20 lecture presentation

7/25/2019 20 Lecture Presentation

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LECTURE PRESENTATIONS

For CAMPBELL BIOLOGY, NINTH EDITIONJane B. Reece, Lisa A. Urry, Micae! L. Cain, "#e$en A. %asser&an, Pe#er '. Minors(y, Ro)er# B. Jac(son

* +-- Pearson E/ca#ion, Inc.

Lectures by

Erin Barley

Kathleen Fitzpatric

Bi!techn!l!"y

Chapter #$



O%er%ie&' The (NA T!!lb!)

• Sequencing of the genomes of more than 7,000species was under way in 2010

• DNA sequencing has depended on advances in

technology, starting with maing recom!inant DNA• "n recombinant DNA, nucleotide sequences from

two different sources, often two species, are

com!ined in vitro into the same DNA molecule




• #ethods for maing recom!inant DNA are centralto genetic engineering, the direct manipulation of

genes for practical purposes

•

DNA technology has revolutioni$edbiotechnology, the manipulation of organisms or

their genetic components to mae useful products

• An e%ample of DNA technology is the microarray,

a measurement of gene e%pression of thousandsof different genes




&igure 20'1



C!ncept #$*+' (NA cl!nin" yiel,s -ultiple

c!pies !. a "ene !r !ther (NA se"-ent• (o wor directly with specific genes, scientists

prepare well)defined segments of DNA in identical

copies, a process called DNA cloning




(NA Cl!nin" an, Its Applicati!ns'

A Preview• #ost methods for cloning pieces of DNA in the

la!oratory share general features, such as the use

of !acteria and their plasmids• Plasmids are small circular DNA molecules that

replicate separately from the !acterial

chromosome

• *loned genes are useful for maing copies of aparticular gene and producing a protein product




• Gene cloning involves using !acteria to maemultiple copies of a gene

• &oreign DNA is inserted into a plasmid, and therecom!inant plasmid is inserted into a !acterialcell

• +eproduction in the !acterial cell results in cloningof the plasmid including the foreign DNA

•

(his results in the production of multiple copies ofa single gene




&igure 20'2Bacterium

Bacterial

chromosome

Plasmid

2

1

3

4

Gene inserted into

plasmidCell containing gene

of interest

ecombinant

DNA !plasmid"

Gene of

interest

Plasmid put into

bacterial cell

DNA of chromosome

!#foreign$ DNA"

ecombinant

bacterium

%ost cell gro&n in culture to

form a clone of cells containing

the #cloned$ gene of interest

Gene of

interest

Protein e'pressed from

gene of interest

Protein har(estedCopies of gene

Basic research

and (arious

applications

Basic

research

on protein

Basic

research

on gene

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up to'ic &aste

Protein dissol(es

blood clots in heart

attac) therapy

%uman gro&th

hormone treats

stunted gro&th



&igure 20'2a

Bacterium

Bacterial

chromosome

Plasmid

2

1 Gene inserted into

plasmidCell containing

gene of interest

ecombinant

DNA !plasmid"

Gene ofinterest

Plasmid put into

bacterial cell

DNA of

chromosome

!#foreign$ DNA"

ecombinant

bacterium



&igure 20'2!

%ost cell gro&n in

culture to form a clone

of cells containing the

#cloned$ gene of interest

Gene ofinterest

Protein e'pressed from

gene of interest

Protein har(estedCopies of gene

Basic research

and (ariousapplications

3

4

Basicresearchon protein

Basicresearchon gene

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up to'ic &aste

Protein dissol(es

blood clots in heart

attac) therapy

%uman gro&th

hormone treats

stunted gro&th



Usin" Restricti!n Enzy-es t! /ae

Rec!-binant (NA• acterial restriction en*ymes cut DNA

molecules at specific DNA sequences called

restriction sites• A restriction en$yme usually maes many cuts,

yielding restriction fragments

• (he most useful restriction en$ymes cut DNA in a

staggered way, producing fragments with -stic)yends'.



http://slidepdf.com/reader/full/20-lecture-presentation 12/127* +-- Pearson E/ca#ion, Inc.

Animation/ +estriction n$ymes

+ight)clic slide select -lay.



