Chapter 20. Biotechnology

Transcription

1 Chapter 20 Bitechnlgy Overview: The DNA Tlbx In 2001, researchers cmpleted a first draft sequence f all 3 billin base pairs f the human genme. By 2010, researchers had cmpleted sequencing mre than 1,000 bacterial, 80 archaeal, and 100 eukarytic genmes, and genme sequencing was under way fr ver 7,000 species. A key advance was the inventin f techniques fr making recmbinant DNA, DNA mlecules frmed when segments f DNA frm tw different surces ften different species are cmbined in vitr. Scientists als have pwerful techniques fr analyzing genes and gene expressin. Human lives are greatly affected by bitechnlgy, the manipulatin f rganisms r their cmpnents t make useful prducts. Bitechnlgy includes such early practices as selective breeding f farm animals and the use f micrrganisms t make wine and cheese. Tday, bitechnlgy als encmpasses genetic engineering, the direct manipulatin f genes fr practical purpses. DNA technlgy is nw applied in areas ranging frm agriculture t criminal law t medical diagnsis, but many f its mst imprtant achievements are in basic research. Using micrarray analysis, researchers can quickly cmpare gene expressin in different samples, such as thse btained frm nrmal and cancerus tissues. Cncept 20.1 DNA clning yields multiple cpies f a gene r ther DNA segment T study a particular gene, scientists develped methds t islate the prtin f a chrmsme that cntains the gene f interest. Techniques fr DNA clning enable scientists t prepare multiple identical cpies f welldefined segments f DNA. One cmmn apprach t clning pieces f DNA uses bacteria, usually Esherichia cli, whse chrmsme is a large circular DNA mlecule. In additin, bacteria have plasmids, small circular DNA mlecules with a small number f genes that replicate independently frm the chrmsme. One basic clning technique begins with the insertin f a freign gene int a bacterial plasmid t prduce a recmbinant DNA mlecule. The plasmid is returned t a bacterial cell, prducing a recmbinant bacterium, which reprduces t frm a clne f genetically identical cells. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-1

2 Every time the bacterium reprduces, the recmbinant plasmid is replicated as well. The prductin f multiple cpies f a single gene is called gene clning. Gene clning is useful fr tw basic purpses: t make many cpies f, r amplify, a particular gene and t create a prtein prduct. Islated cpies f a clned gene may enable scientists t prvide an rganism with a new metablic capability, such as pest resistance. Alternatively, a prtein with medical uses, such as human grwth hrmne, can be harvested in large quantities frm cultures f bacteria carrying the clned gene. A gene makes up nly abut ne millinth f the DNA in a human cell. The ability t amplify such rare DNA fragments is crucial fr any applicatin invlving a single gene. Restrictin enzymes are used t make recmbinant DNA. Gene clning and genetic engineering were made pssible by the discvery f restrictin endnucleases, r restrictin enzymes, that cut DNA mlecules at specific lcatins. In nature, bacteria use restrictin enzymes t cut freign DNA, t prtect themselves against phages r ther rganisms. Restrictin enzymes are very specific, recgnizing shrt DNA nucletide sequences, r restrictin sites, and cutting bth DNA strands at specific pints within these sequences. Bacteria prtect their wn DNA by methylating the sequences recgnized by these enzymes. Each restrictin enzyme cleaves a specific sequence f bases. The mst cmmnly used restrictin enzymes recgnize sequences cntaining fur t eight nucletides. Because such shrt target sequences ccur many times n a lng DNA mlecule, restrictin enzymes make many cuts, yielding the set f restrictin fragments. Restrictin enzymes cut the cvalent sugar-phsphate backbnes f bth strands, ften in a staggered way that creates single-stranded sticky ends. The extensins frm hydrgen-bnded base pairs with cmplementary single-stranded stretches (sticky ends) n ther DNA mlecules cut with the same restrictin enzyme. These DNA fusins can be made permanent by DNA ligase, which seals the strand by catalyzing the frmatin f cvalent bnds t clse up the sugar-phsphate backbne. The ligase-catalyzed jining f DNA frm tw different surces prduces a stable recmbinant DNA mlecule. Eukarytic genes can be clned in bacterial plasmids. Recmbinant plasmids are prduced when restrictin fragments frm freign DNA are spliced int plasmids. The riginal plasmid used t prduce recmbinant DNA is called a clning vectr, defined as a DNA mlecule that can carry freign DNA int a cell and replicate there. Bacterial plasmids are widely used as clning vectrs because they can be islated frm bacteria, manipulated t frm recmbinant plasmids by in vitr insertin f freign DNA, and then intrduced int bacterial cells. Bacterial cells that carry the recmbinant plasmid reprduce rapidly, replicating the inserted freign DNA. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-2

