Ebook Radiobiology for the radiologist (8/E): Part 2

(BQ) Part 2 book “Radiobiology for the radiologist” has contents: Molecular techniques in radiobiology, cancer biology, clinical response of normal tissues, model tumor systems, alternative radiation modalities, the biology and exploitation of tumor hypoxia,… and other contents.

For Students of Radiation Oncology

chapter 17

Radiobiology

Historical Perspectives

The Structure of DNA

RNA and DNA

Transcription and Translation

The Genetic Code

Amino Acids and Proteins

DNA-mediated Gene Transfer

Agarose Gel Electrophoresis

Polymerase Chain Reaction

Polymerase Chain Reaction–mediated Site-directed Mutagenesis

Gene-Cloning Strategies

Genomic Analyses

Mapping

DNA Sequence Analyses

Polymorphisms or Mutations

Comparative Genome Hybridization

Gene Knockout Strategies

Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR Associated Protein Homologous Recombination to Knockout Genes

Knockout Mice

Gene Expression Analysis

Northern Blotting

RNA Interference

Reverse Transcription Polymerase Chain Reaction

Quantitative Real-Time Polymerase Chain Reaction

Genetic Reporters

Promoter Bashing

Chromatin Immunoprecipitation

Protein–DNA Interaction Arrays (Chromatin Immunoprecipitation-Chips)

Microarrays to Assay Gene Expression

RNA-Seq to Assay Gene Expression

Databases and Sequence Analysis

Summary of Pertinent Conclusions

Glossary of Terms

Bibliography

HISTORICAL PERSPECTIVES

Recombinant DNA technology has revolutionized research in biology It allows

questions to be asked that were unthinkable just a few years ago It is also atechnology that is moving so fast that anything written in a book is likely to beout of date before it appears in print This technology is invading every field ofbiologic research, and radiobiology is no exception To keep abreast ofdevelopments in the field, it is essential to know what recombinant DNAtechnology is and how it works A detailed description is beyond the scope ofthis book; for a more extensive account, the interested reader is referred toseveral excellent volumes that have appeared in recent years and are listed in the

“Bibliography.” The goal here is to provide an overview of core recombinanttechniques and core technologies that are commonly used today in radiobiologicresearch

The birth of molecular biology could be ascribed to the one-page publication

in Nature in 1953 by James Watson and Francis Crick describing the structure of

DNA In short order, this work led the way to breaking the genetic code and understanding the process of transcription of DNA to messenger RNA (mRNA) and the translation of mRNA into proteins At about the same time, in

the late 1940s and early 1950s, Linus Pauling realized that three-dimensional

structures were built by amino acids and folded into proteins The whole

concept emerged that the sequence of bases, which coded for a protein,ultimately determined function

These remarkable discoveries were followed by a period of limited progressfocusing mainly on simple systems such as viruses, bacteriophages, and bacteria,until new tools and techniques to work with DNA were perfected

Recombinant DNA technology got its start with the first successful cloningexperiment by Stanley Cohen, in which he joined two DNA fragments together

(a plasmid containing a tetracycline resistance gene with a kanamycin resistance

gene), introduced this recombinant molecule into Escherichia coli and demonstrated that the E coli with the plasmid now had dual antibiotic

resistance.

This simple experiment was only possible because of the simultaneousdevelopment of several techniques for cutting DNA with restriction enzymes,

joining the fragments together with ligases, and using E coli as a host to take up

foreign DNA packaged as plasmid vectors This critical demonstration wasquickly followed by the development of methods to sort pieces of DNA and

RNA by size using gel electrophoresis The stage was set for an explosion ofnew techniques.

What follows is a brief and simplified description of these techniques andtechnologies that followed

THE STRUCTURE OF DNA

The structure of DNA arrived at by Crick and Watson is elegant in its simplicity.The molecule is composed of two antiparallel helices, looking rather like agently twisted ladder The rails of the ladder, which run in opposite directions,contain units of deoxyribose sugar alternating with a phosphate Each rung is

composed of a pair of nucleotides, a base pair, held together by hydrogen bonds

(Fig 17.1) There is a complementary relationship between the bases: Adeninealways pairs with thymine, and cytosine always pairs with guanine

(complementary nucleotides) Thus, the nucleotide sequence of one strand of

the DNA helix determines the sequence of the other

FIGURE 17.1 The DNA double helix is held together by hydrogen bonds

between base pairs These are shown as dotted lines in the figure

This structure explains how a DNA molecule replicates during cell division

so that each progeny cell receives an identical set of instructions The hydrogenbonds between the base pairs break, allowing the DNA ladder to unzip (Fig.17.2) Each half then constitutes a template for the reconstruction of the other

half Two identical DNA molecules result, one for each progeny cell

FIGURE 17.2 The complementary nature of DNA is at the heart of its capacity

for self-replication The two strands of the parental DNA unwind, and thehydrogen bonds break Each strand then becomes a template to specify a newprogeny strand obeying the base-pairing rules

RNA AND DNA

Unlike DNA, which is located primarily in the nucleus, RNA is found

throughout the cell Within the nucleus, RNA is concentrated in the nucleoli, dense granules attached to chromosomes The sugar molecule in RNA is a ribose (hence its name, ribonucleic acid), whereas in DNA, the sugar molecule is a deoxyribose (hence its name, deoxyribonucleic acid) In both DNA and RNA, the bases are made up of two purines and two pyrimidines The two purines, adenine and guanine, as well as the pyrimidine, cytosine, are common to both DNA and RNA However, although thymine is found only in DNA, the structurally similar pyrimidine uracil appears in RNA (Fig 17.3)

FIGURE 17.3 Illustrating the pairing of complementary bases in DNA and

RNA Left: DNA contains the purines adenine (A) and guanine (G) as well as

the pyrimidine’s thymine (T) and cytosine (C) A purine always pairs with a

pyrimidine; specifically, A pairs with T, and G pairs with C Right: RNA

contains uracil (U) instead of thymine In this case, A pairs with U, and G pairswith C

TRANSCRIPTION AND TRANSLATION

The flow of genetic information from DNA to protein requires a series of steps

In the first step, the DNA code is transcribed in the nucleus into mRNA by RNA

polymerase (Fig 17.4) It is at once obvious from a comparison of a maturecytoplasmic mRNA transcript with its parental DNA that the mRNA sequence isnot contiguous with the DNA sequence Some blocks of DNA sequence arerepresented in the mRNA; others are not DNA is transcribed into pre-mRNA

During the process of splicing, large regions called introns are removed and the

remaining exons are joined together into what is termed an open reading frame.

Only the exons of the DNA are translated Almost all genes from higher

eukaryotes contain introns; genes may have only a few or as many as 100introns Typically, introns make up the bulk of the gene For example, in thegene involved with muscular dystrophy, the mRNA consists of 14,000 bases,whereas the gene spans more than 2 million base pairs The mRNA transcriptassociates with a ribosome, at which, with the help of ribosomal RNA and

transfer RNA (tRNA), the mRNA message is translated into a protein.

FIGURE 17.4 Transcription and translation The “information” in DNA is

linear, consisting of combinations of the four nucleotides: adenine, guanine,cytosine, and thymine The information is transcribed into messenger RNA(mRNA), which in turn is a complementary version of the DNA code The

mRNA message is spliced in the nucleus to remove introns and is thentransported to the cytoplasm for translation into protein Triplet RNA codonsspecify each of the 20 amino acids The sequence of amino acids determines theprotein, which ultimately has three-dimensional form.