• Sticy ends can !ond with complementary sticyends of other fragments

• DNA ligase is an en$yme that seals the !onds

!etween restriction fragments




&igure 20'3)1

estriction en*yme

cuts sugar+phosphatebac)bones,

estriction site

DNA

-′

-′

-′

-′

-′

-′

3′

3′

3′

3′

3′

3′

1

.tic)yend

GAA//CC//AAG

C / /AA

G AA/ / C

G



&igure 20'3)3

ecombinant DNA molecule

0ne possible combinationDNA ligaseseals strands

DNA fragment addedfrom another moleculecut by same en*yme,Base pairing occurs,

estriction en*yme

cuts sugar+phosphatebac)bones,

estriction site

DNA

-′

-′

-′

-′

-′

-′

-′

-′

-′-′

-′

-′

-′-′

-′

-′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

2

3

1

.tic)yend

GAA//CC//AAG

C / /AA

G AA/ / C

G

GG

AA/ / C

C/ / AA

G

G

G

G

AA// CAA// C

C //AA C //AA



Cl!nin" a Euary!tic 0ene in a

Bacterial Plas-i,

• "n gene cloning, the original plasmid is called a

cloning (ector

•

A cloning vector is a DNA molecule that can carryforeign DNA into a host cell and replicate there




Producing Clones of Cells Carrying

Recombinant Plasmids• Several steps are required to clone the

humming!ird β)glo!in gene in a !acterial plasmid

4 (he humming!ird genomic DNA and a !acterialplasmid are isolated

4 oth are cut with the same restriction en$yme

4 (he fragments are mi%ed, and DNA ligase is

added to !ond the fragment sticy ends



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Animation/ *loning a 5ene




4 Some recom!inant plasmids now containhumming!ird DNA

4 (he DNA mi%ture is added to !acteria that have

!een genetically engineered to accept it

4 (he !acteria are plated on a type of agar that

selects for the !acteria with recom!inant

plasmids

4 (his results in the cloning of many humming!irdDNA fragments, including the β)glo!in gene


&i 20 6



&igure 20'6

Bacterial plasmid

/C%N

.5/.

ampR gene lacZ gene

estrictionsite

%ummingbird cell

.tic)yends Gene of

interest

%umming+bird DNAfragments

ecombinant plasmids Nonrecombinantplasmid

Bacteria carryingplasmids

Colony carrying non+recombinant plasmid&ith intact lacZ gene

Colony carryingrecombinantplasmid&ith disruptedlacZ gene

0ne of manybacterialclones

&igure 20 6a 1



&igure 20'6a)1

Bacterial plasmid

/C%N

ampR gene lacZ gene

estriction

site

%ummingbird cell

.tic)yends Gene of

interest

%umming+bird DNA

fragments

&igure 20 6a 2



&igure 20'6a)2

Bacterial plasmid

/C%N

ampR gene lacZ gene

estriction

site

%ummingbird cell

.tic)yends Gene of

interest

%umming+bird DNA

fragments


&igure 20 6a 3



&igure 20'6a)3

Bacterial plasmid

/C%N

ampR gene lacZ gene

estriction

site

%ummingbird cell

.tic)yends Gene of

interest

%umming+bird DNA

fragments



&igure 20 6!



&igure 20'6!

.5/.


Colony carrying non+recombinant plasmid&ith intact lacZ gene

Colony carryingrecombinantplasmid&ith disruptedlacZ gene

0ne of manybacterialclones



Storing Cloned Genes in DNA Libraries

• A genomic library that is made using !acteria isthe collection of recom!inant vector clones

produced !y cloning DNA fragments from an entire

genome

• A genomic li!rary that is made using

!acteriophages is stored as a collection of phage

clones


&igure 20



&igure 20'

6oreign genome

Cut &ith restriction en*ymes into either

small

fragmentslargefragments

or

ecombinantplasmids

Plasmidclone

!a" Plasmid library

!b" BAC clone

Bacterial artificialchromosome !BAC"

5argeinsert&ithmany

genes

!c" .toring genome libraries

&igure 20 a



&igure 20'a

!c" .toring genome libraries



• A bacterial artificial chromosome !BAC" is alarge plasmid that has !een trimmed down and

can carry a large DNA insert

• A*s are another type of vector used in DNA

li!rary construction




• A complementary DNA !cDNA" li!rary is made !ycloning DNA made in vitro !y reverse transcription