3 Imagine that researchers are interested in studying the -glbin gene in a hummingbird. The first step is t clne all hummingbird genes, then islate the -glbin gene. Researchers als btain the chsen vectr, a bacterial plasmid frm E. cli cells. The plasmid carries tw useful genes, amp R, which cnfers resistance t the antibitic ampicillin, and lacz, which encdes the enzyme ß-galactsidase that catalyzes the hydrlysis f lactse. ß-galactsidase can hydrlyze a synthetic mimic f lactse called X-gal t frm a blue prduct. The plasmid has a single recgnitin sequence, within the lacz gene, fr the restrictin enzyme used. Bth the plasmid and the hummingbird DNA are digested with the same restrictin enzyme. The fragments are mixed tgether, allwing base pairing between cmplementary sticky ends. DNA ligase is added t permanently jin the base-paired fragments. Many f the resulting recmbinant plasmids cntain hummingbird DNA fragments; ne fragment carries all r part f the -glbin gene. This step als generates ther prducts, such as plasmids cntaining several hummingbird DNA fragments, a cmbinatin f tw plasmids, r a rejined, nnrecmbinant versin f the riginal plasmid. The DNA mixture is mixed with bacteria that have a mutatin in the lacz gene n their wn chrmsme, making them unable t hydrlyze lactse r X-gal. The bacteria take up freign DNA by transfrmatin. Sme cells acquire a recmbinant plasmid carrying a gene, while thers may take up a nnrecmbinant plasmid, a hummingbird DNA fragment, r nthing at all. The transfrmed bacteria are plated n agar cntaining ampicillin and X-gal. Only bacteria that have the ampicillin-resistance (amp R ) plasmid grw. Each reprducing bacterium frms a clne, generating a clny f cells n the agar. The X-gal in the medium is used t identify plasmids that carry freign DNA. Bacteria with plasmids lacking freign DNA stain blue when ß-galactsidase frm the intact lacz gene hydrlyzes X-gal. Bacteria with plasmids cntaining freign DNA inserted int the lacz gene are white because they lack ß-galactsidase. In the final step, thusands f bacterial clnies with freign DNA are srted t find thse that cntain the gene f interest. Clned genes are stred in DNA libraries. In the shtgun clning apprach described abve, a mixture f fragments frm the entire genme is included in thusands f different recmbinant plasmids. A cmplete set f recmbinant plasmid clnes, each carrying cpies f a particular segment frm the initial genme, frms a genmic library. Histrically, bacteriphages were used as clning vectrs fr making genmic libraries. Fragments f freign DNA were spliced int a phage genme using a restrictin enzyme and DNA ligase. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-3

4 The nrmal infectin prcess allwed prductin f many new phage particles, each carrying the freign DNA. Bacterial artificial chrmsmes (BAC) are widely used as vectrs fr library cnstructin. BACs are large plasmids cntaining nly the genes necessary t ensure replicatin and capable f carrying inserts f kb. The very large insert size minimizes the number f clnes that are needed t make up the genmic library, but it makes them mre difficult t wrk with. Clnes are usually stred in multiwelled plastic plates, with ne clne per well. In a genmic library, the clned -glbin gene wuld include nt just exns cntaining the cding sequence, but als the prmter, untranslated regins, and any intrns. A bilgist might be interested in the -glbin prtein itself, asking if this xygen-carrying prtein is different frm its cunterpart in ther, less metablically active species. The researcher culd develp a mre limited gene library by starting with fully prcessed mrna extracted frm cells where the gene is expressed. The enzyme reverse transcriptase is used in vitr t make a single-stranded DNA reverse transcript f each mrna mlecule. The mrna is enzymatically digested, and a secnd DNA strand cmplementary t the first is synthesized by DNA plymerase. This duble-stranded DNA is called cmplementary DNA (cdna). T create a library, cdna is mdified by the additin f restrictin sites at each end and then inserted int vectr DNA. A cdna library represents the subset f a cell s genme that was transcribed in the starting cell frm which the mrna was islated. If a researcher wants t clne a gene but is unsure in what cell type it is expressed r unable t btain that cell type, a genmic library will likely cntain the gene. A researcher interested in the regulatry sequences r intrns assciated with a gene needs t btain the gene frm a genmic library. These sequences are missing frm the prcessed mrnas used in making a cdna library. A cdna library made frm cells expressing the gene (like red bld cells) is ideal fr the study f a specific prtein (like -glbin). A cdna library can als be used t study sets f genes expressed in particular cell types, such as brain r liver cells. By making cdna libraries frm cells f the same type at different times in the life f an rganism, ne can trace changes in the patterns f gene expressin. The researcher screens all the clnies with recmbinant plasmids fr a clne f cells cntaining the hummingbird -glbin gene. One technique, nucleic acid hybridizatin, depends n base pairing between the gene and a cmplementary sequence n a shrt, single-stranded nucleic acid, a nucleic acid prbe. Identifying the sequence f the RNA r DNA prbe depends n knwledge f at least part f the sequence f the gene f interest. A radiactive r flurescent tag is used t label the prbe, which hydrgen-bnds specifically t cmplementary single strands f the desired gene. The clnes in the hummingbird genmic library have been stred in a multiwell plate. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-4

5 If a few cells frm each well are transferred t a defined lcatin n a membrane made f nyln r nitrcellulse, a large number f clnes can be screened simultaneusly fr the presence f DNA cmplementary t the DNA prbe. After the lcatin f a clne carrying the -glbin gene has been identified, cells frm that clny can be grwn in rder t islate large amunts f the -glbin gene. The clned gene can be used as a prbe t identify similar r identical genes in DNA frm ther surces, such as ther species f birds. Eukaryte genes can be expressed in bacterial hst cells. The prtein prduct f a clned gene can be created in either bacterial r eukarytic cells, fr research purpses r fr practical applicatins. Inducing a clned eukarytic gene t functin in bacterial hst cells can be difficult because certain aspects f gene expressin are different in eukarytes and bacteria. One way arund this is t insert an expressin vectr, a clning vectr cntaining a highly active bacterial prmter, upstream f the restrictin site. The bacterial hst cell recgnizes the prmter and prceeds t express the freign gene that has been linked t it. The presence f nncding intrns in eukarytic genes may prevent the crrect expressin f these genes in bacteria, which lack RNA-splicing machinery. This prblem can be surmunted by using a cdna frm f the gene, which includes nly the exns. Mlecular bilgists can avid incmpatibility prblems by using eukarytic cells as hsts fr clning and expressing eukarytic genes. Yeast cells, single-celled fungi, are as easy t grw as bacteria and, unlike mst eukarytes, have plasmids. Scientists have cnstructed recmbinant plasmids that cmbine yeast and bacterial DNA and can replicate in either type f cell. Anther advantage f eukarytic hsts is that they are capable f prviding the psttranslatinal mdificatins that many prteins require. Such mdificatins may include adding carbhydrates r lipids. Sme mammalian cell lines and an insect cell line that can be infected by a Baculvirus virus carrying recmbinant DNA are successful hst cells. Other techniques are als used t intrduce freign DNA int eukarytic cells. In electrpratin, a brief electrical pulse creates a temprary hle in the plasma membrane thrugh which DNA can enter. Scientists can inject DNA int individual cells using micrscpically thin needles. T get DNA int plant cells, the sil bacterium Agrbacterium can be used. Crss-species gene expressin reflects shared evlutinary ancestry. Many genes taken frm ne species functin well when transferred int very different species. These bservatins underscre the shared evlutinary ancestry f species living tday. A gene called Pax-6 has been fund in animals as diverse as vertebrates and fruit flies. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-5