THE GENETIC CODE

The genetic code was cracked by 1966 Triplet mRNA sequences specify each ofthe amino acids Because there are four bases, the number of possibilities for athree-letter code is 4 × 4 × 4, or 64 There are only 20 amino acids, however;consequently, more than one triplet can code for the same amino acid—that is,there is redundancy in the code Because nearly all proteins begin with the amino

acid methionine, the initiation codon (AUG) represents the “start” signal for

protein synthesis Three codons for which there are no naturally occurringtRNAs—UAA, UAG, and UGA—are “stop” signals that terminate translation

(termination codons) Only methionine and tryptophan are specified by a

unique codon; all other amino acids are specified by two or more differentcodons As a consequence of this redundancy, a single-base change in RNA doesnot necessarily change the amino acid coded for

Given the position of the bases in a codon, it is possible from Table 17.1 tofind the corresponding amino acid For example, 5′ CAU 3′ specifies histidine,whereas AUG specifies methionine Glycine is specified by any of four codons:GGU, GGC, GGA, or GGG

Table 17.1 Codes for the Amino Acids

FIRST POSITION (5′

END)

SECOND POSITION

THIRD POSITION (3′ END)

Leu Ser Stop Stop A

Leu Pro Gln Arg A

AMINO ACIDS AND PROTEINS

Most of proteins are composed of a mixture of the same 20 amino acids Eachpolypeptide chain is characterized by a unique sequence of its amino acids.Chain lengths vary from 5 to more than 4,000 amino acids Most proteinscontain only one polypeptide chain, but others are formed through theaggregation of separately synthesized chains that have different sequences.Although most proteins are enzymes (i.e., they act as catalysts, inducingchemical changes in other substances but themselves remaining apparentlyunchanged), many have structural roles as well The essential fabric of bothnuclear and plasma membranes is formed of proteins

Once a polypeptide chain is synthesized from a string of amino acids, it tends

to fold up into a three-dimensional form, the shape of which is governed by theweak chemical interactions between the side groups of the amino acids Eachthree-dimensional shape is unique to the amino acid sequence The shape of aprotein is the key to its function

RESTRICTION ENDONUCLEASES

Restriction enzymes are endonucleases found in bacteria that have the property

of recognizing a specific DNA sequence and cleaving at or near that site Theseenzymes can be grouped into three categories: types I, II, and III The restrictionenzymes commonly used are of type II, meaning that they have endonucleaseactivity only (i.e., they cut the DNA without modification) at a predictable sitewithin or adjacent to the recognition sequence Types I and III have propertiesthat make them impractical for use in molecular biology

More than a thousand type II enzymes have been isolated, and more than 70are commercially available A few examples are shown in Table 17.2 They arenamed according to the following system:

1 The first letter comes from the genus of the organism from which the

enzyme was isolated

2 The second and third letters follow the organism’s species name.

3 If there is a fourth letter, it refers to a particular strain of the organism.

4 The roman numerals, as often as not, refer to the order in which enzymes

were discovered, although the original intent was that it would indicate theorder in which enzymes of the same organism and strain are eluted from achromatography column

Table 17.2 Examples of Type II Restriction Enzymes

BamHI B Genus Bacillus

Restriction endonucleases scan the DNA molecule, stopping if they

recognize a particular nucleotide sequence The recognition sites are short, four

to eight nucleotides, and usually read the same in both directions, forward and

backward, which is termed a palindromic sequence Some endonucleases, such

as PvuII, for example, produce blunt-ended fragments because they cut cleanly

through the DNA, cleaving both complementary strands at the same nucleotideposition, most often near the middle of the recognition sequence Otherendonucleases cleave the two strands of DNA at positions two to fournucleotides apart, creating exposed ends of single-stranded sequences The

commonly used enzymes EcoRI, BamHI, and HindIII, for example, leave 5′

overhangs of four nucleotides, which represent “sticky” ends, very useful for

making recombinant molecules Table 17.3 shows the recognition sequence andpoint of cutting of a dozen commonly used restriction enzymes This specificity

is the same, regardless of whether the DNA is from a bacterium, a plant, or a

human cell

Table 17.3 Specificities of Some Typical Restriction Endonucleases

ENZYME ORGANISM

RECOGNITION SEQUENCEa

RECOGNITION SEQUENCEa

BglfII Bacillus globigii AGATCT Sticky

HindIII Haemophilus influenzae

Sau3A Staphylococcus aureus GATC Sticky

HaeIII Haemophilus aegyptius GGCC Blunt

Most restriction recognition sites have symmetry in that the sequence on one

strand is the same as on the other For example, EcoRI recognizes the sequence 5′ GAATTC 3′; the complementary strand is also 5′ GAATTC 3′ EcoRI cuts the

DNA between the G and A on each strand, leaving a 5′ single-strand sequence ofAATT on each strand The strands are complementary Therefore, all DNA

fragments generated with EcoRI are complementary and can “base-pair” with

each other This is illustrated in Figure 17.5

FIGURE 17.5 Illustration of how some endonucleases cleave each strand of the

DNA off-center in the recognition site, creating fragments with exposed ends ofshort, single-stranded sequences These “sticky” ends are extremely useful inmaking recombinant molecules because they rejoin only with complementarysequences

VECTORS

A vector is a self-replicating DNA molecule that has the ability to carry another

foreign DNA molecule into a host cell In the context of this chapter, the object

of the exercise is usually to insert a fragment of human DNA (perhapscontaining a gene of interest [GOI]) into a bacterium so that it can be replicatedand grown into quantities suitable for study

Over the years, there have been many types of vectors, including plasmids,

bacteriophages (especially bacteriophage λ), bacterial artificial chromosomes

(BACs), and viruses However, the two major vectors that are used today areplasmids and viruses

Plasmids

The simplest bacterial vectors are plasmids, which are circular DNA moleculesthat can exist and replicate inside a bacterium, independent of the hostchromosome A piece of foreign DNA can be inserted into a plasmid, which in

turn is introduced into a bacterium As the bacterium grows and replicates, sodoes the foreign DNA The plasmid also contains a gene for resistance to an

antibiotic (e.g., ampicillin), and if the bacteria are subsequently grown in a

culture medium containing the antibiotic, only those bacteria that have taken upthe plasmid survive and replicate This is illustrated in Figure 17.6

FIGURE 17.6 A plasmid is the simplest bacterial vector—a means of carrying

foreign DNA sequences into bacteria such as Escherichia coli A plasmid is a

circular DNA molecule, capable of autonomous replication, that typically carriesone or more genes encoding an antibiotic resistance Foreign DNA (e.g., from ahuman cell) also can be incorporated into the plasmid If inserted into abacterium, the plasmid replicates along with the main chromosome

It is a relatively simple matter, subsequently, to harvest the recombinantplasmids There are two limitations to this technique First, plasmids are usefulonly for relatively small DNA inserts up to about 10,000 base pairs (bp) Second,the plasmids do not transfect into bacteria with high efficiency

Bacteriophage λ

Bacteriophages are bacterial viruses The bacteriophage most commonly used as

a cloning vector is bacteriophage λ It has two advantages compared with other vectors As a bacteriophage particle, bacteriophage λ can infect its host at a much

higher efficiency than a plasmid, and it can accommodate a larger range of DNAfragments, from a few to up to 24,000 bp, depending on the specific vector used

Many vectors have been derived from bacteriophage λ Some have been

modified to clone small DNAs, usually complementary DNAs (cDNAs) derived

from mRNA, and some have been modified to clone large genomic DNA

molecules If bacteriophage λ is used to clone large DNA molecules, the central

portion of the bacteriophage DNA is deleted This is to allow the foreign DNA to

be accommodated within the bacteriophage particle, which has an upper limit of55,000 bp Once the bacteriophage DNA is ligated with the DNA to be cloned,the total DNA is mixed with extracts containing empty bacteriophage particles

The ligated DNA is taken up into the bacteriophage, which is then used to infect

E coli and form plaques.