of all the m+NA produced !y a particular cell

• A cDNA library represents only part of the

genome8only the su!set of genes transcri!ed

into m+NA in the original cells


&igure 20'9)1



g

DNA innucleus

mNAs incytoplasm

&igure 20'9)2



g

DNA innucleus

mNAs incytoplasm

mNA

e(ersetranscriptase Poly+A tail

DNA

strand

Primer

-′-′

3′3′

A A A A A A

/ / / / /

&igure 20'9)3



g

DNA innucleus

mNAs incytoplasm

mNA


DNAstrand

Primer

-′-′

-′-′

3′3′

3′3′

A A A A A A

A A A A A A

/ / / / /

/ / / / /

&igure 20'9)6



g

DNA innucleus

mNAs incytoplasm

mNA


DNAstrand

Primer

DNApolymerase

-′-′

-′-′

-′ -′

3′3′

3′3′

3′3′

A A A A A A

A A A A A A

/ / / / /

/ / / / /

&igure 20'9)



DNA innucleus

mNAs incytoplasm

mNA


DNAstrand

Primer

DNApolymerase

cDNA

-′-′

-′-′

-′ -′

-′-′

3′3′

3′3′

3′3′

3′3′

A A A A A A

A A A A A A

/ / / / /

/ / / / /



Screening a Library for Clones Carrying a

Gene of Interest • A clone carrying the gene of interest can !e

identified with a nucleic acid probe having a

sequence complementary to the gene

• (his process is called nucleic acid hybridi*ation




• A pro!e can !e synthesi$ed that iscomplementary to the gene of interest

• &or e%ample, if the desired gene is

4 (hen we would synthesi$e this pro!e


-′ 3′⋅⋅

C/CA/CACCGGC⋅⋅⋅

-′

3′

G A G / A G / G G C C G



•(he DNA pro!e can !e used to screen a largenum!er of clones simultaneously for the gene of

interest

• :nce identified, the clone carrying the gene of

interest can !e cultured


&igure 20'7



adioacti(elylabeled probemolecules Gene of

interest

ProbeDNA

.ingle+stranded

DNA fromcell

6ilm

5ocation of DNA &ith thecomplementaryse7uence

Nylonmembrane

Nylon membrane

8ulti&ell platesholding libraryclones

/C%N -′

-′3′

3′

GAG/AG/GGCCG⋅⋅ ⋅ C/CA/CACCGGC⋅⋅ ⋅



E)pressin" Cl!ne, Euary!tic 0enes

• After a gene has !een cloned, its protein productcan !e produced in larger amounts for research

• *loned genes can !e e%pressed as protein in

either !acterial or euaryotic cells




Bacterial !"ression Systems

•Several technical difficulties hinder e%pression ofcloned euaryotic genes in !acterial host cells

• (o overcome differences in promoters and other

DNA control sequences, scientists usually employ

an e'pression (ector , a cloning vector that

contains a highly active !acterial promoter




u#aryotic Cloning and !"ression Systems

•#olecular !iologists can avoid euaryote)!acterialincompati!ility issues !y using euaryotic cells,

such as yeasts, as hosts for cloning and

e%pressing genes

• ven yeasts may not possess the proteins

required to modify e%pressed mammalian proteins

properly

•

"n such cases, cultured mammalian or insect cellsmay !e used to e%press and study proteins




•:ne method of introducing recom!inant DNA intoeuaryotic cells is electroporation, applying a

!rief electrical pulse to create temporary holes in

plasma mem!ranes

• Alternatively, scientists can in;ect DNA into cells

using microscopically thin needles

• :nce inside the cell, the DNA is incorporated into

the cell<s DNA !y natural genetic recom!ination




Cross$S"ecies Gene !"ression and

volutionary Ancestry• (he remara!le a!ility of !acteria to e%press some

euaryotic proteins underscores the shared

evolutionary ancestry of living species

• &or e%ample, Pax-6 is a gene that directs formation

of a verte!rate eye= the same gene in flies directs

the formation of an insect eye >which is quite

different from the verte!rate eye?• (he Pax-6 genes in flies and verte!rates can

su!stitute for each other




A-pli.yin" (NA in %itro' The P!ly-erase

Chain Reacti!n 1PCR2• (he polymerase chain reaction, PC, can

produce many copies of a specific target segment

of DNA

• A three)step cycle8heating, cooling, and

replication8!rings a!out a chain reaction that

produces an e%ponentially growing population of

identical DNA molecules• (he ey to *+ is an unusual, heat)sta!le DNA

polymerase called (aq polymerase'


&igure 20'@ -′

3′/C%N



Genomic DNA

/argetse7uence

Denaturation

Annealing

'tension

Primers

Ne&nucleotides

Cycle 1yields

2molecules

Cycle 2yields

4molecules

Cycle 3yields 9

molecules:2 molecules

!in &hite bo'es"match target

se7uence

-′

-′

-′

3′

3′

3′

2

3

1

&igure 20'@a



Genomic DNA

/argetse7uence

-′

-′

3′

3′

/C%N

&igure 20'@!



