Download Nucleic Acids Research

Volume 12 Number 11 1984
Nucleic Acids Research
Nudeotide sequence of the tmr locus of Agrobactentum tumefaciens pTi T37 T-DNA
S.B.Goldberg, J.S.Flick 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 tizr sequence with the published sequence of the pTi
Ach5 tmz' locus shows over 88% homology in the 240 bases 5' to the translational initiation codon and over 91% homology in the coding sequences. The
3' 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
Agrobacterium 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 timr loci, as the units of
expression responsible for the hormone-independent growth (8-12). Specifically, expression of the tms locus results in elevated auxin (indole acetic
acid) levels in Ti transformed cells while expression of the tmr 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 tms (tumor morphology shooty) locus show large numbers of shoots
appearing on the tumor tissue. Tumors induced by a strain with an inactivated tmr (tumor morphology rooty) locus display excessive root development.
The phenotype of the tumor induced by strains carrying mutations at these
C I R L Press Limited, Oxford, England.
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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 tms mutation on the morphology of
normal cultured tumor tissue (14). From these results, it is evident that
the products of the tms 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 tmr 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 Heidekamp et al. (15). The tmr locus resides in the
DNA region common to both nopaline and octopine type Ti plasmids as determined by DNA heteroduplex analysis, genetic and transcript mapping (16,1012). 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 previously reported pTi Ach5 homologue.
MATERIALS AND METHODS
Bacteria and bacteriophage
The Escherichia coli recipient for plasmid transformation was strain
LE392:F-, hsdR514(rk-, mk+), metBI (17). The host for M13 phage cloning
and growth was JM101 (18). M13 mp8 and mp9 were obtained from BRL (Gaithersburg, MD.)
Enzymes
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 et al. (18)
for M13.
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DNA preparation and sequencing
Plasmid DNA was prepared as described by Davis et al. (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 tmr locus
The tmr 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 HindIII 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
E
f~~~~
pTiT37 Hindil(-22
tm r Coding
Region
8-C8\1
Ee;
8
88r 8
EI
I
#2(1), #3(1)
E
8
8
8
8
8
11
~~~~~I
~
~
~
~ ~~
#1(2), #3(2)
#1(1), #2(1), #3(1)
#3(1)
#1(1)
#1(2)
#4(2)
#5(1)
#3(1)
#1(3)
#8(2)
#2(1)
Figure 1. Restriction endonuclease cleavage map of the pTi T37 HindIII-22
fragment and tmr locus containing 2 kb BamHI to HindIII subfragment. The
major restriction endonuclease cleavage sites are shown for the BamHI-HindIII
subfragment. The arrows beneath the map show the independent clones of
various subfragments, #, and the number of times each was used for sequence
