bab xi vektor kloning

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BAB XI. VEKTOR KLONING

Bab ini akan membahas pengertian dan macam-macam vektor kloning, baik yang

digunakan pada sel inang prokariot maupun eukariot. Setelah mempelajari pokok bahasan

di dalam bab ini mahasiswa diharapkan mampu menjelaskan:

1. pengertian vektor kloning,

2. ciri-ciri plasmid,

3. ciri-ciri kosmid,

4. ciri-ciri bakteriofag, dan

5. ciri-ciri vektor kloning pada khamir dan eukariot tingkat tinggi.

Untuk dapat mempelajari pokok bahasan di dalam bab ini dengan lebih baik

mahasiswa disarankan telah memahami pokok bahasan tentang dasar-dasar teknologi

DNA rekombinan dan konstruksi perpustakaan gen, yang masing-masing telah diberikan

pada Bab IX dan X.

Pengertian dan Macam-macam Vektor Kloning

Pada Bab IX antara lain telah dibicarakan bahwa transformasi sel inang dilakukan

menggunakan perantara vektor. Jadi, vektor adalah molekul DNA yang berfungsi sebagai

wahana atau kendaraan yang akan membawa suatu fragmen DNA masuk ke dalam sel

inang dan memungkinkan terjadinya replikasi dan ekspresi fragmen DNA asing tersebut.

Vektor yang dapat digunakan pada sel inang prokariot, khususnya E. coli, adalah

plasmid, bakteriofag, kosmid, dan fasmid. Sementara itu, vektor YACs dan YEps dapat

digunakan pada khamir. Plasmid Ti, baculovirus, SV40, dan retrovirus merupakan

vektor-vektor yang dapat digunakan pada sel eukariot tingkat tinggi.

Plasmid

Secara umum plasmid dapat didefinisikan sebagai molekul DNA sirkuler untai

ganda di luar kromosom yang dapat melakukan replikasi sendiri. Plasmid tersebar luas di

antara organisme prokariot dengan ukuran yang bervariasi dari sekitar 1 kb hingga lebih

dari 250 kb (1 kb = 1000 pb).

Agar dapat digunakan sebagai vektor kloning, plasmid harus memenuhi syarat-

syarat berikut ini:



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1. mempunyai ukuran relatif kecil bila dibandingkan dengan pori dinding sel inang

sehingga dapat dengan mudah melintasinya,

2.

mempunyai sekurang-kurangnya dua gen marker yang dapat menandai masuk

tidaknya plasmid ke dalam sel inang,

3. mempunyai tempat pengenalan restriksi sekurang-kurangnya di dalam salah satu

marker yang dapat digunakan sebagai tempat penyisipan fragmen DNA, dan

4. mempunyai titik awal replikasi (ori) sehingga dapat melakukan replikasi di dalam sel

inang.

Salah satu contoh plasmid buatan yang banyak digunakan dalam kloning gen

adalah pBR322. Plasmid ini dikonstruksi oleh F. Bolivar dan kawan-kawanya pada tahun1977. Urutan basa lengkapnya telah ditentukan sehingga baik tempat marker maupun

pengenalan restriksinya juga telah diketahui. Sayangnya, tempat pengenalan EcoR I ,

salah satu enzim restriksi yang sangat umum digunakan, terletak di luar marker. Oleh

karena salah satu marker akan menjadi tempat penyisipan fragmen DNA asing, maka

EcoR I tidak dapat digunakan untuk memotong pBR322 di tempat penyisipan tersebut.

Namun, saat ini telah dikonstruksi derivat-derivat pBR322 yang mempunyai tempat

pengenalan EcoR I di dalam marker, misalnya plasmid pBR324 dan pBR325 yang

masing-masing mempunyai tempat pengenalan EcoR I di dalam gen struktural kolisin

dan di dalam gen resisten kloramfenikol.

EcoR I

ampR tet

R

3.147

(ori)

Gambar 11.1. Plasmid pBR322

ampR = marker resisten ampisilin

tetR = marker resisten tetrasiklin

4.363 kb



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Misalnya saja kita menyisipkan suatu fragmen DNA pada daerah marker resisten

ampisilin dengan memotong daerah ini menggunakan enzim restriksi tertentu selain EcoR