To insert itself into the E coli chromosome, the phage DNA circularizes by

the base pairing of the complementary single-strand tails that exist at its two

ends—the cos sites The resulting circular λ DNA then recombines into the E coli chromosome.

If part of the wild-type DNA of the bacteriophage is removed, room can bemade for a piece of human DNA to be inserted, again with a gene that confersresistance to an antibiotic to allow selection The bacteriophage can then be used

to infect bacteria that multiply their own DNA as well as the integrated piece ofhuman DNA (Fig 17.7)

FIGURE 17.7 A bacteriophage is a virus that infects bacteria It represents a

much more efficient means of inserting foreign DNA into a bacterium than using

a plasmid If part of the wild-type DNA is removed, room can be made for apiece of “foreign” DNA, for example, from a human cell, as well as a gene thatconfers resistance to an antibiotic to allow selection The DNA of thebacteriophage replicates along with that of the bacterium

Bacterial Artificial Chromosomes

Sequencing large genomes requires a cloning vector capable of carrying verylarge fragments of DNA BACs are vectors based on a type of plasmid withsequences encoding self-replication while maintaining a low copy number.BACs can accommodate approximately 300 kilobases (kb) of DNA, whereas

plasmids are limited to approximately 10-kb insertions BACs transform E coli

via electroporation more efficiently than comparable but larger constructs,compensating for the reduced amount of inserted DNA that can beaccommodated Because of the defined genetic backgrounds of their bacterial

hosts, BACs are also less prone to recombination events (a common problemwith large DNA vectors) BACs were the primary vector used during thegenome-sequencing projects, mainly because a BAC carrying a GOI is easilyacquired.

Viruses

Viruses are highly efficient vectors for introducing foreign genes intomammalian cells Viral infection of mammalian cells has proved to be aneffective and efficient method for delivering and stably expressing a GOI Thethree main types of viruses used for gene transfer are retroviruses, adenoviruses,

and adeno-associated viruses (AAV) Retroviruses are a type of RNA virus Unlike all other RNA viruses, the RNA genome of retroviruses is transcribed

into DNA, which is then stably integrated into the host genome Retroviruses caninfect virtually every type of mammalian cell, making them very versatile Onewell-known type of retrovirus used for gene transfer is lentivirus They possesshigh rates of infectivity and can infect both dividing and nondividing cells Incontrast to retroviruses, adenoviruses are a type of DNA virus, and they caninfect both proliferative and quiescent cells Adenoviruses can be easily purified

in high titers and the two major strains (Ad2, Ad5) used to make recombinant

viruses can accept 37 kb of foreign DNA Adenovirus gene expression is stable,

but the genome remains epichromosomal AAV are the third major vector familyused They get their name from being identified as contaminants of adenovirusisolates AAV are relatively small viruses, which can package 5 kb of foreignDNA In addition to their use in laboratory research, AAV has also beenapproved for the introduction of foreign DNA into humans for gene therapy

LIBRARIES

Genomic Library

A genomic library is a compilation of DNA fragments that make up the entire

genome Making a genomic library is frequently the starting point of a geneisolation experiment DNA is extracted from a tissue sample or from cultured

cells, and a partial digest is made using EcoRI, for example This enzyme has a

six-nucleotide recognition sequence, so if the digest is complete, it cuts the DNAinto pieces about 4,000 bp long (The probability of cleaving a six base pairsequence is [¼]6, or once every 4,096 bases.) By reducing the enzyme

concentration and incubation time, a partial digest is obtained so that the EcoRI

enzyme cuts at only about one in five restriction sites, resulting in fragments of

about 40,000 bp.

The genomic DNA fragments are then ligated into a suitable vector based on

the size of the DNA-plasmid (up to 10 kb), bacteriophage λ (up to 25 kb), BAC

(up to 300 kb), and yeast artificial chromosome (up to 200 kb) The assembled

particles are used to infect E coli cells or yeast, which are spread on plates and

incubated in growth medium containing the appropriate selection so that only

bacteria or yeast that have taken up the vector grow into colonies Each colony

contains millions of copies of a single genomic DNA insert However, the largerthe fragment of DNA, the more prone to rearrangement in the host bacteria oryeast Therefore, multiple clones overlapping the same genomic region need to

be generated to ensure fidelity in assigning chromosome position or DNAsequence alignment The most common use of genomic libraries was for

“shotgun” DNA sequencing By using this sequence as a probe, overlapping clones can be identified and then sequenced to ultimately develop a “contig,”

which represents a contiguous DNA sequence for a portion of a chromosome

cDNA Library

Sometimes, there is a need to focus only on DNA that will be transcribed intomRNAs cDNA is DNA that is complementary to the mRNA and thereforeincludes only the expressed genes of a particular cell For eukaryotic cells, themRNA is usually much shorter than the total size of the gene because the codingsequences in the genome are split into exons separated by noncoding regions ofDNA called introns

cDNA libraries are made in either plasmids or bacteriophage λ Often, these

vectors have been modified such that the cDNA can be transcribed into mRNA

and then translated into protein Depending on the type of vector, the library can

be screened by oligonucleotide probes or by an antibody that recognizes the protein of the GOI This type of cDNA library is called an expression library.HOSTS

Recombinant DNA molecules can be constructed and manipulated to someextent in the test tube, but amplification and expression ideally require a host

Escherichia Coli

E coli is the most widely used organism in molecular biology because it is

relatively simple and well understood It contains a single chromosomeconsisting of about 5 million base pairs

In addition to their main chromosomes, many bacteria, including E coli,

possess large numbers of tiny circular DNA molecules that may contain only a

few thousand base pairs They are called episomes, a subset of which are known

as plasmids as previously described Plasmids are autonomously replicating

“minichromosomes.” They were first identified as genetic elements separatefrom the main chromosome and carrying genes that conveyed resistance to

antibiotics Foreign DNA can be introduced readily into E coli in the form of

plasmids

Because the DNA of all organisms is made of identical subunits, E coli accepts foreign DNA from any organism The DNA of bacteria, Drosophila,

plants, and humans consists of the same four nucleotides: adenine, cytosine,

guanine, and thymine A foreign gene inside E coli is replicated in essentially

the same way as its own DNA

Yeast

Yeast are simple eukaryotes that have many characteristics in common withmammalian cells but can be grown almost as quickly and inexpensively asbacteria