6 The vertebrate Pax-6 gene prduct (the PAX-6 prtein) triggers a cmplex prgram f gene expressin resulting in frmatin f the vertebrate eye, which has a single lens. The fly Pax-6 gene als leads t frmatin f the cmpund fly eye. Althugh the genetic prgrams triggered in vertebrates and flies generate very different eyes, the tw versins f the Pax-6 gene can substitute fr each ther, evidence f their evlutin frm a gene in a cmmn ancestr. The plymerase chain reactin (PCR) amplifies DNA in vitr. DNA clning in cells remains the best methd fr preparing large quantities f a particular gene r ther DNA sequence. When the surce f DNA is scanty r impure, the plymerase chain reactin (PCR) is quicker and mre selective. This technique can quickly amplify any piece f DNA withut using cells. PCR can make billins f cpies f a targeted DNA segment in a few hurs, a much faster prcess than clning via recmbinant bacteria. In fact, PCR is being used increasingly t make enugh f a specific DNA fragment t insert it directly int a vectr, skipping the steps f making and screening a library. In PCR, a three-step cycle heating, cling, and replicatin brings abut a chain reactin that prduces an expnentially grwing ppulatin f identical DNA mlecules. The reactin mixture is heated t denature (separate) the DNA strands. The mixture is cled t allw annealing (hydrgen bnding) f shrt, single-stranded DNA primers cmplementary t sequences n ppsite sides at each end f the target sequence. A heat-stable DNA plymerase extends the primers in the 53 directin. If a standard DNA plymerase were used, the prtein wuld be denatured alng with the DNA during the first heating step. The key t easy PCR autmatin was the discvery f an unusual DNA Taq plymerase, islated frm a bacterium living in ht springs. The bacterium species, Thermus aquaticus, lives in ht springs, s natural selectin has resulted in a heat-stable DNA plymerase that can withstand the great heat f the prcess. Just as impressive as the speed f PCR is its specificity. Only minute amunts f DNA need be present in the starting material, as lng as a few mlecules cntain the cmplete target sequence. The DNA can be in a partially degraded state. The key t this high specificity is the primers, which hydrgen-bnd nly t sequences at ppsite ends f the target segment. With each successive cycle, the number f target segment mlecules f the crrect length dubles, s the number f mlecules equals 2 n, where n is the number f cycles. After 30 cycles, abut a billin cpies f the target sequence are present! Despite its speed and specificity, PCR amplificatin cannt substitute fr gene clning in cells when large amunts f a gene are desired. Occasinal errrs during PCR replicatin impse limits n the number f gd cpies that can be made. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-6

7 When PCR is used t prvide the specific DNA fragment fr clning, the resulting clnes are sequenced t select clnes with errr-free inserts. Devised in 1985, PCR has had a majr impact n bilgical research and technlgy. PCR has amplified DNA frm a variety f surces: fragments f ancient DNA frm a 40,000- year-ld frzen wlly mammth; DNA frm ftprints r tiny amunts f bld r semen fund at the scenes f vilent crimes; DNA frm single embrynic cells fr the rapid prenatal diagnsis f genetic disrders; and DNA f viral genes frm cells infected with HIV. Cncept 20.2 DNA technlgy allws us t study the sequence, expressin, and functin f a gene Once scientists have prepared hmgeneus samples f DNA, each cntaining a large number f identical segments, they can ask sme interesting questins abut specific genes and their functins. Des the sequence f the hummingbird -glbin gene cde fr a prtein structure that can carry xygen mre efficiently than its cunterpart in less metablically active species? Des a particular human gene differ frm persn t persn? Are certain alleles f that gene assciated with a hereditary disrder? Where in the bdy and when during develpment is a given gene expressed? What rle des a certain gene play in an rganism? T answer these questins, researchers need t knw the nucletide sequence f the gene and its cunterparts in ther individuals and species, as well as its expressin pattern. One methd f rapidly analyzing and cmparing genmes is gel electrphresis. Gel electrphresis separates macrmlecules nucleic acids r prteins n the basis f their rate f mvement thrugh a plymer gel in an electrical field. The rate f mvement f each mlecule depends n its size, electrical charge, and ther physical prperties. Gel electrphresis separates a mixture f linear DNA mlecules int bands, each band cnsisting f many thusands f DNA mlecules f the same length. In restrictin fragment analysis, the DNA fragments prduced by restrictin enzyme digestin f a DNA mlecule are srted by gel electrphresis. When the mixture f restrictin fragments frm a particular DNA mlecule underges electrphresis, it yields a band pattern characteristic f the starting mlecule and the restrictin enzyme used. The relatively small DNA mlecules f viruses and plasmids can be identified simply by their restrictin fragment patterns. The separated fragments can be recvered undamaged frm gels, prviding pure samples f individual fragments. Scientists can use restrictin fragment analysis t cmpare tw different DNA mlecules, such as tw different alleles f a gene. Because the tw alleles differ slightly in DNA sequence, they may differ in ne r mre restrictin sites. Restrictin fragment analysis may be able t distinguish between tw alleles f a gene that differ by nly ne base pair in a restrictin site. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-7