I (mengapa harus selain EcoR I?). Plasmid pBR322 yang tersisipi oleh fragmen DNA

akan kehilangan sifat resistensinya terhadap ampisilin, tetapi masih mempunyai sifat

resistensi terhadap tetrasiklin. Oleh karena itu, ketika plasmid pBR322 rekombinan ini

dimasukkan ke dalam sel inangnya, yakni E. coli, bakteri transforman ini tidak mampu

tumbuh pada medium yang mengandung ampisilin, tetapi tumbuh pada medium

tetrasiklin. Secara alami E. coli tidak mampu tumbuh baik pada medium ampisilin

maupun tetrasiklin sehingga sel transforman dapat dengan mudah dibedakan dengan sel

nontransforman yang tidak mengandung pBR322 sama sekali. Sementara itu, E. coli transforman yang membawa plasmid pBR322 utuh (religasi) mampu tumbuh pada kedua

medium antibiotik tersebut. Jadi, untuk memperoleh sel E. coli transforman yang

membawa DNA rekombinan dicari koloni yang hidup di tetrasiklin tetapi mati di

ampisilin. Secara teknis pekerjaan ini dilakukan menggunakan transfer koloni atau

replica plating (lihat Bab X).

Plasmid yang digunakan pada bakteri gram negatif seperti halnya pBR322 tidak

dapat digunakan pada bakteri gram positif. Namun, saat ini telah tersedia plasmid untuk

kloning pada bakteri gram positif, misalnya pT127 dan pC194, yang dikonstruksi oleh

S.D. Erlich pada tahun 1977 dari bakteri Staphylococcus aureus. Demikian juga, telah

ditemukan plasmid untuk kloning pada eukariot, khususnya pada khamir, misalnya yeast

integrating plasmids (YIps), yeast episomal plasmids (YEps), yeast replicating plasmids

(YRps), dan yeast centromere plasmid (YCps).

Bakteriofag

Bakteriofag adalah virus yang sel inangnya berupa bakteri. Dengan daur hidupnya

yang bersifat litik atau lisogenik bakteriofag dapat digunakan sebagai vektor kloning

pada sel inang bakteri. Ada beberapa macam bakteriofag yang biasa digunakan sebagai

vektor kloning. Dua di antaranya akan dijelaskan berikut ini.

Bakteriofag

Bakteriofag atau fag merupakan virus kompleks yang menginfeksi bakteri E.

coli. Berkat pengetahuan yang memadai tentang fag ini, kita dapat memanfaatkannya



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sebagai vektor kloning semenjak masa-masa awal perkembangan rekayasa genetika.

DNA yang diisolasi dari partikel fag ini mempunyai konformasi linier untai ganda

dengan panjang 48,5 kb. Namun, masing-masing ujung fosfatnya berupa untai tunggal

sepanjang 12 pb yang komplementer satu sama lain sehingga memungkinkan DNA

untuk berubah konformasinya menjadi sirkuler. Dalam bentuk sirkuler, tempat

bergabungnya kedua untai tunggal sepanjang 12 pb tersebut dinamakan kos.

Seluruh urutan basa DNA telah diketahui. Secara alami terdapat lebih dari satu

tempat pengenalan restriksi untuk setiap enzim restriksi yang biasa digunakan. Oleh

karena itu, DNA tipe alami tidak cocok untuk digunakan sebagai vektor kloning. Akan

tetapi, saat ini telah banyak dikonstruksi derivat-derivat DNA yang memenuhi syarat

sebagai vektor kloning. Ada dua macam vektor kloning yang berasal dari DNA , yaitu

(1) vektor insersional, yang dengan mudah dapat disisipi oleh fragmen DNA asing,

(2) vektor substitusi, yang untuk membawa fragmen DNA asing harus membuang

sebagian atau seluruh urutan basanya yang terdapat di daerah nonesensial dan

menggantinya dengan urutan basa fragmen DNA asing tersebut.

Di antara kedua macam vektor tersebut, vektor substitusi lebih banyak

digunakan karena kemampuannya untuk membawa fragmen DNA asing hingga 23 kb.

Salah satu contohnya adalah vektor WES, yang mempunyai mutasi pada tiga gen

esensial, yaitu gen W, E, dan S. Vektor ini hanya dapat digunakan pada sel inang yang

dapat menekan mutasi tersebut.

Cara substitusi fragmen DNA asing pada daerah nonesensial membutuhkan dua

tempat pengenalan restriksi untuk setiap enzim restriksi. Jika suatu enzim restrisksi

memotong daerah nonesensial di dua tempat berbeda, maka segmen DNA di antara

kedua tempat tersebut akan dibuang untuk selanjutnya digantikan oleh fragmen DNA

asing. Jika pembuangan segmen DNA tidak diikuti oleh substitusi fragmen DNA asing,

maka akan terjadi religasi vektor DNA yang kehilangan sebagian segmen pada daerah

nonesensial. Vektor religasi semacam ini tidak akan mampu bertahan di dalam sel inang.

