volume 9 Number 13 1981 Nucleic Acids Research

Rapid and efficient cosmid cloning

D.Ish-Horowicz and J.F.Burke

Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole Lane, London NW7 IAD, UK

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Received 18 May 1981

ABSTRACT
We present a procedure for cosmid cloning that allows rapid and
efficient cloning of individual DNA fragments of between 32kb and 45kb. By
appropriate treatment of the cloning vector, pJB8, we make left-hand and
right-hand vector ends that are incapable of self-ligation but which accept
dephosphorylated insert DNA fragments. The inserted fragments are generated
by partial digestion with Mbol or Satt3A and are dephosphorylated to prevent
ligation and insertion of non-contiguous fragments. The method eliminates
the need to size the insert DNA fragments and prevents formation of clones
containing short or multiple inserts, lpg of target Drosophila DNA gives
about 5xlO5 clones, with an average insert size of 38kb. We also describe
a rapid and efficient method for preparing plasmid and cosmid DNA.

INTRODUCTION
A major advance in the study of eukaryotic gene organisation has been
the development of methods for the purification and propagation in bacteria
of individual DNA fragments. Two major approaches have been adopted: (a)
cloning of double-stranded cDNA copies of mRNA sequences into plasmids;
(b) insertion of genomic DNA fragments into bacterial vectors. The former
technique provides an immediate enrichment for expressed sequences but
the latter permits the study of surrounding (and internal) control regions
that are not necessarily represented in the mature mRNA. Initially,
bacterial vectors (1) were used for genomic cloning but bacteriophage vectors
have become more popular because they accept larger DNA inserts (approx.
15-2Okb) and can be efficiently packaged in vitro into infectious phage
particles (2,3).
Recently, plasmid vectors that can be packaged into \ phage heads
("cosmids") have been constructed (4). Cosmids accept larger inserts than
A phage (3O-45kb) and can be efficiently introduced into host bacteria
through in vitro packaging. Despite these apparent advantages, cosmids
have not been much used, possibly because various side reactions that

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reduce efficiency have been associated with their preparation. Even with
sized chromosomal DNA fragments, cosmids can be recovered that contain non-
contiguous DNA fragments ligated to form a single insert (5), leading to a
distorted view of the original genomic organisation. Although the problem
can be overcome by dephosphorylating the inserted DNA ("target"), this
method i s very sensitive to the exact r a t i o of target and vector DNAs (6)
and tends to give smaller inserts because of vector-to-vector ligation.
In this paper, we present a method for cosmid cloning that overcomes

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both the above problems. Our procedure inserts unique DNA fragments of
about 3O-45kb into cosmid vectors with high efficiency, with none of the
above side reactions. I t also avoids the need to size the target DNA
fragments. In addition, we describe quick and convenient protocols for both
analytical and preparative DNA extractions of plasmids and cosmids.

MATERIALS AND METHODS

Homeri (amp r ) was c o n s t r u c t e d a n d d o n a t e d by W. Chia (7) . I t i s derived
from t h e d i s a b l e d p l a s m i d pAT153 (8). Sail a n d T4 DNA l i g a s e was a g i f t
of Dr. A. udvardy. Unless otherwise indicated, enzymes were/supplied by
New England Biolabs Inc. and used according to t h e i r recommendations.
Nitro cellulose f i l t e r s were purchased from Sartorius GmbH.
Preparation of pJB8. Homeri DNA (lug) was cut with KJORI and the staggered
ends f i l l e d in with T4 DNA-polymerase (20U)in the presence of a l l 4 deoxy-
triphosphates. Synthetic BamHI linker (2.5ug; Collaborative Research) was
phosphorylated with polynucleotide kinase (2U) and ligated to the linearised
Homeri DNA with T4 DNA l i g a s e (0.5U) and RNA ligase (7U) for 20 h a t 16°C.
After phenol extraction, the mixture was digested with BamHI (20U) for 1 h
a t 37 C, and the digested linker removed by chromatography on Sephadex G-150.
The DNA was c i r c u l a r i s e d with T4 DNA ligase and used to transform Ca-treated
HB101. pJB8 was one of the transformants and proved to have a BamHI s i t e
in the position of the ECORI s i t e of Homeri (Fig. 1) .

