A cosmid vector for systematic chromosome walking

Gene, 49 (1986) 9-22

9

Elsevier GEN 01819

A cosmid vector for systematic (Recombinant polymerase;

DNA; cloning; hybridization

chromosome walking

restriction

maps; cos mapping;

promoter;

phage I; phosmids;

SP6 and T7 RNA

probes)

S.H. Cross and P.F.R. Little* Institute of Cancer Research: Royal Cancer Hospital, Chester Beatty Laboratories, Fulham Road, London SW3 6JB (U.K.) Tel. (Ol)-352-8133 (Received

July 4th, 1986)

(Revision

received

(Accepted

September

September

lst, 1986)

24th, 1986)

SUMMARY

We describe the construction polymerase promoter sequences

of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA that are oriented towards and immediately adjacent to Hind111 and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment

of steps in a cosmid

walk.

INTRODUCTION

large (greater than 100000 bp) regions of complex genomes in an ordered array of overlapping recom-

(a) Chromosome walking

binant DNA clones. A walk is comprised of a series of steps and a step consists of the identification of DNA fragments that are located at the extreme left (FL) or right (FR) end of a starting clone (C). FL and FR are then used as probes to screen a library of sequences and isolate new clones that contain FL or FR and additional DNA that is located to the left and right of C. The walk then proceeds in a linear fashion by isolation of new F probes and repeating the process. The technical feasability of walking was first demonstrated by Bender et al. (1983) who cloned 3.15 x lo5 bp of DNA from the ace and rosy locus

Chromosome walking is the name given to a systematic process which allows the isolation of

* To whom correspondence

and

reprint

requests

should

be

addressed. Abbreviations: serum

Ap, ampicillin;

albumin;

C, starting

bp, base pair(s); clone;

phage I; FL, FR, see INTRODUCTION, bases

or 1000 bp; Km,

Klenow (large) fragment of DNA vanadyl

replication; nucleotide

kanamycin;

BSA, bovine

cos, cohesive

nt, nucleotide(s);

ofE. coli DNA polymerase SDS,

complex

0.15 M NaCl, 0.015 M Na,

sodium

dodecyl

(New England citrate,

end

pH 7.8.

site of

section a; kb, kiloPolIk,

I; ori, origin

sulphate;

Biolabs);

VNC,

1 x SSC,

of Drosophila melanogaster in a series of overlapping phage J. clones. Since then there have been several

0378-l 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

10

reports of walking within Drosophila and other higher

radiolabel

eukaryote

analysis

genomes

(for example

1986) as well as many examples

Steinmetz

et al.,

of one- or two-step

‘walks’ within gene families. Chromosome

walking

is a laborious

and time-

consuming

procedure

and this has limited its general

application

and also severely limited the length of a

walk that can be contemplated.

There are, of course,

experimental

limitations

gies because

of their one-dimensional

walk

can

proceed

placed upon walking strate-

faster

than

the

structure: speed

no

of an

individual step. Mapping strategies (Coulson et al., 1986) which allow many clones to be analysed simultaneously are clearly more attractive in this respect. However, mapping is only an experimental option in higher eukaryotes where a small segment of the genome can be isolated with high yield from an heterologous background, and this is often not possible for any given chromosomal locus; walking is, in these circumstances, a major component of the experimental programme. We believe that walking in conjunction with jumping protocols (Collins and Weissman, 1984) may be the most appropriate technical approach to apply to several major eukaryotic cloning problems. Before such a walk could be contemplated, careful attention must be paid to the vectors used for walking and to the experimental protocols employed. The purpose of this article is to detail the construction and use of a cosmid vector that is specifically designed to facilitate the process of walking within the human or other complex genome. (b) Design criteria for a walking vector A walk cannot proceed faster than the speed of each constituent step. It follows therefore that each step should contain as much DNA as possible and for this reason we believe cosmid vectors offer the best cloning system. The deficiencies of these vectors are apparent and discussed below. Each step of a walk comprises the following experimental procedures, if we assume we have an uncharacterized starting clone C 1. (1) Restriction map Cl to identify restriction fragments located at the left and right end of the cloned DNA (FLl, FRl, respectively). Consider only the left fragment FLl. (2) Isolate FL1 by agarose gel electrophoresis,

and

show by genomic

that it is a unique

DNA

Southern

sequence.

(3) Probe the cosmid library to pick colonies that hybridize to FLl. This will inevitably involve a minimum

of one further round

uncontaminated hybridized

secondaries.

of screening Define

to FL1 as likely candidates

contain

Southern

FL1

hybridization

by

restriction

with FLl.

that

for a step.

Call these C2N colonies. (4) Restriction map all C2N cosmids. they

to pick

colonies

Show that

analysis

and

Choose the clone,

say C21, that has the least overlap with Cl and repeat the above process from 2. This protocol can be broken down to three main techniques : restriction mapping, probe preparation and library screening. Vector construction must be centred upon making these procedures as simple as possible. We have achieved this by making a cosmid vector that can be used for either Hind111 or BamHI cloning. We have placed an SP6- and a T7-RNA polymerase promoter on either side of, and with transcription oriented towards, these cloning sites. This allows us, in the absence of prior knowledge of restriction maps, to generate radiolabelled RNA transcripts that are specific for the first 1000 bp of DNA inserted into either of the cloning sites. We have tested this vector system by carrying out a short walk within mouse DNA. Cosmid vectors have significant drawbacks to their use in cloning experiments. There are anecdotal reports of instability of inserted sequences, of overgrowth of cosmid libraries by small vector-sized molecules and of possible examples of unclonable sequences by failure of isolation of specific genes from statistically complete libraries. These problems can in part be overcome by the use of strong recA alleles which stabilize direct repeats in Escherichia coli episomes and possibly by the use of recA _, recBC, sbcBstrains that in principal stabilize inverted repeats in E. coli plasmids (Vapnek et al., 1976; Collins et al., 1982; Wyman et al., 1985). In a previous publication (Little and Cross, 1985), we have described a complementary approach to some of these problems by analysis of the replication properties of a I phage origin of replication cosmid, Loric. This vector, with its enhanced stability, has formed the basis of our construction schemes, there is in principal no reason why our techniques and

11

construction could not be extended to the standard ColEl replicon cosmids or indeed to phage ,?cloning vehicles.

