The biological process by which genetic information is transferred or exchanged between regions of DNA of identical or very similar nucleotide sequence is known as homologous recombination. This natural process of genetic exchange appears to be ubiquitous and the study of its fundamental basis represents an important and exciting area of research in its own right. The potential use of homologous recombination to replace specified genes in both prokaryotic and eukaryotic organisms has resulted however in the investigation of this fundamental process with more applied purposes in

Successful allele replacement promises not only to facilitate studies of gene function and regulation(134) but also to allow the engineering of genetic improvements and the elimination of undesir- able traits from an organism without altering its genetic organisation or constitution.

Applying this technology to plants is highly desirable, but not simple. This is due to the fact that, like in animal cells, foreign DNA introduced by transformation integrates into the plant genome in an apparently random fashion, regardless of homology to endogenous

This hurdle first seemed surmountable when gene targeting to restore a partial non-functional artificial gene was reported in plant protoplasts re- transformed, by direct gene transfer, with homologous

DNA containing the missing region('). (However, in this instance it was not demonstrated unequivocally that it was the targeted locus that was restored. It could well be that the defective gene copy on the incoming plasmid was restored and that this plasmid subsequently integrated at another position in the genome.) Never- theless the most efficient and widely used plant transformation method relies on the natural transform- ation ability of the soil microorganism Agrobacterium tumefaciens (reviewed in ref. 8). The plasmid-encoded T( transfer)-DNA produced in the agrobacteria and subsequently delivered and integrated into the plant genome (via a poorly understood mechanism) rep- resents the centrepiece of this natural transformation system. Thus, one wonders whether T-DNA molecules would be competent for intermolecular, extrachromo- soma1 homologous recombination inside the plant cell nucleus and indeed, whether the properties of such a transformation centrepiece could be exploited for targeted mutagenesis experiments in plants.

A clean answer to these questions is provided in a recent paper ublished in the EMBO Journal by Offringa et al!) They first tested the suitability of T-DNA molecules as intermolecular recombination substrates; here they co-cultivated tobacco mesophyll protoplasts with two Agrobacterium strains each harbouring different T-DNAs encoding a defective selectable marker. Although the T-DNAs contain a region of shared homology (about 0.55 Kb), one has a 5' deletion, the other a 3' deletion of the coding region of this marker. As in previous function restoration of the marker gene was used to select for the occurrence of homologous recombination (Fig. 1A). They found that 1-4% of the cotransformed cells yielded at least one intermolecular recombination event. Analysis of DNA isolated from 11 independent plant lines, regenerated from such recombinant tissue, demonstrated the presence of a restored (wild-type) marker gene; this analysis was done by Southern

A Agrobacteriurn

Fig. 1. Homologous recombination in the plant cell following Agrobacterium transformation. (A) Extrachromosomal recombination: T-DNA molecules from Agrobacterium strains X and Z enter the plant cell and cross the nuclear membrane. Once inside the nucleus molecules x and z recombine extrachromosomally along their shared homology. The recombination product may then integrate into the genome via illegitimate recombination. ( B ) Targeted gene replacement: following transformation a T-DNA molecule recombines with a homologous chromosomal allele. This may result in the replacement of the chromosomal allele by its homologous sequence in the T-DNA.

blotting and PCR amplification. An alternative possi- bility is that the recombination events occurred, not in the plant tissue, but in the agrobacterial cells: first by plasmid conjugation to give agrobacteria harbouring both types of T-DNA, and then homologous recombi- nation in these agrobacteria giving gene restoration prior to T-DNA transfer to plant tissue. This possibility was excluded by highly sensitive bacterial conjugation control experiments conducted in the presence or absence of plant cells. The precise nature of the recombination event leading to marker function restoration could not be elucidated, for example to ask whether recombination occurred either prior to or following nuclear integration of one or other or both the co-transferred T-DNAs. (Because homologous recom- bination between co-transformed T-DNAs is found to occur at high frequencies, it is likely to occur extrachromosomally before integration.) Nevertheless, the experiments furnished proof of the competence of T-DNA molecules to act as homologous recombination substrates inside the plant cell nucleus.