Dengan demikian, ada suatu mekanisme seleksi automatis yang dapat membedakan

antara sel inang dengan vektor rekombinan dan sel inang dengan vektor religasi.



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% total molekul12 pb 15 38 rekombinasi 70 80 85 95 100

5’ dan integrasi 3’

3’ daerah non esensial daerah sint fungsi lisis 5’kepala ekor cI DNA lanjut inang 12 pb

a)

kos

12 pblisis inang kepala

ekor

fungsi lanjut

daerah

sintesis DNA nonesensial

cI

b)

Gambar 11.2. DNA bakteriofag a) konformasi linier (di luar sel inang)

b) konformasi sirkuler (di dalam sel inang)

Bakteriofag mempunyai dua fase daur hidup, yaitu fase litik dan fase lisogenik.

Pada fase litik, transfeksi sel inang (istilah transformasi untuk DNA fag) dimulai dengan

masuknya DNA yang berubah konformasinya menjadi sirkuler dan mengalami

replikasi secara independen atau tidak bergantung kepada kromosom sel inang. Setelah

replikasi menghasilkan sejumlah salinan DNA sirkuler, masing-masing DNA ini akan

melakukan transkripsi dan translasi membentuk protein kapsid (kepala). Selanjutnya, tiap

DNA akan dikemas (packaged) dalam kapsid sehingga dihasilkan partikel baru yang

akan keluar dari sel inang untuk menginfeksi sel inang lainnya. Sementara itu, pada fase

lisogenik DNA akan terintegrasi ke dalam kromosom sel inang sehingga replikasinya

bergantung kepada kromosom sel inang. Fase lisogenik tidak menimbulkan lisis pada sel

inang.



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Di dalam medium kultur, sel inang yang mengalami lisis akan membentuk plak

(plaque) berupa daerah bening di antara koloni-koloni sel inang yang tumbuh. Oleh

karena itu, seleksi vektor rekombinan dapat dilakukan dengan melihat terbentuknya plak

tersebut.

Bakteriofag M13

Ada jenis bakteriofag lainnya yang dapat menginfeksi E. coli. Berbeda dengan

yang mempunyai struktur ikosahedral berekor, fag jenis kedua ini mempunyai struktur

berupa filamen. Contoh yang paling penting adalah M13, yang mempunyai genom

berupa untai tunggal DNA sirkuler sepanjang 6.408 basa. Infeksinya pada sel inang

berlangsung melalui pili, suatu penonjolan pada permukaan sitoplasma.

Ketika berada di dalam sel inang genom M13 berubah menjadi untai ganda

sirkuler yang dengan cepat akan bereplikasi menghasilkan sekitar 100 salinan. Salinan-

salinan ini membentuk untai tunggal sirkuler baru yang kemudian bergerak ke permukaan

sel inang. Dengan cara seperti ini DNA M13 akan terselubungi oleh membran dan keluar

dari sel inang menjadi partikel fag yang infektif tanpa menyebabkan lisis. Oleh karena

fag M13 terselubungi dengan cara pembentukan kuncup pada membran sel inang, maka

tidak ada batas ukuran DNA asing yang dapat disisipkan kepadanya. Inilah salah satukeuntungan penggunaan M13 sebagai vektor kloning bila dibandingkan dengan plasmid

dan . Keuntungan lainnya adalah bahwa M13 dapat digunakan untuk sekuensing

(penentuan urutan basa) DNA dan mutagenesis tapak terarah (site directed mutagenesis)

karena untai tunggal DNA M13 dapat dijadikan cetakan (templat) di dalam kedua proses

tersebut.

Meskipun demikian, M13 hanya mempunyai sedikit sekali daerah pada DNAnya

yang dapat disisipi oleh DNA asing. Di samping itu, tempat pengenalan restriksinya pun

sangat sedikit. Namun, sejumlah derivat M13 telah dikonstruksi untuk mengatasi masalah

tersebut.

Kosmid

Kosmid merupakan vektor yang dikonstruksi dengan menggabungkan kos dari

DNA dengan plasmid. Kemampuannya untuk membawa fragmen DNA sepanjang 32

hingga 47 kb menjadikan kosmid lebih menguntungkan daripada fag dan plasmid.



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Fasmid

Selain kosmid, ada kelompok vektor sintetis yang merupakan gabungan antara

plasmid dan fag . Vektor yang dinamakan fasmid ini membawa segmen DNA yang

berisi tempat att . Tempat att digunakan oleh DNA untuk berintegrasi dengan

kromosom sel inang pada fase lisogenik.