MATERIALS

AND METHODS

(a) Bacterial strains

Non-recombinant I origin cosmids were generally grown in E. coli strain MM294 (Little and Cross, 1985). Fusions of the A origin cosmids and pUC recombinants were selected on E. co& strain C2110 which is E. coli C pot% 1 his &a. LoristB recombinants were grown in ED8767 (all as described in Little and Cross, 1985), L-47.1 is a aimm434cI phage described in Loenen and Brammar (1980). Km-resistant cosmids were selected as described in Little and Cross (1985). Phosmids (cosmids that are linearized at the cos site) were grown and prepared in BHB3175 as defined and described in Little and Cross (1985). (b) Construction

of LoristB

The scheme for the construction of the T7 and SP6 promoter containing cosmids is detailed in Fig. 1. The starting plasmids and cosmids are: Loric (Little and Cross, 1985), pSP64 (Melton et al., 1984), pUC12 (Norrander et al., 1983), and pT7-2 (United States Biochemical Corp., unpublished construction). All manipulations, ligations and bacterial plating methods were essentially as described in Maniatis et al. (1982). Plasmid and cosmid DNA preparations were as described previously (Little and Cross, 1985). Plasmids LdBp12 and LorspT7 were selected by their ability to confer Km and Ap resistance on C2 110. This strain cannot support the growth of ColEl-derived replicons since it is pool and attempts to select on MM294 failed since the I on’ and pUC derivatives are compatible replicons. All constructs were checked by at least two diagnostic restriction enzyme digests. The nt sequence of all intermediates in the construction can be inferred since all starting plasmids are of known sequence. We used the Matilda programs of Shalloway and Deering (1984) and the Microgenie programs of Queen and Korn (1984) to construct

detailed restriction maps of each construct and the final cosmid. These are detailed in Fig. 8. We have, so far, found no anomalous restriction enzyme digestion patterns. The sequence of the BarnHI linker is TAGGATCCTA. When this is ligated at the 5’ end to a PolIk-~lled-in H&d111 site and at the 3’ end to a resected Z%dIII site, the sequence shown in Fig. 2 is generated. This has a Hind111 site recreated one base to the 5’ side of the BamHI site.

(c) Construction

of cosmid libraries in LoristB

Libraries were constructed by insertion of partially ~~~dIII-cleaved eukaryotic DNA into the Hind111 site of LoristB. Mouse DNA was isolated by standard methods from a cell line, IB5, that contained only a single copy of human chromosome 11 (J. Cowell, pers. commun.). We used the method of Seed et al. (1982) to identify partial digest conditions and isolated 25-50-kb partial fragments on sucrose gradients. Centrifugation conditions were: lo-40% sucrose in 1 M NaCl, 100 mM Tris pH 7.5,20 mM EDTA, and 0.3 % (v/v) Sarkosyi. Gradients of 38 ml cont~ning not more than 300 pg partially digested DNA were run for 17 h at 17000 rev./min, 10°C in a Beckman SW27.1 rotor. Vector DNA was prepared for use in the method described by Ish-Horowitz and Burke (1981). Vector ‘arms’ were made by cleavage with either BstEII or SstI followed by dephosphorylation, and cleavage with HindIII. Ligations of vector and eukaryotic DNA were carried out for 2 h at 22 oC in a reaction volume of 20 ~(1containing 0.15 pg each ‘arm’ of vector, 3 pg of eukaryotic 25-50-kb DNA, 5 units of T4 DNA ligase (Boeh~nger) and the m~ufacturer’s recommended buffer. 2-4-~1 aliquots of each reaction were then packaged in vitro using Amersham packaging reactions. Amplification of libraries was as described in Little and Cross (1985). We constructed a library of 285000 events which was stored at -70°C as an amplified library. (d) Screening cosmid libraries

The amplified library was plated out onto 30 P~l/Biodyne 132-mm diameter filters at a concentration of 10000 colonies per filter. Replication of filters, storage of masters and growth conditions

I2

followed Hanahan and Meselson (1980). Colonies were prepared for hybridization using the manufacturers’ recommended conditions. Prehybridization for DNA probes was at 42’ C for at least 4 h and for RNA probes at least 1 h, in a solution containing 50% formamide, 5 x SSC, 250 pg/ml sonicated and denatured salmon sperm DNA, 0.1% (w/v) BSA, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) Ficoll, 50 mM sodium phosphate buffer pH 6.5,O. 1% (w/v) SDS, 10 pg/ml poly(C), 10 pg/ml poly(A), 200 ng/ml Lorist/pUC competitor DNA and 10 mM VNC. Hybridization was carried out for 17 h at 50°C for RNA probes and 42 ‘C for DNA probes in the same solution as described for prehybridization but containing lo6 cpm/ml of the appropriate 32Plabelled RNA or DNA probe. DNA probes were generated by oligolabelling (Feinberg and Vogelstein, 1983), RNA probes by in vitro transcription (see below). After hybridisation, the filters were washed 3 times at room temperature in 2 x SSC 0.1% SDS for 15-20 min and then once at 65 “C in 0.1 x SSC 0.1% SDS for 30’. Filters were exposed to x-ray film (Kodak XAR-5 film) for 17-48 h. Signals under these conditions were intense. (e) Preparation of RNA probes