All the buffer changes and removal of phenol were achieved by spinning
the reactions through Sephadex G-50 (O.8ml) in a lml syringe b a r r e l packed
with polymer wool (9).
Preparation of vector for cosmid cloning. pJB8 (12)jg) was digested with
either / / i n d l l l or Sail (Fig. 2) . Each reaction was terminated by incubating
for 10 min a t 70 with 0.1% diethylpyrocarbonate (Sigma) and spun through
a Sephadex G-50 column equilibrated with H2O. The linear plasmids were
each dephosphorylated with calf i n t e s t i n a l phosphatase (Boehringer; 0.1U)

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for 30 min at 37°C and the enzyme inactivated for 1 h a t 70°C (10). The
efficiency of l i n e a r i s a t i o n and dephosphorylation was checked by l i g a t i o n
and transformation into HB101 and found to be better than 99.5%. (This i s
necessary to eliminate the formation of tandem vectors during l i g a t i o n ) .
The vector DNA was cut with BarrtRI and i t s a b i l i t y to r e l i g a t e was checked
before use. An equimolar mixture of the Sam-cleaved dephosphorylated
tfircdlll-treated pJB8 and Bom-cleaved dephosphorylated SaZ-l-treated pJB8 is
used for cloning (Fig. 2) .

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Target DNA preparation. Drosophila DNA was made from adult f l i e s (11).
About 2CHig DNA (from 1O0 flies) was digested with Mboi in 2O0yl lOmM
Tris pH 7.5, lOmM MgCl,, 20mM NaCl, lOmM 2-mercaptoethanol for 10 min a t
37 C with occasional gentle mixing. The reaction was stopped by heating
to 70 C for 12 min. The extent of the above reaction was assayed by e l e c t r o -
phoresis on a 0.2% agarose gel, supported by a bed of 0.8% agarose. The DNA
was dephosphorylated for 4O min at 37 C with calf i n t e s t i n a l phosphatase
(O.25U), and the phosphatase was inactivated a t 70 C for 1 h. The nucleic
acid was precipitated with ethanol and redissolved in 50pl TE (lOmM Tris
pH 8.0, lmM EDTA).
Ligation and in vitro packaging. Target DNA was ligated with an excess of
the vector mixture (Fig. 2 ) . We used 3.5yl of vector DNA (24O(jg/ml) plus
l y l of target DNA in 8pl for 4 h a t 22 C. In vitro packaging was by a
modification due to V. P i r r o t t a (personal communication) of the method of
Sternberg et at. (12) using s t r a i n s BHB2688 and BHB269O (13), and gives
^5xlo" pfu/yg X DNA. In summary, a freeze-thaw lysate i s made from
BHB2688 and a sonic extract from BHB269O and the DNA packaged in a
putrescine-spermidine buffer. The packaged cosmids are diluted in phage
buffer and stored at 4 C over CHC1,.

Infection of host b a c t e r i a . HB101 {hsdR rec& ) i s grown in L Broth

containing 0.4% maltose u n t i l j u s t s a t u r a t i n g . After harvesting, the
culture i s stored 2-fold concentrated in lOmM MgSO.. I t i s important that
i t s vecA phenotype be tested by assaying UV s e n s i t i v i t y . The cosmids (in
up to 20yl) are added to lopl HB101, and incubated at 37°C for 10 min.
lOOyl L Broth i s added and incubated for a further 30 min at 37°C. The
mixture is spread on a 9cm dish of L Agar containing lOOyg/ml ampicillin.
Single colonies were picked for estimating i n s e r t s i z e . For large scale
screening of cosmid clones, the volumes were doubled and a 14cm dish used.
DNA extraction from plasmid or cosmid c u l t u r e s . We have devised a rapid and
efficient method of making DNA from analytical or preparative plasmid and

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cosmid cultures by combining the methods of Davis et at. (14) with

the alkaline l y s i s method of Birnboim and Doly (15) .
Analytical DNA extractions ("miniprep"). lml of a saturated culture i s
harvested in a 1.5ml microcentrifuge tube (20 sec in a Beckman Microfuge) ,
resuspended in lOOpl 50mM Glucose, 25mM Tris pH 8.0, lOmM EDTA (Solution I)
and incubated for 5 min a t 22°C. 200ul 0.2N NaOH, 1% SDS (Solution I I ;
made weekly from a ION NaOH stock) is added, mixed gently and put on ice
for 5 min. 150yl precooled 5M KDAc pH 4.8 (Solution I I I ; final

