Recombinant DNA.

ANNUAL REVIEWS

Further

Quick links to online content Ann. Rev. Biochem. 1977. 46:415-38 CopyrightS'; 1977 by Annual Reviews Inc. All rights reserved

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RECOMBINANT DNA

+952

Robert L. Sinsheimer Division of Biology, California Institute of Technology, Pasadena, California 91125

CONTENTS PERSPECTIVES AND SUMMARY ....... ..... ... ......... ............. ............ .......................... CLONING ORGANISMS ...... ...................... .... ............. ... ............. ..... ......... ..... ............

415 418

Prokaryotes ... ............................ ...............................................................................

41 S 418

.

Eukaryotes................................................................................................................

CLONING VEHICLES..................................................................................................

418

Plasmids.................................................................................................................... General properties .. .......... .... ...... ........ ..... ........................................................... Cleavage sites .............. ..... ..... .................... ........................................................... Bacteriophage........................................................................................................ Eukaryotic viruses ...................... .... .......... .............. .............................................

418 418 422 423 423 424

DNA SPLICING AND INSERTION ..... ............ ................................ ......................... SELECTION OF CLONED DNAs .... ........ ...... ....... ................... .. ......... ...................... SOME APPLICATIONS................................................................................................

426 428 428

Prokaryotes ....................................... ....... ..... ........... .......................... ......................

Eukaryotes................................................................................................................

429 429 430 431 433 434

SAFETY ............ ............. ......... ......... ..... ........ ..... . ....................... ............. ...... ................

435

.

.

.

Viral Vehicles .................................... ........... ........................................................... .

.

Plasmid structure and function ............................................................................ E. coli chromosome ......... ............ .... ... ...... ... .............................. ............... ............ Eukaryotic DNA .................................... ...... ..................................................... yeast...................................................................................................................... ..

.

.

.

PERSPECTIVES AND SUMMARY The development of recombinant DNA technology permitting the transfer of genetic material between widely divergent species has opened a new era of research into the structure and function of the genome. The transfer of genetic material between individuals by sexual (conjuga tive) means is, by definition, restricted to members of a species and employs 415


416

SINSHEIMER

structures specifically produced for the purpose. Asexual transfer of genetic material as free DNA (transformation) or via viral mediation (transduction, transfection) is well known in microorganisms (for reviews see 1-3) [for transformation of Escherichia coli see (4)] and appears to be well-estab lished in blue-green algae (5) and in Drosophila (6). Such transfer appears to depend upon processes of DNA recombination that require considerable homology between donor and recipient DNA sequences; in the case of virus-mediated transfer, the presence of appropriate cell structures that permit viral penetration, and of DNA sequences appropriate (in each case) for integration, is necessary. Although in some instances, e.g. Mu phage (7), the latter requirement is not very stringent, the combined requirement severely limits the range of species between which DNA may be transferred. The effectiveness of transformation thus falls off rapidly with increasing divergence of the species involved, as the homology of their DNA sequences decreases. Claims of the transformation of the higher organisms (plants) by mi crobial DNAs (8, 9) or even of the sustained expression of microbial DNAs in plant and animal cells (10-)2) remain controversial (13). The recombinant DNA technology that bypasses the restrictions asso ciated with transformation and transduction has resulted from the combi nation of a set of advances in our knowledge: 1. the discovery and application of a wide variety of restriction enzymes [see (14-16) for review, and Table 1 for representative examples] that permit the cleavage of DNA at a small number of reproducible sites (on the order of one site/few thousand base pairs); 2. the development of means for splicing together DNA segments in any combination (see Figure 1); 3. the discovery that the genes and other DNA sequences necessary for the maintenance and replication of bacterial plasmids or the replication (and integration) of certain viruses are clustered and can thus be employed Table 1 Sequence specificities of some corn monly used restriction enzymes

Designation

Source

Sequence

Reference

EcoRI

E. coli RY 13

5' "'T/AG�pAA1TCA/T"'3'

HindIlI

Haemophilus influenzae Rd

5' ···A�pAGCTT .. ·3'

Haell

Haemophilus aegyptius

5' ...PuGCGC�pPy...3'

Hpall

Haemophilus parainfluenzae

Hhal

Haemophilus haemolyticus

5' ... GCG� pC..·3'

Bam HI

Bacillus amyloliquefaciens H

5' .. ·G�pGATCC·..3'

Sail

Streptomyces albus G

?

117

Pstl

Providencia stuartii

?

117

5' ...C�pCGG ...3'

14 111 112,117 113 117 114,117,118

RECOMBINANT DNA

0)

417

Cohesive ends 5' 3'

' G - C-T - G

"

.

.

'C -G-A-C-T-T- A-Ap

+

p A-A-T-T- C-A -T-G'" G-T-A-C'"

3' 5'

,


5' " 'G-C-T-G-A-A-T-T-C-A- T- G '" 3' 3 ' ···C-G-A-C-T-T-A-A-G- T- A-C · · · 5'

b)

dA: dT joins 5'

. " G-C-A-A-A"'A"'A-A

3' " 'C-Gp

pT-G'" +

,

c)

5' " 'G- C -A-A-A"' A" 'A-A-T- G "

·

3'

3'

·

5'

· · ·

3'

T - T · · · T · · ·T - T - T- A - C · · · 5'

C - G - T - T- T · · · T · · · T - T - A - C · ·

Butt joins 5' " 'G-C-T

3' " ' C-G-Ap

+

pG- T- A . . . 3' C-A-T" ' 5'

5 ' " 'G -C-T -G- T- A'" 3'

3' " 'C-G-A-C - A -T'"

Figure

J

5'

Methods of DNA splicing

.

as a unit structure to provide a "vehicle" to which other DNAs may be attached for replication or integration; and 4. the discovery of conditions that permit the introduction of relatively large pieces of DNA. intact into subsequently viable cells of E. coli (17. 18); this tactic made the extensive knowledge of E. coli genetics and biochemistry available for this field of research. •.