Template cosmid DNA was CsCl + EtdBr gradient purified and was used either uncleaved or RsaI-cleaved. It was extracted with phenol and chloroform, isopropanol-precipitated and washed with 70% ethanol before using in a transcription reaction. Both T7 and SP6 transcription reactions were carried out identically in 100 ~1 transcription reactions containing 40 mM Tris pH 7.5, 6 mM MgCI,, 2 mM spermidine, 0.01% BSA (Pentex fraction V), 10 mM dithiothreitol, 400 PM each ATP, CTP and GTP, 30 units RNAsin (Anglian Biotechnology Ltd., UK), 20 pg template cosmid DNA and 40 units of either T7 or SP6 polymerase. 50 PCi [ c(-32P]UTP (800 Ci/mmol Amersham International SP6 grade) was included and ‘cold’ UTP was added at varying concentrations as required. Reactions were incubated at 40” C for 60 min and terminated by chloroform extraction and isopropan01 extraction; the RNA was resuspended in 10 mM Tris pH 7.4, 1 mM EDTA supplemented with 10 mM VNC. We have used SP6 polymerase

from New England Biolabs and Amersham International successfully and T7 polymerase from Boehringer. Incorporation of radioactivity was always more efficient (up to 90 y0) with T7 polymerase than with SP6 polymerase (up to 30%). (f) Cos mapping

Restriction mapping of phosmids using cos linkers (Rackwitz et al., 1984) was carried out as described in Little and Cross (1985). Field inversion gels (Carle et al., 1986) were used to increase resolution of larger DNA fragments. Phosmids were made as described in Little and Cross (1985) in BHB3175. Cosmids were transferred to this strain by in vivo packaging using L-47.1 (Loenen and Brammar 1980) rather than the phage described in Little and Cross (1985).

RESULTSANDDISCUSSION

(a) Characterization

of LoristB

The physical structure and cloning manipulations involved in the construction of LoristB are detailed in Fig. 1 and discussed in MATERIALS AND METHODS, section b. The vector is 5.61 kb and the complete sequence and restriction maps are presented in the APPENDIX. If the SP6 and T7 promoters in LoristB were to be useful they must meet the following design requirements. There must be no significant sequence homology of transcript leader sequences with the vector since this would result in all vector molecules hybridizing to probe RNAs; there must be specificity of the transcription reaction for the DNA immediately adjacent to either promoter, and finally there must be efficient incorporation of radiolabelled nucleotides to generate probe RNAs of high enough yield and specific activities to be used in genomic Southerns and library screening. We will discuss each of these points in turn. The sequence of the promoter initiation and cloning sites is given in Fig. 2. DNA fragments inserted into the Hind111 site would be transcribed to contain 16 nt of vector ‘leader’ sequence with T7 polymerase and 23 nt with SP6 polymerase, whereas sequences cloned into the BamHI site would have 22 nt and 15 nt with T7 and SP6, respectively. We assumed

13

HB

3

51

‘@

LORiSTB

51 re-0

t

Fig. 1. Construction sites relevant

of Lorist B. Plasmid/cosmid

to the preceding

on a 260-bpHindIII-Suu3AI The SP6 promoter

sizes are not to scale. The construct

and following constructions fragment

was removed

are detailed.

treatment

together

from this plasmid

after treatment

with PolIk, creating

resected

with Sl nuclease,

generate

LordelB.

the pUCI2 necessary

pUCl2

as aHind

+ EcoRI (filled-in with PolIk) fragment

position

site removed Abbreviations preceding

cut with EcoRI

and appropriate

was fused, via the PolIk-tilled-in

through

The T7 promoter

by cleavage

with BumHI

B, BumHI;

site tilled-in with PolIk;/T4),

C, &I;

preceding

site in Lordel

then joined

together

to generate

was isolated

in the presence

arrows

x

are destroyed

represent

in the construction.

the T7 promoters.

by

from T7-2 as a HindIII-Hue11

and the pUCl2

linker to

to make LdBP12,

and

Loris. This has the effect of leaving a SsrI site in the

tilling in with PolIk and recircularization ori removed

SI, Sstl; S2, SsrII; H2, HincII; with T4 DNA polymerase;

The short open arrows

The dot marks

the SstI and

was destroyed

of the BarnHI

XbaI site of LordelB fragment

in pUCT7,

to make pUCT7Bd.

the BumHI

Loris was then

by deletion with SstI to generate

LoristB.

Ha, HueII; X, XbaI; Sa, Suu 3AI; E, EcoRI; res, site resected

by S 1 nuclease;

cyclized by ligation at high dilution (note that a Bum HI site tilled in and rejoined makes a Cl01 site); B linker, BumHI with a bold

to make pUCSP.

between the Hind111

was cut with Hind111 and the site either tilled-in with PolIk or

fragments

site resected

the SP6 promoter

of the DNA between

The BamHI

EcoRI site to the PolIk-filled-in

the Hind111 sites to make LorspT7

used: H, HindIII;

introduce

sites ofpUCl2 and inserted

in size by deletion

to make Lordel.

LordelO

with Hind111 and recircularization

for arm preparation.

from this construct

fused to this plasmid

Lorus was reduced

with T4 DNA polymerase,

a CIuI site to make LordelO.

ori deleted by cleavage

name is given within each circle and only those

from Loric to LordelO

isolated from pSP64 and cloned between the Hind111 and BamHI

and Clal (filled-in with PolIk) sites of Loric to make Lorus. SsrII sites, joined

Constructs

mark the location

of the SP6 promoter,

till,

re-0, plasmid

linker. Sites marked

the very short filled-in

the phage i ori sites.

that under the conditions of hybridization we employed (MATERIALS AND METHODS, section d) such a short region would not provide a stable region of duplex. In the experiments reported below (and data not presented), we could see no significant hybridization of probes generated from recombinant vectors to either non-homologous recombinants or vector sequences. We concluded from this that the

transcripts would not significantly cross-hybridize to vector molecules. Specificity of transcription is dependent on the specificity of the polymerase for its promoter, which has been reported to be high for both enzymes (Chamberlin and Ryan, 1982). We were concerned that the greater complexity of recombinant cosmids (average size 45 kb) compared to standard SP6 and

14

T ;L

scribed circular LH2 with either T7 or SP6 polymer-

Hind III BamH I ATAGGGAGACCGG-AGGATCCTATGTATTCTA

ase, we would transcribe much of the template (presumably with decreasing efficiency the farther

-16

downstream -15

from the promoter

of [ 32P]RNA

located).