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concentration = 3M K0AC+2M HOAc) i s added, mixed gently, and after 5 min on
ice, the precipitated protein, dodecyl sulphate and chromosomal DNA removed
by centrifugation for 1 min. 2 vol. EtOH i s added to the supernatant,
incubated for 2 min at room temperature, and the nucleic acid i s
precipitated with a 1 min centrifugation. The p e l l e t i s washed with 70%
EtOH, dried, and taken up in 50pl TE. 2pl portions (containing 100-500ng
of DNA) are digested with restriction enzymes in buffers containing 2yg/ml
boiled RNAase and analysed on horizontal agarose gels in a Tris-Borate-EDTA
buffer. Cosmid DNA i s purified from 5ml cultures using double the above
volumes and p r e c i p i t a t i n g the nucleic acid with 0.6 vol. iso-propanol.
Preparative e x t r a c t i o n s . For 1 l i t r e cultures (or less) : The protocol for
cosraid cultures i s scaled up 40-fold and the DNA purified by isopycnic
centrifugation in a CsCl-ethidium bromide gradient. (Residual acid should
be neutralised with t r i s - b a s e before centrifugation).

We find that yields of plasmid and cosmid DNA are superior to Triton-
l y s i s methods and can be up to lOmg DNA/litre. Chloramphenicol amplified
cultures can be similarly extracted.
High-density plating of cosmid clones. We have modified the screening
method of Hanahan and Meselson (16) . The bacteria are spread on a thick
well-dried L Agar plate (+lO0pg/ml Ampicillin) and the colonies grown to
diameters of 0.2-O.4mm. A dry nitrocellulose filter (not s t e r i l i s e d ) is
carefully layered onto the plate and allowed to wet. After marking the
plate and f i l t e r with a needle, the f i l t e r i s removed - most of the bacteria
should adhere to the f i l t e r . The colonies on the master-plate are grown
up again and the plate stored at 4 C in a sealed bag. The replica filter
i s placed (colonies up) on a chloramphenicol plate (2OOpg/ml in L Agar) and
a second dry f i l t e r carefully placed on top to form a sandwich. The second
filter i s marked and the sandwich incubated overnight at 37 C. The sandwich
i s removed and worked up as follows: 30 sec in 0.5N NaOH; 2M NaCl; >1 min
in 0.5M Tris pH 7.0; 3M NaCl. The 2 f i l t e r s are peeled apart to give 2

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duplicate f i l t e r s and washed s e p a r a t e l y i n 2xSSC, 0.1% SDS (removing

b a c t e r i a l d e b r i s with a t i s s u e ) and 2xSSC ( 5 ) . (SSC i s 15mM t r i - s o d i u m
citrate, 0.15M NaCl). They a r e b l o t t e d dry and baked a t 80°C. Master
p l a t e s can be s t o r e d up t o 6 weeks. I f they a r e not allowed t o d r y , the
filters can be screened with a s u c c e s s i o n of p r o b e s , removing previous
h y b r i d i s a t i o n with 0.1M NaOH (10 min a t room t e m p e r a t u r e ) . Hybridisation
t o n i c k - t r a n s l a t e d probes i s as d e s c r i b e d by J e f f r e y s et al. (17).

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RESULTS

Cosmid v e c t o r pJB8
We have modified t h e 5.3kb cosmid v e c t o r Homer! ( g i f t of W. Chia) by
i n t r o d u c i n g a s y n t h e t i c BamHI l i n k e r i n t o i t s ECORI site (Fig. 1, see
M a t e r i a l s and Methods). This allows i n s e r t i o n of DNA fragments generated
by p a r t i a l cleavage with Mbol or Sau3h. The BamHI l i n k e r has recreated
f l a n k i n g EcoRI s i t e s so t h a t EtooRI e x c i s e s the i n s e r t e d DNA from hybrid
cosmids (Fig. 1) .
F i g . 2 i l l u s t r a t e s our cloning procedure t h a t allows the
dephosphorylation of both t a r g e t and v e c t o r in such a way as to overcome
the s i d e - r e a c t i o n s of: (a) cosmids c o n t a i n i n g non-contiguous inserts, (b)
cosmids c o n t a i n i n g m u l t i p l e v e c t o r s and m u l t i p l e i n s e r t s . If we cut pJB8
with / / - i n d l l l , dephosphorylate, and then c u t with BamHI, we make a left-hand
v e c t o r cos fragment t h a t cannot l i g a t e in tandem b u t which accepts cle-
phosphorylated t a r g e t DNA. Similarly, c u t t i n g pJB8 with Sail, dephos-

Bam Hind m

Pst

Figure 1. R e s t r i c t i o n map of pJB8. The thickened r e g i o n r e p r e s e n t s the

B g l l l fragment from A t h a t c o n t a i n s the cos s i t e ( 7 ) .

