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Heredity, Genes, and DNA

*All living things are able to reproduce (inherit the genetic

information from their parents)

*All cells arise from pre-existing cells (genetic material must bereplicated & passed from parent to progeny cell at each cell

division)

Genes – a pair of inherited factors which determines a trait

Allele- one gene copy specifying each trait is inherited fromeach parent

Genotype- genetic composition (eg. Bb)

Phenotype- physical appearance (eg. straight hair)

Chromosomes- carrier of genes* cells of higher plants and animals are DIPLOID

- containing 2 copies of each chromosome* sperm and egg cell containing only 1 copy of each

chromosome- HAPLOID

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-union of these 2 haploid cells at fertilization creates a new diploid organism

MEIOSIS- formation of the germ cells(the sperm & egg) in which only one member ofeach chromosome pair is transmitted to each progeny cell

Figure 3.1. Inheritance of dominant andrecessive genes

Figure 3.2. Chromosomes at meiosis and

fertilization Two chromosome pairs of ahypothetical organism are illustrated.

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Figure 3.3. Gene segregationand linkage (A) Segregation of

two hypothetical genes forshape ( A/a = square/round)and color (B/b = red/blue)located on differentchromosomes. (B) Linkage oftwo genes located on the same

chromosome.

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Figure 3.4. Genetic recombination

During meiosis, members of

chromosome pairs exchange material.

The result is recombination betweenlinked genes.

Figure 3.5. A genetic map Three genes arelocalized on a hypothetical chromosome

based on frequencies of recombinationbetween them (1% recombination betweena and b; 3% between b and c; 4% between a and c). The frequencies of recombinationare approximately proportional to thedistances between genes on the

chromosome.

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In molecular terms, a gene commonly is defined as the entire nucleicacid sequence that is necessary for the synthesis of a functional polypeptide.

Identification of DNA as the Genetic Material:

Bacterial genes are made of DNA

Figure 4-2. Experimental demonstration thatDNA is the genetic material. Theseexperiments, carried out in the 1940s, showedthat adding purified DNA to a bacteriumchanged its properties and that this change was

faithfully passed on to subsequent generations.Two closely related strains of the bacteriumStreptococcus pneumoniae differ from each otherin both their appearance under the microscopeand their pathogenicity. One strain appearssmooth (S) and causes death when injected intomice, and the other appears rough (R) and isnonlethal. (A) This experiment shows that a

substance present in the S strain can change (ortransform) the R strain into the S strain and thatthis change is inherited by subsequentgenerations of bacteria. (B) This experiment, inwhich the R strain has been incubated withvarious classes of biological molecules obtainedfrom the S strain, identifies the substance as

DNA.Griffith (1923)

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In 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty establishedthat the transforming principle was DNA

Figure 1.3. The transformingprinciple is DNA. Avery and

his colleagues showed that thetransforming principle isunaffected by treatment with aprotease or a ribonuclease, butis inactivated by treatment witha deoxyribonuclease

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Virus genes are made of DNA Figure 1.4. Bacteriophages areviruses that infect bacteria. (A) Thestructure of a head-and-tailbacteriophage such as T2. The

DNA genome of the phage iscontained in the head part of theprotein capsid. (B) The infectioncycle. After injection into anEscherichia coli bacterium, the T2phage genome directs synthesis ofnew phages. For T2, the infectioncycle takes about 20 minutes at 37°C and ends with lysis of the celland release of 250–300 new phages.This is the lytic infection cycle.

Some phages, such as λ, can alsofollow a lysogenic infection cycle,in which the phage genomebecomes inserted into the bacterialchromosome and remains there, inquiescent form, for several

generations of the bacterium(Section 4.2.1).(Hershey and Chase, 1952)

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.glossary.9089


http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.section.6035

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.biblist.5425









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DNA was discovered in 1869 by Johann Friedrich Miescher, a Swiss biochemistworking in Tubingen, Germany (1st using crude extract from human WBC then apurified one from salmon sperm)

Deoxyribonucleic acid (DNA) is the storehouse, or cellular library, that contains allthe information required to build the cells and tissues of an organism.

The full chemical names of the four nucleotides that polymerize to make DNAare:2′-deoxyadenosine 5′-triphosphate2′-deoxycytidine 5′-triphosphate2′-deoxyguanosine 5′-triphosphate2′-deoxythymidine 5′-triphosphate

DNA is a linear, unbranched polymer in which the monomeric subunits are fourchemically distinct nucleotides that can be linked together in any order in chainshundreds, thousands or even millions of units in length.