determinations ( ). The length of the arrow shows the approximate extent of
the sequence data obtained. Continuous sequence through all junctions showed
that no small fragments were lost during subcloning.
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identified the tmr transcript as a 1200 bp mRNA that mapped entirely within
the 2.0 kb BamHI to HindIII segment from the right side of fragment HindIII22 (Fig. 1). This 2.0 kb BamHI-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 HpaI site at approximately 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 tmr 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-
1
GGATCCTGTT ACMGTATTG CACGTTTTAT AAATTGCATA TTAATGCAAT CTTGATTTTC
61 AACMCGAAG GTAATGGCGT AAAAGAAAAA ATGTATGTTA TTGTATTGAT CTTTCATGAT
121
GTTGAAGCGT GCCATAATAT GATGATGTAT AATTAAAATA TTAACTGTCG CATTTTATTG
181 AAATGGCACT GTTATTTCAA CCATATCTTT GATTCTGTTA CATGACACGA CTGCAAGAAG
241 TAAATAATAG ACGCCGTTGT TAAAGAATTG CTATCATATG TGCCTAACTA GAGGGAATTT
301
GAGCGTCAGA CCTAATCAAA TATTACAAAA TATCTCACTC TGTCGCCAGC AATGGTGTAA
361 TCAGCGCAGA CAAATGGCGT AAAGATCGCG GAAAAACCTC CCCGAGTGGC ATGATAGCTG
421
CCTCTGTATT GCTGATTTAG TCAGCCTTAT TTGACTTMG GGTGCCCTCG TTAGTGACAA
481 ATTGCTTTCA AGGAGACAGC CATGCCCCAC ACTTTGTTGA AAAACAAATT GCCTTTGGGfi
541 AGACGGTAAA GCCAGTTGCT CTTCAATMG GAATCTCGAG GAGGCAATAT AACCGCCTCT
601 GGTAGTACAC TTCTCTAATC CAAAAATCAA TTTGTATTCA AGATACCGCA AAAAACTT
659 ATG GAT CTG CGT CTA ATT TTC GGT CCA ACT TGC ACA GGA MG ACG TCG
MET Asp Leu Arg Leu Ile Phe Gly Pro Thr Cys Thr Gly Lys Thr Ser
707 ACC GCG GTA GCT CTT GCC CAG CAG ACT GGG CTT CCA GTC CTT TCG CTC
Thr Ala Val Ala Leu Ala Gln Gln Thr Gly Leu Pro Val Leu Ser Leu
755
GAT CGG GTC CM TGT TGT CCT CAG CTG TCA ACC GGA AGC GGA CGA CCA
Asp Arg Val Gln Cys Cys Pro Gln Leu Ser Thr Gly Ser Gly Arg Pro
803 ACA GTG GM GM CTG AM GGA ACG AGC CGT CTA TAC CTT GAT GAT CGG
Thr Val Glu Glu Leu Lys Gly Thr Ser Arg Leu Tyr Leu Asp Asp Arg
851 CCT CTG GTG AAG GGT ATC ATC GCA GCC MG CAA GCT CAT GAA AGG CTG
Pro Leu Val Lys Gly Ile Ile Ala Ala Lys Gln Ala His Glu Arg Leu
899 ATG GGG GAG GTG TAT MT TAT GAG GCC CAC GGC GGG CTT ATT CTT GAG
MET Gly Glu Val Tyr Asn Tyr Glu Ala His Gly Gly Leu Ile Leu Glu
947 GGA GGA TCT ATC TCG TTG CTC MG TGC ATG GCG CM AGC AGT TAT TGG
Gly Gly Ser Ile Ser Leu Leu Lys Cys MET Ala Gln Ser Ser Tyr Trp
995 AGT GCG GAT TTT CGT TGG CAT ATT ATT CGC CAC GAG TTA GCA GAC GM
Ser Ala Asp Phe Arg Trp His Ile Ile Arg His Glu Leu Ala Asp Glu
1043 GAG ACC TTC ATG AAC GTG GCC AAG GCC AGA GTT AAG CAG ATG TTA CGC
Glu Thr Phe MET Asn Val Ala Lys Ala Arg Val Lys Gln MET Leu Arg
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1091 CCT GCT GCA GGC CTT TCT ATT ATC CM GAG TTG GTT GAT CTT TGG AAA
Pro Ala Ala Gly Leu Ser Ile Ile Gln Glu Leu Val Asp Leu Trp Lys
1139 GAG CCT CGG CTG AGG CCC ATA CTG MA GAG ATC GAT GGA TAT CGA TAT
Glu Pro Arg Leu Arg Pro Ile Leu Lys Glu Ile Asp Gly Tyr Arg Tyr
1187 GCC ATG TTG TTT GCT AGC CAG AAC CAG ATC ACA TCC GAT ATG CTA TTG
Ala MET Leu Phe Ala Ser Gln Asn Gln Ile Thr Ser Asp MET Leu Leu
