Volume 12 Number 11 1984 Nucleic Acids Research Nodeotlde sequence of tht tmr locus of Agmbacterium tumefaciens pTi T37 T-DNA S.B.GoMberg, J.S.Flkk and S.G.Rogers* Monsanto Company, 800 North Lindbergh Boulevard, Saint Louis, MO 63167, USA Received 5 March 1984; Revised and Accepted 16 May 1984 ABSTRACT The nucleotide sequence of the tmr locus from the nopaline-type pTi T37 plasmid of Agrobacterium tumefaciens was determined. Examination of this sequence allowed us to identify an open reading frame of 720 nucleotides capable of encoding a protein with a derived molecular weight of 27025 d. Comparison of the pTi T37 tmr sequence with the published sequence of the pTi Ach5 tmr locus shows over 88% homology in the 240 bases 5' to the transla- tional initiation codon and over 91% homology in the coding sequences. The 3 1 nontranslated regions show less than 50% homology as expected for the 3' regions of divergent related genes. The possible significance of areas of conserved sequences, particularly in the 5' regulatory regions, is discussed. INTRODUCTION AgrobfLCterium tumefaciens causes crown gall disease by transfer of a DNA segment from its large resident Ti plasmid into the plant cell where this DNA is covalently integrated into the genome (1-5). Expression of certain genes located on the transferred DNA (T-DNA) results in in situ neoplastic growth or phytohormone-independent growth of the infected tissue when placed into culture (6-7). Recent results implicate certain specific T-DNA encoded genetic and transcriptional units, the tms and tmr loci, as the units of expression responsible for the hormone-independent growth (8-12). Specifi- cally, expression of the tins locus results in elevated auxin (indole acetic acid) levels in Ti transformed cells while expression of the tor locus elicits increased levels of cytokinins (13) in tumor tissues relative to non-transformed tissues. Mutations at these loci have specific effects on the morphology of the tumors induced by the mutant Ti plasmid (8). Tumors induced by a strain with an inactivated tans (tumor morphology shooty) locus show large numbers of shoots appearing on the tumor tissue. Tumors induced by a strain with an inacti- vated tmr (tumor morphology rooty) locus display excessive root development. The phenotype of the tumor induced by strains carrying mutations at these O IRL Pren Limited, Oxford, England. 4665 Downloaded from https://academic.oup.com/nar/article-abstract/12/11/4665/1109915 by guest on 19 March 2018
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Volume 12 Number 11 1984 Nucleic Acids Research
Nodeotlde sequence of tht tmr locus of Agmbacterium tumefaciens pTi T37 T-DNA
S.B.GoMberg, J.S.Flkk and S.G.Rogers*
Monsanto Company, 800 North Lindbergh Boulevard, Saint Louis, MO 63167, USA
Received 5 March 1984; Revised and Accepted 16 May 1984
ABSTRACTThe nucleotide sequence of the tmr locus from the nopaline-type pTi T37plasmid of Agrobacterium tumefaciens was determined. Examination of thissequence allowed us to identify an open reading frame of 720 nucleotidescapable of encoding a protein with a derived molecular weight of 27025 d.Comparison of the pTi T37 tmr sequence with the published sequence of the pTiAch5 tmr locus shows over 88% homology in the 240 bases 5' to the transla-tional initiation codon and over 91% homology in the coding sequences. The31 nontranslated regions show less than 50% homology as expected for the 3'regions of divergent related genes. The possible significance of areas ofconserved sequences, particularly in the 5' regulatory regions, is discussed.
INTRODUCTION
AgrobfLCterium tumefaciens causes crown gall disease by transfer of a DNA
segment from its large resident Ti plasmid into the plant cell where this DNA
is covalently integrated into the genome (1-5). Expression of certain genes
located on the transferred DNA (T-DNA) results in in situ neoplastic growth
or phytohormone-independent growth of the infected tissue when placed into
culture (6-7). Recent results implicate certain specific T-DNA encoded
genetic and transcriptional units, the tms and tmr loci, as the units of
expression responsible for the hormone-independent growth (8-12). Specifi-
cally, expression of the tins locus results in elevated auxin (indole acetic
acid) levels in Ti transformed cells while expression of the tor locus
elicits increased levels of cytokinins (13) in tumor tissues relative to
non-transformed tissues.