Hybridization

a fashion

would be expected

different cleaved fragments Fig. 2. Sequence T7 polymerase sequence,

transcribes that

before

sequence.

each transcript the transcript

Hind111 or BumHI

regions of LoristB.

the complement

SP6 the displayed

ber of bases sequence

of the T7 and SP6 promoter

of the displayed

will contain

to several

of LH2. In contrast,

if we

restric-

tion enzyme that does not cleave either the T7 or SP6 promoter or any of the DNA between them and the cloning site), we would expect to produce only a

as a 5’ ‘leader’

cloned

were

made in such

to hybridize

first cut LH2 with RsaI (a 4-bp recognition

The bars show the num-

of DNA

the sequences

into either the

single ‘run off transcript that should now be specific for the DNA immediately downstream from the promotor up to the first RsaI site in the mouse DNA. This would be expected to be a small DNA fragment

site.

T7 promoter-containing vectors might allow significant levels of non-specific RNA synthesis. To test this we selected at random a cosmid, LH2, that contained mouse DNA. Cos mapping of LH2 showed that there were 4.8-kb and 2.0-kb Hind111 fragments immediately adjacent to and downstream from the SP6 and T7 promoters, respectively (data not presented). We would predict that if we tran-

and the RNA transcript should be homologous only to a single fragment (or two, depending on the relative position of the RsaI and surrounding sites). Fig. 3A shows the result of this experiment. The lanes labelled + or - are probed with an SP6 or T7 probe made from LH2 cut or uncut with RsaI. There are many fragments that hybridize with the T7 and SP6 probe but with the + probe there is only a single major hybridizing fragment, the size of which

T7

SP6 “RR

HffR

=

o-5

100

m

+

b

-+

a Fig. 3. Specificity

of SP6 and T7 transcription

of a recombinant

cosmid,

Hind111 (H), Hind111 + EcoRI (R/H) or EcoRI (R), run on 1% agarose hybridized

with [a-3ZP]UTP-labe11ed

(R) run on

on probe specificity:

to nitrocellulose

filters.

cosmid LH2 DNA was cut with

to nitrocellulose

using a template

T7 transcriptions

and made with either 100 PM (100) or 0.5 PM (0.5) UTP, were hybridized

1y. agarose gels and transferred

specificity:

gels and transferred

RNA probes made with SP6 or T7 RNA polymerase

&a1 ( + ) or uncut ( - ). (b) Effect of limiting UTP concentrations with [a-32P]UTP

LH2. (a) Promoter

membranes.

These were

of LH2 DNA either cut with

ofuncleaved

LH2 DNA, labelled

to LH2 DNA cut with Hind111 (H) or EcoRI

is consistent with the mapping data of EcoRI and Hind111 sites adjacent to the promoter. We note that there are faint bands above the specific bands in both the T7 and SP6 case. These are in part due to partial digest of the target HindIII-, EcoRI- and doubledigested LH2 DNA but we also see on longer exposures of other experiments faint hybridization of numerous fragments. It is not easy to estimate the level of this hybridization -we would suggest about 1y0 of correctly initiated RNA levels. From these experiments we conclude that the SP6 and T7 RNA polymerase specifically initiate RNA synthesis from their own promoter but also conclude that a low level of more random initiation is likely to occur. Melton et al. (1985) have shown that at low nt concentrations premature termination of SP6 transcription occurs. We reasoned that this may be an alternative way to generate probes specific to the first few thousand bp of inserted DNA. Fig. 3B shows the result of transcribing intact LH2 with T7 polymerase in reactions that contain either 100 PM or 0.5 PM UTP and using these transcripts as probes in Southern hybridization back to EcoRI- or HindIIIdigested LH2. It is apparent that with T7 and SP6 RNA polymerase and limiting UTP concentrations, probes are produced that are a’s specific for the end DNA sequences as those that are generated by RsaI and we find similar results using SP6 RNA polymerase (unpublished). We cannot exclude from these experiments that this only occurs with LH2 because A-rich regions are located close to the promoters, but other clones, as we demonstrate below, have shown similar effects. Transcripts synthesised at low UTP concentrations have an average length of about 800 nt (own unpublished data). We can conclude from these experiments that transcripts can be generated from LoristB recombinants that are highly specific for the ends of DNA fragments cloned in this vector. (b) Use of SP6 and T7 polymerase genome analysis

transcripts

in

For a particular probe to be useful for walking it is necessary that it is unique or of very low repetition within the genome. The most simple test for this is to hybridize SP6 and T7 probes, separately, to Hind111 digests of total mouse DNA. The data presented in Fig. 3 suggest that we should detect in

Fig. 4. (Left panel). [a-32P]UTP-labelled transcripts

of RsaI

cleaved

mouse

DNA

agarose

gels to nitrocellulose

of Iz DNA

digested

digested

cosmid

membranes.

hybridized T7 transcript

to total from

1%

In lanes m are markers

with Hind111 mixed with I DNA cut with

Sizes of mouse fragments labelled,

hybridized

with Hind111 and transferred

Hind111 + EcoRI and cross-hybridizing experiment

SP6 and T7-generated LH2

with LoristB

sequences.

are given in kb. (Right panel) A similar with

a highly

of cosmid

reiterated,

[a-32P]UTP-

H2.

Southern analysis of HindHI-cut mouse DNA a fragment of 4.8 kb with an LH2 SP6 probe and of 2.0 kb with a T7 probe. In Fig. 4 (left panels), we show the results of such hybridization experiments: the appropriate fragments can be detected in the mouse DNA with both enzymes. This shows that both the T7 and SP6 transcripts of LH2 detect unique DNA fragments and would be suitable probes for screening the cosmid library for overlapping fragments. In contrast, Fig. 4 (right panel) shows hybridization of a T7 probe generated from a cosmid, H2, that has a highly reiterated sequence immediately adjacent to this promoter which could not be used for walking. We estimate that the frequency of occurrence of reiterated sequences adjacent to a promoter is about 1 in 6 cosmids and this is discussed in section d below. (c) Use of probes for cosmid library screening

We were able to show that the cosmid LH2 described above was not contained within our IB5

16

library spread on filters (LH2 had been isolated from a trial ligation prior to the full library plating). We picked several cosmids from replicas of filters that had been used to make the library screen filters and selected a cosmid (WI) that had a unique T7 transcript. Southern analysis of WI with the T7 tran-

.’