1235 CAG CTT GAC GCA GAT ATG GAG GAT AAG TTG ATT CAT GGG ATC GCT CAG
Gln Leu Asp Ala Asp MET Glu Asp Lys Leu Ile His Gly Ile Ala Gln
1283 GAG TAT CTC ATC CAT GCA CGC CGA CAA GM CAG AM TTC CCT CGA GTT
Glu Tyr Leu Ile His Ala Arg Arg Gln Glu Gln Lys Phe Pro Arg Val
1331 AAC GCA GCC GCT TAC GAC GGA TTC GAA GGT CAT CCA TTC GGA ATG TAT
Asn Ala Ala Ala Tyr Asp Gly Phe Glu Gly His Pro Phe Gly MET Tyr
1379 TAG
TTTGCACCAG CTCCGCGTCA CACCTGTCTT CATTTGAATA AGATGTTCGC
1432 MTTGTTTTT AGCTTTGTCT TGTTGTGGCA GGGCGGCAAG TGCTTCAGAC ATCATTCTGT
1492 TTTCAAATTT TATGCTGGAG MCAGCTTCT TAATTCCTTT GGAMTAATA GACTGCGTCT
1552 TAAAMTTCAG ATGTCTGGAT ATAGATATGA TTGTAAAATA ACCTATTTAA GTGTCATTTA
1612 GAACATAAGT TTTATGAATG TTCTTCCATT TTCGTCATCG MCGAATAAG AGTAAATACA
1672 CCTTTTTTM CATTATAMT AAGTTCTTAT ACGTTGTTTA TACACCGGGA ATCATTTCCA
1732 TTATTTTCGC GCAAAAGTCA CGGATATTCG TGAAAGCGAC AAAAACTGCG AAATTTGCGG
1792
GGAGTGTCTT CAGTTTGCCT ATTMTATTT AGTTTGACAC TMATTGTTAC CATTGCAGCC
1852 AAGCTCAGCT GTTTCTTTTC TTAAAAACGC AGGATCGAAA GAGCATGACT CGGCAAGGTT
1912 GGCTTGTACC ATGCCTTTCT CATGGCAMG ATGATCAACT GCAGGATGM CTCTCGGAGC
1972 TTTCAAAAGC TT
Figure 2. Nucleotide sequence of the 2 kb BamHI-HindIII pTi T37 tbr locus
containing fragment. The 720 bp open reading frame and derived amino acid
sequence begins at nucleotide 659 with an ATG translation initiator and ends
at 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 subfragments nor in their sequencing.
The final nucleotide sequence appears in Fig. 2. The total sequence extending from the beginning of the BamHI recognition sequence to the end of the
HindIII 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 HpaI cleavage site, preceding nucleotide 1331, where insertions of
foreign DNAs result in inactivation of the pTi T37 tbr 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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419 TGCCTCTGTA TTGCTGATTT AGTCAGCCTT ATTTGACTTA AGGGTGCCCT CGTTAGTGAC
450 TTCCTCTGCA TTGCCAATTT ATTCAGCTTT ATTTGACTTA GGTGTGCCTT CGTTAGCGAC
479
AAATTGCTTT CAAGGAGACA GCCATCCCCC ACACTTTGTT GAAAAACAAA TTGCCTTTGG
510 AAATTGCTTT CAAGGAGACA GCCATCCCCC ACACTTTGTT GAAAAACAAG TTGCCTTTTG
539
GGAGACG1GTA AAGCCAGTTG CTCTTCAATA AGGAATCTCG AGGAGGCAAT ATAACCGCCT
570
GGATACGGTA AAGCCAGTTG CACTTCAATA ATGAATTTCA AGGAGACAAT ATAACCGCCT
599
CTGGTAGTAC ACTTCTCTAA TCCAAAAATC AATTTGTATT CAAGATACCG CAAAAAACTT ATG
630 CTGATAACAC AATTCTCTAA TATAAAAATC AGTTTGTATT CAATATACTG CAAAAAACTT ATG
Figure 3. Comparison of the S' nontranslated regions of the tmr loci from
the T37 (upper lines) and Ach5 (lower lines) Ti plasmids. The underscored
nucleotides are those in the Ach5 sequence that differ from the T37 sequence.
The enclosed nucleotides are regions of potential importance in RNA polymerase II binding and transcription initiation.
termination codon. The derived size for the proposed tmr protein is in
agreement with the bacterial expression and hybrid-selected translation data
of Schroder and his co-workers (29-30) and with the derived octopine tmr
protein size of 27003 d. reported by Heidekamp et al. (15).