Vektor YACs

Seperti halnya kosmid, YACs ( yeast artifisial chromosomes atau kromosom

buatan dari khamir) dikonstruksi dengan menggabungkan antara DNA plasmid dan

segmen tertentu DNA kromosom khamir. Segmen kromosom khamir yang digunakan

terdiri atas sekuens telomir, sentromir, dan titik awal replikasi.

YACs dapat membawa fragmen DNA genomik sepanjang lebih dari 1 Mb. Oleh

karena itu, YACs dapat digunakan untuk mengklon gen utuh manusia, misalnya gen

penyandi cystic fibrosis yang panjangnya 250 kb. Dengan kemampuannya itu YACs

sangat berguna dalam pemetaan genom manusia seperti yang dilakukan pada Proyek

Genom Manusia.

Vektor YEps

Vektor-vektor untuk keperluan kloning dan ekspresi gen pada Saccharomyces

cerevisiae dirancang atas dasar plasmid alami berukuran 2 µm, yang selanjutnya dikenal

dengan nama plasmid 2 mikron. Plasmid ini memiliki sekuens DNA sepanjang 6 kb,

yang mencakup titik awal replikasi dan dua gen yang terlibat dalam replikasi.

Vektor-vektor yang dirancang atas dasar plasmid 2 mikron disebut YEps (yeast

episomal plasmids). Segmen plasmid 2 mikronnya membawa titik awal replikasi,

sedangkan segmen kromosom khamirnya membawa suatu gen yang berfungsi sebagai

penanda seleksi, misalnya gen LEU2 yang terlibat dalam biosintesis leusin. Meskipun biasanya bereplikasi seperti plasmid pada umumnya, YEps dapat terintegrasi ke dalam

kromosom khamir inangnya.

Plasmid Ti Agrobacterium tumefaciens

Sel-sel tumbuhan tidak mengandung plasmid alami yang dapat digunakan sebagai

vektor kloning. Akan tetapi, ada suatu bakteri, yaitu Agrobacterium tumefaciens, yang

membawa plasmid berukuran 200 kb dan disebut plasmid Ti ( tumor inducing atau



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penyebab tumor). Bakteri A. tumefaciens dapat menginfeksi tanaman dikotil seperti tomat

dan tembakau serta tanaman monokotil, khususnya padi. Ketika infeksi berlangsung

bagian tertentu plasmid Ti, yang disebut T-DNA, akan terintegrasi ke dalam DNA

kromosom tanaman, mengakibatkan terjadinya pertumbuhan sel-sel tanaman yang tidak

terkendali. Akibatnya, akan terbentuk tumor atau crown gall .

Plasmid Ti rekombinan dengan suatu gen target yang disisipkan pada daerah T-

DNA dapat mengintegrasikan gen tersebut ke dalam DNA tanaman. Gen target ini

selanjutnya akan dieskpresikan menggunakan sistem DNA tanaman.

Dalam prakteknya, ukuran plasmid Ti yang begitu besar sangat sulit untuk

dimanipulasi. Namun, ternyata apabila bagian T-DNA dipisahkan dari bagian-bagian lain plasmid Ti, integrasi dengan DNA tanaman masih dapat terjadi asalkan T-DNA dan

bagian lainnya tersebut masih berada di dalam satu sel bakteri A. tumefaciens. Dengan

demikian, manipulasi atau penyisipan fragmen DNA asing hanya dilakukan pada T-DNA

dengan cara seperti halnya yang dilakukan pada plasmid E.coli. Selanjutnya, plasmid T-

DNA rekombinan yang dihasilkan ditransformasikan ke dalam sel A. tumefaciens yang

membawa plasmid Ti tanpa bagian T-DNA. Perbaikan prosedur berikutnya adalah

pembuangan gen-gen pembentuk tumor yang terdapat pada T-DNA.

Baculovirus

Baculovirus merupakan virus yang menginfeksi serangga. Salah satu protein

penting yang disandi oleh genom virus ini adalah polihedrin, yang akan terakumulasi

dalam jumlah sangat besar di dalam nuklei sel-sel serangga yang diinfeksi karena gen

tersebut mempunyai promoter yang sangat aktif. Promoter ini dapat digunakan untuk

memacu overekspresi gen-gen asing yang diklon ke dalam genom bacilovirus sehingga

akan diperoleh produk protein yang sangat banyak jumlahnya di dalam kultur sel-sel

serangga yang terinfeksi.

Vektor Kloning pada Mamalia

Vektor untuk melakukan kloning pada sel-sel mamalia juga dikonstruksi atas

dasar genom virus. Salah satu di antaranya yang telah cukup lama dikenal adalah SV40,

yang menginfeksi berbagai spesies mamalia. Genom SV40 panjangnya hanya 5,2 kb.