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ligate to
'dephosphorylated
target DNA
(partial Mbol digest)

target,, <B)

-37-50 kb
ONLY PACK AGE ABLE MOLECULES

Figure 2. Cosmid cloning method

phorylation, and c u t t i n g with BamHI y i e l d s a right-hand v e c t o r COS fragment.

Neither d i g e s t alone can be used a s a c l o n i n g vector as only molecules with
2 COS s i t e s s e p a r a t e d by 37-5Okb (18) can be packaged, but an equimolar
mixture of t h e two d i g e s t s w i l l allow packaging of a p p r o p r i a t e l y sized
t a r g e t fragments (Fig. 2) . Dephosphorylation of the l i n e a r vector
molecules prevents the formation of tandem v e c t o r s and e l i m i n a t e s background
colonies lacking inserts.

Target DNA i s made by p a r t i a l Mbol d i g e s t i o n of l a r g e genomic DNA to a

mean s i z e of 35-45kb followed by dephosphorylation with c a l f intestinal
phosphatase. The frequency of Mbol s i t e s (about every 25Obp on average)
means t h a t such fragments a r e e s s e n t i a l l y random. P a r t i a l d i g e s t i o n w i t h
restriction enzymes recognising a 6bp sequence can lead to s e l e c t i v e loss
of specific fragments ( 6 ) . Ligation of an excess of vector-mixture to
dephosphorylated t a r g e t DNA gives only one kind of packageable molecule

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(Fig. 2) : those containing the left-hand Hindlll-BamHI cos fragment, a

target fragment of 32-45kb and the right-hand Sall-BanMl cos fragment.
Dephosphorylation of both vector and target DNA eliminates a l l the side
reactions described above. Moreover, because of the size-specificity of the
packaging reaction, i t i s not necessary to purify the correct size-fraction
of the target DNA from the p a r t i a l digest.
We have packaged Drosophila DNA prepared from adult flies and made cosmid
"libraries" by infecting HB101. We recover about 5x10^ clones/yg of

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Drosophila DNA. (The estimate of DNA concentration i s only approximate) . As
a guide, we recover about 240,000 clones from 2 fly-equivalents of DMA, an
efficiency of about 105 clones/adult fly.
Characterisation of the cosmids•
We have assayed the integrity of our cosmids by analysing "minipreps"
of randomly picked clones (see Materials and Methods). Fig. 3 shows EcORI
digests of DNA from 16 clones. The EcoRI s i t e s that flank the s i t e of
insertion in pJB8 (Fig. 1) mean that EooRl cuts out a characteristic vector
band and that the insert can be sized by summing the remaining bands.
Fig. 3 shows that each clone has only a single vector fragment and hence
only a single i n s e r t . (Where the vector band i s overly intense, it
separates into 2 bands on a 0.3% gel)..

The clones in Fig. 3 have an average insert-size of 33kb ± 3kb (n=16),

somewhat smaller than expected. This may be due to our failure to check
the veoA phenotype of the HB101 host as recombination within the insert
would give r i s e to smaller cosmids. A subsequent experiment using a pure
reeA HB1O1 stock had inserts sized 38.8kb + 3.7 kb (n=14). This (32kb-45kb)
is the size range expected from the specificity of packaging and suggests
that cosmids are stably propagated.

DISCUSSION
Cosmid cloning allows the isolation of large regions of the genome i n t a c t .
However, previous methods o£ preparing cosmids have tended to give
unsatisfactory clones that were either shorter than expected, or had
potentially misleading non-contiguous i n s e r t s . The cloning protocol
presented in Fig. 2 overcomes both side reactions. I t is designed for the
pJB8 vector but can be adapted for other cosmid vectors. Any two
restriction enzymes that give a left-hand and a right-hand cos fragment
will suffice, e.g. in our scheme, Clal could be substituted for Hindlll and
Aval can be substituted for Sail. As sizing of the i n s e r t DNA i s avoided,