Each nucleotide in a DNA polymer is made up of three components:

1) 2′-deoxyribose2) a nitrogenous base

3) A phosphate group

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The double helix- according to Watson, their work was a desperate race againstAmerican biochemist, Linus Pauling , who initially proposed

an incorrect triple helix model-Rosalind Franklin, whose X-ray diffraction studies provided the bulk ofthe experimental data in support of the double helix and who was herselfvery close to solving the structure.

-double helix, discovered by Watson and Crick on Saturday 7 March 1953,

was the single most important breakthrough in biology during the 20thcentury.

The evidence that led to the double helix

Watson and Crick used four types of information to deduce the double helixstructure:

1. Biophysical data of various kinds. The water content of DNA fibers wasparticularly important because it enabled the density of the DNA in a fiber tobe estimated. The number of strands in the helix and the spacing between thenucleotides had to be compatible with the fiber density. Pauling's triple helixmodel was based on an incorrect density measurement which suggested thatthe DNA molecule was more closely packed than it actually is.





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2. X-ray diffraction patterns (Section 9.1.3), most of which were produced byRosalind Franklin of Kings College, London, and which revealed the helical nature

of the structure and indicated some of the key dimensions within the helix.

3. The base ratios, which had been discovered by Erwin Chargaff of ColumbiaUniversity, New York. Chargaff carried out a lengthy series of chromatographicstudies of DNA samples from various sources and showed that, although thevalues are different in different organisms, the amount of adenine is always the

same as the amount of thymine, and the amount of guanine equals the amount ofcytosine ( Figure 1.10 ). These base ratios led to the base-pairing rules, which werethe key to the discovery of the double helix structure.

4. Model building , which was the only major technique that Watson and Crickmade use of themselves. Scale models of possible DNA structures enabled therelative positioning of the various atoms to be checked, to ensure that pairs ofgroups that formed bonds were not too far apart, and that other groups were not soclose together as to interfere with one another.



http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.5265





http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.5265






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Figure 1.11. The double helix structure ofDNA. (A) Two representations of thedouble helix. On the left the structure isshown with the sugar-phosphate‘backbones' of each polynucleotidedrawn as a red ribbon with the base

pairs in black. On the right the chemicalstructure for three base pairs is given. (B)A base-pairs with T, and G base-pairswith C. The bases are drawn in outline,with the hydrogen bonding indicated bydotted lines. Note that a G-C base pair

has three hydrogen bonds whereas an A-T base pair has just two. The structuresin part (A) are redrawn from Turner etal. (1997) (left) and Strachan and Read(1999) (right).













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The key features of the double helix- double helix is right-handed- two strands run in opposite directions- helix is stabilized by two types of chemical interaction:

1. Base-pairing between the two strands involves the formation ofhydrogen bonds between an adenine on one strand and athymine on the other strand, or between a cytosine and aguanine

2. Base-stacking, sometimes called - interactions, involveshydrophobic interactions between adjacent base pairs and adds

stability to the double helix once the strands have been broughttogether by base-pairing. These hydrophobic interactions arisebecause the hydrogen-bonded structure of water forceshydrophobic groups into the internal parts of a molecule.

The double helix has structural flexibility

-double helix described by Watson and Crick, is called the B-form of DNA-that genomic DNA molecules are not entirely uniform in structure. This ismainly because each nucleotide in the helix has the flexibility to take upslightly different molecular shapes

-Rotations within individual nucleotides therefore lead to major changes in theoverall structure of the helix.













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Figure 3.7. The structure of

DNA (Watson & Crick)

*DNA molecule is a helix thatturns every 3.4 nm.

*distance between adjacentbases is 0.34 nm

*there are ten bases per

turn of the helix.

*the diameter of the helix isapproximately 2 nm, suggestingthat it is composed of not onebut two DNA chains.

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Figure 1.12. Computer-generated images of B-DNA (left), A-DNA (center) and Z-DNA

(right). Reprinted with permission from Kendrew A (ed.), The Encyclopaedia of Molecular

Biology , Plate 1. Copyright 1994 Blackwell Science.