8 Variatins in DNA sequence amng a ppulatin are called plymrphisms, and this particular type f sequence change is called a restrictin fragment length plymrphism (RFLP). If ne allele cntains a RFLP, digestin with the enzyme that recgnizes the site will prduce a different mixture f fragments frm each f the tw alleles. Each mixture will give its wn band pattern in gel electrphresis. A methd called Suthern bltting cmbines gel electrphresis with nucleic acid hybridizatin. Because gel electrphresis yields t many bands t distinguish individually, scientists use nucleic acid hybridizatin with a specific prbe t label discrete bands that derive frm the gene f interest. The prbe is a radiactive, single-stranded DNA mlecule that is cmplementary t the gene f interest. One f this methd s many applicatins is t identify heterzygus carriers f mutant alleles assciated with genetic disease, althugh mre rapid methds invlving PCR amplificatin are currently used fr this. Gene sequencing has been autmated based n a technique called the didexyribnucletide (r didexy) chain terminatin methd. In the last 10 years, techniques have been develped in which a single template strand is immbilized, and reagents are added that allw s-called sequencing by synthesis f a cmplementary strand, ne base at a time. A chemical trick allws electrnic mnitrs t distinguish which f the fur bases is added, allwing determinatin f the sequence. Further technical mdificatins have given rise t third-generatin sequencing, with each new technique being faster and less expensive than the previus. The rapid acceleratin f sequencing technlgy has enhanced ur study f genes and whle genmes. Knwing the entire nucletide sequence f a gene allws researchers t cmpare it t genes in ther species, whse functin may be knwn. If tw genes frm different species are similar in sequence, their gene prducts likely perfrm similar functins. Experimental appraches analyze when and where a gene is expressed. Labeled nucleic acid prbes that hybridize with mrnas can prvide infrmatin abut the time r place in the rganism at which a gene is transcribed. Suppse we wish t find ut hw the expressin f the -glbin gene changes during the embrynic develpment f the hummingbird. In the methd called Nrthern bltting, scientists carry ut gel electrphresis n samples f mrna frm hummingbird embrys at different stages f develpment, transfer the samples t a nitrcellulse membrane, and then allw the mrnas n the membrane t hybridize with a labeled prbe that recgnizes -glbin mrna. If the mrna band is seen at a particular stage, scientists can hypthesize that the prtein functins during events taking place at that stage. A quicker and mre sensitive methd is called the reverse transcriptase-plymerase chain reactin, r RT-PCR. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-8

9 Analysis f hummingbird -glbin gene expressin with RT-PCR begins similarly t Nrthern bltting, with the islatin f mrnas frm different develpmental stages f hummingbird embrys. Next, reverse transcriptase is added t make cdna, which then serves as a template fr PCR amplificatin using primers frm the -glbin gene. When the prducts are run n a gel, cpies f the amplified regin are bserved as bands nly in samples that riginally cntained the -glbin mrna. In the case f hummingbird -glbin, scientists might expect t see a band appear at the stage when red bld cells begin frming, with all subsequent stages shwing the same band. RT-PCR can als be carried ut with mrnas cllected frm different tissues at ne time t discver which tissue is prducing a specific mrna. Specific mrnas can be identified by using labeled prbes in place, r in situ, in the intact rganism. This technique, called in situ hybridizatin, is mst ften carried ut with prbes labeled by the attachment f flurescent dyes. When the entire genmes f a number f rganisms have been sequenced, it is pssible t study the expressin f large grups f genes, t study hw genes act tgether t prduce and maintain a functining rganism. Researchers use genme sequences as prbes t investigate which genes are transcribed in different situatins, such as in different tissues r at different stages f develpment. Researchers als lk fr grups f genes that are expressed in a crdinated manner, with the aim f identifying netwrks f gene expressin acrss an entire genme. The basic strategy in such glbal (genme-wide) expressin studies is t islate the mrnas made in particular cells, use these mlecules as templates fr making the crrespnding cdnas by reverse transcriptin, and then emply nucleic acid hybridizatin t cmpare this set f cdnas with a cllectin f DNA fragments representing all r part f the genme. The results identify the subset f genes in the genme that are being expressed at a given time r under certain cnditins. Genme-wide expressin studies are made pssible by the use f DNA micrarray assays. A DNA micrarray cnsists f tiny amunts f a large number f single-stranded DNA fragments representing different genes fixed t a glass slide in a tightly spaced array, r grid, als called a DNA chip. Ideally, these fragments represent all the genes f an rganism. The DNA fragments n a micrarray are tested fr hybridizatin with cdna mlecules that have been prepared frm the mrnas in particular cells f interest and labeled with flurescent dyes. In ne example f a glbal expressin study, researchers perfrmed DNA micrarray assays n mre than 90% f the genes f the nematde Caenrhabditis elegans during every stage f its life cycle. The expressin f nearly 60% f the genes changed dramatically during develpment. Many genes were expressed in a sex-specific pattern. In additin t uncvering gene interactins and prviding clues t gene functin, DNA micrarray assays may cntribute t a better understanding f certain diseases and suggest new diagnstic techniques r therapies. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc. 20-9