Mutations at these loci have specific effects on the morphology of the tumors
induced by the mutant Ti plasmid (8). Tumors induced by a strain with an
inactivated tans (tumor morphology shooty) locus show large numbers of shoots
appearing on the tumor tissue. Tumors induced by a strain with an inacti-
vated tmr (tumor morphology rooty) locus display excessive root development.
The phenotype of the tumor induced by strains carrying mutations at these
O IRL Pren Limited, Oxford, England. 4665
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loci can be reverted to normal crown gall callus by supplying exogenous
phytohormones. Added cytokinins reverse the effect of the tmr mutation;
added auxins reverse the effect of the tas mutation on the morphology of
normal cultured tumor tissue (14). From these results, it is evident that
the products of the tins and tmr loci are involved in the regulation of
phytohormone levels in the tumor tissue.
As a first step to defining and better understanding the functions of these
genes, we have determined the nucleotide sequence of the tar locus from the
nopaline-type pTi T37 plasmid. During the preparation of this manuscript,
the nucleotide sequence for the tmr locus of an octopine-type pTi Ach5
plasmid was published by Heidetamp et al. (15). The tmr locus resides in the
DNA region common to both nopaline and octopine type Ti plasmids as deter-
mined by DNA heteroduplex analysis, genetic and transcript mapping (16,10-
12). The availability of the DNA sequences of both tmr loci provides a
unique opportunity to examine two functionally related genes for the extent
of similarity or variation in their regulatory and structural regions. Such
a comparison permits insight into the importance of various DNA sequences
within the common regulatory regions and particular amino acids in the
protein encoding regions. In this report we describe the nucleotide sequence
of the pTi T37 tmr locus and compare this sequence with that of the pre-
viously reported pTi Ach5 homologue.
MATERIALS AND METHODS
Bacteria and bacteriophage
The Escherichia coli recipient for plasmid transformation was strain
LE392:F", hsd«514(rk", mk+), awtBl (17). The host for M13 phage cloning
and growth was JM101 (18). M13 mp8 and mp9 were obtained from BKL (Gaithers-
burg, MD.)
All restriction endonucleases were purchased from New England Biolabs
(Beverly, MA) and used according to the manufacturers instructions. See
Roberts (19) for specificity. Bacteriophage T4 DNA ligase was prepared using
a modification of the procedure of Murray et al. (20).
Plasmid and phage DNA reconstructions
Cleavage of DNAs, ligations, and transformations were performed as described
by Taylor et al. (23) for plasmids and as described by Messing ot al. (18)
for M13.
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DNA preparation and sequencing
Plasmid DNA was prepared as described by Davis et aJ. (21). M13 DNA was
prepared by the procedure of Messing et al. (18) and used as template for the
dideoxynucleotide chain termination method described by Anderson (22).
Analysis and assembly of the DNA sequence data was performed using programs
obtained from IntelliGenetics (Palo Alto, CA).
RESULTS
Cloning of the tar locus
The tar locus was first isolated on the 3.8 kb HindIII-22 fragment prepared
by digestion of the nos::Tn7 derivative of pTi T37, pGV3106 (24). This
fragment was inserted into the Hindlll site of pBR327 (25) to yield pMON69.
Restriction mapping showed that the inserted fragment was indeed HindIII-22
by comparison of the internal BamHI cleavage sites with published restriction
cleavage site maps of the pTi T37 plasmid (11-12,26). Transcript mapping
carried out by both Bevan and Chilton (12) and Willmitzer et al. (11) had
§ 8 8 § § 8 g 8
I 1 1i i i
§ 8 8 P I I S 3
I ?!1 U
Figure 1. Restriction endonuclease cleavage map of the pTi T37 HindIII-22fragment and tar locus containing 2 kb BamHI to HindiII subfragment. Themajor restriction endonuclease cleavage sites are shown for the BamHI-Hindlllsubfragment. The arrows beneath the map show the independent clones ofvarious subfragments, jj, and the number of times each was used for sequencedeterminations ( ). The length of the arrow shows the approximate extent ofthe sequence data obtained. Continuous sequence through all junctions showedthat no small fragments were lost during subcloning.