Denaturation

Annealing

'tension

Primers

Ne&nucleo+tides

Cycle 1yields

2molecules

-′

-′

3′

3′

2

3

1

&igure 20'@c



Cycle 2

yields4molecules

&igure 20'@d



Cycle 3yields 9

molecules:2 molecules

!in &hite bo'es"match target

se7uence



C!ncept #$*#' (NA techn!l!"y all!&s us t!

stu,y the se3uence4 e)pressi!n4 an, .uncti!n!. a "ene

• DNA cloning allows researchers to

4 *ompare genes and alleles !etween individuals 4 ocate gene e%pression in a !ody

4 Determine the role of a gene in an organism

• Several techniques are used to analy$e the DNA

of genes




0el Electr!ph!resis an, S!uthern Bl!ttin"

•

:ne indirect method of rapidly analy$ing andcomparing genomes is gel electrophoresis

• (his technique uses a gel as a molecular sieve to

separate nucleic acids or proteins !y si$e,

electrical charge, and other properties

• A current is applied that causes charged

molecules to move through the gel

• #olecules are sorted into -!ands. !y their si$e





Animation/ iotechnology a!


&igure 20'B /C%N



8i'ture of

DNA mol+

ecules of

different

si*es

Po&er

source

Po&er

source

5onger

molecules

Cathode Anode

;ells

Gel

.horter

molecules

.5/.

1

2

&igure 20'Ba/C%N



8i'ture of

DNA mol+

ecules of different

si*es

Po&er source

Po&er source

5onger

molecules

Cathode Anode

;ells

Gel

.horter

molecules

2

1

&igure 20'B!



.5/.



•

"n restriction fragment analysis, DNA fragmentsproduced !y restriction en$yme digestion of a

DNA molecule are sorted !y gel electrophoresis

• +estriction fragment analysis can !e used to

compare two different DNA molecules, such astwo alleles for a gene, if the nucleotide difference

alters a restriction site




•

Cariations in DNA sequence are calledpolymorphisms

• Sequence changes that alter restriction sites are

called 65Ps >restriction fragment length

polymorphisms?


&igure 20'10



Normal β+globin allele

.ic)le+cell mutantβ

+globin allele

5arge

fragment

Normal

allele

.ic)le+cell

allele

2<1 bp1=- bp

3=> bp

!a" DdeI restriction sites in normal and

sic)le+cell alleles of theβ

+globin gene

!b"lectrophoresis of restriction

fragments from normal andsic)le+cell alleles

2<1 bp1=- bp

3=> bp

5arge fragment

5arge fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI

&igure 20'10a



Normal β+globin allele

.ic)le+cell mutantβ

+globin allele

!a" DdeI restriction sites in normal and

sic)le+cell alleles of the β+globin gene

2<1 bp1=- bp

3=> bp

5arge fragment

5arge fragment

DdeI DdeI DdeI DdeI

DdeI

DdeI

DdeI

&igure 20'10!



5arge

fragment

Normal

allele

.ic)le+cell

allele

2<1 bp1=- bp

3=> bp

!b" lectrophoresis of restrictionfragments from normal andsic)le+cell alleles



•

A technique called .outhern blotting com!inesgel electrophoresis of DNA fragments with nucleic

acid hy!ridi$ation

• Specific DNA fragments can !e identified !y

Southern !lotting, using la!eled pro!es thathy!ridi$e to the DNA immo!ili$ed on a -!lot. of gel


&igure 20'11

/C%N



DNA restriction en*yme

321

4

/C%N

I Normal

β+globin

allele

II .ic)le+cell

alleleIII %etero*ygote

estriction

fragmentsNitrocellulose

membrane !blot"

%ea(y

&eight

Gel

.ponge

Al)aline

solution Paper

to&els

III III

III III III III

Preparation of

restriction fragments

Gel electrophoresis DNA transfer !blotting"

adioacti(ely labeled

probe for β+globin

gene

Nitrocellulose blot

Probe base+pairs

&ith fragments

6ragment from

sic)le+cell β+globin allele

6ragment from

normal β+ globin

allele

6ilm

o(er

blot

%ybridi*ation &ith labeled probe Probe detection-



(NA Se3uencin"