The recombinant DNA technology achieves 1. the fractionation of individual DNA components of complex genomes by their individual uptake into single cells (after coupling to an appropri ate reproducing vehicle); 2. the amplification by many orders of magnitude of individual components of complex genomes by the growth of clones from individual trans formed cells; 3. the opportunity to study the expression and control of individual compo nents of complex genomes in quite diverse circumstances; and 4. the potential to create new genetic combinations advantageous for spe cific human purpose.

418

SINSHEIMER

To date (July 31, 1976) most of the literature has been concerned with the development of a recombinant DNA technology. Relatively few signifi cant biological results have appeared, although numerous experiments of considerable potential importance are in progress.


CLONING ORGANISMS Prokaryotes

The only microorganism thus far employed for cloning has been Escherichia coli. Various strains [C600 (19), HB IOI (20, 21), h303 (13), RR I (22)] have been used. It is desirable that the cloning organism lack restriction enzymes that might cleave foreign DNA (such strains usually, but not always, also lack the modification enzymes that protect DNA against restriction; once established in a restrictionless strain, however, the unmodified DNA can, if desired, be subsequently transferred at a low efficiency to a restricted strain where it will also become modified). In order to minimize the likelihood of inadvertent recombinatory events that would impair the integrity of the cloned DNA, the cloning organism is frequently reck-i.e. deficient in the major pathway of DNA recombination. , If expression (transcription and translation) of the cloned DNA is of interest, it is desirable to transfer the recombinant DNA plasmid into a strain that buds off minicells (19, 23). In such strains (24) portions of cytoplasm are budded off which lack chromosomal DNA but may contain plasmids as well as the enzymes and components needed for gene expres sion. In this greatly simplified background mRNAs or proteins derived from the recombinant DNA can be readily detected. Eukaryotes

Recombinant DNA experiments in eukaryotic cells have employed primary and secondary tissue cultures of African Green Monkey kidney cells (2527).

CLONING VEHICLES Plasmids

Various plasmid vehicles have been used (Table 2). Most to date have been derived by additions or deletions from either pSC lOl (28) or ColEl (29). It is desirable that the transformed cell display a new property that permits its selection from the bulk of nontransformed cells. Incorporation in the plasmid of genes providing drug resistance or immunity to specific colicins permits such selection. GENERAL PROPERTIES


RECOMBINANT DNA

419

Introduction of DNA into E coli strains requires the presence of calcium ions to promote penetration (4, 17, 18) and is favored by a short (2-5 min) exposure to elevated temperature (42°C) after prior adsorption. Under favorable conditions approximately one cell in 105 or 106 will produce a transformed clone. From experiments on the frequency of joint uptake of two distinctive plasmids, it is evident that only a small fraction of the treated cells can be successfully transformed (30). These cells can accept more than one plasmid. Cloning of a plasmid that lacks a selective feature can be accomplished by co-transformation with a second plasmid, which both carries a gene permitting selection and is temperature-sensitive with respect to replication (30). If adequate concentrations of plasmid DNA are employed, most of the transformable cells will acquire both plasmids. The selective characteristic of the "indicator" plasmid can be used to select the transformed clone. Growth subsequently at the restrictive temperature will eliminate the car rier plasmid, leaving only cultures that have a high proportion of cells carrying the desired plasmid. Final selection can be made by physical examination of the DNA from a small set of clones. With some of the DNA splicing and insertion procedures (vide infra) a large fraction of the cloned plasmids may not actually contain any inserted DNA and will therefore be of little interest. If the foreign DNA can be inserted at a site such as to destroy a (second) selective characteristic (i.e. a second drug resistance factor), the absence of this characteristic can be used to select those clones with plasmids bearing inserted DNA ("inser tional inactivation") (21, 31, 32). By exposure of the transformants to the drug, those cells which are now sensitive to it will be blocked from growth. Addition of an agent that specifically kills growing cells, such as D-cyclo serine, can then be added to kill off all of the transformants that have not been so inactivated, thus providing a considerable enrichment of the desired clones (33). . The plasmid vehicles commonly employed are in the range of 2-20 X 106 daltons [3, 000-3 0,000 kilobase pairs (kb)]. Since plasmids of over 2 X 108 daltons (300, 000 kb) are known, very large pieces of inserted DNA can be accommodated. pSC101 and its derivatives are "stringent" (31) with respect to DNA replication: exponentially growing cells will have six to eight plasmid mole cules per chromosome (86); replication of the plasmid requires new protein synthesis, and does not require an active DNA polymerase I. ColEl and its derivatives are "relaxed" (31) with respect to DNA replica tion: exponentially growing cells will have 20-30 plasmid molecules per chromosome; replication of the plasmid does not require new protein syn thesis, but does require an active DNA polymerase I. In the presence of an inhibitor of protein synthesis (chloramphenicol) that blocks chromosomal


""'" N 0

Table 2

Vehicle

Properties of pJasmids and other vehiclesa

Size

Selection property

TcR

en

EeoRl cleavage sites

1

b

pSC101

5.8

pSC10S

10.5

X

106d

pSC20S

10.5

X

106d

pSC201

5.8

X

106d

TcR Rep!S

RSF1010

5.5

X

106d

SmR SuR

ColE1

4.2

X

106d

ImmEt

mColEl

2.2

X

106d

ImmEt

1d

pAClOS

1.5

X

106d

ImmEt

pYH51

2.1

X

106d

ImmEt

pML21

6.6 X

106d

X

106d

Z

Number of Derivation

Other cleavage sites

Reference

Plasmids HindIll(1b) BamHI(l C) SalI(1C)

