction t#at results in t#e

attac#ment of t#e carboxyl "roup of an amino acid to t#e


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i.

m-RNA plus t#e cap-bindin" complex combines Cit# t#e ,; preinitiation complex

to produce t#e -9S initi"tion o(&/e.ii. After formation of ,3; initiation complex! it searc#es for precise initiation codon

A6& for met#ionine.iii. Reo%nition o! initi"tion o$on: In eukaryotes! t#e rst amino acid

incorporated is met#ionine (O-A6& codon). 2#is is t#e initiation codon and its

reco"nition by ribosome is facilitated by a specic seEuence of nucleotides. 2#is

marker seEuence for t#e identication of A6& is called as [ozak consensus

seEuences.i8. 2#e ,3; initiation complex noC binds to /?;! C#ic# Cas free after ribosomal

dissociation! forms 3?; initiation complex. 2#is reEuires #ydrolysis of &2% for

ener"y. 2#is reaction t#en results in release of ,3; initiation complex! C#ic# are

t#en recycled for next protein synt#esis.

e. 9+S initi"tion o(&/e:i. 9+S initi"tion o(&/e is noC a complete assembly for protein synt#esis and

contains one small and one lar"e subunit

ii. 7t #as t#ree bindin" sites for tRNA! knoCn as t#e % (peptidyl)! A (aminoacyl)!and L (e@ection) sites.

iii. 2#e p site is #a8in" t#e initiator codon! t#e met-tRNAi and is ready for t#e

elon"ation cycle to commence. Got# t#e A site (aminoacyl or acceptor site) and

L site (deacylated tRNA exit site) are free.

,


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0. E/on%"tion: Llon"ation in8ol8es se8eral steps catalyzed by proteins called elon"ation

factors (Ls). 2#ese steps arei.

Gindin" of aminoacyl-tRNA to t#e A site Cit# proper codon reco"nitionii. %eptide bond formation! andiii. 2ranslocation of t#e ribosome on t#e mRNA.

#. Bin$in% o! "(ino"'/8tRNA:

i. M#en $et-tRNAi is bound to t#e % site! t#e mRNA codon in t#e A sitedetermines C#ic# aminoacyl-tRNA Cill bind to t#at site.

ii. L1A forms a ternary (t#ree) complex Cit# &2% and t#e aminoacyl tRNA to enter

t#e A site Cit# t#e release of L1A &D% and p#osp#ate.iii. L-1A! and &D% are recycled to brin" anot#er aminoacyl-tRNA.

. )e&ti$e #on$ !or("tion:i. 2#e alp#a amino "roup at t#e incomin" amino acid in t#e UAU site forms a

peptide bond (>NB) Cit# carboxyl "roup of t#e peptidyl tRNA occupyin" t#e

U%U site. 2#is reaction is catalyzed by t#e enzyme peptidyl transferase.ii. NoC t#e "roCin" peptide c#ain is occupyin" t#e UAU site and t#e tRNA in t#e %

site is no lon"er contains an amino acid or peptide.i. 2#is is an example of ribozyme C#ere RNA acts as t#e enzyme.ii. 7t is estimated t#at about six amino acids per second are incorporated durin"

t#e course of elon"ation in eukaryotes$. Tr"ns/o"tion:

i. After t#e peptide bond #as been formed! t#e ribosome ad8ances t#ree

nucleotides toCard t#e X end of t#e mRNA. 2#is process is knoCn as

translocation and reEuires t#e participation of eL-' and &2% #ydrolysis to &D%.ii. 2#is causes mo8ement of t#e unc#ar"ed tRNA into t#e ribosomal L site to be

released and mo8ement of t#e peptidyl-tRNA into t#e % site.e. Ter(in"tion:

i. After successi8e addition of amino acids! ribosome reac#es t#e terminator

codon seEuence (6AA! 6A& or 6&A) on t#e mRNA. ;ince t#ere is no tRNA


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bearin" t#e correspondin" anticodon seEuence! t#e UAU site remains free.ii. As t#e termination codon occupies t#e ribosomal A-site! t#e release factor

namely eR reco"nizes t#e stop si"nal. eR-&2% complex! in association Cit#

t#e enzyme peptidyltransferase! clea8es t#e peptide bond betCeen t#e

polypeptide and t#e tRNA occupyin" %-site. ln t#is reaction! a Cater molecule!

instead of an amino acid is added. 2#is #ydrolysis releases t#e protein and tRNA

from t#e %-site.iii. 2#e 3?; ribosome dissociates to form ,?; and /? subunits C#ic# are recycled.

2#e mRNA is also released.

)o/'so(es:

a. $any ribosomes can translate t#e same mRNA molecule simultaneously.

Gecause of t#eir relati8ely lar"e size! t#e ribosome particles cannot attac# to an

mRNA any closer t#an nucleotides apart. $ultiple ribosomes on t#e same

mRNA molecule form a polyribosome! or UpolysomeUb. 7n an unrestricted system! t#e number of ribosomes attac#ed to an mRNA (and

t#us t#e size of polyribosomes) correlates positi8ely Cit# t#e len"t# of t#e

mRNA molecule.

IN;IBITORS O< )ROTEIN SYNT;ESIS

1. $a@ority of t#e antibiotics interfere Cit# t#e bacterial protein synt#esis and are #armlessto #i"#er or"anisms.