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Feature B-DNA Conformation A-DNA Z-DNA

Type of helix Right-handed Right-handed Left-handed

Helical diameter (nm) 2.37 2.55 1.84

Rise per base pair

(nm)

0.34 0.29 0.37

Distance per complete

turn (pitch) (nm)

3.4 3.2 4.5

Number of base pairs

per complete turn

10 11 12

Topology of major

groove

Wide, deep Narrow, deep Flat

Topology of minor

groove

Narrow, shallow Broad, shallow Narrow, deep

Table 1.1. Features of different conformations of the DNA double helix





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Figure 3.9. Experimental demonstrationof semiconservative replication

(Meselson and Stahl, 1958)

Figure 3.8. Semiconservative replication of DNA The two strands of parental DNA separate, and each

serves as a template for synthesis of a new daughterstrand by complementary base pairing.

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Expression of Genetic Information

Colinearity of Genes and Proteins

Figure 3.10. Colinearity of genesand proteins A series of mutations(arrowheads) were mapped in theE. coli gene encoding tryptophansynthetase (top line). The amino

acid substitutions resulting fromeach of the mutations was thendetermined by sequence analysisof the proteins of mutant bacteria(bottom line). These studiesrevealed that the order ofmutations in DNA was the sameas the order of amino acidsubstitutions in the encodedprotein.

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Central dogma of molecular biology:

1. Transcription - a process where RNA molecules are synthesized from DNA

templates

2. Translation- proteins are synthesized from RNA templates

1 2

Figure 3.11.

Synthesis of RNAfrom DNA The two

strands of DNA

unwind, and one is

used as a template

for synthesis of a

complementarystrand of RNA.

Figure 3.12. Function of

transfer RNA Transfer

RNA serves as an

adaptor during protein

synthesis. Each amino

acid (e.g., histidine) is

attached to the 3′ end of

a specific tRNA by anappropriate enzyme (an

aminoacyl tRNA

synthetase). The charged

tRNAs then align on an

mRNA template by

complementary basepairing.

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=cooper.glossary.2886






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The Genetic Code

-the correspondence between nucleotide triplets and amino acids in proteins.-it is a triplet code (43) or 64 codons-of the 64 codons, 61 specify an amino acid; the remaining three (UAA, UAG,and UGA) are stop codons that signal the termination of protein synthesis.-the code is degenerate(meaning, many amino acids are specified by more than

one codon)

-experiment done byMarshall Nirenberg andHeinrich Matthaei,

Figure 3.14. The triplet UUU encodes phenylalanine In vitro translation ofa synthetic RNA consisting of repeated uracils (a poly-U template) results inthe synthesis of a polypeptide containing only phenylalanine.

S d iti

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Second position

First position U C A G Third position

Phe Ser Tyr Cys U

U Phe Ser Tyr Cys C

Leu Ser stop stop A

Leu Ser stop Trp G

Leu Pro His Arg U

C Leu Pro His Arg C

Leu Pro Gln Arg A

Leu Pro Gln Arg G

Ile Thr Asn Ser U

A Ile Thr Asn Ser C Ile Thr Lys Arg A

Met Thr Lys Arg G

Val Ala Asp Gly U

G Val Ala Asp Gly C

Val Ala Glu Gly A

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RNA Viruses and Reverse Transcription

The synthesis of DNA from RNA, now called reverse transcription.

-by RNA tumor viruses (now called

retroviruses)-enzymes that catalyze RNA-directed DNA synthesis (reverse transcriptases) can

be used experimentally to generate DNA copies of any RNA molecule.

Recombinant DNA(revolutionized the understanding of cellbiology)

-provided scientists with the ability to isolate, sequence, and manipulate individual

genes derived from any type of cell.

Restriction Endonucleases

- enzymes that cleave DNA at specific sequences- identified in bacteria




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Enzymea Source Recognition siteb

BamHI Bacillus amyloliquefaciens H GGATCC

EcoRI Escherichia coli RY13 GAATTC

HaeIII Haemophilus aegyptius GGCC HindIII Haemophilus influenzae Rd AAGCTT

HpaI Haemophilus parainfluenzae GTTAAC

HpaII Haemophilus parainfluenzae CCGG

MboI Moraxella bovis GATC

Not I Nocardia otitidis-caviarum GCGGCCGC

SfiI Streptomyces fimbriatus GGCCNNNNNGGCC

TaqI Thermus aquaticus TCGA

a Enzymes are named according to their species of isolation, followed by a number to distinguish

different enzymes isolated from the same organism (e.g., HpaI and HpaII).b

Recognition sites show the sequence of only one strand of double-stranded DNA. “N”

represents any base.