21,23,28,39,95

9.2 kb TcR KmR

TcRKmR Repts

+ KmR

fragment

30

2

pSC101

2

By mutation from pSC105

30

Deletion of KmR fragment from

30

pSC205

KmRImmEt

I

d

29,31,100

HindIll(O) Diminutive form of ColE1

29,77

1

Diminutive form of ColE1

119

1

Indirectly from ColEl

2

HindIII(1e) SalI(l) BamHI(O)

pVH51 + Km R fragment of pSC 105

40 21,40,85,95

10.5 kb pGM16

-20 kb

TcRKmRImmEt

pML2

8.7

X

106d

KmR lmmEt

pCRl

8.7

X

106d

KmRImmEt

RSF2124

7.3x 106d

ApR

Bam HI (1f) 2

pSC101 + pML21 ColEl

+ KmR

fragment of pSCI05

Deletion of one EcoR l site from pML2 ColEI + TnA fragment carrying ApR

21,95 29 41,75,81 32

en :r:

tTl

�

tTl :::c


Table 2

(continued)

pMB9

3.5 x l06d

pBR3l3

5.8

X

l06d

R Tc lmmEI + R Tc ApR

RK2

20

X

106d

R Tc KmR ApR

RP4

30

X

106d

Agt-AC

39.3 kb

2

42

Agt-AB

38.4 kb

2

47

SV40GTl

-3000 bp

I

SV40a

1670-bp monomer

0

BamH l(l)

54

SV40d

880-bp monomer

0

HindIlI(1) HpaII(J)

25

HindIIl(l C);SalI(JC);BamI(JC)

ColEl +

R Tc from pSClOl

33

HindIIl(1 C);Sal(1 C) ;Bam(l C)

Derived from pMB9 + TnA fragment

33

Pst(l g)

of R SF2124

Tc RICm R Ap R

P-type plasmid

34

P-type plasmid

35-38

Bacteriophage

SV40 derivatives 27

a Abbreviations used: d, daltons; kb, kilobase pairs; bp, base pairs; T

respect to replication; Sm

R

�, resistant to tetracycline; KmR, resistant to kanomycin; Rep!!, temperature-sensitive with

, resistant to streptomycin; Su R, resistant to sulfanilimide; ImmE , confers resistance to colicin E1; ApR, resistant to ampiCillin; TnA,

!

translocation fragment A. bNo effect of cleavage on replication; may affect TcR. R eNo effect of cleavage on replication; inactivates Tc . d No effect of cleavage on replication or immunity to colicin E1; inactivates colicin El synthesis. R eNo effect of cleavage on replication or immunity to colicin E1; inactivates Km . R R f No effect of cleavage on replication, Km or immunity to colicin E1; inactivates Tc . R gNo effect of cleavage on replication; inactiv ates Ap .

:::t'

8==

t:C

Z

> Z >-l o Z >

t


422

SINSHEIMER

DNA replication, several hundreds of plasmid molecules will accumulate in the cell until 40--50% of the cellular DNA is plasmid DNA (29). To a first approximation, the smaller the ColEl-type plasmid, the larger the number of copies will be, both in exponentially growing cells and in the presence of chloramphenicol. pSClOl and ColE1 plasmids and their derivatives are non-self-transmissi ble-i.e. they can only be passed from cell to cell via conjugation if they are "mobilized" by the presence of another plasmid. For some purposes it might be desirable to use a self-transmissible plasmid. RK2 (34) and RP4 (35-38)-members of the P class of plasmids, which are widely transmissi ble to many bacterial types-have been proposed for this purpose. For purposes of insertion of a foreign DNA it is desir able that the plasmid vehicle have one cleavage site for the restriction enzyme employed. Cleavage and insertion at this site must not inactivate the "replicator" region essential for the propagation of the plasmid or the feature that permits selection of the transformed cell. Thus pSClOI is cleaved at one site by EcoRI; insertion at this site does not affect replication of the plasmid but may affect expression of the plasmid gene that provides the tetracycline resistance by which transformants are selected [(28); S. Cohen, personal communication]. pSClOl is similarly cleaved at one site by HindIII (39) without effect on either function. However, BamHI or Sail, which likewise cleave pSC101 at one site, do so in a region such that insertion prevents expression of the tetracycline resistance factor (21) (Fig ure 2).

CLEAVAGE SITES

f1 Eco"[! RI :t

pSCIOI (9200bp)

Figure

2

pBR313

(9000bp)

Cleavage sites of pSClO1 (21, 39) and pBR313 (33).