'. ;treptomycin 7nitiation of protein synt#esis is in#ibited by streptomycin. lt causes

misreadin" of mRNA and interferes Cit# t#e normal pairin" betCeen codons and

anticodons.

. 2etracycline lt in#ibits t#e bindin" of aminoacyl tRNA to t#e ribosomal complex and can

also block eukaryotic protein synt#esis. 2#is! #oCe8er! does not #appen since eukaryotic

cell membrane is not permeable to t#is dru".

,. %uromycin 2#is #as a structural resemblance to aminoacyl tRNA. %uromycin enters t#e A

site and "ets incorporated into t#e "roCin" peptide c#ain and causes its release. 2#is

antibiotic pre8ents protein synt#esis in bot# prokaryotes and eukaryotes.

. #loramp#enicol lt acts as a competiti8e in#ibitor of t#e enzyme peptidyltransferase and

t#us interferes Cit# elon"ation of peptide c#ain.

/. Lryt#romycin lt in#ibits translocation by bindin" Cit# ?; subunit of bacterial ribosome.

0. Dip#t#eria toxin lt pre8ents translocation in eukaryotic protein synt#esis by inacti8atin"

elon"ation factor eL'.

)OST.TRANSLATIONAL MODI


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(B;%s). Any stress to t#e cell includin" #eat! toxins! #ea8y metals! free radicals!

radiation! bacteria! etc. Cill cause increased production of B;%s! and #ence t#ey

are more correctly termed as stress proteins.d. #aperonopat#ies are disorders resultin" from Usick c#aperonesU. 2#ese diseases

pro"ress Cit# a"e.') )roteo/'ti /e"v"%e $odication of polypeptides by partial proteolysis! e.". con8ersion

of pro-insulin to insulin.) /ntein s&/iin% 7nteins are inter8enin" seEuences in certain proteins. 2#ese are

comparable to introns in mRNAs. lnteins #a8e to be remo8ed! and exteins li"ated in t#e

appropriate order for t#e protein to become acti8e.-? Mo$i6"tions o! "(ino "i$s

1. &amma carboxylation of "lutamic acid residues of prot#rombin! under t#e inuence

of 8itamin [.'. Bydroxylation of proline and lysine in colla"en Cit# t#e #elp of 8itamin . %#osp#orylation of #ydroxyl "roups of serine! t#reonine or tyrosine by kinases! e.".

"lyco"en p#osp#orylase.,. otranslational "lycosylation arbo#ydrates are attac#ed to serine or t#reonine

residues t#rou"# >-"lycosidic linka"es and to aspara"ine or "lutamine residues

t#rou"# N-"lycosidic linka"es.

) Su#unit "%%re%"tion

Lxamples are immuno"lobulin! #emo"lobin and maturation of colla"en. ailure of post-

translational modication a4ects t#e normal function of many proteins. or example!

poor cross-linkin" of colla"en in scur8y! since ascorbic acid is reEuired for t#e

#ydroxylation of proline and lysine.

)ACAGING O< DNA

1. hih &rotein is invo/ve$ in DNA &"3in%. )on "&r *+++*. Ro/e o! histones in ("int"inin% DNA inte%rit'. )on M"' *++

1. DNA molecules reEuire special packa"in" to enable t#em to reside Cit#in cells becauset#e molecules are so lar"e.

'. DNA consists of a double #elix! Cit# t#e tCo strands of DNA Crappin" around eac# ot#er

to form a #elical structure. 2o be compact! t#e DNA molecule coils about itself to form a

structure called a supercoil.. Bistones 2#ey are proteins containin" unusually #i"#er

concentration of basic amino acids. 2#ere are classes* B1! B'A!

B'G! B and B,. 2#e B1 #istone is loosely attac#ed to t#e DNA,. Lukaryotic DNA binds to an eEual Cei"#t of #istones! C#ic# are

small basic proteins containin" lar"e amounts of

ar"inine and lysine. 2#e complex of DNA and proteins

is called c#romatin and appears as beeds on a strin".

. 2#e beads Cit# DNA protrudin" from eac# end areknoCn as nucleosomes! and t#e beads t#emsel8es

are knoCn as nucleosome cores./. 2Co molecules of eac# of four core #istone classes (#istones B'A! B'G! B! and B,) form

t#e center of t#e core around C#ic# DNA are Cound. 2#e DNA @oinin" t#e cores is

complexed Cit# t#e ft# type of #istone! B1.0. >t#er types of proteins are also associated Cit# DNA in t#e nucleus. 2#ese proteins are

called 9non#istone c#romosomal proteins.:

NUCLEIC ACID METABOLISM

0


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1. Disuss "#out nu/ei "i$s un$er !o//o2in% he"$in%s: >MGR U?

"? T'&es #? n t#e ribosomes! t#e mRNA

and tRNA molecules interact to translate into a specic protein molecule

information transcribed from t#e "ene. Durin" periods of acti8e protein synt#esis!

many ribosomes can be associated Cit# any mRNA molecule to form an assembly

called t#e &o/'so(e

c. Tr"ns!er RNA >tRNA). 2#ere are about /? di4erent species present. 2#eyconstitute about 1T of t#e total RNA in t#e cell. 2#ey are 8ery stable.2#ey

transfer amino acids from cytoplasm to t#e ribosomal protein synt#esizin"

mac#inery.d. S("// RNA.

i. $ost of t#ese molecules are complexed Cit# proteins to form

ribonucleoproteins and are distributed in t#e nucleus! t#e cytoplasm! or

bot#. 2#ey constitute about 1-'T of total RNA in t#e cell. 2#ere are about

? di4erent 8arieties.ii. S("// Nu/e"r RNAs >snRNAs?: snRNAs! a subset of t#e small RNAs! are

3


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si"nicantly in8ol8ed in rRNA and mRNA processin" and "ene re"ulation.

