New and versatile cloning vectors with kanamycin-resistance marker

Gene, 56 (1987) 309-312

309

Elsevier GEN 02079

New and versatile (Recombinant

DNA;

cloning

vectors

polycloning

with kanamycin-resistance

marker

sites)

Raymond David Pridmore Ciba-Geigy AG, Department of Biotechnology, CH-4002 Base1 (Switzerland) Received

31 March

Accepted

15 April 1987

1987

SUMMARY

Described here is a pair of small multi-copy kanamycin-resistance plasmids, containing the pUC IucZcrcomplementation peptide and the pUC18 and pUC19 multiple cloning site. These plasmids and their derivatives allow simple and rapid transfer of inserts from one replicon to another without the necessity of purifying the insert from vector.

INTRODIJCTION

In molecular biology the cloning and subcloning of DNA fragments is important for the dissection and analysis of functional units. This may include nucleotide sequencing, site-specific mutagenesis, or gene expression, and many plasmid and phage systems have been developed for specific tasks (Sanger et al., 1980; Nilsson

et al., 1983). Nevertheless

Correspondence to: Mr. R.D. Pridmore, ment

of

Ciba-Geigy

CH-4002

Biotechnology,

subcloning

of DNA fragments remains important and time-consuming and many techniques have been introduced to improve the efficiency. The pUC plasmids (Vieira and Messing, 1982), because of their small size, high copy number and versatile MCS have become popular for subcloning and in the construction of vectors. Here I report a pair of new plasmids with ah advantages of the pUC plasmids, while carrying a KmR marker. Some uses are also discussed.

AC, Depart-

Base1

(Switzerland)

Tel. (06 1) 366484. EXPERIMENTAL Abbreviations: IPTG.

bp,

base

pair(s);

EtdBr,

ethidium

bromide;

kb,

kilobase

isopropyl-b-D-thiogalactopyranoside;

pair(s); Km, kanamycin;

LB, Luria broth; MCS, multiple cloning

site; ori, origin of DNA replication; ment of,!?. coli DNA polymerase 4-chloro-indolyl-fi-o-galactopyranoside.

PolIk, Klenow

I; R, resistance;

(a) Construction

AND

DISCUSSION

of pK18 and pK19 plasmids

(large) frag-

XGal, 5-bromo-

The KmR gene pBRNeo (Southern

037X-I Il9,‘X7/$03 50 0 1987 Elsevier Science Pubhshers B.V (Bmmedical Division)

was taken from the plasmid and Berg, 1983; a kind gift of

GGCAG-3’

pier

2500

(PstI)and

CCCCGAC-3’ duplex plasmids

5’-CAAGGCGCGGATG-

(SphI),

method

(Morinaga

by

the

et al., 1984). This gave

pK18 and pK19 (Figs.

(b) Applications Described

plasmid-gapped 1 and 2).

and advantages of the plasmids

is a pair of small plasmids,

with a Km’

marker and MCS array in the ZucZ%-complementing peptide. It is intended to be used either alone or in conjunction with the similar and highly successful pUC plasmids.

The availability

of a second marker

allows the rapid transfer of inserts from one plasmid

Fig. 1. Physical

map of the pK plasmids

showing

1acZz and KmR gene are represented direction

of transcription,

Construction:

The

NurI-AkaIII

1985) was ligated pBRNeo.

and

pUCi9

repair

containing

Yanisch-Perron

to the 1.33-kb CluI-SmaI

the

by a bar.

ori-IarZa

(bp 238-1565;

The extra pBRNeo-derived

by Pollk

showing

while ori is represented

pUCI8

fragment

the MCS. The

by arrows

et al.,

KmR fragment

of

Hind111 site was removed

of the Hind111 hnearised

plasmid

(marked

by

+ + + +

; bp 6-10 in Fig. 2). The Pstl site in the KmK gene was

mutated

from CTCCAG

mutated

from

nucleotides by asterisks

GCATGC

described

to CTGAAT. to GGATGC

while the SphI site was with the oligodeoxy-

in section a. These changes

at positions

are indicated

536, 538 and 895 in Fig. 2.

Dr. F. Asselbergs) as a 1.33-kb Ctai-SmaI fragment, and ligated to the ori-facZ 1.3-kbNarI-DraI fragment of pUC18 and pUC19 (Norrander et al., 1983). This ligation mixture was transformed (Hanahan, 1983) into the Escherichia coli strain JM109 (YanischPerron et al., 1985), expressed for 2 h at 37 “C and then plated on to LB plates supplemented with 50 pg/ml of Km, 30 pg/ml of XGal and 7 pg/ml of IPTG. All blue Km-resistant colonies analysed were identical. The extra Hind111 site originating from the transferred KmR fragment was removed by linearising the plasmid with Hind111 in 50 pg EtdBr/ml (Osterlund et al., 1982) and end-repairing with PolIk (Maniatis et al., 1982). Km-resistant blue colonies were analysed with the enzyme NheI, the site created by the Hind111 fill-in. The PstI and SphI sites of the KmR gene were mutated with the oligodeoxynucleotides 5’-CCCTGAATGAACTCCAAGACGA-