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identified the tar transcript as a 1200 bp mlUJA that mapped entirely within
the 2.0 kb BamHI to Hindlll segment from the right side of fragment Hindlll-
22 (Fig. 1). This 2.0 kb B«mHI-HindIII fragment was isolated from pMON69 and
inserted into similarly cleaved pBR327 to yield pMON99. The 2.0 kb insert
was mapped by cleavage with various restriction endonucleases to provide the
detailed map in Fig. 1. The presence of the unique Hpal site at approxi-
mately nucleotide 1350 served to locate the active portion of the pTi T37
tmr gene since insertion of DNA fragments encoding antibiotic resistance at
this site results in the tmr phenotype and inactivates the gene (27-28).
Nucleotide sequence determination of the tar locus
The resulting restriction map (Fig. 1) provided a large number of cleavage
sites all of which were used, alone or in combinations, to obtain sub-
Figure 2. Nucleotide sequence of the 2 kb Baniil-Hindlll pTi T37 tmr locuscontaining fragment. The 720 bp open reading frame and derived amino acidsequence begins at nucleotide 659 with an ATG translation initiator and endsat nucleotide 1378 adjacent to a TAG translational terminator.
fragments that were cloned into M13 mp8 or mp9 for subsequent di-deoxy
sequencing. The strategy for the subcloning and sequencing appears in Fig.
1. No difficulty was encountered in obtaining clones of any of the sub-
fragments nor in their sequencing.
The final nucleotide sequence appears in Fig. 2. The total sequence extend-
ing from the beginning of the BanMl recognition sequence to the end of the
Hindlll recognition sequence comprises 1983 nucleotides. An open reading
frame of 720 nucleotides sufficient to encode a protein of derived molecular
weight 27025 d was found. Significantly, this open reading frame includes
the Hpal cleavage site, preceding nucleotide 1331, where insertions of
foreign DNAs result in inactivation of the pTi T37 tmr locus (27-28). This
coding sequence starts with an ATG initiator codon beginning at nucleotide
659 and ends at nucleotide 1378 which is adjacent to a TAG translational
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Figure 3. Comparison of the 5' nontranslated regions of the tmr loci fromthe T37 (upper lines) and Ach5 (lower lines) Ti plasmids. The underscorednucleotides are those in the Ach5 sequence that differ from the T37 sequence.The enclosed nucleotides are regions of potential importance in RNA poly-merase 11 binding and transcription initiation.
termination codon. The derived size for the proposed tmr protein is in
agreeoent with the bacterial expression and hybrid-selected translation data
of Schroder and his co-workers (29-30) and with the derived octopine tar
protein size of 27003 d. reported by Heidekamp et ai. (15). Further
similarities to the octopine tar protein will be discussed in the comparison
of the coding sequences below.
Examination of the DNA sequences immediately preceding the coding sequence
reveal the features expected for an RNA polymerase II recognition and tran-
scription initiation region. These include a 5'-TATAA- sequence beginning at
nucleotide 588. This canonical "TATA box" is preceded at nucleotide 545 by
the sequence S'-GGTAAAG- which was also identified by Heidekamp et aJ. (15),
bears some resemblance to the canonical "CAAT box" (5'-GGC/TCAATCT-)
described for non-plant eucaryotic RNA polymerase II recognition regions
(31). Based upon our current understanding of plant gene regulatory elements
(38), it is possible that plant gene promoters do not contain this feature.
Although we have not performed SI digestion analysis to precisely locate the
5' end of the transcript, the similarity of the signals just described for
the pTi T37 tor gene and those of the pTi Ach5 tmr gene discussed below
suggest strongly that these signals are indeed those recognized during
transcription of the pTi T37 tmr gene in transformed plant cells.