Table 3.2. Recognition Sites of Representative Restriction Endonucleases

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Figure 3.16. EcoRI digestion and gel

electrophoresis of λ DNA EcoRI cleaves λ DNA

at five sites (arrows), yielding six DNA

fragments. These fragments are then

separated by electrophoresis in an agarose gel.The DNA fragments migrate toward the

positive electrode, with smaller fragments

moving more rapidly through the gel.

Following electrophoresis, the DNA is stained

with a fluorescent dye and photographed. The

sizes of DNA fragments are indicated.

Gel Electrophoresis

-method of

separation based

on the rates of theirmigration in an

electric field

Figure 3.17. Restriction maps of λ and adenovirus DNAs

The locations of cleavage sites for BamHI, EcoRI, and

HindIII are shown in the DNAs of E . coli bacteriophage λ(48.5 kb) and human adenovirus-2 (35.9 kb).

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Generation of Recombinant DNA Molecules

Molecular cloning is to insert a DNA fragment of interest (e.g., a segment of humanDNA) into a DNA molecule (called a vector) that is capable of independent

replication in a host cell.

Figure 3.19. Joining ofDNA molecules

Figure 3.18. Generationof a recombinant DNA

molecule Figure 3.20. cDNA cloning



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Vectors for Recombinant DNA

Figure 3.21. Cloning inbacteriophage λ vectors Figure 3.22. Cloning inplasmid vectors Figure 3.23. Cloning incosmid vectors

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Cosmid and yeast artificial chromosome (YAC) vectors-clone even larger fragments of DNA: cosmid (45kb)

YAC (hundreds of kilobases)- which replicate as

chromosomes in yeast cells.DNA Sequencing Figure 3.24. DNA sequencing by the Sanger

procedure Dideoxynucleotides, which lack

OH groups at the 3′ as well as the 2′ position

of deoxyribose, are used to terminate DNA

synthesis at specific bases. These molecules

are incorporated normally into growing DNA

strands. Because they lack a 3′ OH, however,

the next nucleotide cannot be added, so

synthesis of that DNA strand terminates.

DNA synthesis is initiated with a radioactive

primer. Four separate reactions are carried

out, each containing one dideoxynucleotide

mixed with its normal counterpart as well as

the three other normal deoxynucleotides.

When the dideoxynucleotide is

incorporated, DNA synthesis stops, so each

reaction yields a series of products

extending from the radioactive primer to the

base substituted by a dideoxynucleotide.

Products of the four reactions are separated

by electrophoresis and analyzed by

autoradiography to determine the DNAsequence.





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Figure 3.25. Automated DNAsequencing Four separatesequencing reactions areperformed, each containing onechain-terminatingdideoxynucleotide and a primerlabeled with a distinct fluorescenttag. The products are then pooledand subjected to gelelectrophoresis. As the DNAstrands migrate through the gel,they pass through a laser beam thatexcites the fluorescent label. Theemitted light is detected by aphotomultiplier, which is

connected to a computer thatcollects and analyzes the data.

Use to determine completegenome sequences of bacteria,yeast, C. elegans, and Drosophila,

human

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Expression of Cloned Genes

Figure 3.26. Expression ofcloned genes in bacteria Expression vectors containpromoter sequences (pro) thatdirect transcription of inserted

DNA in bacteria and sequencesrequired for binding of mRNAto bacterial ribosomes (Shine-Delgarno [SD] sequences). Aeukaryotic cDNA insertedadjacent to these sequences can

be efficiently expressed in E.coli, resulting in production ofeukaryotic proteins intransformed bacteria

Amplification of DNA by the Polymerase

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Figure 3.27. Amplification ofDNA by PCR The region ofDNA to be amplified is flankedby two sequences used to primeDNA synthesis. The startingdouble-stranded DNA is heatedto separate the strands and thencooled to allow primers (usually

oligonucleotides of 15 to 20bases) to bind to each strand ofDNA. DNA polymerase fromThermus aquaticus (Taq polymerase) is used tosynthesize new DNA strands

starting from the primers,resulting in the formation of twonew DNA molecules. Theprocess can be repeated formultiple cycles, each resulting ina twofold amplification of DNA.

Amplification of DNA by the Polymerase

Chain Reaction

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-the formation of double stranded DNA and/or RNA molecules bycomplementary base pairing.

Nucleic Acid Hybridization

Figure 3.28. Detection

of DNA by nucleicacid hybridization

ssDNA or ssRNA probecan be used to detectDNA (in Southern

hybridization) or RNA (inNorthern hybridization)of complementarysequences.