•+elatively short DNA fragments can !e sequenced!y the dideo%y chain termination method, the firstautomated method to !e employed

• #odified nucleotides called dideo%yri!onucleotides>ddN(? attach to synthesi$ed DNA strands ofdifferent lengths

• ach type of ddN( is tagged with a distinct

fluorescent la!el that identifies the nucleotide atthe end of each DNA fragment

• (he DNA sequence can !e read from the resultingspectrogram


&igure 20'12

DNA

!t l t t d"

/C%N

/

Primer 3

Deo'yribonucleotides Dideo'yribonucleotides

!fluorescently tagged"



!template strand"-′

3′

C

C

C

C

/

//

G

G

A

AAA

G//

/

DNA

polymerase

-′

3′

P P P

0%

G

dA/P

dC/P

d//P

dG/P


P P P

%

G

ddA/P

ddC/P

dd//P

ddG/P

-′

3′

C

C

C

C

/

//

G

G

A

AAA

DNA !template

strand"5abeled strands

.hortest 5ongest-′

3′

ddCddG

ddAddA

ddA

ddG

ddG

dd/ddC

G//

/ G//

/

C

G//

/

C

/ /

G

G//

/

C

/

GA

G//

/

C

/

GAA

G//

/

C

/

GAAG

G//

/

C

/

GAAG/

G//

/

C

/

GAAG/C

G//

/

C

/

GAAG/CA

Direction

of mo(ement

of strands

5ongest labeled strand

Detector

5aser .hortest labeled strand

.5/.

5ast nucleotide

of longest

labeled strand

5ast nucleotide

of shortest

labeled strand

G

G

G

A

AA

C

C

/

&igure 20'12a



DNA

!template strand"

/C%NPrimer Deo'yribonucleotides Dideo'yribonucleotides


DNA

polymerase

-′

-′

3′

3′

0% %

GG

dA/P

dC/P

d//P

dG/P

P P P P P P

ddA/P

ddC/P

dd//PddG/P

/

//

G

G

G

C

C

C

C/

//

A

A

AA

&igure 20'12!

/C%N !continued"



DNA !template

strand"5abeled strands

.hortest 5ongest

Direction

of mo(ement

of strands5ongest labeled strand

Detector


/C%N !continued"

-′

3′

G

G

C

C

C

C/

//

A

A

A

A

/

/

/

G

ddC

ddC

ddG

ddG

ddG

ddAddA

ddA

dd/

3′

-′

/

/

/

G

C/

/

/

G

CG

/

/

/

G

CGA

/

/

/

G

CGAA

/

/

/

G

CGAAG

/

/

/

CGAAG/

/

/

/

CGAAG/

CA

/

/

/

CGAAG/

C

G G G

&igure 20'12c



.5/.

5ast nucleotide

of longest

labeled strand

5ast nucleotide

of shortest

labeled strand

G

G

G

A

AA

C

C

/

Direction

of mo(ement

of strands5ongest labeled strand

Detector




Analyzin" 0ene E)pressi!n

•

Nucleic acid pro!es can hy!ridi$e with m+NAstranscri!ed from a gene

• ro!es can !e used to identify where or when a

gene is transcri!ed in an organism




Studying t&e !"ression of Single Genes

•

*hanges in the e%pression of a gene duringem!ryonic development can !e tested using

4 Northern !lotting

4 +everse transcriptase)polymerase chain reaction

• oth methods are used to compare m+NA from

different developmental stages




•

Northern blotting com!ines gel electrophoresisof m+NA followed !y hy!ridi$ation with a pro!e on

a mem!rane

• "dentification of m+NA at a particular

developmental stage suggests protein function atthat stage




•

e(erse transcriptase+polymerase chainreaction !/+PC" is quicer and more sensitive

!ecause it requires less m+NA than Northern

!lotting

• +everse transcriptase is added to m+NA to maecDNA, which serves as a template for *+

amplification of the gene of interest

•

(he products are run on a gel and the m+NA ofinterest is identified


&igure 20'13

cDNA synthesis NA1

/C%N



cDNA synthesis

PC amplification

Gel electrophoresis

mNAs

cDNAs

Primers

β

+globingene

mbryonic stages1 2 3 4 - >

2

3

1

.5/.