The study of yeast has frequently provided insights into similar phenomenaand functions in mammalian cells that are much more difficult to address Yeasthave been of particular value in radiobiology because the availability of a widearray of mutants that are sensitive to ultraviolet or ionizing radiations has madethe study of the genes responsible for radiosensitivity and radioresistance muchsimpler than if studies were conducted only in mammalian cells.Complementation of many yeast mutants with mammalian genes has proved to

be a powerful screening method to identify mammalian genes that affectradiosensitivity

Yeast have also proved to be good systems for studying cell-cycle control.Because it appears that the cell-cycle machinery of all eukaryotes is very similar,

it makes sense to concentrate on the simplest and most easily manipulatedsystem The availability of temperature-sensitive yeast mutants is of particular

value The yeast Saccharomyces cerevisiae (budding yeast, baker’s yeast, or

brewer’s yeast) and Schizosaccharomyces pombe (fission yeast) have been used

widely They grow rapidly and have been well characterized genetically

An approach has been developed for analyzing large numbers of uniquely

tagged yeast deletion strains by hybridization to high-density oligonucleotide

arrays Deletion strains are generated using a polymerase chain reaction targeting

strategy, and each deletion strain is specifically labeled with two 20-base tagsequences Each molecular tag is important, as it serves as a “bar code,” allowingapproximately 4,500 deletion strains to be pooled and analyzed simultaneously

by hybridization to an array The abundance of a given strain is reflective of itshybridization to its corresponding complementary sequence on a high-densityoligonucleotide array The yeast deletion strains and oligonucleotide arrayscontaining complementary sequences for each deletion mutant are commerciallyavailable

Mammalian Cells

The limited number of cell systems used in radiation and chemical

transformation studies can be separated broadly into two categories The first

category includes short-term explants of cells derived from rodent or humanembryos with a limited life span These include:

Hamster embryo cells

Rat embryo cells

Human skin fibroblasts

Human foreskin cells

Human embryo cells

These cell assay systems can be used to assess the expression or activity offoreign genes transfected into them, or they may be used in studies of oncogenictransformation induced by radiation or chemicals

In practice, the bulk of the experimental work has been performed withhamster or rat embryo cells One advantage of such systems is that they consist

of diploid cells so that parallel cytogenetic experiments can be performed Cellsurvival and cell transformation can be scored simultaneously in the same dishes.The experimental methodology is illustrated in Figure 17.8 Cells are seeded

at low density into dishes or flasks and treated with radiation or chemicals Theyare allowed to grow for 8 to 10 days, and the resultant colonies are fixed andstained Transformed colonies are identifiable by dense multilayered cells,random cellular arrangement, and haphazard cell-to-cell orientation accentuated

at the colony edge Normal counterparts are flat, with an organized cell-to-cellorientation and no piling up of cells An example of the contrast between anormal and a transformed colony is shown in Figure 17.9

FIGURE 17.8 Protocol for the assay of oncogenic transformation in hamster

embryo cells by radiation Midterm hamster embryos are removed, minced,enzymatically dissociated, and seeded as single cells on feeder layers They arethen treated with either radiation or chemicals, and the resultant colonies (normaland transformed) are scored after 8 to 10 days of incubation

FIGURE 17.9 A: A normal untransformed colony of hamster embryo cells The cells are orderly and show contact inhibition B: A colony of radiation-

transformed hamster embryo cells Note the densely stained, piled up cells andthe crisscross pattern at the periphery of the colony

The second category of experimental systems includes established cell linesthat have an unlimited life span The karyotype of these cells shows variouschromosomal rearrangements and heteroploidy The two most widely usedestablished cell lines for transformation studies are the BALB/C-3T3 cell lineand the C3H 10T1/2 cell line Both originated from mouse embryos, aretransformable by various oncogenic agents, and have been used extensively in

transformation studies The advantage of these established cell lines lies in thefact that they are “immortal” so that a particular passage can be used over a longperiod of time and maintained in banks of frozen cells The transformation assay

is a focal assay Cells are treated with radiation or chemicals and then allowed togrow for 6 weeks The “normal” cells stop growing after confluence is reached,and transformed foci can be identified against a background of the contact-inhibited normal cells because they are densely stained, tend to pile up, and show

a crisscross random pattern at the edge of the focus Transformed cells, identified

by their characteristic morphology, grow in soft agar, which indicates that theyhave lost anchorage dependence, and produce fibrosarcomas if injected intosuitably prepared animals This is illustrated in Figure 17.10

FIGURE 17.10 A: A type III transformed focus of C3H 10Tl/2 cells induced bythe hypoxic cell sensitizer etanidazole (SR 2508) Note the multilayered growthand the crisscrossing of cells at the periphery of the clone over a contact-

inhibited background of nontransformed cells B: Cells from the focus shown in

A were plucked, expanded in culture, and plated into semisolid medium; theyformed colonies, indicating that they had lost anchorage dependence This is an

indication of malignancy C: The ultimate test of malignancy is whether cells

from a type III transformed clone injected into a suitably prepared animalproduce a tumor (a fibrosarcoma) that eventually kills the animal

The in vitro assay systems based on mammalian cells have two quite

different uses in radiobiology First, they may be used to accumulate data andinformation that are essentially pragmatic in nature; for example, they may be

used to compare the oncogenic potential of various chemical and physicalagents As such, they occupy a useful intermediate position between the bacterialmutagenesis assays, which are quick and inexpensive but score mutagenesisrather than carcinogenesis, and animal studies, which may be more relevant tohumans but are quite cumbersome and inordinately expensive Second, the assaysystems can be used to study the mechanisms of carcinogenesis In this context,

transformation assays have played a vital role in unfolding the oncogene story

because transfecting DNA from human tumors into established rodent cell linesused for transformation, and observing the induction of transformed foci is oneway to detect the expression of an oncogene (see Chapter 18 for more details)

DNA-MEDIATED GENE TRANSFER

Gene transfer is now a routine tool for studying gene structure and function.Because gene transfer into mammalian cells is an inefficient process, anabundant source of starting cells is necessary to generate a workable number oftransfected cells—that is, cells containing a transferred gene

Mammalian cells do not take up foreign DNA naturally; indeed, they try toprotect themselves from invading DNA Consequently, one of several techniquesmust be used to bypass natural barriers:

1 Microinjection: This is the most direct but the most technically demanding

procedure to accomplish DNA can be injected directly into the nucleus of acell through a fine glass needle

2 Calcium phosphate precipitation: Cells take up DNA relatively efficiently in

the form of a precipitate with calcium phosphate The efficiency variesmarkedly from one cell line to another For example, NIH 3T3 cells areparticularly receptive to foreign DNA introduced by this technique High-molecular-weight DNA is mixed with insoluble calcium phosphate as acarrier and layered onto cells in petri dishes Typically, a plasmid containing

a selectable marker, such as G418 resistance, is copipetted and

cotransfected into cells In this way, cells that take up DNA can be selected

Of the cells that take up DNA, only a small percentage ultimately integratethe DNA into their genomes (are stably transfected) For example, if afragment of DNA containing an activated oncogene is transfected into NIH3T3 cells, morphologic transformation of the cell occurs, leading to loss ofcontact inhibition, and acquires the ability to grow as tumors if injected intoimmune-suppressed animals

3 Cationic lipids: Cationic lipids offer some of the highest transfection

efficiencies and expression levels to a wide variety of cells, both insuspension and attached The protocol is very simple in that you mix lipidand DNA, vortex gently, centrifuge, and allow to sit at room temperature for