10 Cmparing patterns f gene expressin in breast cancer tumrs and nncancerus breast tissue, fr example, has already resulted in mre infrmed and effective treatment prtcls. Scientists disable genes and bserve the cnsequences t determine the functin f the genes. In an applicatin called in vitr mutagenesis, specific mutatins are intrduced int the sequence f a clned gene, and then the mutated gene is returned t a cell in such a way that it disables ( kncks ut ) the nrmal cellular cpies f the same gene. If the intrduced mutatins alter r destry the functin f the gene prduct, the phentype f the mutant cell may help reveal the functin f the missing nrmal prtein. A newer methd fr silencing the expressin f selected genes explits the phenmenn f RNA interference (RNAi). This apprach uses synthetic, duble-stranded RNA mlecules that match the sequence f a particular gene t trigger the breakdwn f the gene s messenger RNA r t blck its translatin. RNAi was used t prevent the expressin f 86% f the genes in early nematde embrys, ne gene at a time. Analysis f the phentypes f the wrms that develped frm these embrys enabled the researchers t classify mst f the genes int a small number f grups by functin. In humans, ethical cnsideratins prhibit kncking ut genes t determine their functins. Researchers carry ut large-scale genetic analyses, genme-wide assciatin studies, f large numbers f peple with a certain phentypic cnditin r disease, such as heart disease r diabetes. They test fr genetic markers, DNA sequences that vary in the ppulatin. Amng the mst useful f these genetic markers are single base-pair variatins in the genmes f the human ppulatin. A single base-pair site where variatin is fund in at least 1% f the ppulatin is called a single nucletide plymrphism (SNP). A few millin SNPs ccur in the human genme, n average abut nce in 100 t 300 base pairs f bth cding and nncding DNA sequences. SNPs can be detected by very sensitive micrarray analysis r by PCR. Once a regin is fund that has a SNP shared amng affected but nt unaffected peple, researchers sequence that regin. In mst cases, the SNP itself des nt cntribute t the disease. In fact, mst SNPs are in nncding regins. If the SNP and a disease-causing allele are clse enugh, crssing ver between the marker and the gene is very unlikely during gamete frmatin. Therefre, the marker and gene will almst always be inherited tgether, even thugh the marker is nt part f the gene. SNPs have been fund that crrelate with diabetes, heart disease, and several types f cancer. The search is n fr genes that might be invlved. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

11 Cncept 20.3 Clning rganisms may lead t the prductin f stem cells fr research and ther applicatins Scientists can nw clne multicellular rganisms frm single cells. This is called rganismal clning t distinguish it frm gene clning and cell clning, the divisin f an asexually reprducing cell int a cllectin f genetically identical cells. Organismal clning has the ptential t generate stem cells, which can develp int many different tissues. The clning f plants and animals was first attempted mre than 50 years ag in experiments designed t determine whether all the cells f an rganism have the same genes (a cncept called genmic equivalence) r whether cells lse genes during the prcess f differentiatin. Whle plants have been clned frm single differentiated cells since the 1950s. Whle plants were successfully clned frm single differentiated cells during the 1950s by F. C. Steward f Crnell University. Steward and his students fund that cultured, differentiated rt cells culd grw int nrmal adult plants, each genetically identical t the parent plant. This demnstrated that differentiatin des nt cause irreversible changes in the DNA. In plants, mature cells can dedifferentiate and then give rise t all the specialized cell types f the rganism. Any cell with this ptential is said t be ttiptent. Plant clning is nw used extensively in agriculture t reprduce plants that have valuable characteristics, such as the ability t resist a plant pathgen. Nuclear transplantatin was used in animal experiments. Differentiated cells frm animals d nt readily divide in culture. Early researchers investigating whether differentiated animal cells can be ttiptent remved the nucleus f an unfertilized frg s egg r zygte and replaced it with the nucleus f a differentiated cell frm a tadple, a prcedure called nuclear transplantatin. If the nucleus frm the differentiated dnr cell retains its full genetic capability, then it shuld be able t direct develpment f the recipient egg int all the tissues and rgans f an rganism. The transplanted nucleus was ften able t supprt nrmal develpment f the egg int a tadple. The ptential f a transplanted nucleus t direct nrmal develpment was inversely related t the age f the dnr: The lder the dnr nucleus, the lwer the percentage f nrmally develping tadples. Clearly, smething in the nucleus des change as the tadple s cells differentiated. In frgs and mst ther animals, nuclear ptential tends t be increasingly restricted as embrynic develpment and cell differentiatin prgress. In 1997, Scttish researchers captured newspaper headlines when they annunced the birth f Dlly, a lamb clned frm an adult sheep by nuclear transplantatin frm a differentiated cell. These researchers cultured dnr mammary cells in a nutrient-pr medium and then fused these cells with enucleated sheep eggs. The resulting diplid cells divided t frm early embrys, which were implanted int surrgate mthers. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