•

In situ hybridi*ation uses fluorescent dyesattached to pro!es to identify the location of

specific m+NAs in place in the intact organism


&igure 20'16



-<µ

m



Studying t&e !"ression of Interacting

Grou"s of Genes

• Automation has allowed scientists to measure

the e%pression of thousands of genes at one

time using DNA microarray assays

• DNA microarray assays compare patterns of

gene e%pression in different tissues, at different

times, or under different conditions


solate mNA1

/C%N&igure 20'1



solate mNA,

2

1

3

4

8a)e cDNA by re(ersetranscription? usingfluorescently labelednucleotides,

Apply the cDNA mi'ture to amicroarray? a different genein each spot, /he cDNA hybridi*es

&ith any complementary DNA onthe microarray,

inse off e'cess cDNA: scan microarrayfor fluorescence, ach fluorescent spot!yello&" represents a gene e'pressed

in the tissue sample,

/issue sample

mNA molecules

5abeled cDNA molecules!single strands"

DNA fragmentsrepresenting aspecific gene

DNA microarray

DNA microarray&ith 2?4<<human genes

&igure 20'1a



DNA microarray

&ith 2?4<<human genes

( t i i 0 F ti



(eter-inin" 0ene Functi!n

•

:ne way to determine function is to disa!le thegene and o!serve the consequences

• sing in vitro mutagenesis, mutations are

introduced into a cloned gene, altering or

destroying its function

• Ehen the mutated gene is returned to the cell, the

normal gene<s function might !e determined !y

e%amining the mutant<s phenotype




•

5ene e%pression can also !e silenced using NAinterference !NAi"

• Synthetic dou!le)stranded +NA molecules

matching the sequence of a particular gene are

used to !rea down or !loc the gene<s m+NA




•

"n humans, researchers analy$e the genomes ofmany people with a certain genetic condition to try

to find nucleotide changes specific to the condition

• 5enetic marers called .NPs >single nucleotide

polymorphisms? occur on average every 1004300 !ase pairs

• SNs can !e detected !y *+, and any SN

shared !y people affected with a disorder !ut notamong unaffected people may pinpoint the

location of the disease)causing gene


&igure 20'19



DNA

.NP

Normal allele

Disease+causing

allele

/

C



• :rganismal cloning produces one or more

organisms genetically identical to the -parent. that

donated the single cell

C!ncept #$*5' Cl!nin" !r"anis-s -ay

lea, t! pr!,ucti!n !. ste- cells .!rresearch an, !ther applicati!ns


Cl i Pl t Si l C ll C lt



Cl!nin" Plants' Sin"le6Cell Cultures

•

:ne e%perimental approach for testing genomicequivalence is to see whether a differentiated cell

can generate a whole organism

• A totipotent cell is one that can generate a

complete new organism

• lant cloning is used e%tensively in agriculture


&igure 20'17



Crosssection of carrot root

2+mgfragments

6ragments &erecultured in nu+

trient medium:stirring causedsingle cells toshear off intothe li7uid,

.ingle cellsfree in

suspensionbegan todi(ide,

mbryonicplant de(eloped

from a culturedsingle cell,

Plantlet &ascultured on

agar medium,5ater it &asplanted in soil,

Adultplant



Cl!nin" Ani-als' Nuclear Transplantati!n

•

"n nuclear transplantation, the nucleus of anunfertili$ed egg cell or $ygote is replaced with the

nucleus of a differentiated cell

• %periments with frog em!ryos have shown that a

transplanted nucleus can often support normaldevelopment of the egg

• Fowever, the older the donor nucleus, the lower

the percentage of normally developing tadpoles


6rog embryo 6rog egg cell 6rog tadpoleP8N/&igure 20'1@



@

5ess differ+entiated cell

Donor

nucleus

trans+

planted

nucleated

egg cell

6ully differ+

entiated!intestinal" cell

Donor

nucleus

trans+

plantedgg &ith donor nucleus

acti(ated to begin

de(elopment

8ost de(elop

into tadpoles,

8ost stop de(eloping

before tadpole stage,

.5/.



&igure 20'1B

8ammary

/C%N

gg cell



cell donor

21

3

4

-

>

.5/.

Culturedmammarycells

ggcell from

o(ary

donor

Nucleusremo(edCells fused

Gro&n in culture

mplanted in uterusof a third sheep

mbryonicde(elopment

Nucleus frommammary cell

arly embryo

.urrogatemother

5amb !#Dolly$" geneticallyidentical to mammary cell donor



Nucleus from

&igure 20'1B!