Further
similarities to the octopine tmr 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 transcription initiation region. These include a 5'-TATAA- sequence beginning at
nucleotide 588. This canonical "TATA box" is preceded at nucleotide 545 by
the sequence 5'-GGTAAAG- which was also identified by Heidekamp et al. (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 tmr gene and those of the pTi Ach5 tbr 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% homoInterestingly, the region with the greatest continuous
logy (Fig. 3).
conserved sequence falls between bases 477 and 526 which are approximately 130 to 180 nucleotides 5' to the ATG initiation codon. Whether
this has significance with respect to promoter function will await
Of
deletion or site-directed mutagenesis analysis of these sequences.
the 27 base changes that occur in this 240 nucleotide segment, most
(17 of 27) are transitions which preserve a purine or pyrimidine, respectively, 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 et al. found two mRNAs from the pTi Ach5 tmr locus: a minor, "long
start" transcript (5' end:nucleotides 646-651, Ach5; nucleotides 615-620,
T37) and a major, "short start" transcript (5' end:nucleotides 679-683, Ach5;
nucleotides 649-653, T37). Each of these starts is preceded by a canonical
"TATA box" approximately 30 nucleotides upstream. Significantly, the "TATA
box" (5'-TATAAA) for the "short" transcript has been mutated at nucleotides
620 and 621 of the pTi T37 sequence to become 5'-TCCAAA presumably eliminating this transcript of the pTiT37 tmr locus. As mentioned previously, we
have not mapped the transcription start of the pTi T37 tmr RNA and cannot say
for -certain that the T37 gene will show only one mRNA equivalent to the
longer of the two transcripts described for the Ach5 tmr gene. Certainly
that would be the prediction based on studies which demonstrate the
importance of the "TATA box" in positioning the start point for transcription
of other eucaryotic genes (31). The answer to this question awaits further
experimental analysis.
If only the "long start" is used during pTi T37 tmr transcription, then
the transcription start should lie between nucleotides 615 and 620 of the
T37 sequence as shown by Heidekamp and co-workers (15) for Ach5. This means
that 5 out of the 27 changes have occurred in the 5' nontranslated leader
(nucleotides 617-659) of the tmr gene and confirms the variability seen in
the 5' nontranslated sequences of other plant gene transcripts of different
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659 ATG GAT CTG
MET Asp Leu
C
Asp
CGT CTA ATT TTC GGT CCA ACT TGC ACA GGA MG ACG TCG
Arg Leu Ile Phe Gly Pro Thr Cys Thr Gly Lys Thr Ser
A
A
His
Thr
707 ACC GCG GTA GCT CTT GCC CAG CAG ACT GGG CTT CCA GTC CTT TCG CTC
Thr Ala Val Ala Leu Ala Gln Gln Thr Gly Leu Pro Val Leu Ser Leu
A
A
T
Ile
Thr
Leu
755
GAT CGG GTC CM TGT TGT CCT CAG CTG TCA ACC GGA AGC GGA CGA CCA
Asp Arg Val Gln Cys Cys Pro Gln Leu Ser Thr Gly Ser Gly Arg Pro
C
A A
Gln Leu
Cys
803 ACA GTG GM GAA CTG MA GGA ACG AGC CGT CTA TAC CTT GAT GAT CGG
Thr Val Glu Glu Leu Lys Gly Thr Ser Arg Leu Tyr Leu Asp Asp Arg
C
Leu
851 CCT CTG GTG MG GGT ATC ATC GCA GCC MG CAA GCT CAT GM AGG CTG
Pro Leu Val Lys Gly Ile Ile Ala Ala Lys Gln Ala His Glu Arg Leu
G
C T
Glu
His
899 ATG GGG GAG GTG TAT MT TAT GAG GCC CAC GGC GGG CTT ATT CTT GAG
MET Gly Glu Val Tyr Asn Tyr Glu Ala His Gly Gly Leu Ile Leu Glu