12 Out f several hundred implanted embrys, ne successfully cmpleted nrmal develpment, and Dlly was brn. Later analyses shwed that Dlly s chrmsmal DNA was identical t that f the nucleus dnr. Dlly s mitchndrial DNA came frm the egg dnr. In 2003, at age 6, Dlly suffered cmplicatins frm a lung disease usually seen in nly much lder sheep, and she was euthanized. Dlly s early death led t speculatin that her cells were nt as healthy as thse f a nrmal sheep, perhaps reflecting incmplete reprgramming f the riginal transplanted nucleus. Since 1997, reprductive clning the prductin f new individuals has been demnstrated in many ther mammals, including mice, cats, cws, hrses, pigs, dgs, and mnkeys. Clned animals f the same species d nt always lk r behave identically. In a herd f cws clned frm the same line f cultured cells, certain cws are dminant and thers are mre submissive. The first clned cat, named CC fr Carbn Cpy has a calic cat, like her single female parent, but the clr and pattern are different because f randm X chrmsme inactivatin, which is a nrmal ccurrence during embrynic develpment. Identical human twins, naturally ccurring clnes, always differ smewhat. Envirnmental influences and randm phenmena play a significant rle during develpment. The successful clning f s many mammals has led t speculatin abut the clning f humans. Nuclei frm differentiated human cells have been transplanted int unfertilized enucleated eggs, and the eggs stimulated t divide. In 2001, a research grup in Massachusetts bserved a few early cell divisins in such an experiment. A few years later, researchers at Seul Natinal University in Suth Krea reprted clning embrys t an early stage called the blastcyst stage, but the scientists were later fund guilty f research miscnduct and data fabricatin. This episde sent shck waves thrugh the scientific cmmunity. The first primate (macaque) embrys was clned by researchers at the Oregn Natinal Primate Research Center in This has mved the field ne step clser t human clning, the prspect f which raises unprecedented ethical issues. There are prblems assciated with animal clning. In mst nuclear transplantatin studies, very few clned embrys develp nrmally t birth and many f thse exhibit defects. Even clned animals that appear nrmal likely have subtle defects. In the nuclei f fully differentiated cells, the expressin f many genes is repressed as a result f epigenetic changes in chrmatin, such as acetylatin f histnes r methylatin f DNA. During the nuclear transfer prcedure, many f these changes must be reversed in the later-stage nucleus frm a dnr animal in rder fr genes t be expressed r repressed apprpriately in early stages f develpment. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

13 DNA in embrynic cells frm clned embrys, like DNA f differentiated cells, tends t have mre methyl grups than DNA in equivalent cells frm unclned embrys f the same species. This suggests that the reprgramming f dnr nuclei requires chrmatin restructuring, which des nt ccur fully during clning prcedures. Because DNA methylatin helps regulate gene expressin, misplaced methyl grups in the DNA f dnr nuclei may interfere with the pattern f gene expressin necessary fr nrmal embrynic develpment. The success f clning may depends n whether r nt the chrmatin structure in the dnr nucleus can be restred t that f a newly fertilized egg, a prcess we d nt yet understand. Stem cells can be used t treat human diseases. Scientists majr aim in clning human embrys is the prductin f stem cells fr treating human diseases. A stem cell is an unspecialized cell that can reprduce itself indefinitely and, under apprpriate cnditins, differentiate int specialized cells f ne r mre types. Stem cells are able bth t replenish their wn ppulatin and t generate cells that travel dwn specific differentiatin pathways. Many early animal embrys cntain stem cells capable f giving rise t many types f differentiated embrynic cells. Embrynic stem (ES) cells can be islated frm early embrys at the blastula r blastcyst stage. The adult bdy als has adult stem cells, which give rise t many, but nt all, cell types. Fr example, bne marrw cntains several types f stem cells, including ne that can generate all the different kinds f bld cells and anther that can differentiate int bne, cartilage, fat, muscle, and the linings f bld vessels. Even the adult brain cntains stem cells that prduce certain kinds f nerve cells. Scientists are learning t identify and islate stem cells frm varius tissues and, in sme cases, t grw them in culture. With the additin f specific grwth factrs, cultured stem cells frm adult animals have been made t differentiate int multiple types f specialized cells. This technlgy may prvide cells fr the repair f damaged r diseased rgans, such as insulin-prducing pancreatic cells fr peple with type 1 diabetes r certain kinds f brain cells fr peple with Parkinsn s disease r Huntingtn s disease. Embrynic stem cells ffer mre ptential than adult stem cells fr medical applicatins, because ES cells are pluriptent, capable f differentiating int different cell types. Deriving embrynic stem cells frm human embrys raises ethical and plitical issues. Embrynic stem cells are currently btained frm embrys dnated by patients underging infertility treatment r frm lng-term cell cultures riginally established frm dnated embrys. If scientists can clne human embrys t the blastcyst stage, they might be able t use such clnes as the surce f ES cells. A dnr nucleus frm a patient with a particular disease culd allw the prductin f ES cells fr treatment that match the patient and are nt rejected by the immune system. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

14 Althugh mst peple believe that the reprductive clning f humans is unethical, pinins vary abut the mrality f therapeutic clning, whse majr aim is t prduce embrynic stem cells t treat disease. Researchers have been able t turn back the clck in fully differentiated cells, reprgramming them t act like ES cells. They transfrmed muse skin cells int ES cells by using retrviruses t intrduce extra clned cpies f fur stem cell master regulatry genes. All tests carried ut suggested that the transfrmed cells, which are knwn as induced pluriptent stem (ips) cells, culd d everything ES cells can d. It is impssible t test all capabilities, hwever. Research grups have recently fund sme differences between ips and ES cells in gene expressin and ther cellular functins such as cell divisin. There are tw majr ptential uses fr human ips cells. Cells frm patients suffering frm diseases can be reprgrammed t becme ips cells, which can act as mdel cells fr studying the disease and ptential treatments. Human ips cell lines have been develped frm individuals with type 1 diabetes, Parkinsn's disease, and a dzen ther diseases. In the field f regenerative medicine, a patient s wn cells culd be reprgrammed int ips cells and then used t replace nnfunctinal tissues. Develping techniques t direct ips cells t becme specific cell types fr this purpse is an area f intense research that has already seen sme success. These cell types culd eventually prvide tailr-made replacement cells fr patients withut using any human eggs r embrys, thus eliminating ethical bjectins. Cncept 20.4 The practical applicatins f DNA technlgy affect ur lives in many ways DNA technlgy is reshaping medicine and the pharmaceutical industry. Mdern bitechnlgy is making enrmus cntributins bth t the diagnsis f diseases and in the develpment f pharmaceutical prducts. The identificatin f genes whse mutatins are respnsible fr genetic diseases may lead t ways t diagnse, treat, r even prevent these cnditins. Susceptibility t many nngenetic diseases, frm arthritis t AIDS, is influenced by a persn s genes. Diseases f all srts invlve changes in gene expressin within the affected genes and within the patient s immune system. Using DNA micrarray assays and ther techniques t cmpare gene expressins in healthy and diseased tissue can identify these changes and lead t the develpment f targets fr preventin r therapy. PCR and labeled nucleic acid prbes can track dwn the pathgens respnsible fr infectius diseases. Fr example, RT-PCR can amplify and thus detect HIV DNA in bld and tissue samples, detecting an therwise elusive infectin. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