-

*

14

*5

Fig. 5. Screening

ofrepresentative

library filters. All probes were

labelled with [cz-~‘P]UTP. Each library filter contains 10000 recombinant on

filters,

Hanahan

cosmid-containing

replica-plated

ization

solution

RIALS

AND

hybridized

and conditions

of specific

Lower

cosmid-generated

in MATEwere

solution.

arc displayed.

with RraI-cut

Wl T7

Middle row: filters 2 and 14 hybridcosmid

with this probe

7 SP6 probes. nor does

row: left filter 14 hybridized probe

1.5 is arrowed

hybridized

of interest

in

and hybrid-

of Wl on filter 2, and 7 on filter 3 are

in white lettering.

does not hybridize

cosmid

as described

were as described

filters

of

grown

sections d and e. 30 filters

ized with low-UTP-generated (arrowed).

in situ

filters 2 and 3 hybridized

The location

indicated

lysed

replicas

colonies

in a final volume of 50 ml of hybridization

row:

probes.

ED8767

(1980). Probe preparation

METHODS,

Autoradiographs Upper

and

and Meselson

script generated from RsaI-cleaved template indicated that the Hind111 fragment adjacent to the T7 promoter was 0.95 kb and that judged by genomic Southern analysis this was a single copy sequence (data not presented). In Fig. 5, we show the results of screening of the library filters with a T7 probe generated from R&-cut WI. Filter 2, which we know contains the original WI, shows hybridization at the predicted location for WI and filter 3 and others had additional positives, 35 in total. Wl was represented at higher frequency than expected for a single copy DNA sequence (about 5 expected). We think that this is due to differential bacterial growth since some other single-copy genes have been shown to be over-represented in similarly amplified libraries (unpublished data). We selected three positives (7, 1 I and 12), rescreened at lower colony density and picked single colonies. These were grown up for small-scale DNA preparations and analysed by Hind111 digestion: in each case we found that the positive clones contained the 0.95kb fragment seen in WI and additional DNA. Southern

WI

SP6.

The

and hybridizes

and does not hybridize

with this probe.

Fig. 6. DNA fragment

pattern

ofHindIII-digested

12 (lanes W, 7, 11 and 12) run on a 1% agarose

with Iow-UTP-

gel transferred

colony

strongly.

with low-UTP- WI T7. The location

Note Wl

1.5 on filter 14 containing

Right filter 14 of I .5 is arrowed

to nitrocellulose

the [c+~‘P]UTP fragment

marked

labelled

membrane

T7 transcript

and common

WZ, 7, II and gel and the same

and hybridized

with

of WI. Note the 0.95-kb

to all four cosmids.

m is a marker

lane of i DNA cut with Hind111 mixed with I DNA cut with Hind111 + EcoRI.

17

Fig. 7. Restriction map of a region ofmouse DNA defined by the overlapping cosmid clones, 7, II, 12, WI and 1.5 detailed in RESULTS AND DISCUSSION, section c. Only Hind111 sites are shown and these are marked by vertical lines showing regions of cosmid homology. The horizontal bracket marks a region of ambiguous mapping. Symbols s and t specify SP6 and T7 promoter locations at ends of inserted DNA. Numbers on the top line are lengths in kb.

analysis of these clones with WI T7 RNA as a probe showed that this fragment strongly hybridized in each case (Fig. 6). We made Hind111 restriction maps of the selected cosmids (Fig. 7). Clones 7 and 12 overlapped WI by only 950 bp. Clone I I had substantial similarity to 7, 12 and WI : all EcoRI + Hind111 double-digest fragments of 11 were contained in 7, 12 or WI, but the map structure could only be interpreted by suggesting that the DNA cloned in 11 was an unequal recombination product of the 16.8 and 23-kb DNA fragments. The T7 11 probe hybridizes to the 7 and 12 16.8kb fragment (not presented). It is likely, but not proven, that we are mapping alleles of the mouse DNA, one chromosome of which is represented by clone 11, the second chromosome being represented by clone 12 or 7. The low UTP SP6 transcript of Wl is also unique and homologous to a 8.8-kb fragment in mouse DNA. We used this to probe a set of library filters and identified positive colonies that hybridized to Wl SP6 but not to WI T7 or 7 SP6 probes (Fig. 5). These should formally represent a step to the right of Wl. The Hind111 map of one of these cosmids, 1.5, shows overlap with WI of the 0.67- and 8.8-kb fragment (Fig. 7). The final map comprises 114 kb of DNA contained in three cosmids. (d) Conclusions

We have previously described the use and properties of a cosmid, Loric, that has made some con-

tribution to ease of handling and stability of cosmid libraries (Little and Cross, 1985). LoristB has identical growth and replication properties to Loric and can also be readily used in cos mapping protocols. We and others have been able to show that cosmid vectors do not clone sequences in a statistically random fashion (Coulson et al., 1986; T. Gibson and P.F.R.L., unpublished). This implies that some DNA sequences will be ‘unclonable’ or so rarely cloned in a library as to be functionally unclonable. LoristB appears to be significantly different in the spectrum of sequences it clones from ColEl cosmids such as pJB8 (Ish-Horowitz and Burke, 1984). Modification of LoristB, described by T. Gibson, A.R. Coulson, J.E. Sulston and P.F.R.L. (in preparation) further extends the randomness of clones isolated in Lorist vectors. The use of LoristB cosmids can substantially improve speed and ease of walking procedures, since it eliminates the need for detailed restriction mapping to identify ‘end’ restriction fragments (step 1 in the INTRODUCTION, section b walking scheme) and fragment isolation (step 2) for probe preparation. Both these procedures are time-con&ming. An obvious difficulty with all walking procedures is the presence of repeat sequences in the probe. Our preliminary experiments (unpublished) suggest that such repeats occur once in about 6 cosmid transcripts. It is difficult to estimate the frequency with which repetitive sequences should be adjacent to the promoters. The transcripts are about 800 nt in length and reassociation analysis (McConaughy and