RECOMBINANT DNA

423

Cleavage of ColE1 or derivatives with EeoRI is at one site and does not interfere with replication, but it prevents expression of the gene for produc tion of the EI colicin. However, the plasmid still confers immunity to the EI colicin and this property can be used for selection (29). Composite plasmids made by deliberate insertion of DNA fragments carrying drug resistance factors [as, for instance, pML2 (29) or pML21 (40)] will consequently have two cleavage sites for the restriction enzyme used in their composition. Partial digestion may then be employed to obtain singly cleaved plasmid molecules for insertion, or derivatives in which one of the restriction sites has been deleted [pCRI (41)] can be obtained. A systematic procedure (101) to obtain such derivatives involves partial diges tion of the parent plasmid by the restriction enzyme to obtain singly cleaved molecules, limited degradation of the resultant 5'-ends of the DNA strands with a 5'-exonuclease, electrophoretic fractionation to separate the linear DNA molecules from uncleaved rings, and transformation with the linear form. These linear forms can be converted to circles in vivo and can subse quently be propagated with a small deletion in the region of the former restriction site. The efficiency of such transformation is about 1/1000 of that normally obtained. Viral Vehicles BACTERIOPHAGE Appropriately constructed mutants of the bacterio phage A can be used as a cloning vehicle. Large portions of A DNA are not essential for lytic growth or for integration into the E coli chromosomes. Such portions can be replaced by the foreign DNA for cloning. Wild-type A (46.5 kb DNA) has five sites of cleavage by EcoRI (42-45). By recombinations between appropriate deletion mutants and by the use of procedures for the selection of mutants lacking EeoRI sites (42, 43, 46) derivatives can be obtained with one or two or three restriction enzyme sites, all in nonessential regions. For cloning purposes the use of A deriva tives with two restriction sites permits the excision of a central, nonessential region and its replacement by the foreign DNA. Transformation by the recombinant DNA is accomplished as with plasmid transformation, using nonrestricting host cells. Phage A DNA cannot be encapsulated into virus particles if it is less than 75% in length of the full genome (or more than 109%) (42, 47). By the use of appropriate deletions a A genome can be constructed with two EeoRI sites, so that after excision and removal of the central fragment the sum of the two end fragments is only 71 % of genome length. Thus only recombi nant DNA molecules into which at least a small fragment of foreign DNA has been inserted will give rise to progeny DNA of sufficient length to be encapsulated and thus give rise to a viral plaque; Foreign DNA of up


424

SINSHEIMER

to 17, 000 kb in length can be inserted into such recombinants (42) (see Figure 3). The integration proficiency of the recombinant viral DNA is dependent upon the residual presence of the necessary gene set (aU, in!, xis). If the particular A used as the cloning vehicle cannot integrate by itself, a helper phage that is proficient in integration can be used (48) to form a double lysogen. Formation of such double Iysogens, involving one phage genome lacking the viral attachment site, is mediated by general bacterial recombination at about 11100 the usual frequency. Appropriate genetic markers can be used to distinguish the helper from the recombinant phage if it is subsequently desired to differentiate them. Wild-type A DNA has six restriction sites for HindIII (49) that could furnish the basis for construction of a cloning vehicle for HindIII DNA fragments. In some A-type phages (434) the immunity region has the cleav age site for EcoRI (and also one for HindIII). Insertion at such a site would inactivate the repressor so that the recombinant phage particles could be distinguished from parental particles by their clear plaques (50). Lambda dv plasmids that contain portions of the A genome might also be used as cloning vehicles. All A dv's examined (51) have one EcoRI site, which is, however, in the replication region. Lambda dv021 has one HindIII site, however, which could be used to clone HindIII DNA fragments. Lambda dv plasmids confer resistance to Avir phage of the same immunity type. To date the only cloning vehicles employed in eukaryotic cells have been derivatives of the oncogenic virus simian virus

EUKARYOTIC VIRUSES

I

+ wt lambda

0 I

IA

0.1 I

E

0.2

0.3

V

K

I

A

I

I

B I

9.8

+ 0.9

0.8

0.7

I

I

I

cI p

C

D

E

11.3

15.4

12.1

Introduce

Eco RI I

Q RI I F I 6.8

nin-5 deletion (6.1%) in

Delete fragment

X

B

I •

(O)

l(

genes

EcoRI

fragments

E

C

nin-5

I

sites

1.0

Delete sites 4 and 5

--- 44.5 ----·-·9.8·

Figure 3

+

exo§.

0/1

j A

I

J

44.5

Agt - AB

0.6

I

5 0.931

+

+ 0.5

0.4

I

4 0.810

3

2

0.445 0.543 0.636

�

28.3-

Derivation of Agt-AB cloning vehicle from wt A (42, 45, 47).

RECOMBINANT DNA

425


£Co RI

SV40

.5

T antigen? Figure 4

Genetic map and cleavage sites of SV40 DNA (-5000 bp) (52, 115).

40 (SY40) (52), which contains about 5 kb of double-stranded, circular DNA (Figure 4). Mutants of SY40 with intact origin (OR!) region and A gene, but defec tive in the B, C, or D gene regions, can replicate their DNA and can transform host cells, but they can only form progeny virus particles with the aid of helper virus (53). Genomes with a length of 70-100% of wild-type SY40 can be encapsulated (55). Deletions in the B, C, and D gene regions of 1300 base pairs (26) (from the Hpall site to the EcoRl site) and of 2 000 base pairs (27) (from the Hpall site to the Bam HI site) have been replaced by foreign DNAs to produce genomes of over 90% of the original length. Such genomes could then be propagated with the aid of a helper SY40 DNA containing a ts mutant in gene A; with this combination, at the restrictive temperature, only coinfected cells would yield progeny virions. To date there is no evidence for a restriction enzyme system in the monkey cells employed in these experiments (54). After repeated passage of SY40 stocks at high multiplicity, reiteration mutants are found (55-57). Genomes of these mutant forms are composed of tandem reiterations of a segment of DNA that may range from 8% to 30% of the size of the SY40 genome and that always includes the ORI region of SY40 at which replication is initiated. The number of reiterations (three to nine) is sufficient to bring the DNA to a size that allows encapsula tion. The size of the necessary SY40 region may be as small as 150 base pairs (57) (the remainder is host-cell DNA).


426

SINSHEIMER

The replication of such reiteration mutants requires the presence of helper viral DNA with intact A gene function (and B, C, and D function if the DNA is to be encapsulated). Because of their multiple sites of initia tion of DNA replication, these mutants tend to outgrow the helper virus. Alternatively, the origin of SV40 replication can be isolated by the action of a single-strand specific nuclease on DNA heteroduplexes formed between the strands of two deletion mutants chosen to have mismatched regions flanking the ORI region (58). When the duplex DNA fragment (about 13% of the SV40 genome) thus obtained is added to host cells together with helper SV40 DNA, complete DNA circles are formed, composed of several tandem reiterations of the fragment, and are packaged into progeny parti cles. The monomeric units of tandem reiteration mutants can be used as cloning vehicles for foreign DNA (25, 54). By coupling a foreign DNA to such a monomer and coinfecting host cells with either the linear or the circular forms of the recombinant, virus-sized genomes are produced in tracellularly with tandem repeats of the recombinant DNA.

