Gene, 102 (1991) 139-140 0

1991 Elsevier

GENE

Science

Publishers

139

B.V. 0378-l 119/91/$03.50

05005

Brief Notes Plasmids with easily excisable cat gene cartridges* (Recombinant

DNA;

chloramphenicol

acetyltransferase;

kanamycin

resistance;

polylinker;

vector

construction;

gene

regulation) Peter Dobrowolski Institute of Biotechnology, 7050 Leipzig (F.R. G.) Received by A.J. Podhajska: 5 February Revised: 19 December 1990 Accepted: 12 March 1991

1990

SUMMARY

New cat-cassette-containing vectors based on kanamycin-resistance-encoding They were designed to facilitate the subcloning of the promoterless cut gene with an ampicillin-resistance marker. In plasmids pCATlO-pCAT40, the sequences and can be excised by double digestion with pairs of appropriate in all three reading frames are located upstream from the AUG start codon excised by a single digestion with the enzymes, SafI, PstI, or HindIII.

The cut cartridges (Close and Rodriguez, 1982) have been frequently used in transcriptional mapping of DNA fragments, analysis of promoter strength and gene expression of both prokaryotic and eukaryotic DNA, as well as for various vector constructions. Most of the available cat gene cartridge vectors have a limited range of restriction sites suitable for isolation of the gene cassette and are based on plasmids with ApR as the selection marker. To facilitate subcloning procedures, particularly into frequently used ApR vectors like pBR322, pUC-plasmids and their derivatives we designed new vectors with different polylinker sequences located at both ends of the cut gene and KmR as the marker. As shown in Fig. 1, pCAT10, pCAT20, and pCAT30 contain BumHISulI, BarnHI-HindIII, and SalI-Hind111 cat cassettes, Correspondenceto: Dr. P. Dobrowolski, Permoserstrasse Tel. (37)2392428;

15, O-7050

Institute Leipzig (F.R.G.)

Fax (37)23 13950.

* On request, the author will supply detailed the conclusions reached in this brief note. Abbreviations:

of Biotechnology,

Ap, ampicillin;

ferase; cat, gene encoding for KmR; kb, kilobase resistant.

experimental

bp, base pair(s);

evidence

for

CAT, Cm acetyltrans-

CAT; Cm, chloramphenicol; or 1000 bp; Km, kanamycin;

km, gene coding

R, resistance/

plasmids, pHSG298/299, were constructed. into frequently used plasmids, including those cat gene cartridges are flanked by polylinker restriction enzymes. Translation stop codons of the pCAT40 cartridge; the latter can also be

respectively, with several additional unique restriction sites. In pCATl1, pCAT21, and pCAT3 1 these unique sites are at the opposite ends of the cut gene. From these plasmids a wide variety of cat gene cartridges can be excised by double restriction. All plasmids contain the cut gene inserted in an orientation opposite to the P,,= promoter. The gene cartridge is transcribed from the km promoter. Obviously, under the selection conditions used, this is the favoured orientation of the cut cassette yielding stable recombinant plasmid molecules. Vector pCAT40 is a derivative of vectors pCAT10 and pKK232-8 (Brosius, 1984) containing translation stop signals in all three reading frames between the polylinker sequence located at the 5’ end of the cassette and the cat-AUG start codon. The advantage of such cassette is the possibility of precise promoter-activity measurement because no fusion proteins can be produced. The pCAT40 gene cartridge can be isolated by a single digestion with enzymes SalI, PstI, or Hind111 or by double digestion with restriction enzymes having cleavage sites located at opposite ends of the cassette. In this respect, the plasmid is similar to vector pJS 133 (Shiau and Smith, 1988). But, like most cut cartridge vectors, pJS 133 contains ApR as the only selectable resistance marker.

140

Fig. 1. Structure shaded

boxes

positions

of pCAT-plasmid represent

and transcriptional

pHSG298 with

box) are indicated.

digested

DNA (Takeshita

cut gene cartridges

and Rodriguez,

The polylinker

the

sequences

et al., 1987) was ligated

of Sal1 +EcoRI-digested obtained

and

BarnHI

+ EcoRI-

from pCM1 and pCM4 (Close

1982). The ligation mixtures

E. coli strain HBlOl

The

and the corresponding restriction sites are of pCAT vectors, Sal1 + BarnHI-digested

and pHSG299

a mixture

sequences.

of the P,,,, promoter,

the KmR gene (kun), and the cat

(oriV),

flanking the cat cartridge shown. For construction

The thin lines and the

pHSG298/299

orientations

origin of DNA replication gene (open arrowed

vectors.

plasmid

were used to transform

with selection for KmR (50 pg Km/ml) and CmR

(25 pg Cm/ml) on L-agar plates (Miller, 1972). The resulting plasmids, pCATI0

and pCATl1,

contain

Sal1 sites and the remaining In an analogous cartridge, respectively.

of KmR as the only resistance

marker and

ACKNOWLEDGEMENTS

Hind111

having

a GUI

of pCATl0

was obtained

after replacing

by the corresponding

the small

EcoRI fragment

of

with stop codons in all three frames between the upstream

polylinker

and the AUG start codon of cat (Brosius,

1984). CAT (in

= cat.

REFERENCES Brosius,

J.: Plasmid

vectors

for the selection

of promoters.

Gene

27

(1984) 151-160. Close, T.J. and Rodriguez,

R.L.: Construction

and characterization

chloramphenicol-resistance

gene cartridge:

transcriptional mapping (1982) 305-316.

of extrachromosomal

Miller, J.H.: Experiments Laboratory, Shiau,

The author is grateful to Dr. Koki Horikoshi, in whose laboratory this work was carried out, for his interest in the project and helpful discussion. This work was a part of the ‘Horikoshi Superbugs Project’ of the Japanese Research Development Corporation.

the pCM7-derived were obtained

and

vector.

pKK232-8

open arrows)

The presence

by BumHI

ofthe pHSG

and Hind111 or Sal1 and Hind111 sites,

Vector pCAT40

EcoRI fragment

the variety of polylinker sequences flanking the newly constructed cassettes makes pCAT plasmids a convenient tool for use in vector construction and for analysis of uncharacterized DNA fragments.

and pCAT30/31

by BamHI

flanked

sequence

way, using additionally

pCAT20/21

gene flanked

cut cassettes

polylinker

A. and Smith,

analysis Takeshita,

in Molecular

Cold Spring Harbor,

and genetic

Genetics.

elements.

J.M.: Improved constructions.

mentation

and low-copy-number and chloramphenicol-

Gene 61 (1987) 63-74.

to the Gene

Cold Spring

20

Harbor

NY, 1972. cat gene cassette

for promoter

Gene 67 (1988) 295-299.

S., Sato, M., Toba, M. and Hashimoto-Gotoh,

number

ofthe

a new approach

plasmid

vectors

T.: High-copyfor [acZa-comple-

or kanamycin-resistance

selection.