Genom ini mengalami kesulitan dalam pengepakan (packaging) sehingga pemanfaatan

SV40 untuk mentransfer fragmen–fragmen berukuran besar menjadi terbatas.



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Retrovirus mempunyai genom berupa RNA untai tunggal yang ditranskripsi

balik menjadi DNA untai ganda setelah terjadi infeksi. DNA ini kemudian terintegrasi

dengan stabil ke dalam genom sel mamalia inang sehingga retrovirus telah digunakan

sebagai vektor dalam terapi gen. Retrovirus mempunyai beberapa promoter yang kuat.

Lecture 15, MCDB 2150, Fall 2000

Vectors for large inserts, cDNA, Libraries, Probes, Expression vectors

Textbook assignment: Chapter 9, Pages 264-277

Major concepts .

Vectors for cloning large DNA inserts

o Vectors derived from bacteriophage lambdao Cosmids

o Bacterial artificial chromosomeso

Yeast Artificial chromosomes

M13 vectors (not in textbook)o replicative cycle of single stranded DNA viruses

o site-directed mutagenesis cDNA

o Isolation of polyadenylated mRNA

o

Reverse transcriptaseo

Removal of RNA templateo

Second strand synthesis

o cDNA clones Recombinant DNA libraries.

o "Shotgun" cloningo Hybridization probes

o Selection of clones contianing specific sequences

Expression vectorso

Promoter supplied by vectoro

Shine and Delgarno sequence supplied by vectoro

Control of promoter activityo Modifying eukaryotic cDNAs for expression in prokaryotic hosts.

Vectors for large DNA inserts: As shown in table 9.3 of the textbook, plasmid vectorsdo not work well for DNA inserts longer than about 10 kb in length The abbreviation

"kb" stnds for sequence length in kilobases (actually kilobase pairs for double strandedDNA). Many different types of vectors have been developed for the cloning of longer

DNA inserts. This lecture examines four such vectors:

1. Modified bacteriophage lambda, in which the genes that are only needed forlysogeny are replaced with a cloned insert of 12-20 kb;



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2. Cosmids, which combine features from plasmids and bacteriophage lambda andcan be used for inserts up to 46 kb;

3. Bacterial artificial chromosomes, which are highly engineered F factors that can

carry up to 300 kb of inserted DNA;4. Yeast artificial chromosomes, which have all essential elements of eukaryotic

chromosomes, and can carry up to 500 kb

Lambda phage vectors: The life cycle of bacteriophage lambda was described on pages

202-205 and control over the choice between lysis and lysogeny was described on pages233-237 (which we skipped over this year). In the replicative form, the bacteriophage

lambda genome is a cl osed circle containing about 50 kb of DNA. However, before the

genome is packed into phage heads, it is cut once by an endonuclease to generate a linear

DNA with cohesive (sticky) ends, which can be rejoined to form a new circle when the bacteriophage injects its DNA into a new host cell. The cohensive ends are called "cos-

sites". The cut site essentially separates the early and late parts of the right operon, suchthat in the linearized genome, genes for head and tail protein are at the left, genes related

to lyosegny are near the middle, and genes related to DNA synthesis and lytic cycle on

the right. The lambda genome is converted to a vector by removing the genes related to

lysogeny and replacing them with a "stuffer" sequence of about 15 kb that contains

selectable markers, as well as restriction endonuclease cut sites at both ends (figure 9.10).

The vector is no longer capable of lysogeny, but can readily initiate a lytic infection.Techniques have been developed for inserting the vector genome into phage heads,

making it easy to infect bacteria with lambda phage vectors containing either the stuffer

DNA or a cloned insert.

Cloning in lambda vectors: For cloning, the stuffer in removed and replaced with aDNA insert that has been cut with the same restriction endonuclease. The two arms andthe insert are simply ligated together and packed into phage heads (figure 9.11).

Packaging of the DNA into a phage head only works when the total length of the DNA is between 38 and 51 kb. The size of the DNA insert that can be successfully clones in a

lambda phage vector is dependent on the amount of phage DNA that has been replacedwith the stuffer, with an upper limit of about 23 kb. Because successful packaging does

not occur when there is not enough DNA, no infectious particles are obtained that lack aninsert or contain an insert that is below a lower size limit for the particular vector. Inserts

that cause the total amount of DNA to be too large are also not successfully cloned. For atypical lambda phage vector, the insert usually must be at least 12 kb and not over about

20 kb. This size range makes lambda phage vectors particularly useful for genomiclibraries (collections of phage containing all of a genome in 12-20 kb fragments).