4

-

>

.5/.

Gro&n in culture

mplanted in uterusof a third sheep

mbryonicde(elopment

mammary cell

arly embryo

.urrogatemother

5amb !#Dolly$" geneticallyidentical to mammary cell donor



•

Since 1BB7, cloning has !een demonstrated inmany mammals, including mice, cats, cows,horses, mules, pigs, and dogs

• ** >for *ar!on *opy? was the first cat cloned=

however, ** differed somewhat from her female-parent.

• *loned animals do not always loo or !ehavee%actly the same


&igure 20'20



Problems Associated wit& Animal Cloning



Problems Associated wit& Animal Cloning

•

"n most nuclear transplantation studies, only asmall percentage of cloned em!ryos have

developed normally to !irth, and many cloned

animals e%hi!it defects

• #any epigenetic changes, such as acetylation ofhistones or methylation of DNA, must !e reversed

in the nucleus from a donor animal in order for

genes to !e e%pressed or repressed appropriately

for early stages of development


Ste- Cells !. Ani-als



Ste- Cells !. Ani-als

•

A stem cell is a relatively unspeciali$ed cell thatcan reproduce itself indefinitely and differentiate

into speciali$ed cells of one or more types

• Stem cells isolated from early em!ryos at the

!lastocyst stage are called em!ryonic stem >S?cells= these are a!le to differentiate into all cell

types

•

(he adult !ody also has stem cells, which replacenonreproducing speciali$ed cells


&igure 20'21mbryonicstem cells

Adultstem cells



Cultured

stem cells

Differentcultureconditions

Differenttypes of differentiatedcells

Cells generatingall embryoniccell types

Cells generatingsome cell types

5i(er cells

Ner(ecells

Bloodcells



•

+esearchers can transform sin cells into S cells!y using viruses to introduce stem cell master

regulatory genes

• (hese transformed cells are called iS cells

>induced pluripotent cells?• (hese cells can !e used to treat some diseases

and to replace nonfunctional tissues


&igure 20'22



emo(e s)in cells

from patient, 2

1

3

4

eprogram s)in cells

so the cells become

induced pluripotentstem !iP." cells,

Patient &ith

damaged heart

tissue or other

disease

eturn cells to

patient? &here

they can repair

damaged tissue,

/reat iP. cells so

that they differentiate

into a specific

cell type,



C!ncept #$*7' The practical applicati!ns !.

(NA techn!l!"y a..ect !ur li%es in -any&ays

• #any fields !enefit from DNA technology and

genetic engineering


/e,ical Applicati!ns



/e,ical Applicati!ns

•

:ne !enefit of DNA technology is identification ofhuman genes in which mutation plays a role in

genetic diseases


Diagnosis and (reatment of Diseases



Diagnosis and (reatment of Diseases

•

Scientists can diagnose many human geneticdisorders using *+ and sequence)specific

primers, then sequencing the amplified product to

loo for the disease)causing mutation

• SNs may !e associated with a disease)causingmutation

• SNs may also !e correlated with increased riss

for conditions such as heart disease or certaintypes of cancer


)uman Gene (&era"y



)uman Gene (&era"y

•

Gene therapy is the alteration of an afflictedindividual<s genes

• 5ene therapy holds great potential for treatingdisorders tracea!le to a single defective gene

• Cectors are used for delivery of genes intospecific types of cells, for e%ample !one marrow

• 5ene therapy provoes !oth technical and ethicalquestions


&igure 20'23 Cloned gene

1 nsert NA (ersion of normal allele



2

1

3

4

etro(irus

capsid

Bone

marro&

cell from

patient

@iral NA

Bone

marro&

nsert NA (ersion of normal allele

into retro(irus,

5et retro(irus infect bone marro& cells

that ha(e been remo(ed from the

patient and cultured,

@iral DNA carrying the normal

allele inserts into chromosome,

nect engineered

cells into patient,

P&armaceutical Products



P&armaceutical Products

•

Advances in DNA technology and geneticresearch are important to the development of new

drugs to treat diseases




• (he drug imatini! is a small molecule that inhi!its

overe%pression of a specific leuemia)causing

receptor • harmaceutical products that are proteins can !e

synthesi$ed on a large scale

.ynthesis of .mall 8olecules for se asDrugs




• Fost cells in culture can !e engineered to secrete

a protein as it is made, simplifying the tas of

purifying it

• (his is useful for the production of insulin, human

growth hormones, and vaccines

Protein Production in Cell Cultures




• /ransgenic animals are made !y introducinggenes from one species into the genome ofanother animal

• (ransgenic animals are pharmaceutical -factories,.producers of large amounts of otherwise raresu!stances for medical use

Protein Production by #Pharm$ Animals


&igure 20'26



&igure 20'26a



&igure 20'26!



