Construction of β-lactamase-encoding ApR gene cassettes for rapid identification of cloned genes

Gene, 170 (1996) 143 144 © 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00

143

G E N E 09528

Construction of [3-1actamase-encoding Ap R gene cassettes for rapid identification of cloned genes (DNA sequencing; pUC plasmid primer; recombinant DNA tool)

T o m o k i Yokochi, Jun-ichi K a t o and Hideo Ikeda Department of Molecular Biology, Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan Received by Y. Sakaki: 7 September 1995; Revised/Accepted: 12 October/14 October 1995; Received at publishers: 20 November 1995

SUMMARY

We have constructed two Escherichia coli plasmids, pYK18 and pYK19, from which the BamHI, SmaI or EcoRI DNA fragments containing the Ap s gene, conferring resistance to ampicillin, can be excised. The Ap R cassettes have an annealing site for the sequencing primer of pUC plasmids at each end. Therefore, when the cassette is inserted into a gene, we can determine the nucleotide sequence of the gene from the insertion site using the sequencing primers of the pUC plasmids. This method is useful for identifying a cloned gene.

To identify a gene cloned on a plasmid, we usually localize the region necessary for the gene and determine its nt sequence (Sambrook et al., 1989). For these steps, several rounds of subcloning are usually needed and they are time-consuming. Since many nt sequence data have been accumulated, it is important to determine whether the cloned gene is new, since it is otherwise not necessary to determine the complete nt sequence. For rapid identification of a cloned gene, we have constructed gene cassettes (Fig. la), that carry an ApR gene and annealing sites for sequencing primers of pUC plasmids. The gene cassette is inserted into the target gene, and the plasmids with the insertion, which inactivates the target gene, are selected (Fig. lb). Then the nt sequence of the target gene is determined starting from the inserted fragment (Fig. lb).

Correspondence to: Dr. H. Ikeda, Department of Molecular Biology, Institute of Medical Science, University of Tokyo, P. O. Takanawa, Tokyo 108, Japan. Tel. (81-3) 5449-5325; Fax (81-3) 5449-5422. Abbreviations: Ap, ampicillin;bp, base pair(s);kb, kilobase(s) or 1000 bp; Kin, kanamycin; nt, nucleotide(s); ori, origin of DNA replication; Plac, lac promoter; R, resistant/resistance. SSDI 0378-1119(95)00826-8

ACKNOWLEDGEMENT

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

REFERENCES Kato, J., Nishimura, Y., Yamada, M., Suzuki, H. and Hirota, Y.: Gene organization in the region containing a new gene involved in chromosome partition in Escherichia coll. J. Bacteriol. 170 (1988) 3967-3977. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Vieira, J. and Messing, J.: The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19 (1982) 259 268. Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33 (1985) 103-119.

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Fig. 1. Ap R cassettes and their use in gene identification. (a) pYK18 and pYK19 plasmids. Plasmids pYK18 and pYK19 were constructed by inserting a Km R marker into pUC18 and pUC19, respectively. The PvuII fragment with a Km R marker of pYS8 (Kato et al., 1988) was inserted into the HincII site of pUC8 (Vieira and Messing, 1982) to obtain pJK274. Sequencing analysis confirmed that the direction of transcription of the Km e gene is the same as that of the lac promoter of pUC8. The PvuII fragment containing the Km R marker of pJK274 was excised and inserted into one of the DraI sites (the DraI site at the intervening region between the Ap R gene and the ori) of the plasmids of pUC18 and pUC19 (Yanisch-Perron et al., 1985) to construct pYK18 and pYK19, respectively. From pYK18 and pYK19, Ap R cassettes, which carry annealing sites for primers of pUC plasmids at the each end, can be excised with BamHI, Sinai or EcoRI. On the both plasmids, the direction of the Km R marker is the same as that of the Ap R gene. U and R represent the annealing sites of the commercially available universal primer and reverse primer of pUC plasmids, respectively. E, EcoRI; B, BamHI; H, HindIII. (b) Rapid identification of the cloned gene. The plasmid with a target gene (,4) is partially digested with the restriction enzyme Sau3AI. The DNA fragments are separated by 0.7% agarose-gel electrophoresis and those cut at one Sau3AI site are extracted from the gels. The Sau3AI digestion should not be exceeded to avoid contamination of the smaller fragments, that are cut at two or more Sau3Al sites, especially in the case of the large plasmid. The extracted linear fragments are ligated with the Ap e BamHI casette of pYK 18 or pYK 19. Among the resultant Ap e plasmids, plasmids lacking the activity of the target gene by insertion of the cassettes are selected. To avoid the plasmid with deletion of the target gene or insertion of two or more copies of the Ap R cassettes, the plasmid DNA should be analyzed by restriction enzyme digestion. Using the obtained plasmids as template DNA and sequencing primers, universal (U) and reverse (R) primers, of pUC plasmids, the nt sequence of the target gene is determined from the Sau3AI site, at which the cassette is inserted, since the cassettes have annealing sites for the primers of pUC plasmids at the ends (panel a}. By this method, we have succeeded in sequencing and identifying many genes. The cassettes can be excised by Sinai, which produces blunt ends, thereafter they can be inserted into a blunt-end site produced by, for example, AccII, Afal, Alul or HaeIII. The BamHI cassette of pYK18 has unique HincII, HindIII, PstI, SalI, Sphl, Sinai and XbaI sites, and the BamHI cassette of pYK19 has unique KpnI and SacI sites (panel a). Using these restriction sites on the cassettes and those sites on the cloned region, deletion derivatives of the target plasmids can be obtained and the nt sequence from the restriction sites on the cloned region can be determined. The BamHI cassette of pYK19 has two Sinai sites and two EcoRI sites, and the Ap R gene can be removed by Sinai or EcoRI digestion. When the cassette is inserted into the upstream region of an operon containing a target gene in one direction (the direction of the operon is opposite to that of the lac promoter of the cassette), the expression of the downstream gene (a target gene) can be inactivated, leading to misinterpretation that the upstream region is the target gene. To avoid this type of misinterpretation, the plasmid with the insertion of the cassette in the other direction should also be isolated (the direction of the target gene is the same as that of the lac promoter of the cassette).