Lambda phage vectors of this sort are sometimes called Charon vectors, a reference to

Greek mythology, in which the boatman Charon ferries the souls of the dead across theRiver Styx.

Selective markers for lambda phage vectors: Wild-type bacteriphage lambda form

turbid plaques because some of the infected bacteria enter the lysogenic state and are notlysed. However, lambda phage vectors always form clear plaques because the genes for

lysogeny have been removed to make room for the temporary "stuffer" sequence. The



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stuffer contains an IPTG-inducible beta-galactosidase gene, which is eliminated from thevector when the stuffer is replaced with a cloned DNA insert. When induced with IPTG,

plaques formed by vectors that still contain their stuffers turn blue with X-gal, whereas

those with cloned DNA inserts remain colorless. Thus, cloned inserts will be found onlyin clear paques that do not turn blue with X-gal.

Cosmids: Linearized lambda-phage genomes contain cos sites at their ends (cohesiveends). It is possible to create an artificial plasmid consisting of two cos sites plus a

plasmid origin of replication and up to 46 kb of foreign DNA. This will replicate as a plasmid and then can be packaged into lambda-phage heads for infection into bacteria.

These highly engineered vectors are a sort of cross between a plasmid and a lambda

phage and are capable of carrying 30-46 kb of foreign genes with only a little genetic

material of their own (figure 9.13).

Bacterial artificial chromosomes (BAC): The F factor plasmid has the ability tocontinue to function even when integrated into a complete bacterial chromosome. Highly

modified F plasmids have been generated that are capable of cloning very large inserts of

up to 300,000 base pairs. One interesting feature is the incorporation of cut sites for

restriction endonucleases with eight base cut sites. Such endonucleases cut DNA less

frequently and thus generate larger fragments for cloning. Bacterial artificialchromosomes are sometimes introduced into their host cells by electroporation, which

consists of a brief treatment with high voltage electric current that momentarily disrupts

the cell membranes and facilitates entry of large DNA molecules. Once in the cell, the

BAC replicate like F plasmids.

Yeast Artificial Chromosomes (YAC): Our textbook delays a discussion of yeastartificial chromosomes until after it has presented a general discussion of eukaryotic

chromosomes. In brief summary, a yeast artificial chromosome (YAC) contains a yeastorigin of replication, a centromere, a telomere at each end, and a large inserted DNA

sequence of up to about 500 kb (figure 10.25, page 311). Prior to insertion of the foreign

DNA, the essential components of the YAC are maintained in bacterial cells as circular

plasmids.

The material that follows on single-stranded cloning vectors and site-directed

mutagenesis is not described in our current textbook. You can find a brief

discussion of M13 vectors on pages 434-436 of Klug and Cummings, Concepts of

Genetics, 5th Edition (Norlin Reserve) and a discussion of site-directed mutagenesison pages 414-415 of that book. However, it is not necessary for you to know more

about these topics than is discussed in these notes.

Bacteriophage M13: : Bacteriophage M13 has a single-stranded DNA genome. After

M13 infection into a bacterial cell, a complementary DNA strand is synthesized,generating a double-stranded replicative form (RF) of the bacteriophage genome. The

complementary strand then serves as a template for synthesis of new single-stranded viral

DNA by a rolling circle mechanism (like figure 2.30, except that the second strand is not

synthesized until a new cycle of replication is intiated). The single stranded DNA is cut



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into genome length fragments and extruded from the cell. This single-stranded DNA isuseful in sequencing studies on foreign DNA cloned into M13, as will be discissed in

Lecture 17. In addition, M13 clones can easily be subjected to site-directed mutagenesis,

as described below.

Cloning into an M13 vector: M13 vectors have been engineered to contain restriction

endomuclease cut sites in the double-stranded replicative form. For cloning, thereplicative form can be isolated from infected bacteria or generated artificially from the

single-stranded form with a complementary synthetic oligonucleotide primer and DNA polymerase plus ligase. The double stranded form is cut with a restriction endonuclease

and the DNA is inserted much like any other cloning procedure. The double-stranded

replicative form with the insert is infected into bacteria and generates single stranded

clones by the normal process of single-stranded genomic replication.