DNA SPLICING AND INSERTION Three general methods have been developed for the joining of DNA frag ments (see Figure I). 1. Cohesive ends. Many of the restriction enzymes make cuts (Table 1), leaving extended single-stranded termini at the 5'-ends of both strands of the fragments. The unpaired regions may be two to five nucleotides in length. Since the termini at the two ends of any fragment are complemen tary, under appropriate conditions, such single-stranded termini can pair up to form a portion of the customary DNA double helix (28, 59). A fragment may thus be converted to a circle [the minimum size for circle formation is about 250-300 base pairs (60)]; fragment oligomers may be formed; or DNA molecules from different sources but bearing the same termini may be joined. In particular, if a circular DNA molecule (i.e. a plasmid) bearing one such restriction site is cleaved to provide two cohesive ends, a fragment of any other DNA bearing the same cohesive ends can cohere to the two ends of the open circle to reform a now larger circle (28). With but a few base pairs such hydrogen-bonded fragments will melt apart at a low temperature unless the fragments are covalently joined by the action of a DNA ligase (E. eoli or phage T4) (61, 62). For ends formed by EeoRI, which contain four A-T base pairs, the Tm is 5-6°C. As a compro mise between ligase activity and stability of the hydrogen-bonded frag ments, covalent joining is usually performed in the temperature range of 10-15°C (28, 60). Single-stranded termini with more G-C pairs (produced


RECOMBINANT DNA

427

by other restriction enzymes) form more stable complexes and can be joined at a higher temperature. Because of the symmetry of the DNA structure, any fragment produced by restriction enzyme cleavage can be inserted in either of two orientations. Whether the orientation affects its subsequent properties (transcription, translation) depends upon the presence and strand location of neighboring promoters, terminators, etc. , If a fragment is excised from a ring DNA by the successive action of two different restriction enzymes, leaving two different cohesive ends, only a fragment of foreign DNA with two similar ends can be inserted and then only in a unique orientation (70). 2. dA:dTjoints. Another procedure for joining DNA fragments uses the enzyme terminal deoxynucleotidyl transferase (63) to add (for instance) a chain of dA residues (50-200 nucleotides long) to the 3'-ends of the strands of one fragment and a chain of the complementary dT residues (50-200 nucleotides long) to the 3'-ends of the strands of the other fragment. Such fragments obviously cannot form circles or oligomers by themselves, but when the two differently extended fragments are mixed, under appropriate conditions, they are joined with the formation of an intervening segment of dA:dT (64, 65). If one of the fragments used is a singly cleaved DNA ring (plasmid), addition of the second DNA fragment can reform a new, larger DNA ring. With tens of dA:dT pairs the hydrogen-bonded recombinant DNA is rela tively stable. It can be converted to a covalently closed form by filling in the gaps with deoxyribonudeotides in the presence of DNA polymerase I, followed by covalent joining with DNA ligase. Usually this is not necessary, as the transformed cell will perform the same steps. Ordinarily terminal deoxynucleotidyl transferase will only add nucleo tides to a single-stranded 3'-terminus. For this reason, DNA fragments obtained by restriction enzyme cleavage have most often been treated with a 5'-exonuclease [usually that coded by the bacteriophage A (66)] to expose single-stranded 3'-ends before addition of the transferase enzyme. More recently it has been found (67) that in the presence of cobalt ions, trans_ferase is less fastidious and will add nucleotides readily to 3'-termini without prior removal of that portion of the complementary strand. With dA:dT joints a fragment can be inserted in either of two orienta tions. With this method, however, a cleaved plasmid DNA cannot reform a ring without insertion of the foreign DNA, thus improving the proportion of transformed clones that are of interest. The presence of the dA:dT sequence in the resultant recombinant DNA may or may not affect its function (68). 3. Butt joints. It has been observed (69) that under appropriate condi tions the DNA ligase coded by the bacteriophage T4 can join butt ends of

428

SINSHEIMER


DNA (with no unpaired nucleotides), provided only that the 5'-ends have terminal phosphate groups and the 3'-hydroxyl groups are not blocked. This simple method can be used to join fragments unselectively. As with cohesive ends, large fragments can be converted to circles and inserts can take either possible orientation.

SELECTION OF CLONED DNAs In some instances selection can be made prior to cloning. Thus DNA can be prepared from Xenopus laevis oocytes enriched in the 5S RNA gene (39, 70) or from X laevis highly enriched in the genes coding for ribosomal RNA (19, 71, 72). By appropriate sedimentation techniques recombinant plasmids can be selected to have a desired sedimentation rate or buoyant density indicative of the nature of the inserted DNA (73, 74). When reasonably pure messenger RNA is available (e.g. globin mRNA), partial or full-length cDNA can be synthesized with the aid of reverse transcriptase (75, 76) and converted to double-stranded DNA with DNA polymerase I (75, 76). Such double-stranded DNA can then be inserted into a plasmid vehicle by means of dA:dT joints or butt joints. [In an ingenious variation of this procedure (77) the globin mRNA is first bonded to the opened plasmid DNA through formation of base pairs between the poly(A) at the 3'-end of the mRNA at a dT segment added to the 3'-end of the plasmid DNA. Reverse transcriptase is then used to add a globin cDNA to the 3'-end of the dT segment. Through appropriate steps this globin cDNA can then be built into the recircularized plasmid ring.] The resultant clones can then be screened for those containing the globin DNA by assay of the ability of their plasmids to hybridize with labelled globin mRNA. Simplified procedures have been developed for screening clones in situ, whenever a labelled RNA or DNA that will hybridize to the plasmids of the desired clone is available (20, 78-80). "Sib selection" procedures that make use of combinatorial tactics can be used to reduce markedly the task of screening large numbers of clones for those with the desired characteris tic indicating the presence of a particular cloned DNA (20, 48). Com plementation assays can be used to select clones that provide a particular function in the transformed cell [as for DNA ligase (47), tryptophan (81), arabinose (81), or histidine (48)].

