University of Groningen Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene Heidekamp,

The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined. The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant cells. The nucleotide sequence of the tmr gene displays a continuous open reading f rame specif ying a polypeptide chain of 240 amino acids. The 5'terminus of the polyadenylated tmr mRNA isolated from octopine tobacco tumor cell lines was determined by nuclease S1 mapping. The nucleotide sequence 5'-TATAAAA-3', which sequence is identical to the canonical "TATA" box, was found 29 nucleotides upstream from the major initiation site for RNA synthesis. Two potential polyadenylation signals 5'-AATAAA-3' were found at 207 and 275 nucleotides downstream from the TAG stopcodon of the tmr gene. A comparison was made of nucleotide stretches, involved in transcription control of T-DNA genes. INTRODUCTION Tumor-inducing (Ti) plasmids harbored by Agrobacterium tumefaciens cause neoplastic cell growth called crown galls on most dicotyledonous plants (for recent reviews see ref s. 1,2). Tumor formation originates from the transfer of a specific Ti plasmid DNA segment, designated T-region, from the bacteria to the plant cells. Beside this T-region, a second portion of the Ti plasmid, the virulence region, is required for tumor induction (3,4). Upon transfer, the T-region is covalently integrated into plant nuclear DNA (5,6,7). Ti plasmid transformed plant cells are characterized by unlimited cell proliferation and the ability to grow in the absence of phytohormones like a cytokinin and an auxine. The tumor cells synthesize opines which are catabolized by the bacteria (8,9,10). Depending on the type of opine(s), produced in crown galls, the Ti plasmids have been classified in three major groups: octopine Ti plasmids, nopaline Ti plasmids and agropine Ti plasmids (11). In octopine tumors it has been shown that the enzyme octopine synthase (LpDH) is encoded by T-DNA © I R L Press Limited, Oxford, England. 6211 Nucleic Acids Research (12,13). These findings have prompted the development of Ti plasmidderived vectors for the genetic manipulation of plant cells. The extent of T-DNA and the T-DNA organization in octopine tumor tissues have been investigated (6,7,14). In octopine tumor cells the T-DNA consists of either one or two segments which originate from adjacent regions in the Ti plasmid DNA. All tumor cell lines investigated to this date contain the left part of the T-DNA (TL-DNA), whereas the right part of the T-DNA (TR-DNA) is present only in a limited number of tumor cell lines. Hybridization studies have shown that the TL-DNA in octopine tumor cells encodes eight polyadenylated mRNAs (15,16,17). At least f ive and probably six of these transcripts have been shown to be involved in the process of tumor formation and maintenance (18,19,20,21). In this paper the nucleotide sequence of the octopine T-region, encoding the tmr gene, is presented. This cistron specif ies one of the abovementioned transcripts (viz. transcript 4); presumably its product inhibits root formation of the tumors on certain plant species and appears to play a role in the cytokinin-independent growth of transformed cells (17,21). The 5'terminus of the polyadenylated tmr mRNA, isolated from various octopine tumor cell lines, was determined by nuclease Si mapping. A comparison is made between nucleotide stretches, involved in regulation of transcription of the tmr gene, of the octopine and nopaline synthase genes (22,24,25) and of the region which encodes octopine transcript 7 (23). MATERIALS AND METHODS Enzymes The various restriction endonucleases indicated in this paper were obtained from New England Biolabs, Inc. (Beverly, MA) with the exception of BamHI, PstI, ClaI and HpaII which were purchased from Boehringer (Mannheim) and Sau3AI which was bought from Amersham International Ltd. (Amersham, UK). Assay conditions for restriction endonucleases were as described by the manufacturers . T4 DNA ligase, T4 polynucleotide kinase and calf intestine alkaline phosphatase were purchased from Boehringer (Mannheim); DNA polymerase I (large fragment) was from New England Biolabs, Inc. (Beverly, MA) and nuclease S1 was from Sigma (St. Louis, MO). Recombinant plasmids and preparation of DNAs Subfragments of the restriction DNA fragment EcoRI-7 from pTiAch5, on