10 to 30 minutes before adding to cells Although cationic lipids producehigh transfection efficiencies, some cells are sensitive to these lipids

4 Electroporation: This technique is useful for cells that are resistant to

transfection by calcium phosphate precipitation Cells in solution aresubjected to a brief electrical pulse that causes holes to open transiently inthe membrane, allowing foreign DNA to enter

AGAROSE GEL ELECTROPHORESIS

The purpose of agarose gel electrophoresis is to separate pieces of DNA of

different size This technique is based on the fact that DNA is negativelycharged Under the influence of an electrical field, DNA molecules move fromnegative to positive poles and are sorted by size in the gel In a given time, smallfragments migrate through the gel farther than large fragments

The technique, illustrated in Figure 17.11, is as follows: Molten agarose ispoured into a tray in which a plastic comb is suspended near one end to formwells in the gel after it has solidified like gelatin The concentration of theagarose is varied according to the size of the DNA fragment to be separated:high concentration for small fragments, lower concentration for larger fragments.The solidified gel is immersed in a tray containing an electrolyte to conductelectricity The DNA samples, mixed with sucrose and a visible dye, are pipetted

into the wells, and the electrical field is connected Electrophoresis is monitored

by observing the movement of the dye in the electrical field After separation is

complete, the gel is soaked in ethidium bromide, which intercalates into DNA

and fluoresces under ultraviolet light to make the position of the DNA visible.The smaller DNA fragments migrate farther on the gel than the larger fragments,

as illustrated in Figure 17.12 In fact, the distance migrated is directly related toDNA size (Illustrations of several examples appear elsewhere in this chapter.)

FIGURE 17.11 Illustration of agarose gel electrophoresis DNA is negatively

charged so that under the influence of an electrical field, it migrates toward theanode During electrophoresis, DNA fragments sort by size, small moleculesmoving farther than larger molecules Because smaller molecules move fartherthan larger molecules in a given time, polyacrylamide gel electrophoresis often isemployed to separate smaller DNA fragments with greater resolution than withagarose

FIGURE 17.12 A: Illustration of the separation of λ DNA after digestion with

EcoRI Agarose gels separate DNA fragments that can be quite large By varying

the percentage of agarose, the resolution of different-sized fragments can be

maximized B: Regardless of change in agarose concentration, the distance

migrated is directly proportional to DNA size

POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) technique uses enzymatic amplification

to increase the number of copies of a DNA fragment The principle is based on

primer extension by DNA polymerases, which was discovered in the 1960s.

First, primers that are complementary to the 5′ end of the double-stranded DNAsequence to be amplified are synthesized The two primers are mixed in excesswith a sample of DNA that includes the fragments to be amplified, together with

a heat-stable DNA polymerase The four deoxyribonucleotide triphosphates arealso included in excess; one or more of them may be radioactively labeled Thechoice of the heat-stable DNA polymerase depends on the size of the DNA to be

amplified, Taq polymerase is inexpensive and effective up to 500 bp but has a

high error rate Vent polymerase is considered a workhorse and is used to

amplify DNA in the 1- to 5-kb range At the high end of polymerases is

Phusion, very high processivity and low error rate but very expensive Heatstable polymerases such as Phusion are used to amplify DNA in the 20-kb range.The PCR technique is illustrated in Figure 17.13

FIGURE 17.13 The polymerase chain reaction (PCR) for the amplification of

DNA fragments The number of DNA molecules is doubled in each cycle, whichtakes about 7 minutes so that in a matter of several hours, millions of copies of aDNA fragment can be made (Courtesy of Dr Greg Freyer.)

The amount of the sequence is doubled in each cycle, which takes about 7minutes During each cycle, the sample is heated to about 94° C to denature the

DNA strands, then cooled to about 50° C to allow the primers to anneal to the

template DNA, and then heated to 72° C, the optimal temperature for Taq

polymerase activity In a matter of a few hours, a million copies of the DNAfragment can be obtained in an essentially automated device PCR has foundmany applications in both basic research and clinical settings For example, ithas been used to detect malignant cells in patients with leukemias that arecharacterized by consistent translocation breakpoints Primers that span thebreakpoint are added to a bone marrow sample and subjected to multiple cycles

of PCR Even one cell in a million with the translocation can be detected

Polymerase Chain Reaction–mediated Site-directed Mutagenesis

PCR-mediated site-directed mutagenesis is a technique used to create mutationssuch as nucleotide replacements, insertions, or deletions at a desired location inthe gene or its flanking sequences to investigate the relationship between genesequence and gene function The starting material is usually a double-strandedDNA vector with the gene or nucleotide of interest acting as the template forPCR In this technique, two complementary oligonucleotides containing the

mutation of interest are used as the primers for PCR The mutant strand of DNA

is synthesized by denaturing the DNA template, annealing mutagenic primers tothe DNA template, and extending the primers using a high-fidelity DNApolymerase to reduce the chance of unwanted random errors being introducedduring replication Bacteria modify DNA by methylation to prevent it from beingdigested by restriction endonucleases This modification of DNA is then used toeliminate the starting DNA from the PCR-amplified DNA by digestion with a

methylation-specific endonuclease (Dpn I), leaving only the mutated DNA This

DNA is then used to transform competent bacterial cells so that it can be used forfurther study

GENE-CLONING STRATEGIES

Gene-cloning strategies in the past have revolved around choosing a source ofDNA, either genomic or cDNA, constructing a library (a collection of DNAfragments in an appropriate vector), and screening the library to locate the GOI.This labor-intensive approach has now been supplanted with both publicrepositories of mammalian genes established by the National Institutes of Health(NIH) and commercial repositories Even if you cannot find your GOI, PCRwould be a rapid means of obtaining your gene

GENOMIC ANALYSES

The complete sequencing of the human genome as well as the genomes of otherorganisms has led to the interdisciplinary field of genomics During the lastdecade, DNA sequence information has made the task of investigating DNAmuch easier This revolution has been brought about by two major approaches tosequencing: (1) hierarchical shotgun sequencing of cloned DNA fragments asdescribed earlier and (2) whole genome shotgun sequencing in which DNAsequences of random, uncloned DNA fragments are obtained and aligned usingcomputer algorithms Although sequencing DNA fragments is straightforward,there is a great deal of effort in filling in the gaps between sequences (Fig.17.14)

FIGURE 17.14 Illustration of the technique of “shotgun sequencing” to obtain

contiguous sequences spanning the complete length of chromosomes In thistechnique, which was successfully used to generate an almost complete sequence

of the human genome, random fragments of DNA are generated in an unorderedfashion and sequenced in a random fashion Hence, the term shotgun sequencing

as the sequence analysis is random as a shotgun blast covers a large target in arandom fashion Once the sequences of the random fragments are obtained,computer algorithms are used to align the random sequences Then, overlappingsequences are aligned into a contiguous sequence

Although it is extremely useful to have “a complete genomic DNAsequence” for humans, there is significant heterogeneity between individuals.Recognition of the importance of this heterogeneity between individualsstimulated the development of the Hap Map project, whose goal is to identifygenome differences between individuals that can result in disease or even cancer

In fact, the differences between cancer genomes and cancer-free individualsstimulated “The Cancer Genome Atlas” (TCGA) project by the National CancerInstitute (NCI) and National Human Genome Research Institute (NHGRI) Theinitial thoughts were to sequence the genomes of cancers grouped by histologictype Because genomic sequencing has become more cost effective, the ability tosequence the genomes of every cancer patient could become feasible in thefuture Sequence analysis of a cancer genome has brought forth the concept of