Site-directed mutagenesis: A synthetic oligonucleotide is prepared that iscomplementary to the region that is to be mutated except that it contains the desired

mutation (usually a one nucleotide change). The oligonucleotide containing the mutation

is hybridized to the single-stranded cloned gene and used as a primer to make a complete

complementary strand that is identical to complementary strand of the replicative form,

except for the mutation introduced by the synthetic primer. The double strandedreplicative form with the mutation in its complementary strand is then infected into E.

coli, where the modified complementary strand serves as a template for production of

new single-stranded vectors carrying the mutated cloned insert. If desired, the phage

DNA can be made double-stranded and the mutated insert can be cut out of the M13

vector, and put into any other convenient double-stranded DNA vector. The net result of

this procedure is to selectively change one base pair in the coding sequence and thus tochange a single amino acid in the coded protein (see figure 14.19 of Klug andCummings). This is an extremely powerful tool for detailed analysis of the roles of

individual amino acids in the overall function of a protein.

cDNA cloning: An alternative cloning procedure called complementary DNA (cDNA)

cloning uses mRNA (usually of eukaryotic origin) as a starting point for cloning a codingsequence. The first step is to isolate the mRNA. This is often done by using a column

containing immobilized poly (dT), which anneals to the poly (A) tails of most mRNAs.

The mRNA can then be eluted by modifying the conditions such that the base pairing nolonger holds the mRNA immobilized. The next step is to make a DNA copy of the RNA.

The first strand of DNA is templated from the RNA by a viral enzyme known as reverse transcriptase. The RNA template is then digested away with ribonuclease H , which isspecific for digesting the RNA part of an RNA:DNA hybrid. It is also possible to use

selective alkaline hydrolysis with NaOH to remove the RNA without damaging the DNA,which is more resistant. The 3'-end of the single-stranded DNA tends to fold back on

itself and find enough complementary base pairing to form a hairpin loop, which servesas a primer for second strand synthesis. The Klenow fragment of DNA polymerase I

(which lacks endonuclease activity) is then used to synthesize the second DNA strand



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and the loop is cut with S1-nuclease. The so-called cDNA (complementary DNA) is thenligated into a vector. This can be done either by blunt end ligation or by adding sticky

ends artificially. cDNA cloning is often done in an expression vector that contains a

promoter that allows synthesis of mRNA and expression of the coded protein in the hostcell, as will be discussed later in this lecture.

Libraries: As an alternative to selective cloning of specific DNA sequences, it is

possible to construct a library of cloned sequences that is sufficiently complex so that it is

statistically expected to include all of the sequences in the DNA that was used as a

starting point. This can be the entire genome of an organism or all of the genes carried onone particular chromosome, or all of the mRNA sequences in a particular tissue that have

first been converted to cDNA as described above. Libraries are typically constructed in

vectors that accept only a limited range of sizes of inserts. To avoid cutting some genesinto fragments that are too small to be cloned in such vectors, the digestion process is

often stopped before it has been carried to completion. This generates cloned sequences

that still contain some cut sites for the enzyme used for the cloning procedure, and

usually results in overlapping clones, which are very useful for locating adjacent

sequences. Alternatively, it is sometimes possible to use enzymes with less frequent cutsites if the sizes of their digestion fragments match the range that can be cloned in the

vector that is being used.

Hybridization probes Complementary strands of DNA, RNA, or DNA plus RNAhybridize readily to form double stranded helical structures when placed under suitable

annealing conditions. This property is used extensively in molecular genetics to identifyspecific nucleic acid sequences. A probe consisting of radioactively labeled DNA (or

RNA) is hybridized to denatured DNA (or naturally single-stranded RNA) immobilizedon a support, such as a nitrocellulose membrane. Hybridization is normally done at a

temperature about 25°C below the melting (denaturation) temperature for the DNA.

Probe sequences that do not hybridize because there is no immobilized complementary

strand for them are washed off. The sites that contain sequences capable of hybridization

are now radioactive and can be detected by autoradiography or by direct counting with

scanning counters. When combined with electrophoretic separation of DNA (or RNA)

fragments by size, hybridization probes play major roles in many different molecular

biology procedures, including Northern and Southern blotting (lecture 17), DNA

fingerprinting, etc.

Cloned DNA probes: Any DNA (or RNA) that can be prepared as a single uniform

sequence can be used as a probe. Cloned DNA is particularly useful, because it consistsof multiple copies of a single sequence, and is generally carried in a vector that is

sufficiently "foreign" so that it will not react with any of the DNA that is being analyzed.

Screening a library: It is possible to transfer replicas of bacterial colonies containing

cloned vectors to a nitrocellulose membrane, followed by lysis of the cells and fixing the

DNA onto the membrane. Hybridization with a radioactive probe followed by washing



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off of unhybridized probe then reveals which colonies contain the desired cloned DNA.From the position of the radioactivity, which is detected by laying an X-ray film over the

membrane and then developing it after an appropriate exposure period, it is possible to go

back to the original plate containing the colonies of bacteria and pick the colony (orcolonies) that contain plasmids with the desired cloned DNA inserts (figure 9.15). It isalso possible to transfer bacteriophage from plaques onto nitrocellulose membranes and

use a similar hybridization process to identify those plaques that contain specific clonedDNA sequences.