20

10 BTC61(lCC66

6CbAC61T61

30 T6CCAlT6Cl

40 50 60 70 60 90 100 110 120 6CA6666666 6666666666 666666666T TGICTTCCIIT TGTTCATTCC AC66ACAllRA ACA6P6AAA6 6ARAC6ACA6 b66CCAMAb

130 140 150 lb0 170 180 190 200 210 220 230 240 GCCTCGCTTT C(I6CACCTGT CGTTTCCTTT CTTlTCAlAO GBTATTTTAA ATAMAACAT lAA6TTATGA C61116AA6AA C66CIAACGCC lTtIAACC66A AAlTTllCbT MATAGCGAR 250 260 270 260 290 300 310 320 330 340 350 360 AACCCGCGA6 GTCGCCGCCC 6lAACCl6TC 66AlCACC66 AAA66ACCC6 TAAAGTGATII IITGAllbTCb TCTACATATC RCAAC616C6 T66h66CCRT CMACCACGT ClARlAATCll 370 360 390 400 410 420 430 440 450 460 470 460 ATTATGACGC 866lATCGlb lT(IATT6BTC TGCATCMCT TAACGTAAAA ACAACTlCA6 ACAATACPAP lCA6CGllCBC T68ATAC666 GCAACCTCAT GTCAACCCCC CCCCCCCCCC 490 500 510 520 530 540 550 560 570 560 591) 600 CCCCCCCCCC ClGCA66C66 bGAPCT66TA 66lAl66AA6 ATClCTb686 MTTCGAGCT CGCCC6666A TCGRTCCTCT AGAGTCCTGA lGCG6lRTll TCTCCTTACG CRTCTGTGCG 610 620 630 640 650 660 670 660 690 700 710 720 GTPTTTCACB CCGCATPTGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATkGTTAh GCCAGTATAT PCACTCCGCT ATCGCTICGT GbCT666TCA 16GClGC6CC CCGbCACCCG 730 740 750 760 770 760 790 600 610 620 630 640 CCAACCICCCG CTGACGCGCC Cl6ACGGGCT TGTCTGCTCC CGGCEITCCGC TTACAGllCAR GCTGTGRCCG lCTCC666116 CTGCATGTGT CbGAG6llTT CACCGTCATC ACCGllARC6C 650 660 870 660 890 900 ?lO 920 930 940 950 960 GCGPGGCCCP GCTGGCTTbT CGAAATTAAT ACGPCTCACT AlbG66668C C66AAGCllR GBATCCTRTG TATTCTATAG TGTCACCTRA ATCGTRTGTG TATGATACAT dA6GlTllT61 970 960 990 1000 1010 1020 1030 1040 1050 1060 1070 1060 ATTAATTGTA GCCGCGTTCT AACGK#tTA TGTACAAGCC TAATTGTGTA GCATCTGGCT TACTGARGCA GACCCTATCA TCTCTCTCGT AAACTGCCGT CA6A6TC66T 1166lTG6AC 1090 1100 1110 1120 1130 1140 1150 1160 1170 1160 1190 1200 GAACCTTCTG AGTllCl66T AACGCCGTCC CGCACCCG611 RATGGTCAGC GAACCAATCA GCA666TCAT CGCTAGCCAG ATCCCC666C GA6Cl6C66C CTGATTTIITG Cl6GTlACTG 1210 1220 1230 1240 1250 1260 1270 1260 1290 1300 1310 1320 TTGCGCCTGT TCIGCGC6GCA fXGTCCGGC6 CACA6lbGCl ATTATGCGTC CCCAGGTMT GllAlACITTGC CTCTTTGCCC GTCATACACT TGCTCCTTTC AGTCCGhXl TAGCTTTGAT 1330 1340 1350 1360 1370 1360 1390 1400 1410 1420 1430 1440 TTCTGCGATC TTCGCCAGAG CCTGTGCACG ATTTAGAGGT CTACCGCCCA 16ACAGGM6 TTGTTTTACT GGTTCb6GGA TCGCCTCACC AC66lTAATl ClCGCA6lCB lAlG6dCl)bG 1450 1460 1470 1400 1490 1500 1510 1520 1530 1540 1550 1560 CTCATCTGCG GCCTlACG6C GTMTTCCGC PTC16lAb6C GCATl66CCC GCATGTTCTG ATRCAGGTTG GTAICCAGCC R6TdGTGC6C GTTTGRTTTC CAC66ATBAG ACTCCGCRTC 1570 1560 1590 1600 1610 1620 1630 1640 1650 1660 1670 1660 CG6ATIICAG6 CCTCGCTTCC 66CARTFiClC GTAAACCATA TCAPCCAGCT CGCTGACGTT TGGCAGTCCG GCGGTAACG6 ATGCTTCTTC CCGGCACCAT GClRCddACl GCCC66GTGtl 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 TGGCAGAAAT GGTCGATTCT GCCGACGGGC TACGCGCATT CCTGCGTTAA CCTGTTCCAT CGT66lGAlC CCGTTTTCCC 6RAAAGCCA6 AllCCCAClGG CGRC6611111 CGTTCACTTC 1610 1620 1630 1640 1650 1660 1670 1660 1690 1900 19!0 GTlCT6GTCb CG6TTk6CCA GGCTCGCCFG GAM6TT6CC fl6TAACTGGC TGMCACACC 6Tl6AT6ATC TGCGCTACCT GCTGTACCTG CGGCTTTTCG TCGTKTGTT

1920 CC6GCAl611

1930 1940 1950 1960 1970 1960 1990 2000 2010 2020 2030 2040 6TTGGCGATC CGRCGCATCT GCTCK6GTC AAAGTTBACC ATCl6TGC66 C6Al6TlllT CAlIGATCCd CCCCGTMAT CCA6lClGl6 lTl6lCAG6T C6A6lTTTG6 lTlGCl66Cl 2050 2060 2070 2080 2090 2100 2110 GTCACOCCTG CCl6Tl6Cll 6TlAC6GlTG BlTTCGbGTl G66lCCbCTl AlC6CG6A6T TlG6CCGG6C 2170 ACACACATGT