SOME APPLICATIONS In general, it is now established that foreign DNA incorporated into bac teria as inserts in plasmid or viral DNAs can be maintained without major change of nucleotide sequence over hundreds of bacterial generations, even


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429

in the absence of any selective process (19, 71, 82, 83). Functional expres sion in E. eali of prokaryotic DNA from species that are not known to exchange DNA with E. eoli seems to occur readily (120, 121). Transcrip tion of eukaryotic DNA in prokaryotes has been demonstrated (19, 20, 23), as has transcription of prokaryotic DNA in eukaryotes (106). Translation of eukaryotic DNA in prokaryotes also occurs (23), although its relation to normal translation of such DNA remains to be clarified (116). In at least one instance (48) complementation of a deficient prokaryote function by the presence of eukaryotic (yeast) DNA in the cell has been established.

Prokaryotes PLASMID STRUCTURE AND FUNCTION By mixing together all of the EeoRI fragments of a plasmid of interest (F' lae, R6-5) with a plasmid derived EeoRI fragment that is incapable of replication by itself but carries a specific drug resistance marker (ampicillin, kanomycin), and then anneal ing and ligation, a new set of recombinant DNA rings can be produced. If these are used to transform an appropriate host, which is then grown in a medium containing the specific drug, growth will be possible for only those clones containing a plasmid that is composed (at the minimum) of the replicator region of the plasmid of interest coupled to the fragment coding for drug resistance (84, 85). In this way the EeoRI fragment of F'lac and of R6-5, requisite for replication of each plasmid, has been identified and isolated. These studies have demonstrated that one DNA region of 5-7 X 106 daltons carries the replication genes, together with the genes determin ing plasmid incompatibility and the elements determining stringent or re laxed replication. A hybrid plasmid containing two independent replicator regions, one from pSC101 (stringent) and one from ColEl (relaxed), normally uses the ColEl replicator (86) but can use the pSC lOl replicator when the other is not functional (absence pol I) (31, 86). The fact that such a plasmid is incompatible with other ColEI or other pSC101 plasmids argues in favor of a negative control of plasmid DNA synthesis by means of a replicator specific, diffusible plasmid gene product. [That one replication site in a plasmid may not always be dominant over the other is, however, demonstrated by experiments with a naturally occur ring R plasmid containing two independent origins of replication (87). In this case either origin may be used (never both simultaneously), although external circumstances may alter the relative frequency of their use.] The F plasmid (94.5 kb) is cleaved by EeoRI into 19 fragments. In an extensive analysis (88-90) fragments of F derived by partial EeoRI diges tion have been cloned with the pSC101 vehicle. By analysis of the complete EeaRI digest of the cloned DNAs bearing the partial digestion fragments,


430

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the physical order of the fragments has been determined. By investigation of the properties of transformed cells carrying portions of the F plasmid, and by determination of the ability of the specific hybrid plasmids to com plement specific genetic defects in the original F plasmid, the genetic map of F has been superimposed on the physical map. In this way the locations of the origins for vegetative and transfer replication have been ascertained, as have been the locations of the genes for replication and incompatibility, for pilus formation, for susceptibility to male-specific phage infection, for exclusion of female-specific phage, for surface exclusion (reduction of DNA transfer from one F"carrying cell to another), for immunity to lethal zygosis (reduced viability after excessive mating), and for various control functions. Flagellar assembly and function in E. coli involves at least 20 chromoso mal genes. Most, if not all, of these can also be carried on a large plasmid, F1338. EcoRI digestion of F1338 yields 33 fragments. By cloning these fragments with the Agt-AC vehicle and using the resultant clones to comple ment mutant strains defective in one or another of the flagellar genes, it is possible to map these genes on the EcoRI fragments and to confirm the previously established gene order (91). By studying the effects of deletions in the cloned DNA fragments upon the expression of the flagellar proteins made in recombinant-infected, ultraviolent-treated cells, it has been possible to identify the gene (hag) that codes for flagellin (the major flagellar protein) and to demonstrate that expression of the hag gene depends upon the presence of the flal gene product. Genes coding for the synthesis of enterotoxins are carried in large plas mids of some E. eali strains. By cleaving one of these with EeaRI, cloning the fragments with the pSC10 1 vehicle, and assaying the tetracycline-resist ant transformant for toxin production, one clone was obtained (out of 72 assayed) that produced the ST (heat stable) toxin. The transforming plas mid contained a 5.7 X 106-dalton DNA fragment, which could be further transferred, together with the toxin-synthesizing capacity, to the RSF2124 vehicle (92). The entire chromosome of E. coli is about 4 X 103 kb and (assuming quasi-random nucleotide sequence) EeaRI cleavage sites would be spaced on the average about 4 kb apart. Such a complete EeaRI digest of E. eali chromosome would produce about 1000 fragments, all of which could be cloned. Given an appropriate selection procedure and assuming the gene (or operon) had not been fragmented by the cleavage, clones bearing any desired genes could be isolated (81). In this way a 9.1-kb fragment bearing the gene for E. coli ligase (47), an l1-kb fragment bearing the Irp operon (81), and the 14-kb fragment bearing both the ara and leu operons (81) have been isolated. Operons containing E. COLI CHROMOSOME