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Comparison of the nucleotide sequences of the pTi T37 tmr and pTi Ach5
tmr loci
The 5' regions
The sequences of the 240 nucleotides preceding the ATG initiator codon
of both the pTi T37 and pTi Ach5 tmr genes show greater than 88% homo-
logy (Fig. 3). Interestingly, the region with the greatest continuous
conserved sequence falls between bases 477 and 526 which are approxi-
mately 130 to 180 nucleotides 5' to the ATG initiation codon. Whether
this has significance with respect to promoter function will await
deletion or site-directed mutagenesis analysis of these sequences. Of
the 27 base changes that occur in this 240 nucleotide segment, most
(17 of 27) are transitions which preserve a purine or pyrimidine, respec-
tively, at the site of the change. Of the transversions that have occurred,
most of these have been of the G+T type when comparing the pTi T37 to the pTi
Ach5 sequence. Without quantitative comparison of transcription levels from
the two tmr loci, it is not possible to assess the overall effects of these
base changes on relative promoter strength.
Heidekamp at ai. found two mRNAs from the pTi Ach5 tmr locus: a minor, "long
Figure 5. Comparison of the 3' nontranslated regions of the tmr loci fromthe T37(upper lines) and Ach5 (lower lines) Ti plasmids. Spaces have beeninserted into both sequences to achieve maximal alignment. The nucleotidenumbering is the same as in Fig. 2 and has been adjusted for the insertedspaces in the pTi T37 tmr sequence. The underscored nucleotides arepotential poly-adenylation signals.
5'-G/AATAA- (38). These sites are marked on Fig. 5. It is interesting that
both the pTi T37 and the pTi Ach5 tmr loci show a consensus poly-adenylation
signal near to the coding sequence (nucleotide 1416; 5'-AATAA- for T37 and
5'-GATAA for Ach5). The significance of these signals approximately 36
nucleotides from the translational termination codon is not known but they
have been found in most of the plant 3' nontranslated sequences examined
(38). In addition to these "close-in" poly-adenylation signals both the pTi
T37 and pTi Ach5 3' regions show consensus plant signals at similar locations
at approximately 200 and 270 nucleotides downstream from the translation
terminator. The pTi T37 sequence shows two additional consensus poly-
adenylation signals one of which is located 155 nucleotides from the termina-
tor codon and the other of which occurs approximately 300 nucleotides from
the terminator. The relative utilization of these various signals in post-
transcriptional modification of the respective tmr mRNAs awaits further
experimentation.
DISCUSSION
In this paper we report the nucleotide sequence of the pTi T37 tar locus and
compare and contrast this with the sequence of the pTi Ach5 tmr locus. The
results raise many basic questions concerning plant gene expression as have
previous reports describing and comparing nucleotide sequences in the absence
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of experimental manipulation of these DNAs. The existence of two func-
tionally identical but structurally different DNAs has allowed us to reach
the following conclusions concerning the significance of, in particular, the
conserved sequences. We suggest that the extreme conservation of sequences
located 130 to 180 nucleotides 51 of the translational start signal indicates
a more significant role of these distal sequences in proper binding and
interaction with the plant cell RNA polymerase II complex than is usually
presumed. The importance of these regions might be assessed by experimental
analysis. The significance of the pTi T37 single "TATA box" versus two such
signals and two different mRNAs for the pTi Ach5 promoter can only be
assessed by quantitation of the total amount of transcription from the two
different tmr gene promoters.
Fortunately, all of the questions raised are answerable. We now have access
to the nucleotide sequences and the means to alter and re-introduce modified
DNAs into plant cells to assay the effects of our manipulations (39-40). In
addition, the availability of the coding sequence permits us to modify the
pTi T37 tar gene for expression in Escherichia coll. This will enable us to
obtain the product free from contaminating plant proteins and be able to
perform assays for the possible cytokinin biosynthetic enzyme activities of
this protein (42). Such experiments are currently in progress.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. J. Schell for plasmid pGV3106. The authors
wish to thank Ms. P. Guenther for exceptional patience during the preparation
of the figures and text of this manuscript and to Drs. R. Fraley, R. Horsch,
G. Barry and A. Levine for their critical reading of this manuscript.
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