2#ey are in8ol8ed in mRNA splicin" and take part in t#e formation of

spliceosomes. All of t#em are located in t#e nucleus. 2#ey complex Cit#

specic proteins! to form small nuclear ribonucleoprotein particles (;nRN%s).

7t is pronounced as U;nurpsU. %roduction of autoantibodies a"ainst 9;nurps:

cause systemic lupus eryt#ematosis (;HL)! a fatal autoimmune disease.

iii. Miro8RNA >(iRNA). 2#ey alter t#e function of mRNA. 2#ey aremoderately stable. miRNAs cause in#ibition of "ene expression by

decreasin" specic protein production.iv. S("// Inter!erin% RNAs >siRNAs?: duplexes betCeen siRNA and mRNA

results inreduced specic protein production. siRNAs are freEuently used to

decrease or Uknock-doCnU specic protein le8els in experimental contexts in

t#e laboratory.

Co(&onents o! nu/ei "i$s:

1) Nucleic acids are t#e polymers of nucleotides (polynucleotides) #eld by < and <

p#osp#ate brid"es') A nucleotide is made up of components

a. Nitro"enous base! (a purine or a pyrimidine)

b. %entose su"ar! eit#er ribose or deoxyribose*c. %#osp#ate "roups esteried to t#e su"ar.) M#en a base combines Cit# a pentose su"ar! a nucleoside is formed. M#en t#e

nucleoside is esteried to a p#osp#ate "roup! it is called a nucleotide or nucleoside

mono-p#osp#ate. 2#e nucleic acids (DNA and RNA) are polymers of nucleoside

monop#osp#ates.,) 2#ere are four di4erent types of nucleotides found in DNA! di4erin" only in t#e

nitro"enous base.) 2#e purine bases present in RNA and DNA are t#e same* adenine and "uanine./) 2#e pyrimidine bases present in nucleic acids are cytosine! t#ymine and uracil.

ytosine is present in bot# DNA and RNA. 2#ymine is present in DNA and uracil in RNA.

Ch"r %"s ru/e o! DNA o(&osition:

1) LrCin #ar "a4 in late 1=,?s Euantitati8ely analysed t#e DNA #ydrolysates fromdi4erent species. Be obser8ed t#at DNA #ad eEual numbers of adenine and t#ymine

residues (A 2) and eEual numbers of "uanine and cytosine residues (& ). 2#is is

knoCn as #ar"a4


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polynucleotide strands of DNA mo8e in a someC#at


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DNA polymerase 777 is a #i"#ly 9processi8e: enzymeQt#at is! it remains bound to t#e

template strand as it mo8es alon"! and does not di4use aCay.

)roo! re"$in%:

1. 2#e nucleotide seEuence of DNA Cill be replicated Cit#out errors. $isreadin" of t#e

template seEuence could result in mutations.'. DNA polymerase 777 #as a 9proofreadin": acti8ity t#rou"#


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(partial)b. Deletion of a codon! e.". cystic brosis (one amino acid! ?3t# p#enyl alanine is

missin" in t#e 2R protein.c. Deletion of a sin"le base! C#ic# Cill "i8e rise to frames#ift e4ect.

-. Insertion: 7nsertions or additions or expansions are subclassied into

a. ;in"le base additions! leadin" to frames#ift e4ect.b. 2rinucleotide expansions. 7n Buntin"ton


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can lead to t#alassemia due to premature c#ain termination and polypeptide t#at are

non-functional.

7. Con$ition"/ Mut"tions: $ost of t#e spontaneous mutations are conditional* t#ey are

manifested only C#en circumstances are appropriate.". Gacteria acEuire resistance! if treated Cit# antibiotics for a lon" time. 2#is is

explained by spontaneous conditional mutations. 7n t#e normal circumstances!

Cild bacilli Cill "roC. 7n t#e medium containin" antibiotic! t#e resistant bacilliare selected.#. 7n a tuberculous patient! a lun" ca8ity may #arbor about 1?1' bacilli. 2#is may

contain about 1?/ mutations! out of C#ic# a feC could be streptomycin

resistant. 2#erefore if only one dru" is "i8en! t#ere Cill be o8er"roCt# of dru"

resistant bacilli. 2o a8oid t#is! a combination of tCo antituberculous dru"s is

"i8en. ;o! dru"-1-resistant mutants are killed by dru"-' and dru"-'-resistant

mutants are remo8ed by dru"-1. 2#e statistical probability of a sin"le bacillus

acEuirin" resistance a"ainst bot# dru"s is ne"li"ible.9. Mut"%ens "n$ Mut"%enesis:

". Any a"ent C#ic# Cill increase DNA dama"e or cell proliferation can cause

increased rate of mutations also. ;uc# substances are called muta"ens.