The question of whether T-DNA molecules can be used to target specified sequences already resident in the plant nuclear genome was approached as in previous gene targeting studies(337). Here, a transgenic plant line was constructed containing a defective selectable marker inactivated by a deletion of the 3‘ end of the coding region; fortuitously this line contained an inverted repeat (i.e. two copies of the construct). This plant line was re-transformed with a T-DNA construct containing another defective copy of this selectable marker, but here, inactivated by a non-overlapping deletion of the 5’ end of the coding region and including the promoter region (Fig. 1B). Surprisingly, a high frequency of drug resistant transformants were re- covered; however, DNA analysis, by PCR amplifi- cation and Southern blotting, demonstrated that only a minute proportion of these (1/213 analysed) resulted from correct replacement at the target locus; two other drug resistant individuals arose from the correction of the incoming T-DNA molecule (presumably by recom- bination with its chromosomal homologue) followed by subsequent integration elsewhere in the genome. The remaining drug resistant calli were postulated to have arisen by formation of translational fusions with active endogenous genes following T-DNA integration via illegitimate recombination. In this experiment, the 5‘ deletion in the marker was very small, only 34 bp of the coding region. The enormous number of ‘false positives’ observed highlights the need to use a recombination substrate that is sufficiently defective to preclude restoration of gene function by such events. Other refinements might be to include a homologous recombination-enriching strategy in the selection proto- col (see for example ref. 10) or else of developing plant gene replacement techniques that rely on screening rather than on selection (see for example ref. 11). It is interesting and noteworthy that, in addition to the demonstration of a bona fide gene targeting event, the

experiments also show that the incoming T-DNA can exchange information with an integrated homologous sequence but then integrate elsewhere by illegitimate recombination.

The potential use of Agrobacterium-mediated gene replacement in plants is not limited to artificial loci. This is illustrated in another recent paper published in The Plant Cell by Lee et ul.(l2). Lee’s experiments concerned the acetolactate synthase (ALS) endogenous gene which appears to be located, in two copies, at two genetically unlinked loci (SuRA and SuRB) in Nicotiana tabacum. ALS participates in the biosynthesis of branched-chain amino acids in plants and is the target of action of sulfonylurea herbicides. A single point mutation that makes a single amino acid change in the protein makes it resistant to the herbicide chlorosul- furon and thus provides a selection system for the detection of SURA/SURB targeted mutagenesis. Lee et al. transformed chlorosulfuron sensitive tobacco proto- plasts with an Agrobacterium strain carrying an ALS gene inactivated by a deletion of the 5‘ region of the coding region (the deletion included the promoter region) but carrying this point mutation. Seven herbicide-resistant individuals were recovered and, although no direct, unequivocal proof of a gene targeting event was furnished, DNA restriction analysis and genetic studies suggested that formation of a hybrid ALS gene by homologous recombination occurred in three of these resistant lines. The frequency of endogenous gene replacement observed by Lee et al.(I2) compares to those reported by Offringa et ~ 1 . ( ~ ) and by Paszkowski et d7) for artificial loci. Taken together, the results demonstrate the suitability of the natural plant transformation system for gene replacement studies in plant species susceptible to Agrobacteriurn infection.

Much ignorance remains regarding the mechanism of T-DNA transfer from the agrobacteria to the plant cell nucleus (reviewed in ref. 13). However, it is now clear that agrobacterial proteins associated with the T-DNA during transfer do not preclude T-DNA interaction with the enzymatic apparatus responsible for homolo- gous recombination in the plant cell nucleus. Whether this is because such associated proteins dissociate from the T-DNA molecule upon entry into the nucleus or because they only protect the ends of the DNA molecules leaving most of their length susceptible to ‘attack’ by the recombination machinery of the cell has yet to be established. It may even be that the T-DNA associated proteins do not hinder but indeed promote or assist in the interaction of T-DNA molecules with the recombination proteins that lead to their integration into the plant genome (whether as an illegitimate or as a homologous process). What is now clear is that plant scientists and genetic engineers will soon benefit from replacing plant genes nature’s way.