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Figure 3. EcoRI-digests of cosmid DNA. 2pl of a n a l y t i c a l - s c a l e DNA
p r e p a r a t i o n s (Materials and Methods) from randomly picked cosmid clones were
d i g e s t e d with EaoRX and run on a 0.8% agarose g e l . Tracks containing marker
DNA fragments are i n d i c a t e d by the hollow arrowheads. The pJB8 band i s
i n d i c a t e d by t h e s o l i d arrowhead. Larger DNA fragments were s i z e d an a 0.3%
agarose g e l .

lowered e f f i c i e n c y of cloning due to l o s s or breakage of the t a r g e t DNA i s

minimised. Moreover, t h e complex manipulations necessary when using
sheared DNA fragments as t a r g e t (19) are avoided by the use of Mbol d i g e s t i o n
t o give e s s e n t i a l l y random fragments.
The lack of s i d e - r e a c t i o n s and e l i m i n a t i o n of background transformants
mean t h a t cosmid c l o n i n g i s an a l t e r n a t i v e to phage X for the recovery of
chromosomal DNA fragments. The choice between the two w i l l depend on
circumstances, b u t we note some advantage of using cosmids. Fewer colonies
need be s c r e e n e d . The high density screening method of plasmids (16) AS
fully a p p l i c a b l e to cosmids so t h a t a complex e u k a r y o t i c genome can be
screened on a few f i l t e r s . For Drosophila, 4300 c o l o n i e s a r e a genome-
e q u i v a l e n t and the p r o b a b i l i t y of d e t e c t i n g a s p e c i f i c sequence i s 0.95 if

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13,000 clones are screened. The packaged cosmids are stable refrigerated
for many months and our preliminary experiments suggest that they can be
quick frozen and stored at -70°C in the presence of 10% glycerol without
loss of infectivity. Using a simplification of the Hanahan-Meselson colony
screening method (Materials and Methods), we have recovered several cosmid
clones derived from the 87A7 and 87C1 heat-shock loci and from the 63F
ecdysterone-induced locus (unpublished results). They have inserts sized
between 32kb and 38kb and appear to propagate stably (unpublished

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observations). The high efficiency of cosmid production allows direct
screening of recombinants, avoiding the loss of poorly growing clones that
occurs during amplification. The clones can be stored frozen on filters
(16), or freshly screened as required. Because of the larger insert,
cloning of large chromosome regions ("walking") can be more rapidly
achieved than with X. Single cosmids can give up to 45kb of DNA and it is
easy to prepare end fragments as probes for walking because the insert can
be excised exactly with EcoRl.
Our experience has been that cosmids can propagate without noticeable
instability. Thus, we find only appropriate-sized inserts and no evidence
of reduction in size during propagation. The use of a recA host is clearly
important and we can say that most cloned sequences appear stable. Moreover,
clones containing up to four copies of SV40 (7)or two 6 globin sequences (5)
appear to propagate without excision. Instability during propagation in
phage X has been reported for duplicated a-globin or polyoma sequences
(20,21,22) .
The analytical and preparative DNA isolation procedures described give
high yields of both cosmid and plasmid DNA. It takes about 60 min to make
DNA from 2O cultures suitable for restriction enzyme digestion. After phenol
extraction and gel filtration, the DNA can be used for subcloning or in situ
hybridisation to polytene chromosomes (unpublished observations). The same
method can be used to purify any covalently closed circular DNA and has been
used to prepare extrachromosomal circular molecules from Drosophila tissue
culture cells (A.J. Flavell and D. Ish-Horowicz, manuscript in preparation).
A final advantage of the use of cosmids lies in the desire to achieve
in vivo expression of cloned DNA. Experiments on the nuclease sensitivity
of native chromatin suggest that differences in chromatin configuration of
active and inactive genes extend outside the directly transcribed regions
(23). If correct chromatin packaging requires the flanking regions, the
integrity of large DNA fragments may be necessary for correct expression.

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Indeed many eukaryotic genes extend over long regions due to their
interruption by non-coding intervening regions. In such cases, correct
in vivo expression of cloned DNA may demand the use of the long DNA
fragments that are only obtainable with cosmid cloning.

ACKNOWLEDGEMENTS
We thank Dr. A . J . Leigh Brown who a s s i s t e d i n the i n i t i a l s t a g e s of t h i s
work. We a l s o thank our colleagues for h e l p f u l c r i t i c i s m of t h e manuscript.

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We are g r a t e f u l t o Dr. w. Chia f o r the g i f t of Homer I, and D r s . F.Grosveld
and H-H.M. Dahl f o r d i s c u s s i o n on cosmid s c r e e n i n g methods.

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