#. J-ray! "amma-ray! 6S ray! acridine oran"e! etc. are Cell knoCn muta"ens.c. 2#e rate of mutation Cas proportional to t#e dose of irradiation.d. 2atum (Nobel %rize! 1=3) s#oCed t#at a mutation of a sin"le "ene resulted

only in a sin"le c#emical reaction! C#ic# "a8e e8idence to t#e concept of Uone

"ene! one enzymeU.

M"ni!est"tions o! Mut"tions

1. Het#al $utations 2#e alteration is incompatible Cit# life of t#e cell or t#e or"anism.

or example! mutation producin" alp#a-, Bb is let#al! and so t#e embryo dies.'. ;ilent $utations Alteration at an insi"nicant re"ion of a protein may not #a8e any

functional e4ect.. Genecial $utations Alt#ou"# rare! benecial spontaneous mutations are t#e basis of

e8olution. ;uc# benecial mutants are articially selected in a"riculture. Normal maize

is decient in tryptop#an. 2ryptop#an-ric# maize 8arieties are noC a8ailable forculti8ation. $icroor"anisms often #a8e anti"enic mutation. 2#ese are benecial to

micro-or"anisms (but of course! bad to #uman bein"s).,. arcino"enic L4ect 2#e mutation may not be let#al! but may alter t#e re"ulatory

mec#anisms. ;uc# a mutation in a somatic cell may result in uncontrolled cell di8ision

leadin" to cancer. Any substance causin" increased rate of mutation can also increase

t#e probability of cancer. 2#us all carcino"ens are muta"ens.. Ame


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extended by DNA%. After replication! one strand is normal and t#e ot#er strand

contains t#e mutation at t#e specic site. 2#is alloCs study on t#e e4ect of t#at

particular mutation.

CELL CYCLE

rite " note on e// '/e. )on Nov *+1+

1. ;omatic cells are "enerated by t#e di8ision of existin" cells. 2#ey duplicate t#eir contents andt#en di8ide to produce tCo identical dau"#ter cells . 2#is seEuence of duplication is knoCn

as t#e cell cycle'. 7t refers to t#e e8ents occurrin" durin" t#e period betCeen tCo mitotic di8isions.. 2#e cell cycle may be broadly di8ided into t#ree sta"es

i. 7nterp#ase! mitosis! and cytokinesis.ii. 7nterp#ase can be furt#er subdi8ided into t#ree p#ases called &1 p#ase ! ;

p#ase ! and &' p#ase.iii. $itosis ($) can be di8ided into 8e distinct p#ases called prop#ase !

prometap#ase ! metap#ase ! anap#ase ! and telop#ase.i8. 2#e t#ird sta"e! cytoplasmic di8ision or cytokinesis! culminates Cit# t#e

separation into tCo distinct dau"#ter cells,. RNA and protein synt#esis also take place durin" &1 p#ase. 7n addition! or"anelles and

intracellular structures are duplicated and t#e cell "roCs durin" t#is p#ase. cells in &1

p#ase t#at are not committed to DNA synt#esis are in a specialized restin" state called &?

p#ase. 7n a normal cell population! most of t#e cells are in &o p#ase. &eneral metabolic

e8ents are takin" place in &o p#ase.. ;ynt#esis of nuclear DNA! also knoCn as DNA replication ! occurs durin" ; p#ase/. &'! is a time of preparation for t#e nuclear di8ision of mitosis and p#asei s c#aracterizedb y

enlar"ement of cytoplasm and t#is is folloCed by t#e actual cell di8ision t#at occurs in t#e

mitotic p#ase.0. ell ycle re"ulators

i. yclins 2#e cyclins! cate"orized as cyclins D! L! A! or G! are a family of cell

cycle re"ulatory proteins. Di4erent cyclins are expressed to re"ulate speci c

p#ases of t#e cell cycle.ii. yclin-dependent kinases cyclin-D[ complex catalyzes t#e p#osp#orylation

of substrate proteins on serine and t#reonine amino acid residues. ;uc#

alteration of re"ulatory proteins alloCs for initiation of t#e next p#ase of t#e

cell cycle.3. #eckpoint re"ulation

i. #eckpoints placed at critical points in t#e cell cycle monitor t#e completion of

critical e8ents and! if necessary! delay t#e pro"ression to t#e next sta"e of t#e

cell cycle. 2#ey are key re"ulators t#at ensure t#at &1 is completed prior to

t#e start of ; p#ase.ii. 2umor suppressor proteins normally function to #alt t#e cell cycle pro"ression

Cit#in &1 C#en t#e cell s#ould not continue past t#e restriction pointiii. p 2#is tumor suppressor protein plays a ma@or re"ulatory role in &1i8. Retinoblastoma (RG) protein 2#is tumor suppressor functions to #alt a cell in

t#e restin" or &1 p#ase of t#e cell cycle.

Buman papilloma8irus binds Cit# RG and p and inacti8ate t#em leadin" to unre"ulated cell

cycle pro"ression and mali"nancy.

