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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-amino- imidazole 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
Bypoxant#ine "uanine p#osp#o ribosyl transferase
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.
Bypoxant#ine "uanine p#osp#o ribosyl transferase
i. Gunine )R)) 8F Gu"nosine (ono &hos&h"te
))i
Bypoxant#ine "uanine p#osp#o ribosyl transferase
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 (
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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.
=
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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"#
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'. 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
,'
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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
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;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
,/
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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
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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
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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
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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.
?
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,. ;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
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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