to another without gel purification (Smith, 1980), simply by selecting for the recipient plasmid. This may be by force-cloning (Messing and Vieira, 1982) and/or by the lac colour reaction. With this method I have only recovered the insert of interest, and never the donor plasmid, even though this is theoretically possible as a tandem repeat. I have used this system successfully for the transfer of the 3.0-kb BglII-StuI CDC9 fragment (Barker et al., 1985; a kind gift of Dr. L.H. Johnston) from pUC18 into a pK19 plasmid carrying ARSl/ CENl4/URA3, for transfer and analysis in Saccharomyces cerevisiae. But this system is of special interest in cases where the insert is the same size or larger than the original vector. With gel purification, contamination of insert by vector (about 5--loo/,) may be bypassed by techniques such as phosphatase treatment (Chaconas and Van de Sande, 1980) or a second digestion to remove the vector. This is time-consuming and not always possible and convenient restriction sites are not always available. The pK plasmids and their derivatives offer a simple and fast alternative. Two observations are noteworthy. (i) The Km resistance does not result in the formation of ‘satellite’ colonies after extended growth, and (ii) in a standard DNA preparation the pK plasmids yield consistently about three times as much DNA than do the pUC plasmids. This may be due to the unterminated Km transcript running into the ori and possibly ‘driving’ the ori.

++++

250

1870

970

1170

TGCTGCTTGCAAACA

1490

1390

1510 1530 CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGRGCCTC

1410 1430 1450 CCACTGAGCGTCAGACCCCGTAG~GATC~GGATCTTCTTGAGATCCTTTTTTTCTGCGCGT~TC

1350 1370 CCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATCGTGAGTTTTCGTT

1270 1290 1310 ATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACC

1210 1230 1250 AGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATG-GGTTGGGCTTCGGA

1150 1130 CGCATCGCCTTCTATCGCCTTCTTGRCGRGTTCTTCTGAGTGACCGACCA

1070 1090 1110 TGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAG

990 1010 1030 TCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGC

930 950 GATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATA~GGCCGCTTTTCTGGA~

1470

1330

1190

1050

from bp 1333-2661 ofthe pBRNeoHindlI1

nucleotide

changes

site, while the asterisks

pK18.

Sequences

from

described

in section a.

536,538 and 895 indicate

added by the PolIk repair

with the MCS from bp 2443-2499. nucleotides above the sequences

bp 6-10 indicates

plasmid

2650

2590

2450

2310

2170

2030

1890

1750

1610

with the KmR gene from bp 365-1156.

2661.bp pBRNeo,

of the

are derived from pUC18, The + + + + above the sequence

Sequences

sequence from the plasmid

nucleotide bp 1-1332 are derived

Fig. 2. Complete

G

2610 2630 TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC~CAGTTGCGCAGCCTG~TGGCG~TGGC

2530 2550 2570 ACGTCGTGACTGGGAe9ACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGC

2470 2490 2510 GCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGC~GCTTGGCACTGGCCGTCGTTTTACA

2430

2390 2410 TGTTGTGTGGAATTGTGAGCGGATAACAATTTTCACAGGTTCGA

910

850 670 890 CGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCGGCGCGGATGCCCGACGGCGAG

*

2330 2350 2370 AACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTA

790 810 830 AAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGG~GCCGGTCTTGTCGATCAGGATGATCTGGA

2250 2270 2290 CGCGCGTTGGCCGATTCATTAATGCAGCTGCAGCTGGCACGACAGGTTTCCCGACTGG-GCGGGCAG~GAGCGC

770

2230

710 730 750 GTATCCATCATGGCTGATGCAATGCGGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACC

2210

2190 GAACGACCGAGCGCAGCGAGTCAGTGAGCGGACCGCCTCTCCC

650 670 690 GGACTGGCTGCTATTGGGCGAAGTGCCGGGGGCAGGATCTCCTGTCATCTCACC~TGCTCCTGCCGAG~

2110 2130 2150 GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC

570 590 610 GGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTG~GCGGG~G

630

2050 2070 2090 AAACGCCAGC~CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT

510 530 l l 550 GGGCGCCCGGTTCTTTTTGTCAAGACCGACCGACCTGTCCGG~GCCCTG~TG~CTCC~GACGAGGCAGCGC

1970 1990 2010 TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGG~

490

1830 1850 CCTACAGCGTGAGCATTGAGAAAGCGCGCCACGAAGC

430 450 470 ATTCGGCTATGACTGGGCACRACAGACAGAC~TCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAG

350

1770 1790 1810 GGTCGGGCTGRRCGGGGGGTTCGTGCRCACRGCCCAGCTTGGAGCGAACGACCTRCRCCGARCTGAGATA

1910 1930 1950 GGCAGGGTCGGRACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG~CGCCTGGTATCTTTATAGTCCTG

270

370 390 410 GAGGATCGTTTCGCATGATTGAACAAGATGGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCT

290 310 330 AACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGAT

230 ATGGACAGCAAGCGAACCGGRRTTGCCAGCTGCCAGCCCGTA

1690 1710 1730 GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC~GACGATAGTTACCGGAT~GGCGCAGC

210

1570 1590 1550 TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC~TACTGTCCTTCTAGTGTAGCCGTAGTT

150 170 190 ACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGC~AGACTGGGCGGTTTT

70

1670 1650 1630 AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT~TCCTGTTACCAGTGGCT

50

90 110 130 AAAGCCAGTCCGCAGRRACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAA

30 CGATAAGCTAGCTTCACGCTGCCGC~GCRCTCAGGGCG~AG

312

Norrander,

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