2120 2130 2110 TC86CACGTl ACCGGRCCAG AAGTTGTCCT

2150 2160 GGCATGCCCA 6C6GMCAGC

2160 2190 2200 2210 2220 2230 2240 2250 2260 2270 2260 CGC6GTG611 ACGTCCGTCA CGTTCACGCR lCAGGC66Al ATCGTTAGCC CbCCCllGCAl bAllCG6TTl lCl6GCT6Al 6616CGlllAG TCllCACClll GTCAAACATC

2290 2300 2310 2320 2330 2340 2350 2360 2370 2360 2390 2400 CRCTClGCG6 C66TCAGGlC TTCTGCTGTC CCCCACTTGC TGCCGCTCTG AATTGCAGCII TCCGGTTTCA CCACBGAAAG GlC6llllCT 66CT66TCA6 A6GdTTC6CC AGBIITTCTCT 2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 6AC6MlAAl CTTTTCTTTT llClTlT6TA AlA6T6lClT TTGTGTCCCC Cl6llTT6A6 66AlAGCM CCCCCAATTT 61166GATGTl TTATCCCTCG lTllR6666d

2520 TTTTCCCTCG

19

2530 2540 2550 2560 2570 2580 2590 2600 2610 2620 2b30 2640 TTTTGnGG6n TGCnCCnTTC TGAGATGTTT TTnTTT66TC CnnnCITGCC GCCTTGCTGC TTGnTnnTnT TCnTTCTGnC GAGTTCTMC TT66CTTCnT TGCACCGTTT 6nC1166TAnC 2650 2660 2670 2660 2690 2700 2710 2720 2730 2740 2750 2760 TTTGTnnTCT C6CTnn6TTG nGAnTC66TG nTTCT6TCCn TTGBTTTnTT CCRCCCnTn6 GTTTTICGCA 6nnTG6Cnn6 CAGCnCTTTn nnCT6TCGCT T66TCnGnTC T6C6CCC6nn 2770 2760 2790 2600 2610 2620 2630 2640 2650 2660 2670 2660 TAnGCCTCAn GCAGCATATT T6ATn6TCTG 6C6TAnCCnT CnTCGnGnTC TGCCACATTA CGCTCCTGTC CGGCnnn6TT nCCTCTGCC6 nn6TT6116Tn TTTTTGCTGT nTTT6TCnTn 2690 2900 2910 2920 2930 2940 2950 2960 2970 2960 2990 3000 ATGPCTCCTG TTGnTnGnTC CnGTnnTGnC CTCAGAACTC CnTCT66nTT TGTTCAGRLC 6CTCG6TT6C CGCCG6GC6T TTTTTnTT66 T6n6nnTC6C RGCAACTTGT CGCGCCnnTC 3010 686CCAT6TC 3130 TCTTCTCAGT

3020 GTCGTCAACG 3140 TCCnnGCATT

3030 3040 3050 3060 3070 3060 3090 3100 3110 nCCCCCCnTT CMGAACRGC AnGCA6CnTT Gn6nnCTTT6 GnnTCCn6TC CCTCTTCCAC CTGCTGnTCT GCGnCTTnTC AnCGCCCnCn

3120 GCTTCCGCTG

3150 3160 3170 3160 3190 3200 3210 3220 3230 3240 GCGnTTTTGT TnnGCnnCGC ICTCTCGATT C6TnGn6CCT CGTTGCGTTT GTTT6CnC6n RCCnTRTGTn AGTnTTTCCT TAGnTnnCnn TTGATTBnnT