15 Medical scientists can use DNA technlgy t identify individuals wh have genetic diseases befre the nset f symptms, even befre birth. PCR can be used t identify symptmless carriers f ptentially harmful recessive alleles. Genetic disrders are diagnsed by using PCR and primers crrespnding t clned disease genes, and then sequencing the amplified prduct t lk fr the disease-causing mutatin. Clned disease genes include thse fr sickle-cell disease, hemphilia, cystic fibrsis, Huntingtn s disease, and Duchenne muscular dystrphy. Genme-wide assciatin studies have fund SNPs (single nucletide plymrphisms) that are linked t disease-causing alleles. Sme SNPs are crrelated with increased risk fr cnditins such as heart disease, Alzheimer s, r varius types f cancer. Cmpanies that ffer individual genetic testing fr health risk factrs lk fr the presence f previusly identified, linked SNPs. Such genetic tests reflect crrelatins and d nt make predictins. By analyzing the expressin f many genes amng breast cancer patients, researchers carrying ut ne genme-wide assciatin study were able t identify 70 genes whse expressin pattern culd be crrelated with likelihd that the cancer wuld recur. Since lw-risk patients have a 96% survival rate ver a ten-year perid, this allws dctrs and patients access t valuable infrmatin when they are cnsidering treatment ptins. The future may ffer persnalized medicine in which a persn s genetic health prfile can infrm them abut diseases r cnditins fr which they are especially at risk, and help them make treatment chices. A genetic prfile currently means the cmbinatin f a set f genetic markers such as SNPs, but culd mean the cmplete DNA sequence f each individual nce sequencing becmes inexpensive enugh. Human gene therapy hlds great ptential. Gene therapy intrducing genes int an afflicted individual fr therapeutic purpses hlds great ptential fr treating the few genetic disrders that are caused by a single gene. In thery, a nrmal allele f the defective gene culd be inserted int dividing smatic cells f the tissue affected by the disrder, such as bne marrw cells. Bne marrw cells, which include the stem cells that give rise t all the cells f the bld and immune system, are prime candidates fr gene therapy. Severe cmbined immundeficiency (SCID) caused by a single defective gene has been treated in gene therapy trials. If successful, gene therapy cures the patient, whse bne marrw cells then prduce the missing prtein. In 2000, ten yung children with SCID were treated by gene therapy. After tw years, nine f these patients shwed significant imprvement. Hwever, three f the patients subsequently develped leukemia and ne died. Tw factrs may have cntributed t the develpment f leukemia: The insertin f the retrviral vectr near a gene invlved in the prliferatin f bld cells and an unknwn functin f the replacement gene itself. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

16 Tw ther genetic diseases have recently been treated with gene therapy: ne causing prgressive blindness and the ther leading t degeneratin f the nervus system. The successful trials invlve very few patients, but are still cause fr cautius ptimism. Gene therapy als raises many technical questins. Hw can the activity f the transferred gene be cntrlled s that cells make apprpriate amunts f the gene prduct at the right time and in the right place? Hw can scientists be sure that the insertin f the therapeutic gene des nt harm sme ther necessary cell functin? In additin t these technical challenges, gene therapy raises prvkes ethical questins. Sme critics believe that tampering with human genes in any way is immral. Others see n difference between the transplantatin f genes int smatic cells and the transplantatin f rgans. The treatment f human germ-line cells in the hpe f crrecting a defect in future generatins raises ethical questins. N scientists are currently pursuing this gal. Under what circumstances, if any, shuld we alter the genmes f human germ lines? Wuld this inevitably lead t the practice f eugenics, a deliberate effrt t cntrl the genetic makeup f human ppulatins? The pharmaceutical industry uses bitechnlgy t develp new drugs. Determining the sequence and structure f prteins crucial fr tumr cell survival has led t the identificatin f small mlecules that cmbat certain cancers by blcking the functin f these prteins. One drug, called imatinib, is a small mlecule that inhibits a specific receptr tyrsine kinase. The verexpressin f this receptr, resulting frm a chrmsmal translcatin, is instrumental in causing chrnic myelgenus leukemia (CML). Patients in the early stages f CML wh have been treated with imatinib have exhibited nearly cmplete, sustained remissin frm the cancer. Similar drugs have als been used t treat a few types f lung and breast cancers, whse mlecular basis is fairly well understd. DNA clning and gene expressin systems can prduce large quantities f prteins that are present naturally in nly minute amunts. The hst cells used in these expressin systems can be engineered t secrete a prtein as it is made, thus remving the need fr its purificatin. Amng the first pharmaceutical prducts manufactured in this way were human insulin and human grwth hrmne (HGH). Sme 2 millin peple with diabetes in the United States require daily insulin injectins. Human grwth hrmne has been used t treat children brn with a frm f dwarfism caused by inadequate amunts f HGH. Anther imprtant pharmaceutical prduct created by genetic engineering is tissue plasmingen activatr (TPA). If administered shrtly after a heart attack, TPA helps disslve bld clts and reduces the risk f subsequent heart attacks. Prteins are prduced by pharm animals. In sme cases, whle animals are used t prduce prteins, instead f cell systems. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