F!rensic E%i,ence an, 0enetic Pr!.iles



•

An individual<s unique DNA sequence, or geneticprofile, can !e o!tained !y analysis of tissue or

!ody fluids

• DNA testing can identify individuals with a high

degree of certainty• 5enetic profiles can !e analy$ed using +&

analysis !y Southern !lotting




•

ven more sensitive is the use of genetic marerscalled short tandem repeats !./s", which arevariations in the num!er of repeats of specificDNA sequences

•

*+ and gel electrophoresis are used to amplifyand then identify S(+s of different lengths

• (he pro!a!ility that two people who are notidentical twins have the same S(+ marers is

e%ceptionally small


&igure 20'2 /his photo sho&s;ashington ust beforehis release in 2<<1?

!a"



after 1= years in prison,

!b"/hese and other ./ data e'onerated ;ashingtonand led /insley to plead guilty to the murder,

.emen on (ictim

arl ;ashington

enneth /insley

1=?1

1>?19

1=?1

13?1>

14?1-

13?1>

12?12

11?12

12?12

.ource of sample

./mar)er 1

./mar)er 2

./mar)er 3

&igure 20'2a



/his photo sho&s

;ashington ust beforehis release in 2<<1?after 1= years in prison,

!a"

En%ir!n-ental Cleanup



p

•

5enetic engineering can !e used to modify themeta!olism of microorganisms

• Some modified microorganisms can !e used to

e%tract minerals from the environment or degrade

potentially to%ic waste materials


A"ricultural Applicati!ns



" pp

•

DNA technology is !eing used to improveagricultural productivity and food quality

• 5enetic engineering of transgenic animals speeds

up the selective !reeding process

• eneficial genes can !e transferred !etweenvarieties or species




•

Agricultural scientists have endowed a num!er ofcrop plants with genes for desira!le traits

• (he /i plasmid is the most commonly used vector

for introducing new genes into plant cells

• 5enetic engineering in plants has !een used totransfer many useful genes including those for

her!icide resistance, increased resistance to

pests, increased resistance to salinity, and

improved nutritional value of crops


&igure 20'29 /C%N Agrobacterium tumefaciens



Plant &ith ne& trait

.5/.

/iplasmid

.ite &hererestrictionen*yme cuts

DNA &iththe geneof interest

ecombinant

/i plasmid

/ DNA

Sa.ety an, Ethical 8uesti!ns Raise, by



Sa.ety an, Ethical 8uesti!ns Raise, by

(NA Techn!l!"y

• otential !enefits of genetic engineering must

!e weighed against potential ha$ards of

creating harmful products or procedures

• 5uidelines are in place in the nited States

and other countries to ensure safe practices for

recom!inant DNA technology




•

#ost pu!lic concern a!out possi!le ha$ardscenters on genetically modified !G8" organisms

used as food

• Some are concerned a!out the creation of -super

weeds. from the transfer of genes from 5# cropsto their wild relatives

• :ther worries include the possi!ility that

transgenic protein products might cause allergic

reactions




• As !iotechnology continues to change, so does its

use in agriculture, industry, and medicine

• National agencies and international organi$ations

strive to set guidelines for safe and ethical

practices in the use of !iotechnology


&igure 20'N03



3′

3′ 3′

3′

-′

-′

-′

-′C//AA

AA//CG

G

.tic)y end

&igure 20'N06



DNA fragments from genomic DNA

or cDNA or copy of DNA obtainedby PC

Cloning

(ector

8i' and ligate

ecombinant DNA plasmids

&igure 20'N0

- 3/CCA/GAA//C/AAAGCGC//A/GAA//CACGGC



Aard(ar) DNA

Plasmid

-′

3′ -′

3′/CCA/GAA//C/AAAGCGC//A/GAA//CACGGC

AGG/AC//AAGA///CGCGAA/AC//AAG/GCCG

G

G

A A

AA / /

/ / C C

&igure 20'N09



&igure 20'N07



&igure 20'N0@

20 lecture presentation

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