LACTOSE >LAC? O)ERON

1. Analysis of Hactose $etabolism in L oli Hed to t#e >peron Bypot#esis. Hac operon

explains re"ulation of "ene expression.'. 2#e concept of operon Cas introduced by `acob and $onod in 1=/1 based on t#eir

/,


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obser8ations on t#e re"ulation of lactose metabolism in L. coli. 2#is is popularly knoCn

as lac operon.. 2#e "enes t#at encode proteins are called structural "enes. 7n bacteria! t#e structural

"enes t#at code for proteins in8ol8ed in a particular metabolic pat#Cay are "rouped on

t#e c#romosome alon" Cit# t#e re"ulatory elements t#at determine t#e transcription

of t#ese "enes. 2#ese units are called operons. 2#e "enes in an operon are expressed

coordinately* t#at is! t#ey are eit#er all 9turned on: or all 9turned o4.:,. 2#e lac operon consists of a re"ulatory "ene (l* 7 for in#ibition)! operator "ene (>) and

t#ree structural "enes (\! ! A). Gesides t#ese "enes! t#ere is a promoter site (%)! next

to t#e operator "ene! C#ere t#e enzyme RNA polymerase binds. 2#e structural "enes

\! and A respecti8ely! code for t#e enzymes b-"alactosidase! "alactoside permease

and "alactoside acetylase. b &alactosidase #ydrolyses lactose to "alactose and

"lucose C#ile permease is responsible for t#e transport of lactose into t#e cell.. A sin"le polycistronic mRNA is produced t#at codes for all t#e proteins of t#e operon.

An mRNA codin" for more t#an one protein is knoCn as polycistronic mRNA. 2#e

transcription of t#ese "enes start from a common promoter (%)! located close to t#e \

"ene. 2#e RNA polymerase binds to t#e promoter and transcribes t#ese structural

"enes as a sin"le mRNA.

/. 2#e re"ulatory 7 "ene in lac operon produces a repressor molecule. M#en t#ere is

absence of lactose in L. coli! t#is molecule pre8ents t#e bindin" of t#e enzyme RNA

polymerase to t#e promoter site (%)! t#ereby blockin" t#e transcription of structural

"enes ('! and A) . 2#is is called repression of lac operon.0. M#en lactose is present! it binds Cit# repressor molecule and alloCs t#e "enes to

prduce lactase enzyme. 2#is is called depression of lac operon C#ere lactose acts byinacti8atin" t#e repressor molecules.3. M#en "lucose is t#e only su"ar a8ailable ! t#e lac operon is repressed (turned o4).

M#en only lactose is a8ailable! t#e lac operon is induced (maximally expressed or

turned on).=. M#en L coli is exposed to bot# lactose and "lucose as sources of carbon! t#e

or"anisms rst metabolize t#e "lucose and t#en temporarily stop "roCin" until t#e

"enes of t#e lac operon become induced to pro8ide t#e ability to metabolize lactose as

a usable ener"y source.1?. 2#is is explained by t#e formation of A%-cA$%. 2#e attac#ment of RNA polymerase to

t#e promoter site reEuires t#e presence of a cata#olite "ene acti8ator protein (A%)

bound to cyclic A$%. 2#e presence of "lucose loCers t#e intracellular concentration of

cA$%. Due to t#e diminis#ed le8els of cA$%! t#e formation of A%-cA$% is loC and t#e#ence t#e transcription are almost ne"li"ible in t#e presence of "lucose. 2#us! "lucose

interferes Cit# t#e expression of 7ac operon by depletin" cA$% le8els.11. A%-cA$% acts as a positi8e re"ulator for t#e "ene expression. lt is! t#erefore! e8ident

t#at lac operon is sub@ected to bot# positi8e (by repressor) and ne"ati8e re"ulation.1'. linical Applications

1. Hactase in #uman intestine is an inducible enzyme.'. 7n #umans! examples of derepression include induction of tryptop#an pyrrolase!

and transaminases by "lucocorticoids* as Cell as AHA synt#ase by barbiturates.

/


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RECOMBINANT DNA TEC;NOLOG AND GENE T;ERA)Y

Questions:

1. Reo(#in"nt DNA8A&ri/ *+++*. Desri#e in $et"i/ the ste&s invo/ve$ in reo(#in"nt DNA tehno/o%'.0. Desri#e the "&//i"tions o! reo(#in"nt DNA tehno/o%'. )on Nov *+11 )on

Nov *+1+-. h"t is restrition en$onu/e"ses4 )on M"' *++0,. Use o! &/"s(i$s in %eneti en%ineerin%8A&ri/ *++15. h"t is the !untion o! ),0 %ene4 )on M"' *++57. h"t is the &rini&/e o! %ene ther"&'. )on M"' *++59. Chi(eri DNA. h"t is /onin%4 Mention the v"rious t'&es o! /onin%.1+. C'/i AM)

RECOMBINANT DNA TEC;NOLOGY

1. Reo(#in"nt DNA8A&ri/ *+++*. Desri#e in $et"i/ the ste&s invo/ve$ in reo(#in"nt DNA tehno/o%'.0. Desri#e the "&//i"tions o! reo(#in"nt DNA tehno/o%'. )on Nov *+11 )on

Nov *+1+-. h"t is restrition en$onu/e"ses4 )on M"' *++0

,. Use o! &/"s(i$s in %eneti en%ineerin%8A&ri/ *++15. Chi(eri DNA7. h"t is /onin%4 Mention the v"rious t'&es o! /onin%.