Reverse genetics with degenerate oligonucleotide probes: In certain cases, it is

possible to generate a hybridization probe for a coding sequence based on the amino acid

sequence of the coded protein. This is dependent on finding a stretch of at least six amino

acids whose codon redundancies are relatively low. The procedure is to artificiallysynthesize a mixture of all of the possible nucleotide sequences that could code for the

amino acid sequence in question, and to use that mixture as a hybridization probe (see boxed example 9.4). One of the members of the mixture is expected to contain the exact

coding sequence and thus to hybridize stringently with the message sequence. In addition,

others with a single base mismatch may hybridize sufficiently so that they can be seen to

be associated with the message sequence if the stringency of the hybridization reaction is

reduced somewhat by adjusting temperature and/or salt concentrations so that slightly

mismatched probes are not washed off of their immobilized target sequences.

Expression vectors: It is often desirable to be able to obtain expression of the protein

coded by a cloned gene or cDNA. Expression vectors are useful for production of thecoded protein in various types of host organisms, including commercial production of

proteins that are difficult to obtain in adequate quantities from natural sources. They also permit the proteins that are produced to be used to identify the cloned genes that code for

those proteins. They can also be used to produce fusion proteins that are useful in the

isolation of previously unknown protein products.

Expression vectors of many different types have been designed for use in various types of

host cells. Typically they contain either constituitively strong promoters or promoterconstructs that are capable of regulated expression. They also usually contain a ribosome-

binding site, such as the bacterial Shine-Dalgarno sequence, to insure vigorous translation

of the transcripts that are produced from them. In many cases, they also contain an ATGstart codon, followed by a few amino acids from a host protein. In such cases, the cloned

gene must be in-frame and inserted in the right direction. In cases where one wishes to

produce a eukaryotic protein using a bacterial vector, it is necessary to start with a cDNAclone, which already has intron sequences spliced out, since splicing does not occur in

prokaryotic cells.

Inducible expression: Production of large amounts of a foreign protein can be toxic to a

host cell. Many expression vectors have been designed with inducible expression to allow

the vector to be grown up to a high level in the cell without expression, followed by a



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burst of intense expression to generate as much as possible of the product before the hostcell ceases to function. The host cell and the vector are often engineered to work together.

One example is use of a bacterial strain that produces large amounts of the lac repressor

protein and a vector with the cloned gene under the control of the lac promoter/operatorsystem. The repressor inhibits expression while the vector population is being expanded.Expression is activated at the appropriate time by adding a synthetic analog of allo-

lactose called IPTG that is not metabolized by the cells and thus provides a strong stableinduction signal. Another control mechanism that can be utilized involves the lambda left

promoter, which when combined with a temperature sensitive lambda repressor (cI) gene,allows large amounts of bacteria containing vectors to be grown up at a permissive

temperature, followed by massive expression of the cloned gene when the temperature israised enough to inactivate the heat-sensitive lambda repressor protein, as described in

the textbook for the pPLa2311 expression vector. Promoters that respond to hormones or

to heavy metals are often used for inducible expression in eukaryotic cells. We will

revisit expression vectors in the lecture on biotechnology near the end of the semester.

Use of antibodies to identify cloned genes: Cloned genes (or cDNAs) that code for

proteins that can be identified with antibodies are frequently detected through the use of

an expression vector derived from bacteriophage lambda. It is called lambda-gt11

(usually written with the Greek letter lambda). This vector produces a fusion protein

under control of the inducible promoter for the E. coli lactose (lac) operon. The vectorlyses the bacterial cells, forming plaques on a lawn of E. coli. When expression is

induced with IPTG at a critical time during plaque formation, the fusion protein is

released into the plaques and can be detected by blotting onto nitrocellulose, followed by

binding of antibody and then binding of radioactive Protein A, which attaches to the

antibody. This allows plaques that express proteins capable of binding to the antibody to be located by autoradiography. The detection procedure is similar to that used in Western blots (Lecture 16).

Heteroduplex analysis: Hybridization of two nucleic acids that contain a mixture of

complementary and non-complementary sequences will cause the formation of a series ofloops that are similar in principle to the loops that are formed when processed mRNA is

hybridized to genomic DNA that contains introns (figure 3.22) Such structures can bevisualized quite readily with an electron microscope. Thus, it is possible to see a cloned

DNA sequence in a vector that has been denatured and hybridized with a vector that lacksthe insert.

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