3250 3260 3270 3260 3290 3300 3310 3320 3330 3340 3350 3360 6TnTGCAMT AnnTGCnTnC nCCnTA66TG TGGTTTRATT TGATGCCCTT TTTCAG6GCT 66nnT616Tn AGA6C6666T TnTTTnTGCT GTTGTTTTTT TGTTnCTC66 Gnn666CTTT 3370 3360 3390 3400 3410 3420 3430 3440 3450 3460 3470 3460 ACCTCTTCCG CnTnAACGCT TCCRTCAGCG TTTATAGTTA AnnnnnTCTT TC66CCTGCn TGnnTG6CCT TGTT6nTCGC GCTTTGnTAT nCGCCGnGnT CTTTnGCTGT CTT66TTTGC 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600 CCnnnGCGCn TTGCATRATC TTTCnG66TT ATGCGTTGTT CCnTACnnCC TCCTTRGTAC nT6CnnCCAT TATCACCGCC n6666Tlnnn TnGTCnnCnC 6CnC66T6TT RGnTnTTTnT 3610 3620 3630 3640 3650 3660 3670 3660 3690 3700 3710 3720 CCCTTGCG~T 6nTn6nTTTA nc6TnTGnGc ncnnnnnn6n nnccnTTnnc ncnn6nGcn6 cTTGnGGncG cnc6Tc6ccT TnnnGcnnTT TnTGnnnnnn n6nnnnnT6n ncTT6GcTTn 3730 3740 3750 3760 3770 3780 3790 3800 3610 3620 3630 3640 TCCChGGnAT CT6TCGCn6n Cnn6nTGGGG ATGG6GCnGT CnGGC6TTGG TGCTTTnTTT BnT6GCATCll ATGCnTTnnn TGCTTnTnnC GCCGCATTGC TTnCnnnnnT TCTCnnnGTT 3650 3660 3670 3660 3890 3900 3910 3920 3930 3940 3950 3960 nGCGTTGnR6 MTTTnGCCC TTCnnTCGCC nGn6AnnTCT K6nGATGTA TGnn6C6GTT AGTnTGCn6C CGTCRCTTIIG nnGT6116TnT Gn6TnCCCTG TTTTTTCTCn T6TTCn66Cn 3970 3980 3990 4000 4010 4020 4030 4040 4050 4060 4070 4060 666nT6TTCT CACCTGAGCT TnGMCCTTT nCCLnnG6TG nTGC66n6AG nT666TnAGC nCAnCCAAnn Rn6CCn6T61 TTCTGCnTTC T66CTT6nG6 TT6nn66Tnn TTCCnTGnCC 4090 4100 4110 4120 4130 4140 4150 4160 4170 4160 4190 4200 GCACCnnCnG GCTCCnnGCC nn6CTn6CTT CnCGCTGCCG CnnGCnCTCn G66C6CAnG6 GCTGCTRnnG GnA6C66nnC nC6Tn6nnn6 CCnGTCCGCn 6nnnC6GT6C TGnCCCC66n 4210 4220 4230 4240 4250 4260 4270 4260 4290 4300 4310 4320 T6nnT6TCR6 CTnCTG66CT ATCTBlACnA 66GnAnX6C An6C6Cnnn6 n6nnnGCn66 Tn6CTT6CnG TGG6CTTnCn T66C6nTA6C Tn6nCT666C 66TTTTnTG6 nCn6Cnn6C6 4330 4340 4350 4360 4370 4360 4390 4400 4410 4420 4430 4440 AACCGGnnTT GCCnGCT66G GCGCCCTCTG 6Tnn66TT66 GAAGCCCTGC nnnGTnnnCT 6GnT6GCTTT CTTGCCGCCI n66nTCT6AT 06C6Cn666G nTCnA6nTCT 6nTCnn6n6n 4450 4460 4470 4460 4490 4500 4510 4520 4530 4540 4550 4560 CnG6ATGn66 ATCGTTTCGC AT6ATT6AnC AnGnT6GnTT GCnCGCnGGT TCTCCGGCCG CTT666T66n GAGGCTATTC 66CTnT6MT 606CICnnCn GACnnTC66C TGCTCTGRTG 4570 4580 4590 4600 4610 4620 4630 4b40 4650 4660 4b70 4660 CCGCCGTGTT CC6GCT6TCA 6CGCn666GC GCCCG6TTCT TTTTGTCMG nCC6nCCTGT CCGGTGCCCT GAnTGnnCTG CR6GACGn6G CnGCGC66CT nTC6T66CTG 6CCnC61C66 4b90 4700 4710 4720 4730 4740 4750 4760 4770 4760 4790 4600 GCGTTCCTTG CGCnGCT6TG CTCGACGTTG TCnCTGnnGC G6GAA66GnC T66CTGCTnT TGG6CGRn6T GCCG666CAG 6nTCTCCTGT CnTCTCnCCT TGCTCCTGCC 6116nnn6TbT 4610 4620 4630 4640 4650 4660 4670 4680 4690 4900 4910 4920 CCATCATGBC TGnTGCnnTG CG6CGGCT6C PTACGCTTGA TCC66CTnCC TGCCCATTCG ACCACCnnGC 6nnnCnTCGC nTCGA6C6116 CnCGTnCTC6 6nT66nn6CC G6TCTT6TC6 4930 4940 4950 4960 4970 4960 4990 5000 5010 5020 5030 5040 ATCAGGATGA TCT6GAC6nA GAGCnTCnGG G6CTCGCGCC nGCCGnACT6 TTCGCCnG6C TCAn6GC6C6 CnTGCCCGnC 66CGn66nTC TCGTCGTGAC CCnT66C6nT GCCTGCTTGC

5120 5130 5140 5150 5160 5050 SO60 5070 5080 5090 5100 5110 CGbBTATCAT 66T66RAdAT GGCCGCTTTT CTllATTCAT CWT6T66C C66CT666T6 TGGC6ACC6C TATCA6611CA TA6C6TT66C TACCCGTGbT ATT6CT6AA6 A6CTT66C66 5240 5250 5260 5270 5260 5170 5160 5190 5200 5210 5220 5230 C6AbT666CT GACCGCTTCC TCGTGCTTTA C66TRTCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG RCGPGTTCTT CT6A6C666A CTCT6666TT C6AbAT6ACC 5360 5370 5360 5390 5400 5290 5300 5310 5320 5330 5340 5350 6ACCRA6C61 C6CCCAACCT 6CCATCK6A GATTTCGATT CCACCGCCGC CTTCTATGAA A66TT666CT TC66MTC6T TTTCC66GM 6CC66CT66A TGhTCCTCCb 6C6C6666AT 5460 5490 5500 5510 5520 5410 5420 5430 5440 5450 5460 5470 CTCAT6CT66 AGTTCTTCGC CCACCCC666 CTCGtTCCCC TC6C6A6TT6 GTTCAGCTGC TGCCTG866C T66ACGllCCT C6C6686TTC TACC66CA6T GCAMTCCGT C66CATCCA6 5600 5610 5530 5540 5550 5560 5570 5560 5590 6AAACCA6C1 6C66CTATCC GCGCATCCAT GCCCCCGMC T6CA66AGT6 666866CAC6 AT66CCGCTT T66TC66ATC AATTCGCGCG ACCG

21

McCarthy, 1970) suggests that 25 % of mouse DNA molecules of about this size should be middle or highly repetitive, but we do not know if this is true for all 800 bp sequences that are bordered at an end by a Hind111 site (the normal case for probes generated from our cosmid libraries). We observe in both mouse and human DNA HindIII-resistant DNA that are presumably tandem repeat arrays and these will not be contained within our libraries, which will reduce the frequency of highly reiterated sequences. The use of cosmids that terminate at different places within the genome will allow a reasonable probability for making a step from a different location within the cluster of overlapping clones. We would stress that we have not demonstrated in this paper the utility of walking as a strategy for cloning large regions of the genome; indeed our own experience with the short ‘walk’ of 2 steps show that this can be a formidable problem. We believe that it will be necessary to use either homozygous material for library construction or a hybrid cell line that contains only a single human chromosome as the source DNA for any walking protocol. We have prepared such a library from a hybrid cell line which contains only a single human chromosome 11 and in this paper have detailed and demonstrated the general methods that we will use to attempt a walk. This will no doubt identify further problems but .we believe that it is in principal possible to perform one step per month using our procedures, and this would correspond to about 25-40 kb per step or perhaps 250 kb per year. LoristB and derived vectors, host and in vivo packaging strains, and complete instructions for construction of cosmid libraries are available from the authors upon request.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Cancer Research Campaign and Medical Research Council (U.K.).

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