C A
Ile Glu
C
His
A
Asn
947 GGA GGA TCT ATC TCG TTG CTC MG TGC ATG GCG CM AGC AGT TAT TGG
Gly Gly Ser Ile Ser Leu Leu Lys Cys NET Ala Gln Ser Ser Tyr Trp
C C
Ser Thr
C
Asn
C
G A
Arg Asn Ser
995 AGT GCG GAT TTT CGT TGG CAT ATT ATT CGC CAC GAG TTA GCA GAC GM
Ser Ala Asp Phe Arg Trp His Ile Ile Arg His Glu Leu Ala Asp Glu
A
CC
A
C
Ala
Lys
Pro
Gln
1043 GAG ACC TTC ATG MC GTG GCC AAG GCC AGA GTT MG CAG ATG TTA CGC
Glu Thr Phe MET Asn Val Ala Lys Ala Arg Val Lys Gln MET Leu Arg
A C
G A
Lys Ala
Leu His
1091 CCT GCT GCA GGC CTT TCT ATT ATC CM GAG TTG GTT GAT CTT TGG AM
Pro Ala Ala Gly Leu Ser Ile Ile Gln Glu Leu Val Asp Leu Trp Lys
C
A
T
T
T
Pro
His
Ile
Asn
Tyr
1139 GAG CCT CGG CTG AGG CCC ATA CTG MA GAG ATC GAT GGA TAT CGA TAT
Glu Pro Arg Leu Arg Pro Ile Leu Lys Glu Ile Asp Gly Tyr Arg Tyr
A
T
Glu
Ile
1187 GCC ATG TTG TTT GCT AGC CAG MC CAG ATC ACA TCC GAT ATG CTA TTG
Ala MET Leu Phe Ala Ser Gln Asn Gln Ile Thr Ser Asp MET Leu Leu
G G A
Thr Ala
1235 CAG CTT GAC GCA GAT ATG GAG GAT MG TTG ATT CAT GGG ATC GCT CAG
Gln Leu Asp Ala Asp MET Glu Asp Lys Leu Ile His Gly Ile Ala Glri
A
Asn
A G
Glu Gly
A
Asn
1283 GAG TAT CTC ATC CAT GCA CGC CGA CAA GM CAG AM TTC CCT
Glu Tyr Leu Ile His Ala Arg Arg Gln Glu Gln Lys Phe Pro
T
A
G
G
C
Phe
Ala
Gln Gln
Pro
CGA GTT
Arg Val
A
Gln
1331 MC GCA GCC GCT TAC GAC GGA TTC GAA GGT CAT CCA TTC GGA ATG TAT TAG
Asn Ala Ala Ala Tyr Asp Gly Phe Glu Gly His Pro Phe Gly MET Tyr
T
G
Phe
Pro
Figure 4.
Comparison of the nucleotide and derived amino acids sequences of
the tmr loci from the T37 (upper lines) and Ach5 (lower lines) Ti plasmids.
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members of the same functional gene family, such as those of the pea small
subunit of ribulose bis-phosphate carboxylase family (32-33).
The coding sequence
The 723 nucleotides that comprise the coding sequence and termination codons
of both tmr loci are shown in Fig. 4, and the deduced amino acid sequence for
each is also presented. There is greater than 91% nucleotide homology. Of
the 54 nucleotide changes that occur, 21 are third position changes which do
not alter the amino acid at this position. Nine of the remaining changes
result in substitution of similar amino acids such as the change of a
threonine for a serine at amino acid 16. The remaining changes result in
substantial differences of the amino acid; for example, the replacement of
lysine by glutamate at amino acid 68 or the replacement of aspartate by
tyrosine at amino acid 157. Overall, these changes result in a net negative
charge of -2 for the T37 tmr protein versus a net negative change of -5 for
the Ach5 protein. These changes have not substantially altered the basic
function of the resulting tmr proteins since genetic evidence suggest that
each performs the same function in T37 or Ach5 transformed tissues. What
effects these substitutions might have on the efficiency with which each of
the respective tmr proteins fulfills its intracellular role awaits identification of biological activity and comparison of the two purified proteins.
It should be noted that there is no great difference in the codon usage for
the two coding sequences. Codons that appear infrequently in either of the
tmr genes are not under represented in the codon usage of both the octopine
and nopaline synthase proteins (34-35) and probably represent only random
variation in codon usage in the smaller tmr proteins with their fewer number
of codons.