“personalized medicine” where the goal is to match a therapy with a genomicalteration For example, a patient with breast cancer is found to have a mutation

in the BRCA1 gene This patient would be a candidate for poly adenosinediphosphate-ribose polymerase (PARP) inhibitors or a platinum drug that willkill BRCA1 mutated cells defective in homologous recombination.Unfortunately, most of the mutations identified in cancer genomes are

“inactionable,” meaning that there is not a specific therapy targeting the

mutation Furthermore, most of the mutations identified do not necessarily have

a clear impact on radiosensitivity or radioresistance One example wheregenomic analysis has been informative in regard to radiation resistance isdeletion/inactivation of the Keap1 gene Keap1 inhibits Nrf2, which promotesresistance to oxidative stress Genome deletion of Keap1 increases the cancercell’s resistance to oxidative stress and decreases the efficacy of low–linearenergy transfer (LET) radiation Therefore, for patients with the loss of Keap1,the use of high-LET radiotherapy is potentially warranted

Deletions or mutations of DNA repair genes detected by genomic analysisshould theoretically increase the radiosensitivity of tumors However, there havebeen few studies investigating the effect of DNA repair mutations on localcontrol by radiotherapy Such a study would seem an additional approach to

to orient physical markers, such as restriction fragment length polymorphisms, on adjacent fragments so that they can be lined up and the

nucleotide sequence can be made continuous If, for example, restrictionfragments from a DNA library are sequenced, relating these sequences to knownphysical markers eventually can produce the nucleotide sequence of the entiregenome This is the goal of the Human Genome Project, but the task is somassive that it cannot be accomplished without the development of automatedsequencing technology and sophisticated computer strategies to store and handlethe data

DNA Sequence Analyses

DNA sequencing is the process of identifying the exact sequence of nucleotides

in a given DNA sample, whether it is a particular gene or an entire genome.DNA sequencing is achieved by the dideoxynucleotide chain termination method(the Sanger method), which exploits the fact that DNA is composed ofdeoxynucleotides Primers are used to amplify a given target of the single-stranded DNA to be sequenced Dideoxynucleotides lack a hydroxyl (OH) group

at the 3′ position Thus, when a dideoxynucleotide is added, the chain isterminated Using conditions similar to PCR, multiple rounds of primerextension incorporate deoxynucleotides and labeled dideoxynucleotides on thesequenced strand In manual sequencing, four separate reactions are run in whichonly one radiolabeled dideoxynucleotide is added to the reaction Each of thefour dideoxynucleotides is electrophoresed on a gel and visualized byautoradiography A more recent (and commonly used) innovation is automatedsequencing, in which a single reaction is run with a mixture of all fourdideoxynucleotides, each carrying a different-colored fluorescent label Theproducts are electrophoresed in one lane, separating the replication productsbased on size A laser scans the bottom of the gel, detecting the four fluorescenttags of the dideoxynucleotide from which the original strand sequence ofnucleotides can be determined The sequencing method described earlier isknown as Sanger sequencing The latest revolution in sequencing is known as

“next generation sequencing” (NGS) or deep sequencing Although differentplatforms for NGS have been used, they all involve sequencing of millions ofsmall fragments of DNA in parallel The sequence information is then stitchedtogether using bioinformatic programs run on very powerful computers Thereason NGS is also known as deep sequencing is because the same genomicDNA from the normal or tissue material is sequenced multiple times to provideaccurate sequence readouts NGS can be used to sequence the entire humangenome (~3 × 109 billion bases) or complete exome sequencing for all codinggenes (~25,000 genes)

Polymorphisms or Mutations

Restriction Fragment Length Polymorphisms

Relatively small differences in similar DNA sequences (alleles), DNA polymorphisms, as they are called, may result from point mutations, deletions

or insertions, or varying numbers of copies of a DNA fragment (so-called

tandem repeats) A Southern blot analysis can be used to detect DNA polymorphisms by using a probe that hybridizes to a polymorphic region of the

DNA molecule Other techniques such as polymerase chain reaction–restrictionfragment length polymorphism (PCR-RFLP) can also be used to determinegenetic variations that result in single nucleotide polymorphisms (SNPs)

If a particular restriction enzyme is used to cut human DNA, a polymorphiclocus yields restriction fragments of different sizes These are called restrictionfragment length polymorphisms Deletions, insertions, or tandem repeats

involving more than about 30 nucleotides can be detected as recognizable shifts

in the Southern blot hybridization pattern or PCR products Even a point mutation can be detected if the resultant change in sequence removes or adds a

new recognition site at which a restriction endonuclease cuts Using differentrestriction enzymes will generate a unique pattern of DNA fragments that act as

a fingerprint for normal cells and tumor cells

Comparative Genome Hybridization

Genomic DNA is harvested from a tumor or cancer cell line and digested with

DpnII (a restriction endonuclease that cleaves DNA approximately every 256

bp), creating a random pool of DNA fragments DNA is amplified with randomprimers and nucleotides labeled with the fluorescent dye Cy5 (red) As a control,DNA from a normal cell line is also digested and then amplified with thefluorescent dye Cy3 (green) The two samples are mixed and hybridized to a

DNA microarray composed of cDNAs The microarray is scanned and the ratio

of Cy5 to Cy3 (red to green) fluorescence calculated for each cDNA spot togenerate a baseline fluorescent ratio Individual spots with a Cy5:Cy3 ratiosignificantly higher than background correspond to regions of the genome that

are amplified (gene amplification) in that specific cancer cell, whereas those

with lower than baseline correspond to chromosomal deletions (also called loss

of heterozygosity) Spots corresponding to contiguous chromosomal regionsshould show similar alterations in enrichment and define a common locus Thegenes residing in the altered loci are identified from the human genome database

to generate a list of potential tumor suppressors or oncogenes (Fig 17.15)

FIGURE 17.15 Illustration of the technique of comparative genomic

hybridization (CGH) Tumor and reference (normal) DNAs are fragmented andamplified with random primers and nucleotides labeled with Cy5 and Cy3 Thetwo samples are mixed hybridized to a DNA microarray of cloned fragmentscontaining cDNAs The microarray is scanned, and the ration of Cy5 to Cy3fluorescence is calculated for each spot on the microarray Spots with higher Cy5

to Cy3 compared to baseline represent amplifications, and spots with lower Cy5

to Cy3 represent deletions The genes residing in these regions are thenidentified in a human database to generate potential oncogenes or tumorsuppressors (Modified from Helixio, with permission.)