431


RECOMBINANT DNA

an EcoRI site (e.g. lac) might be isolated by another restriction enzyme or by a shearing method. Similarly, a 2 X 106-dalton EcoRI fragment of E. coli DNA on a pSClOl vehicle can, in a transformant, revert several of the symptoms of capR mutants (excess colanic acid production, killing by plating in complex medium after growth in simple medium) but not some of the other charac teristics of these mutants (high sensitivity to ultraviolet and ionizing radia tion and to nitrofurantoin) (93): The original mutation is evidently more complex than initially presumed. In the sea urchin (Strongylocentrotus purpuratus) the histone genes are repeated about 400-500 times and appear to be clus tered into a few chromosomal sites. They comprise approximately 0.250.3% of the genome. By cloning an EcoRI digest of sea urchin DNA and employing sib selection techniques to select out those clones carrying DNA hybridizable to histone mRNA, it wa� determined that two EcoRI sites define a 6.6-kb repeat period of histone genes and provide two fragments of 2.0 and 4.6 kb (20, 94-97; see also 98, 99). By further degradation of these fragments with other restriction enzymes (HindIII, BamHI, Hha I, and SalI), by hybridization of fragments to fractionated histone mRNA, and by partial DNA sequence analysis of the ends of plasmid DNA-cut at sites known to lie within a histone gene [with subsequent correlation of the DNA sequence with known histone amino acid sequences (96)]-the order and locations of the five histone genes and intervening spacers within the repeat period have been determined (95) (Figure 5). The spacer regions are not homologous and are not related to DNAs found anywhere else in the sea urchin genome. All of the genes are transcribed from the same DNA strand.

EUKARYOTIC DNA

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(95).

Genetic map of histone DNA region of S. purpuratus. Adapted from


432

SINSHEIMER

When transferred into minicells, the histone gene sequences of the plas mid are transcribed. In X laevis there are about 20,000 copies of the gene for the oocyte ribosomal SS RNA (120 bp) clustered at the telomeres of most of the IS haploid chromosomes (39, 70). There is one HindIII cleavage site in the SS DNA region, which defines a repeat unit of approximately 700 base pairs. This can be cloned in pSC101 and the fragment further analyzed by Hae III digestion, which cleaves it at three sites. In this way the repeat unit has been mapped to contain, in the S'�3' direction, an A-T-rich region of variable length from 320 to S lO base pairs, the SS gene (120 base pairs), and a G-C-rich region of ISO base pairs. The A-T-rich variable region appears to be composed of integral num bers of a repeat unit containing 14 or 15 base pairs. The lengths of adjacent variable regions (from studies of clones of partial digests of the 5S DNA) have the same standard deviation as the lengths of all repeats; this result argues against the master-slave or contraction-expansion models for the formation of repeat gene sequences and in favor of a nonreciprocal recombi nation mechanism (39). Also in X laevis there are about 500 copies of the DNA coding for the 28S and 18S ribosomal RNAs, clustered in one chromosomal region at the nucleolar organizer. In the oocyte, however, thousands of copies of "ampli fied" ribosomal DNA molecules are made to serve as templates for rRNA synthesis. There are two EcoRI cleavage sites within the ribosomal DNA region (19, 71) yielding two fragments that have been cloned with the pSC101 vehicle. One, which is 3.0 X 106 daitons, codes principally for the 2SS RNA. The other is of variable length (3.S-7.S X 106 daltons) and codes for most of the ISS RNA, the "transcribed spacer" adjacent to the ISS RNA, and a variable nontranscribed spacer region (71). Heteroduplex analysis of the nontranscribed spacer region from different clones indicates that it is com posed of two constant regions interspersed with two variable regions. In the chromosomal rDNA the lengths of adjacent variable regions are highly variable, even within one anima:. The distribution of spacer lengths does vary from individual to individual (72). The spacer regions in any one molecule of amplified rDNA are uniform in length, suggesting that the amplified DNA is produced by a "rolling circle" process from one repeat unit of the chromosomal rDNA. The distri bution of spacer lengths among the amplified rDNA molecules does not parallel that of the chromosomal rDNA in the same individual, suggesting a selection of those rDNA segments that will be amplified (72). Drosophila DNA fragments obtained by shear (82) or by digestion with EcoRI (22, 73, 1(0), BAMHI (21), or Sall (21) have been cloned with appropriate vehicles. Different fragments representative of different aspects


RECOMBINANT DNA

433

of the genome have been obtained. Several contain "single copy" DNA sequences present once in the genome; these cloned DNAs can be shown by in situ hybridization to derive from specific chromomeres of the Drosoph ila genome. Another appears to contain the rDNA genes and hybridizes to the nucleolar organizer (22, 73). Another clone represents a sequence re peated about 90 times in the genome and hybridizes to about 15 different chromomeres as well as to the chromocenter (82). In principle, by cloning the fragments produced by the use of two or more restriction enzymes, a physical map of the entire Drosophila genome could be made. About 50,000 chimeric clones would be needed (21). Studies of clones of repetitive DNA sequences from sea urchin DNA indicate that neighboring repetitive sequences are unrelated and that the variability (approximately 10% average mismatch) within an individual family of a repetitive DNA sequence is similar to that observed for the overall set of repetitive DNA families (101). Mouse mitochondrial DNA is cleaved into three fragments by EcoRI (83.). These have been cloned in various combinations and spatial relation ships with the pSClOl vehicle (23, 83). Heteroduplex analysis by both electron microscopy and thermal denaturation measurements indicates that the mitochondrial DNA sequences are maintained with high fidelity over more than 125 cell generations (in HBI 01, a recA- strain). The mitochondrial DNA in eukaryotic cells normally has a small number (10-30) of ribonucleotides incorporated into the covalently closed ring and is therefore alkali-labile. However, plasmids incorporating mtDNA do not have ribonucleotides (23, 83). The D-Ioop structure characteristic of repli cating mtDNA is not seen; rather, the chimeric plasmids are observed to use the pSClO1 origin. Transfer of chimeric plasmids containing mtDNA into certain minicell producing strains resulted in aberrant plasmid structures, but transfer to the strain X1274 was satisfactory (23). In the minicells budded off from this strain, RNA-largely complementary to the L strand of the mtDNA-was produced, independent of the orientation of the mtDNA within the plasmid ring. This result suggests the use of a promoter within the mtDNA itself (23). (Within the eukaryotic cell the H strand of mtDNA is preferentially transcribed.) Four small peptide components ranging from 2500 to 5000 daltons were observed to be attributable to the presence of the mtDNA in the plasmid (23). These do not correspond to known mitochondrial proteins. Closed circular DNA rings 2.0 p,m in circumference (6.8 X 1 06 daltons) of unknown function are found in yeast (Saccharomyces cere visiae) (102). Renaturation kinetics indicate that all of the rings have the same base sequences. They have two EcoR l cleavage sites and three Bin YEAST