RECOMBINANT DNA TEC;NOLOGY

1. M#en a "ene of one species is transferred to anot#er li8in" or"anism! by articial means!

it is called recombinant DNA tec#nolo"y. 7n common parlance! t#is is knoCn as "enetic

en"ineerin".'. Applications of Recombinant 2ec#nolo"y

a. Gy means of recombinant tec#nolo"y! #ormones like "roCt# #ormone can be

produced in lar"e scale.b. Saccines like Bepatitis G can be prepared in pure forms

c. ;pecic %robes for Dia"nosis of Diseases ;pecic probes are useful fori. Antenatal dia"nosis of "enetic diseases. e.". cystic brosis! p#enyl

ketonuria! etc.ii. 2o identify 8iral particles or bacterial DNA in suspected blood and tissue

samples.iii. 2o demonstrate 8irus inte"ration in transformed cells.i8. 2o detect acti8ation of onco"enes in cancer.8. 2o pinpoint t#e location of a "ene in a c#romosome.

8i. 2o identify mutations in "enes ;ickle cell disease is an example of point

mutationd. &ene 2#erapy Normal "enes could be introduced into t#e patient so t#at "enetic

diseases can be cured.e. ;pecial tec#niEues #a8e led to remarkable ad8ances in forensic medicine.

0. )rini&/es o! reo(#in"nt DNA tehno/o%':i. &eneration of DNA fra"ments and selection of t#e desired piece of DNA (e.". a

#uman "ene).ii. 7nsertion of t#e selected DNA into a clonin" 8ector (e.". a plasmid) to create a

recombinant DNA or c#imeric DNA (#imera is a monster in &reek myt#olo"y t#at

#as a lion


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-. As&ets o! Reo(#in"nt DNA tehno/o%':

i. $olecular tools of "enetic en"ineerin" (Restriction endonucleases).ii. Bost cells-t#e factories of clonin".iii. Sectors-t#e clonin" 8e#icles.i8. $et#ods of "ene transfer.8. &ene clonin" strate"ies.

RESTRICTION ENDONUCLEASES: >Short notes?

1. 2#ese are enzymes t#at can cut DNA from any source at specic sites. 2#ey Cere rst

disco8ered in L.coli restrictin" t#e replication of bacteriop#a"es by cuttin" t#e 8iral

DNA. 2#us! t#e enzymes t#at restrict t#e 8iral replication are knoCn as restriction

enzymes or restriction endonucleases.'. A restriction enzyme is named accordin" to t#e or"anism from C#ic# it Cas isolated.

2#e rst letter of t#e name is from t#e "enus of t#e bacterium. 2#e next tCo letters are

from t#e name of t#e species. An additional letter indicates t#e type or strain! and a

nal number is appended to indicate t#e order in C#ic# t#e enzyme Cas disco8ered in

t#at particular or"anism.i. Lco R7 is isolated from Lsc#eric#ia coli R1 strain.ii. Bae777 is t#e t#ird restriction endonuclease isolated from t#e bacterium

Baemop#ilus ae"yptius.. Restriction enzymes clea8e dsDNA so as to produce a R; Sectors are t#e DNA molecules! C#ic# can carry a forei"n DNA fra"ment to be

cloned

1. %lasmids %lasmids are extrac#romosomal! doublestranded! circular! self-replicatin"

DNA molecules. Almost all t#e bacteria #a8e plasmids. pGR'' of L.coli is t#e most

popular and Cidely used plasmid 8ector.'. Gacteriop#a"es are t#e 8iruses t#at replicate Cit#in t#e bacteria. %#a"e 8ectors can

accept s#ort fra"ments of forei"n DNA into t#eir "enomes.. osmids are t#e 8ectors t#at possess t#e c#aracteristics of plasmid and

bacteriop#a"e.,. Buman articial c#romosome synt#etically produced 8ector DNA! possessin" t#e

c#aracteristics of #uman c#romosome.. east articial c#romosomes synt#etic DNA t#at can accept lar"e fra"ments of

forei"n DNA./. Gacterial articial c#romosomes is based on one -plasmid C#ic# is lar"er t#an t#e

ot#er plasmids used as clonin" 8ectors

)ROCEDURE O< DNA RECOMBINATION:

1. %reparation of ;pecic Buman &ene cDNA (complementary copy DNA) is prepared

/0


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from mRNA usin" re8erse transcriptase'. %reparation of Chi(eri DNA $olecules (Short notes)

a. #imera is t#e &reek myt#olo"ical monster Cit# a lion


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b. 2#is insertional inacti8ation of mr "ene is t#e marker for #ybrid DNA./. Lxpression Sectors

a. 2o produce t#e #uman proteins! L. coli carryin" t#e 8ector Cit# t#e insert is

alloCed to "roC! Cit#out any protein in#ibitors.b. ;uc# a 8ector carryin" t#e forei"n "ene! C#ic# is translated into a protein! is

called expression 8ector. 2#e #uman proteins can be #ar8ested from t#e

bacterial culture.

GENE CLONING

h"t is /onin%4 Mention the v"rious t'&es o! /onin%.h"t is /onin%4 Mention the v"rious t'&es o! /onin%.Desri#e in $et"i/ the ste&s

invo/ve$ in reo(#in"nt DNA tehno/o%'.

1. %ropa"ation of t#e #ost or"anism containin" t#e recombinant DNA forms a set of

"enetically identical or"anisms! or a clone. 2#is process is #ence knoCn as DNA

clonin".

'. 2#ere are t#ree di4erent types of clonin"

a. &ene clonin"! C#ic# creates copies of "enes or se"ments of DNA

/=


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b. Reproducti8e clonin"! C#ic# creates copies of C#ole animalsc. 2#erapeutic clonin"! C#ic# creates embryonic stem cells.