In situ hybridization

-used to detect homologousDNA or RNA sequences inchromosomes or intact cells

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Southern and NorthernBlotting

Blotting: transfer of nucleic acids orproteinsfrom a gel to a membrane or filter.

Western blotting (immunoblotting) fordetection of proteins with antibody

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Figure 3.31. Western blotting

Antibodies as Probes for Proteins

Figure 3.32. Immunoprecipitation -the use of antibodies to

isolate proteins



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Probes for Screening Recombinant DNA Libraries

Recombinant DNA libraries—collections of clones that contain all the genomicor mRNA sequences of a particular cell type

Genomic library -A collection of recombinant DNA clones that collectivelycontain the genome of an organismcDNA library -A collection of recombinant cDNA clones.

Variety of probes can be used for screening recombinant libraries:

1. a cDNA clone can be used as a probe to isolate thecorresponding genomic clone2. a gene cloned from one species (e.g., mouse) can be used to isolate a

related gene from a different species (e.g., human).3. aside from isolated DNA fragments, synthetic oligonucleotides can

be used as probes, enabling the isolation of genes on the basisof partial amino acid sequences of their encoded proteins.

4. use of antibodies as probes to screen expression libraries

DNA libraries

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DNA libraries

Construction of cDNAfrom mature mRNA

Genomic DNA library cDNA library

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Figure 3.33. Screening a recombinant library by hybridization

Fragments of cell DNA are cloned in a bacteriophage λ vector

and packaged into phage particles, yielding a collection of

recombinant phage carrying different cell inserts. The phages

are used to infect bacteria, and the culture is overlaid with a

filter. Some of the phages in each plaque are transferred to

the filter, which is then hybridized with a radiolabeled probe

to identify the phage plaque containing the desired gene. The

appropriate phage plaque can then be isolated from the

original culture plate.

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Gene Function in Eukaryotes

-understanding the function of a gene, however, requires analysis of thegene within cells or intact organisms—not simply as a molecular clone inbacteria.

Genetic Analysis in Yeasts Figure 3.34. Cloning of yeast genes (A) A yeast vector.The vector contains a bacterial origin of replication (ori)and an ampicillin resistance gene ( Ampr), allowing it tobe propagated as a plasmid in E. coli. In addition, thevector contains a yeast origin of replication and amarker gene (LEU2), allowing the selection of

transformed yeast. The LEU2 gene encodes an enzymerequired for synthesis of the amino acid leucine, sotransformation of yeast strains lacking this enzyme canbe selected for by growth on medium lacking leucine.(B) Isolation of a yeast gene. A gene of interest isidentified by a temperature-sensitive mutation, whichallows yeast to grow at 25°C but not at 37°C. To isolatea clone of the gene, the temperature-sensitive yeasts aretransformed with a plasmid library containing acollection of genes encompassing the entire yeastgenome. All yeasts transformed by plasmid DNAs areable to grow on media lacking leucine at 25°C, but onlythose yeasts transformed by a plasmid carrying anormal copy of the gene of interest are able to grow at37°C. The desired plasmid can be isolated from

transformed yeasts that form colonies at thenonpermissive temperature.

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Gene Transfer in Plants and Animals

Figure 3.37. Production oftransgenic mice DNA is microinjected

into a pronucleus of a fertilized mouse

egg (fertilized eggs contain twopronuclei, one from the egg and one

from the sperm). The microinjected

eggs are then transferred to foster

mothers and allowed to develop. Some

of the offspring (transgenic) have

incorporated the injected DNA into their

genome.

Figure 3.38.Introductionof genes into

mice viaembryonalstem cells

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Transgenic plants

Plants cells can dedifferentiate,

proliferate and redifferentiate

into other cell types.

Callus single cells are

totipotent

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Mutagenesis of Cloned DNAs-In classical genetic studies (e.g., in bacteria or yeasts), mutants are the key toidentifying genes and understanding their function by observing the alteredphenotype of mutant organisms.

Classical genetics (forward genetics)

Random mutagenesis Natural mutants

An altered phenotype is screened (genetic screening).

A gene mutation is mapped.

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Reverse geneticsA gene is identified by recombinant DNA technology and genome sequencing.

Mutants of the gene are generated.

Altered phenotypes of mutant cells or organisms are identified to reveal thefunction of the gene.

in vitromutagenesis

Figure 3.40. Deletion mutagenesis of a cloned gene

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Site-directedmutagenesis

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