434

SINSHEIMER

d-3 cleavage sites (103). By cloning the partially EcoRI-digested (to provide full-length linear molecules) yeast episomes with the peRl vehicle, it has been demonstrated that there are two classes of rings, which differ by the inversion of a 0.8-fLm sequence located between two 0.23-fLm segments comprising a noncontiguous reverse duplication sequence. The most definitive evidence for functional expression of the eukaryotic gene in a prokaryote is derived from complementation experiments (48). A bacterial mutant lacking hisB function (imidazole-glycerol-phosphate dehydratase) and believed to be a deletion (revertants are never seen) was lysogenized with A-carrying EcoRI fragments of the yeast genome. Trans formants that had lost the requirement for histidine were selected. If the phages are "cured" from the strain, the histidine requirement is regained. Lambda phage derived from a transformant can similarly relieve a hisB gene defect in other E. coli strains. The A phage derived from the transformed strain has a 10.3 kb-DNA insert. RNA complementary to this DNA insert will hybridize with a 1001 l -kb fragment of yeast DNA, demonstrating that the lysogenized cell does carry a piece of yeast DNA. Transformation works just as well if the yeast DNA is inserted into A in the opposite orientation, suggesting that the origin of transcription is in the eukaryotic DNA. It is of some interest that no similar prototrophic trans formants could be obtained with A phage carrying EcoRI DNA fragments, from E. coli, Klebsiella strains, or Dictyostelium-presumably because of the presence of an inactivating EcoRI cleavage site in the histidine genes of these organisms (48). By coupling a DNA fragment to a well-defined bacterial promoter such as that for lac (104, 105) before transformation, transcription could be assured and some of the ambiguities inherent in experiments concerned with gene expression might be removed.

Eukaryotes Portions of A bacteriophage DNA have been coupled to monomeric seg ments from SV40 reiteration mutants (25, 54, 55), or have been inserted into SV40 genomes from which portions of the late genes have been deleted (27), and then propagated in eukaryotic cells with the aid of helper virus. It has been shown that such A segments retain their accustomed cleavage sites for restriction enzymes and the binding site for A repressor (54). The A insert did not appear to be transcribed (27), although this may be attributable (106) to the dA:dT joints used in its construction. An E coli suppressor gene coding for tRNATyrsu+HI has been inserted, in unique orientation, into a deletion in the late genes of SV40 (26, 106) and propagated with the aid of helper virus. The prokaryotic DNA fragment appears to be transcribed within a long SV40 transcript (late mRNA), and


RECOMBINANT DNA

435

the transcript is comparably stable to ordinary SV40 mRNA. However. no mature. suppressor tRNA capable of being acylated could be found, pre sumably because of the lack of the tRNA-processing enzymes found in E. coli. Drosophila DNA fragments can be replicated in monkey cells in the same way (25), as can globin genes or other fragments of eukaryotic DNA for analysis of their regulation and structure (106).

SAFETY The problem of hazard in research with organisms containing recombinant DNA is extensively discussed in the well-known NIH guidelines (107) and elsewhere (108). The use of "crippled" mutant strains (108, 10 9) with reduced likelihood of survival outside of laboratory conditions is required for certain types of experiments. Other techniques that simplify laboratory operations of hybridization (79) or minimize the handling of live cells (110) could reduce the potential for escape of such organisms from the laboratory proper. NOTE ADDED IN PROOF More recent papers (August-December 1976) demonstrate the continuing rapid advance in this field. Plasmids have been described that contain the lac promoter and operator (122, 123). By insertion of foreign DNA (Xenopus laevis rDNA or AcI repressor DNA, respectively) so that its expression is regulated by the lac region, extraordinarily large synthesis of the desired transcription or trans lation product can be obtained. The construction of plasmids bearing a chemically synthesized lac operator region has also been described (124, 125). Under specific denaturing conditions, the dA:dT joints used in some procedures to insert the foreign DNA can be rendered specifically suscepti ble to endonuclease SI> thus permitting specific excision of the inserted fragment (126). An additional method for synthesis of globin DNA and its insertion into plasmids by dA:dT joints has been described (127). General methods for the cloning of fragments representing the entire genome of E. coli have been described (128, 129), as has the construction of specific clones bearing the structural gene of E. coli exonuclease I (130). the EcoRI fragment.s of A (131), and calf thymus satellite DNA I (132). The structure of the sea urchin histone gene cluster has been further elaborated (133-136) by the use of cloned fragments together with other techniques of nucleic acid analysis. The structure of the 2-fLm episome found in yeast has been elucidated by restriction fragment analysis, cloning, and electron microscopy (137, 138).

436

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