The plasmids pRT100-pRT104. carrying the 35S promoter and the polyadenylation signal of CaMV strain Cabb B-D (corresponding to bp 7016-7434 and 7436-7639 of CM 1841') were constructed in modified polylinkers of pUC18/192. The DNA sequence of pRT100 between its two HindllI sites is shown; capital letters indicate CaMV sequences. TATA-box. ATG. and polyadenylation signal (uirnderlined) as well as the putative transcriptiohal start (-.) and stop (-) are marked (ref.3).

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. transcription from promoters in the intergenic region (IR) (4) and rolling-circle replication (5-8). Apart from one single viral protein, geminivirus DNA multiplication entirely relies on the DNA-replication apparatus of the host plant, representing a simple system to study DNA replication in the plant nucleus. The origin of viral-strand DNA replication is located in the IR and has been mapped to the conserved nonanucleotide sequence TAATATTAC, flanked by inverted repeats potentially forming a stem-loop (9, 10).Only one viral protein is indispensable for replication (11). This replication protein (AL1, Cl, or ORFIII/IV protein) is encoded by either one or two open reading frames. In the latter case, the mRNA spanning the overlapping reading frames is spliced to give rise to a single polypeptide chain (12,13 (27) was cloned in pGEX-3X (29). Alternatively to the factor Xa cleavage site of pGEXC1, the amino acid sequence-PGSGSGDDDK-specifying an enterokinase cleavage site (30) was engineered between the glutathione S-transferase (GST) and the Rep domains. The following oligonucleotides, 5'-GT GGGATCCCGGGCTCCGGCTCCGGCGACGACG-ACGACAAAATGCCAAGATCAGGTCGTTT-3' and 5'-C-ACCCTCAATCACTATACTCACCGG-3', were used for PCR (31) on pGEXC1 DNA, yielding the expression vector pGEXEC1.Expression of Rep Protein in E. coli and Purification. A 1:10 dilution of E. coli DH5 a (pGEXC1 or pGEXEC1) in NZCY medium (29) containing 100 ,tg of ampicillin per ml was grown for 1 hr at 28°C, and protein expression was induced by 0. (Fig. 1A) were done with the GST-Rep fusion of pGEXC1; all other experiments were done with the GSTRep fusion protein expressed by pGEXEC1. Approximately 300 ng of GST-Rep protein were incubated with substrate DNA in a total volume of 20 ,ul for 30 min at 37°C in cleavage buffer (25 mM Tris-HCl, pH 7.5/75 mM NaCl/5 mM MgCl2/ 2.5 mM dithiothreitol/0.5 mM EDTA), containing 50 fmol of each oligonucleotide or 100 fmol of double-stranded BspEIBamHI IR fragment. The reaction was stopped by 2 ,ul of 0.5 M EDTA, lyophilized, resuspended in loading buffer [98% (vol/vol) formamide] and analyzed on 12% sequencing gels, which were dried and autoradiographed. The cleavage products were excised from the gels and quantified by liquid scintillation counting.Abbreviations: GST, glutathione S-transferase; IR, intergenic region; Rep protein, replication initiator protein; TYLCV, tomato yellow leaf curl virus. tTo whom reprint requests should be addressed.

The nucleotide sequence of the lacY gene coding for lactose permease (M protein) in Escherichia coli has been determined. The sequence includes the intergenic regions between the lacZ (beta-galactosidase) and lacY genes as well as the region between the lacY and lacA (transacetylase) genes. Lactose permease is predicted to consist of 417 residues (71% nonpolar), resulting in a protein with a molecular weight of 46,504. The reading frame was confirmed by the sequence of a nonsense mutation changing codon 33 from UGG to UAG.

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