References 1 GARFINKEL. D. J . , SIMPSON, R. B., REAM, L. W.. WHITE. F. F . . GORDON, M.

P. A N D NESTER, E. W. (1981). Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27, 143-153. 2 STRUHL. K. (1983). The new yeast genetics. Nature 305, 391-397. 3 THOMAS. K. R., FOLGER, K. R. A N D CAPECCHI, M. R. (1986). High frequency targeting of genes to specific sites in the mammalian genome. Cell 44,419-428. 4 THOMAS, K. R. AND CAPECCHI, M. R. (1990). Targeted disruption of the murine int-/ proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346, 847-850. 5 PASZKOWSKI. J.. SHII.I.ITO. R. D. . SAUL, M., MANDAK, V. . HOHN. T.. HOHN. B . A N D POTRYKUS. I. (1984). Direct gene transfer to plants. EMBO Journal 3. 2717-2722. 6 CHYI, Y.-S., JORGENSEN. R. A, . GOLDSTEIN, D., TANKSLEY. S . D. AND LOAIZA-FItiuERoA, F. (1986). Location and stability of Agrobacterium-mediated T-DNA inscrtions in the Lycopersicum genome. Mol. Gen. Genet. 204, 64-69. 7 PASZKOWSKI, J.. BAUR, M., BOCUCKI, A. AND POTRYKUS. I. (1988). Gene targeting in plants. EMBO Journal 7, 4021-4026. 8 GASSER. C. S . AND FRALEY, R. T. (1989). Genetically engineering plants for crop improvement. Science 244, 1293-1299. 9 OFFRINCA, R.. DE GROOT. M. J. . HAAGSMAN, H. J., DOES, M. P., VAN DEN EI.ZEN. P. J. A N D HOOYKAAS, P. J . (1990). Extrachromosomal homologous

recombination and gene targeting in plant cells after Agrobucferium mediated transformation. EMBO J. 9, 3077-3084. 10 MANSOUR, S . L., THOMAS, K. R. ANDCAPECCHI, M. R. (1988). Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336. 348-352. 11 ZIMMER, A. A N D GRUSS, P. (1989). Production of chimaeric mice containing embryonic stem (ES) cells carrying a homeobox H o x - / . / allclc mutated by homologous recombination. Nature 338. 150-153. 12 LEE. K. Y . , LUND. P. , LOWE, K. A N D DUNSMUIR. P. (1990). Homologous recombination in plant cells after Agr-obacferium-mcdiated transformation. The Plant Cell 2, 415-425 13 ZAMBRYSKI, P. (1988). Basic processes underlying Agrohacterium-mediated DNA transfer to plant cells. Annu. Rev. Genet. 22. 1-30

Jorge Tovar and Conrad Lichtenstein are at the Centre for Biotechnology, Imperial College of Science, Technology & Medicine, London SW7 2AZ, UK.

SPONSORED BY THE NEW YORK ACADEMY OF SCIENCES

Second International Conference on

Sodium/Calciurn Exchange

April 8-10, 1991

Baltimore Marriott Inner Harbor, Baltimore, MD

The plasma membrane sodium/calcium exchange system regulates intracellular calcium levels in many kinds of cells. It controls cardiac and smooth muscle contractility, visual adaptation in retinal photoreceptors, and calcium homeostasis in neurons, secretory cells and transporting epithelia. It may also participate in the pathogenesis of salt-dependent hypertension. The conference will emphasize properties, physiological significance and pathophysiological roles of sodium/calcium exchangers from various tissues.

Conference Chairmen:

Dr. Mordecai P. Blaustein Dr. Reinaldo Dipolo Department of Physiology Centro de Biofisica y Bioquimica University of Maryland Instituto Venezola de Molecular Biology School of Medicine Investigaciones Cientificas (IVIC) Roche Research Center 655 W. Baltimore Street Apartado 21827 Nutley, NJ 07110 Baltimore, MD 21201 Caracas 1020A

Dr. John P. Reeves Roche Institute of

Venezuela

For further information contact: Conference Department, The New York Academy of Sciences, 2 East 63rd Street, New York, NY 10021, USA

TEL: 212-838-0230, FAX: 212-888-2894