. 2#ere are tCo types of "ene clonin"a. 7n 8i8o! C#ic# in8ol8es t#e use of restriction enzymes and li"ases usin" 8ectors

and clonin" t#e fra"ments into #ost cells (as can be seen in t#e ima"e abo8e).b. 7n 8itro type C#ic# is usin" t#e polymerase c#ain reaction (%R) met#od to

create copies of fra"ments of DNA.

,. $olecular clonin" alloCs for t#e production of a lar"e number of identical DNAmolecules! C#ic# can t#en be c#aracterized or used for ot#er purposes. 2#is tec#niEue

is based on t#e fact t#at c#imeric or #ybrid DNA molecules can be constructed in

clonin" 8ectorsQtypically bacterial plasmids! p#a"es! or cosmidsQC#ic# t#en continue

to replicate in a #ost cell under t#eir oCn control systems. 7n t#is Cay! t#e c#imeric

DNA is amplied.. ;teps of clonin"

a. &eneration of desired DNA fra"ments.b. 7nsertion of t#ese fra"ments into a clonin" 8ector.c. 7ntroduction of t#e 8ectors into #ost cells.d. ;election or screenin" of t#e recipient ells for t#e recombinant DNA molecules

/. lonin" can start by selection of DNA fra"ment from "enomic DNA or usin" mRNA. 2#e

farmer is ideal but t#e later is easier.0. %lasmid 8ector DNA and t#e selected #uman DNA are incubated to"et#er so t#at

annealin" takes place formin" #imeric DNA Cit#in t#e plasmid 8ector.3. Next step is transfection of Sector into t#e Bost ell e". L.oli=. 2#e #ost cells proba"ate in appropriate culture media and bacterial colonies containin"

t#e desired 8ector are identied by t#e tec#niEue of insertional inacti8ation of

Antibiotic Resistance &enes in bacterial colonies.1?. A collection of t#ese di4erent recombinant clones is called a library. A 8ariety of

molecules can be used to UprobeU libraries in searc# of a specic "ene or cDNA.11. Glottin" tec#niEues are used for t#e specic identication of desired DNA or RNA

fra"menfs from t#ousands of molecules in library. Glottin" refers to t#e process of

immobilization of sample nucleic acids on solid support (nitrocellulose or nylon

membranes) 2#e most comonly used blottin" tec#niEues area. ;out#ern blottin" (for DNA)b. Nort#ern blottin" (for RNA)c. Dot blottin" (DNAKRNA)

1'. Determination of nucleotide seEuence in a DNA molecule is important to understand

t#e functions of "enes! and basis of in#erited disorders. urt#er! DNA clonin" and "ene

nianipulation in8ariably reEuire knoCled"e of accurate nucleotide seEuence. $anual I

Automated 2ec#niEues Are A8ailable to Determine t#e ;eEuence of DNA

1. Applications of DNA clonin"

a. lonin" #as led directly to t#e elucidation of t#e complete DNA seEuence of t#e

"enomes of a 8ery lar"e number of speciesb. $any useful proteins are currently a8ailable as recombinant products. 2#ese

includeQi. Recombinant factor S777ii. 2issue plasmino"en acti8ator! used to treat strokes

iii. Recombinant subunit 8accines! (e.". Bepatitis G 8accine)i8. Recombinant proteins as standard material for dia"nostic laboratory

tests.c. loned "enes may be inserted into or"anisms! "eneratin" trans"enic or"anismsd. lonin" tec#niEues are part of "ene t#erapy

Use o! &/"s(i$s in %eneti en%ineerin%8A&ri/ *++11. $ost species of bacteria normally contain small! extrac#romosomal DNA molecules

0?

http://www.nlm.nih.gov/medlineplus/stemcells.htmlhttp://en.wikipedia.org/wiki/List_of_recombinant_proteinshttp://en.wikipedia.org/wiki/List_of_recombinant_proteinshttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Tissue_plasminogen_activatorhttp://en.wikipedia.org/wiki/Hepatitis_B_vaccinehttp://www.nlm.nih.gov/medlineplus/stemcells.htmlhttp://en.wikipedia.org/wiki/List_of_recombinant_proteinshttp://en.wikipedia.org/wiki/Factor_VIIIhttp://en.wikipedia.org/wiki/Tissue_plasminogen_activatorhttp://en.wikipedia.org/wiki/Hepatitis_B_vaccine


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called plasmids.'. %lasmid DNA under"oes replication t#at may or may not be sync#ronized to

c#romosomal di8ision. %lasmids may carry "enes t#at con8ey antibiotic resistance to

t#e #ost bacterium! and may facilitate t#e transfer of "enetic information from one

bacterium to anot#er.. %lasmids can be readily isolated from bacterial cells! t#eir circular DNA clea8ed at

specic sites by restriction endonucleases.,. 2#e recombinant plasmid can be introduced into a bacterium! and lar"e numbers of

copies of t#e plasmid produced. 2#e bacteria are "roCn in t#e presence of antibiotics!

t#us selectin" for cells containin" t#e #ybrid plasmids! C#ic# pro8ide antibiotic

resistance.. %lasmids #a8e se8eral properties t#at make t#em extremely useful as clonin" 8ectors.

a. 2#ey exist as sin"le or multiple copies Cit#in t#e bacterium and replicate

independently from t#e bacterial DNA.b. 2#e complete DNA seEuence of many plasmids is knoCn* #ence! t#e precise

location of restriction enzyme clea8a"e sites for insertin" t#e forei"n DNA is

a8ailable.c. %lasmids are smaller t#an t#e #ost c#romosome and are t#erefore easily

separated from t#e latterd. 2#e desired plasmid-inserted DNA is readily remo8ed by cuttin" t#e plasmid

Cit# t#e restriction enzyme.e. 2#ey bear a special re"ion of DNA called an ori%in o! re&/i"tion. 7t confers

on t#e plasmid t#e property t#at 7t Cill replicate its oCn DNA as Cell as any

passen"er DNA.f. %lasmid DNAs replicate independently and t#ey can be easily separated from

#ost bacteria.