Comparative genomic hybridization (CGH) has also been combined withSNP technology, which is commercially available through Agilent Sure PrintHuman Genomic CGH and SNP microarrays In this technique, a similarprotocol as described previously for CGH is used except that the Cy5 and Cy3labeled DNAs from tumor and normal reference material are hybridized to theAgilent CGH and SNP oligonucleotide arrays The combination of techniques isuseful in showing copy number gain and loss both at the chromosome level aswell as the gene level

GENE KNOCKOUT STRATEGIES

Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR Associated Protein

Clustered regularly interspaced short palindromic repeats (CRISPR) and

CRISPR associated (Cas) genes have evolved to allow cells to respond andeliminate invading genetic material In lower eukaryotes such as bacteria, viralinfection activates the CRISPR pathway and a byproduct of CRISPR activation

is short DNA sequences from the viral genome integrated into the bacterialgenome, which serve as a physical reminder of the past viral infection A newinfection by a similar virus activates complementary CRISPR RNA (crRNA) toidentify a homologous sequence The Cas nuclease introduces a double-strandbreak at the “foreign” DNA sequence In fact, to achieve specificity in DNArecognition and cleavage, the Cas nuclease forms a complex with a crRNA and atrans-activating crRNA (tracrRNA) which has complementarity to the crRNA

The Cas9 nuclease cuts both DNA strains, generating double-strand breaks

which are defined by 20-nucleotide “target” sequences within the crRNA (Fig.17.16)

FIGURE 17.16 Illustration of clustered regularly interspaced short palindromic

repeats (CRISPR) and CRISPR associated protein (Cas9) targeting In lowereukaryotes, short interspersed sequences (approximately 20 base pairs) fromviral infections or plasmids serve as a reminder of a previous infection This isprobably the same for mammalian cells as well The system is relativelystraightforward to use in gene editing because it requires a Cas nuclease (Cas9)and a guide RNA (gRNA) to the target sequence of choice The gRNA contains

a tracrRNA that is necessary for nuclease activity Cas9-RuvC cleaves the DNAstrand not complementary to the guide RNA Cas9-HNH cleaves the DNA strandcomplementary to the guide RNA Specificity of DNA binding is due to thegRNA and a three-nucleotide NGG sequence called the protospacer adjacentmotif (PAM) sequence Today, plasmids are commercially available that containall in one Cas9-CRISPR components with codon-optimized Cas9 protein and aguide RNA Furthermore, unique CRISPR sites have been developed to reducethe problem of off-target cutting for several genomes, including human.(Modified from Sigma-Aldrich, with permission.)

The power of CRISPR lies in the use of only three components—Cas9,crRNA, and trRNA Nowadays, a ready-to-use CRISPR system is composed oftwo plasmids—one containing the Cas9 nickase and a second plasmidexpressing a guide RNA (gRNA) under the control of the U6 promoter.Alternatively, CRISPR lentiviral vectors also are available which incorporatesboth gRNAs and the Cas9 genes These are the basic workhorse components, butmutant forms of Cas9 have been developed to increase precision of cutting ortargeting

Homologous Recombination to Knockout Genes

Homologous recombination is the cellular process that allows the eukaryotic cell

to repair damage to one chromosome with the DNA from the homologous sisterchromosome acting as a template (see Chapter 2) This process also occursduring meiosis, where the DNA from the parental chromosomes is shuffledbefore segregation to gametes Homologous recombination can be used toselectively delete or alter the endogenous gene in a cell line A simplified version

of this process is illustrated in Figure 17.17 Cloned DNA from a GOI ismodified to delete a key functional domain from the middle of the sequence and

replace it with a cDNA for neomycin resistance (neoR) A cDNA (tk) conferring

sensitivity to another drug (ganciclovir) is ligated to the end of the GOI-neoconstruct The linearized construct is then transfected into cells, and the cells aretreated with both neomycin and ganciclovir If the construct is randomly inserted

into the cell’s genome (a common event), both neoR and tk are inserted and

expressed These cells are resistant to neomycin but will be killed by ganciclovir

If homologous recombination occurs (a rare event), the GOI sequences flanking

the neoR cassette will be recognized by the cellular recombination machinery and replace the original region of GOI with the neoR cassette but will not incorporate the tk gene These cells will then be neomycin resistant and survive

ganciclovir The same process is repeated with a different antibiotic resistance

marker to delete the other copy of the GOI PCR or Southern blotting is then

used to verify loss of the native gene and insertion of the neoR gene A similar

process can be used to swap native parts of a GOI with mutant forms

FIGURE 17.17 Use of homologous recombination to delete function in a gene

of interest (GOI) Exon 2 of GOI is known to be responsible for GOI’s biologicactivity A knockout construct is cloned from a genomic fragment spanning GOI

by replacing exon 2 with the neomycin antibiotic resistance gene (neoR) The gene for thymidylate kinase (tk) at the 5′ end of the construct is cloned Cells are

transfected with the knockout construct and treated with neomycin andganciclovir to select for homologous recombinants and against randomintegrants The surviving colonies are screened by PCR or Southern blotting toverify deletion of exon 2

Knockout Mice

Using homologous recombination, one copy of a GOI is deleted in mouseembryonic stem (ES) cells The neomycin- and ganciclovir-resistant ES cells areinjected into early mouse embryos The resultant chimeric mice that are

heterozygous for GOI in their gametes are bred to generate male and female GOIheterozygous mice The heterozygotes are subsequently bred, and of theiroffspring, one in four should be homozygous knockouts for GOI PCR orSouthern blotting is used to confirm homozygous GOI deletion Moresophisticated methods use specialized recombination systems to delete a GOI in

a tissue-specific and/or temporally regulated manner

GENE EXPRESSION ANALYSIS

Northern Blotting

The name Northern blot was coined to describe the technique for separatingRNA by gel electrophoresis and is analogous to the Southern blot technique used

to study DNA Both abundance and turnover of specific RNAs can be detected

by Northern blot Total cellular RNA is harvested from tissue or cells and thendenatured to prevent hydrogen bonding between base pairs Denatured RNA is in

a linear, unfolded conformation, which allows fragments to be separated by gelelectrophoresis according to size This is then transferred to a membrane, made

of either nitrocellulose or nylon, which can then be hybridized by a specificDNA probe This technique is rarely used today because of the advent of reversetranscription (RT) PCR described in the following text

RISC to its complementary RNA target sequence, resulting in cleavage and

subsequent silencing of the target RNA Similar expression vectors can be used

to introduce shRNAs, which are then cleaved into double-stranded RNAs byDicer, a cytoplasmic nuclease These RNAs of 21 to 25 nucleotides can theninteract with RISC, leading to silencing of target RNA sequences In general,siRNAs are useful for transient gene silencing, whereas shRNAs are more usefulfor long-term gene inactivation due to their inherent low rate of degradation

FIGURE 17.18 Short double-stranded RNA molecules (short-interfering RNAs

[siRNA]) are transfected into a cell as chemically synthesized oligo duplexes orhairpin expression vectors Cellular RNA degradation machinery incorporatesthe siRNA to form the RNA-induced silencing complex (RISC) The sequencesincorporated cause the RISC complex to selectively recognize thecomplementary sequences on target messenger RNAs (mRNAs) RISC cleavesthe mRNA, leading to degradation and a functional loss of protein expression.(Adapted from Brummelkamp TR, Bernards R New tools for functional

mammalian cancer genetics Nature Rev Cancer 2003;3:781–789.)