17 Using experimental methds, scientists intrduce a gene frm ne animal int the genme f anther animal, which is then called a transgenic animal. T d this, scientists first remve eggs frm a female and fertilize them in vitr. They inject the clned DNA frm anther rganism directly int the nuclei f the fertilized eggs. Sme f the cells integrate the freign DNA, the transgene, int their genmes and are able t express the freign gene. The engineered embrys are then surgically implanted int a surrgate mther. If an embry develps successfully, the result is a transgenic animal. Assuming the intrduced gene encdes a prtein desired in large quantities, these transgenic animals can act as pharmaceutical factries. Fr example, a transgene fr a human bld prtein such as antithrmbin has been inserted int the genme f a gat s that antithrmbin is secreted in the animal s milk. The prtein can then be purified frm the milk. Researchers have als engineered transgenic chickens that express large amunts f the transgene s prduct in eggs. The human prteins prduced by farm animals may differ in sme ways frm the crrespnding natural human prteins, s they must be tested carefully t ensure that they will nt cause allergic reactins r ther adverse effects in patients wh receive them. Genetic prfiles may be imprtant frensic evidence. Bdy fluids r small pieces f tissue may be left at the scene f a vilent crime r n the clthes f the victim r assailant. If enugh bld, semen, r tissue is available, frensic labratries can determine the bld type r tissue type by using antibdies t detect specific cell-surface prteins. Such tests require fairly fresh samples in relatively large amunts, hwever, and can serve nly t exclude a particular suspect. DNA testing, in cntrast, can identify the guilty individual with a high degree f certainty because the DNA sequence f every persn is unique (except fr identical twins). Genetic markers that vary in the ppulatin can be analyzed fr a given persn t determine that individual s unique set f genetic markers, r genetic prfile. The FBI started applying DNA technlgy in frensics in 1988, using RFLP analysis by Suthern bltting t detect similarities and differences in DNA samples. This methd required very small samples f bld r tissue nly abut 1,000 cells. Tday, in place f RFLPs, frensic scientists usually use an even mre sensitive methd, which takes advantage f genetic markers called shrt tandem repeats (STRs). STRs are variatins in the lengths f certain repeated base sequences in specific regins f the genme. The number f repeats present in these regins is highly plymrphic, varying frm persn t persn and, fr ne individual, in the tw alleles f an STR. Fr example, ne individual may have the sequence ACAT repeated 30 times at ne genme lcus and 15 times at the same lcus n the ther hmlg, whereas anther individual may have 18 repeats at this lcus n each hmlgs. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc

18 PCR is used t amplify particular STRs, using sets f primers that are labeled with different clred flurescent tags; the length f the regin, and thus the number f repeats, can then be determined by electrphresis. The PCR step allws the methd t be used even when the DNA is in pr cnditin r available in nly minute quantities. A tissue sample cntaining as few as 20 cells can be sufficient fr PCR amplificatin. In a murder case, fr example, this methd can be used t cmpare DNA samples frm the suspect, the victim, and a small amunt f bld fund at the crime scene. Frensic scientists test nly a few selected prtins f the DNA abut 13 STR markers. The prbability that tw peple wh are nt identical twins wuld have exactly the same set f STR markers is vanishingly small. The Inncence Prject, a nnprfit rganizatin dedicated t verturning wrngful cnvictins, uses STR analysis f archived samples frm crime scenes t revisit ld cases. As f 2010, 250 inncent peple had been released frm prisn as a result f frensic and legal wrk by this grup. In anther example f genetic prfiling, a cmparisn f the DNA f a mther, her child, and the purprted father can cnclusively settle a questin f paternity. Smetimes paternity is f histrical interest: Genetic prfiles prvided strng evidence that Thmas Jeffersn r ne f his clse male relatives fathered at least ne f the children f his slave, Sally Hemings. Analyzing genetic prfiles can als identify victims f mass casualties. After the Wrld Trade Center attack in 2001, mre than 10,000 samples f victims remains were cmpared with DNA traces n persnal items prvided by families. These cmparisns led t the identificatin f nearly 3,000 victims. Just hw reliable is a genetic prfile? The greater the number f markers examined in a DNA sample, the mre likely it is that the prfile is unique t ne individual. In frensic cases using STR analysis with 13 markers, the prbability f tw peple having identical DNA prfiles is smewhere between ne chance in 10 billin and ne in several trillin. The exact prbability depends n the frequency f thse markers in the general ppulatin. Infrmatin n hw cmmn varius markers are in different ethnic grups is critical because these marker frequencies may vary cnsiderably amng ethnic grups and between a particular ethnic grup and the ppulatin as a whle. With the increasing availability f frequency data, frensic scientists can make extremely accurate statistical calculatins. Genetic prfiles are nw accepted as cmpelling evidence by legal experts and scientists. Micrrganisms can be used fr envirnmental cleanup. As a grup, micrrganisms have a remarkable ability t transfrm chemicals. Many bacteria can extract heavy metals, such as cpper, lead, and nickel, frm their envirnments and incrprate the metals int cmpunds such as cpper sulfate r lead sulfate, which are readily recverable. Lecture Outline fr Campbell/Reece Bilgy, 9 th Editin, Pearsn Educatin, Inc