/. 2ypes of plasmidsa. plasmids (;ex plasmids) 2#is plasmid transfers a

replica of t#e plasmid from a donor (+) cell to a recipient () cell Cit#out t#e + cell

losin" its plasmid.b. R plasmids! dru" resistance plasmids 2#ese plasmids carry "enes conferrin"

resistance to one or more antibiotics and usually can transfer t#is resistance to an R-

free recipient cell.c. ol plasmids or colicino"enic factor plasmids 2#ey carry "enes for t#e synt#esis of a

protein knoCn as colicins.d. Lnt plasmids %lasmids called Lnt are responsible for tra8ellerOs diarr#oea and some

types of dysentery.e. %lasmid 8ector %GR '' #as bot# tetracycline (tet) and ampicillin (amp) resistance

"enes.

h"t is the &rini&/e o! %ene ther"&'. )on M"' *++5

1. 2#e ultimate cure for "enetic diseases is to introduce normal "enes into indi8iduals

C#o #a8e defecti8e "enes.'. &ene t#erapy consists of intracellular deli8ery of "enes to "enerate a t#erapeutic

e4ect by correctin" an existin" abnormality. >nly somatic "ene t#erapy! by insertin"

t#e neC "ene into somatic cell of t#e patient is under trial. &erm cell "ene t#erapy is

01


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considered as unet#ical.. %rinciples of t#e %rocedure

i. 7solate t#e #ealt#y "ene alon" Cit# t#e seEuence controllin" its expression.ii. 7ncorporate t#is "ene into a carrier or 8ector as an expression cassette.iii. inally deli8er t#e 8ector to t#e tar"et cells.

,. $et#ods of 7ntroduction of t#e &enesa. E vivo str"te%'

i. lsolate cells Cit# eenetic defect from a patient.ii. roC t#e cells in culture.iii. 7ntroduce t#e t#erapeutic "ene to correct "ene defect.i8. ;elect t#e "enetically corrected cells (stablet ransformantsa) nd "roC.8. 2ransplant t#e modied cells to t#e patient.

8i. 2#is tec#niEue is not associated Cit# ad8erse immunolo"ical responses

after transplantin" t#e cellsb. In situ str"te%' C#en t#e expression cassette is in@ected to t#e patient eit#er

intra8enously or directly to t#e tissuec. In vivo str"te%'! C#ere t#e 8ector is administered directly to t#e cell! e.". (cystic

brosis) "ene to t#e respiratory tract cells.

The etors:

1. Di4erent 8ector (carrier) systems used for "ene deli8ery area. Siruses Retro8iruses! adeno8iruses! adeno associated 8iral 8ectors and #erpes

simplex 8iruses.b. Non-8irus systems include liposomes!

plasmids and p#ysical met#ods.'. Siruses

a. 2#e 8ectors freEuently used in "ene

t#erapy are 8iruses! particularly

retro8iruses. RNA is t#e "enetic

material in retro8iruses.b. 2#e "a"! pol! en8 "enes are deleted

from t#e Cild type retro8irus! renderin"

it incapable of replication inside #uman

body. 2#en t#e #uman "ene is inserted into t#e 8irus.a. As t#e retro8irus enters t#e #ost cell! it synt#esizes DNA from RNA (by re8erse

transcription).b. 2#e normal #uman "ene can noC express .

. %lasmid Hiposome omplex Hiposomes are articial lipid bilayers! C#ic# could be

incorporated Cit# plasmids carryint#e normal #uman DNA. 2#e complexes can enter

into t#e tar"et cells by fusin" Cit# t#e plasma membrane. Hiposomes can form

complexes spontaneously Cit# DNA.,. &ene &un $et#od 2un"sten particles are coated Cit# plasmid DNA! and accelerated by

#elium pressure disc#ar"e. 2#is enables particles to penetrate t#e tar"et tissues.

Suess stories o! %ene ther"&':

Disease &ene transferred by

;e8ere combined

immunodeciency

Adenosine deaminase enzyme in c#romosome

1 and '? into (;7D) lymp#ocytes* by

retro8irusDuc#enne muscular dystrop#y Dystrop#in "ene on s#ort arm of J

c#romosome* by retro8irusystic brosis 2R "ene on c#romosome 0 to bronc#ial

epit#elium* adeno8irus

Bemop#ilia A and G "enes for factor S777 and

0'


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7J into broblasts* retro8irus

ancer Acti8ation of p (tumor suppressor "ene) by

liposome

STEM CELLS T;ERA)Y:

1. ;tem cells are totipotential cells from embryonic tissue.'. ;tem cells #a8e t#e ability to di8ide for an indenite period. 2#ey c

Documents

19. Molecular Biology Copy