Reverse Transcription Polymerase Chain Reaction

Although the Northern blot is the classical method to assay gene expression, itrequires a rather large amount of RNA (5 to 10 mcg), particularly if the RNA toassay is in low abundance To circumvent this limitation, RNA can be amplifiedusing RT followed by amplification of the resulting cDNA with conventionalPCR techniques Several retroviruses express DNA polymerases that can reversetranscribe viral RNA into DNA prior to genomic insertion RNA purified from

cells is annealed with random primers and incubated with a reverse transcriptase (Moloney Murine Leukemia Virus RT or a commercial variant is

most common) and deoxynucleotides The cDNA is amplified by PCR withprimers specific for the GOI and separated by electrophoresis for imaging It isimportant to use linear PCR conditions and to also amplify a control RNA (actin,glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) for normalization,relative quantities can be determined and used to assay changes in geneexpression However, because this technique is subject to variation from primerefficiencies and PCR amplification, it is often referred to as semiquantitative RT-PCR

Quantitative Real-Time Polymerase Chain Reaction

The need to verify gene expression data from microarray studies led to the

widespread use of quantitative real-time PCR (qRT-PCR) Originally

developed for preimplantation diagnosis in fertility clinics, qRT-PCR makes itpossible to accurately quantify RNA from very small amounts of a startingsample Amplification and measurement take place in the same reaction vessel,enabling high-throughput analysis The qRT-PCR thermocyclers have extremelylarge dynamic ranges (greater than five orders of magnitude), allowing forquantitation of both high- and low-abundance RNAs from the same sample.RNA from cells or tissue is reverse transcribed into cDNA and then amplifiedwith specific primers in the presence of a fluorescent oligo specific for a GOI(TaqMan, minor groove binder [MGB] probes, etc.) or a fluorescent intercalatingdye (SYBR Green) At each PCR cycle, the change in fluorescence is recorded.After normalizing amplification of a GOI against that of a control RNA, it isthen possible to calculate the differences in gene expression among samples

those that code for β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, Renilla luciferase, and a host of fluorescent proteins from aquatic

organisms (green fluorescent protein [GFP], red fluorescent protein [RFP],

etc.) There are many different types of promoter sequences that can be used toregulate the expression of a reporter gene: Some promoters are constitutivelyexpressed (from housekeeping genes or viral genes), whereas others are artificialcreations of a particular transcriptional regulatory sequence (i.e., the p53 tumor-suppressor binding sequence) and a basal promoter element For example, toinvestigate the regulation of p53 in irradiated cells using a reporter gene, apromoter consisting of the p53 binding site and a basal regulatory element would

be more appropriate than a large promoter that contains other responsive regulatory factors in addition to p53

radiation-Promoter Bashing

A very common use of biologic reporters is the “promoter bash,” where apromoter is dissected to determine which region is responsible for an interestingactivity (e.g., as induction during ionizing radiation or hypoxia) Usingconventional techniques, the promoter is cloned upstream of a bioluminescentreporter gene, such as the gene coding for firefly luciferase, and transfected into

cells with a control plasmid constitutively expressing Renilla luciferase This

approach makes use of the differing substrate dependencies of luciferase

enzymes from fireflies (Photinus pyralis) and jellyfish (Renilla reniformis).

Using two distinct reagents, light emitted by the firefly construct that reflectspromoter activity in response to ionizing radiation can be normalized with the

signal from the Renilla construct that reflects promoter expression in unstressed

cells to correct for transfection efficiency By systematically deleting parts of the

flanking region of the promoter, one can isolate the DNA sequence that

regulates promoter activity (Fig 17.19)

FIGURE 17.19 Example of a promoter-bashing experiment PPARγ2 expression

has been shown to be repressed by the transcription repressor DEC1 665 (the

span from −603 to +62) base pairs of the PPARγ2 promoter are cloned upstream

of a firefly luciferase reporter (FL) and transfected into NIH3T3 mouse

fibroblasts with either a DEC1 expression construct or an empty vector The

activity of the FL construct is repressed to approximately 50% by DEC1 The FLconstruct was progressively truncated with restriction enzymes (constructs 1 to3), demonstrating that deletion of a fragment spanning −285 through −116(construct 3) removes the ability of DEC1 to repress transcription Luciferasedriven by the −285 through −116 fragment in isolation (construct 4) is stillrepressed by DEC1, confirming the location of a repressed region This isconfirmed by deletion of the region from the full-length promoter (construct 5),which is no longer repressed by DEC1 (From Yun Z, Maecker HL, Johnson RS,

et al Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia Dev Cell.

2002;2:331–341.)

Chromatin Immunoprecipitation

Methods to study transcriptional regulation in mammalian cells are generallylimited to biochemical assays (cell-free transcription, electrophoretic mobilityshift assays, etc.) or cellular assays (transiently transfected reporter assays).Although both approaches are useful, they are limited in that they can onlyapproximate the interactions that occur in the cell, primarily because neither can

completely duplicate the chromatin environment as it exists in the cell On the other hand, in vivo footprinting can demonstrate that something is bound to

native chromatin but cannot identify the specific protein involved Chromatin immunoprecipitation (ChIP) is a revolutionary advance that determines

transcription factor interactions with a target promoter in the native chromatinenvironment (Fig 17.20) Cells, tissues, or tumors are fixed with formaldehydeand sonicated to shear DNA into short fragments (typically 1 kb or less).Promoters and other regulatory regions are then immunoprecipitated with anantibody against a protein thought to bind to that region DNA can then beidentified and quantified by PCR or microarray analysis Using this technique, it

is possible to correlate the DNA-binding activity of a given transcription factorwith corresponding changes in the surrounding chromatin environment and geneexpression in cells exposed to different hormones, cellular stresses, anddifferentiation programs

FIGURE 17.20 Illustration showing key steps in chromatinimmunoprecipitation 1 Fix cells with formaldehyde 2 Sonicate chromatin into500- to 1,000-bp fragments 3 Immunoprecipitate transcription factor (TF) with

a specific antibody 4 De-crosslink DNA, use polymerase chain reaction (PCR)

to verify specific interaction with target promoter and visualize specificamplification by agarose electrophoresis

Protein–DNA Interaction Arrays (Chromatin Immunoprecipitation-Chips)

Surveying all of the interactions of the transcription apparatus with the humangenome is a daunting task ChIP-chips combine the power of the ChIP assaywith microarray technology to investigate these interactions (the “Chips” are

chips of glass slides on which the ChIP is placed) Immunoprecipitatedchromatin is ligated to universal primers and amplified by PCR to generate apool of DNA associated with a given transcription factor DNA is fluorescentlylabeled, mixed with amplified and labeled unprecipitated DNA (input), andhybridized to a microarray of regulatory DNA Higher relative fluorescence fromthe immunoprecipitated DNA on a spot compared with input indicates aninteraction of the protein with that regulatory sequence This technique has beenused primarily to map transcription factor binding and histone modificationacross the entire yeast genome The vastly larger size of the human genome hasmade it necessary to focus on more specific regions using arrays of either clonedpromoter fragments or CpG islands (sequences of DNA that cluster aroundpromoters and other regulatory regions) Production of high-density tiledmicroarrays spanning all promoters in the human genome will soon makepossible the correlation of transcription factor binding, chromatin structure, andgene expression.

Microarrays to Assay Gene Expression

With the sequencing of the human genome, studying global changes in geneexpression became possible in principle Efforts were being made to study theexpression of as many genes as possible without resorting to individual Northernblots for each identified gene In this way, the microarray was born Expressionarrays work like an inverse Northern blot: Cellular RNA is reverse transcribed,amplified with modified nucleotides that allow fluorescent detection, andhybridized to an array of sequences corresponding to the genes in an organism(Fig 17.21) The arrays used for detection fall into two types: spotted and oligoarrays

FIGURE 17.21 Oligonucleotide microarray analysis A: Silicon wafers are