The 3' region
The sequences of nearly 360 nucleotides from the 3' end of the pTi T37 and
pTi Ach5 tmr genes appear in Fig. 5. We have attempted to align these so
that the maximum homology has been shown. This has been accomplished by
including spaces in both sequences where insertions or deletions appear to
have occurred. As has been described for the 3' nontranslated regions of
different members of a gene family (33,36-37), a great amount of variability
exists between the two tbr genes. The overall homology is only about 50%.
Because neither we nor Heidekamp and his co-workers (15) have performed Si
analysis to accurately determine the location of the 3' end of the respective
tbr mRNAs, the following conclusions will be based entirely on inspection of
the sequences for the presence of the canonical plant poly-adenylation site
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1382 TTTGCACCAG CTCCGCGTCA CACCTGTCTT CATTTGAATA AGATGTTCGC AATTGTTTTT
1413 GTTACGCCAG CCCTGCGTCG CACCTGTCTT CATCTGGATA AGATGTTCGT AATTGTTTTT
1447 AGCTTTGTCT TGTTGTGGCA GGGCGGCAAG TGCTTCAGAC ATCATTCTG TTTTCAAAT
1473 GGCTTTGTCC TGTTGTGGCA GGGCGGCAAA TACTTGCGAC AATCCATCGT GTCTTCAAAC
1500 TTTATGCTGG AGAACAGCTT CTTAATTCCT TTGGAAATAA TAGACTGCGT CTTAAAATT
1533 TTTATGCTGG TGAACAAGTC TTAGTTTCCA CGAAAGTA TTATGTTAAA TTTTAAAATT
1559 CAGATGTCTG GATATAGATA TGATTGTAAA ATAACCTATT TAAGTGTCA TTTAGMCAT
1591 TCGATGTATA ATGTGGCTAT AATTGTAAAA ATAAACTATC GTAAGTGTGC GTGTTATGTA
1618 AAGTTTTATG AATGTTCTTC CATTTTCGTC ATCGAACGAA TAAGAGTAAA TACACCTTTT
1651 TAATTTGTCT AAATGTTTAA TATATATCAT AGAACGCAAT AAATATTAAA TATAGCGCTT
1678 TTAACAT TA TAAATAAGTT CTTATACGTT GTTTATACAC CGGGAATCAT TTCCATTATT
1711 TTATGAAATA TAAATACATC ATTACAAGTT GTTTATATTT CGGGTACCTT TTCCATTATT
Figure 5. Comparison of the 3' nontranslated regions of the tmr loci from
the T37 (upper lines) and Ach5 (lower lines) Ti plasmids. Spaces have been
inserted into both sequences to achieve maximal alignment. The nucleotide
numbering is the same as in Fig. 2 and has been adjusted for the inserted
spaces in the pTi T37 tmr sequence.
The underscored nucleotides are
potential 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't 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 polyadenylation signals one of which is located 155 nucleotides from the terminator codon and the other of which occurs approximately 300 nucleotides from
the terminator. The relative utilization of these various signals in posttranscriptional modification of the respective tmr mRNAs awaits further
experimentation.
DISCUSSION
In this paper we report the nucleotide sequence of the pTi T37 tmr 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 functionally 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 5' 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 tmr gene for expression in Escherichia coli. 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.
*To whom correspondence should be addressed
ABBREVIATIONS
nos
nopaline synthase
kb
kilobases, 1000 bases
REFERENCES
1. Chilton, M.-D., Drummond, M. H., Merlo, D. J., Sciaky, D., Montoya, A.
L., Gordon, M. P. and Nester, E. W. (1977) Cell 11, 263-271.
2. Zaenen, I., Van Larebeke, N., Teuchy, H., Van Montagu, M. and Schell, J.
(1979) J: Mol. Biol. 86, 109-127.
3. Chilton, M.-D. (1982) in Molecular Biology of Plant Tumors, Kahl, G. and
Schell, J. Eds., Academic Press, New York.
4675
Nucleic Acids Research
Holsters, M., Hernalsteens, J.-P. Van Montagu, M. and Schell, J. (1982)
in Molecular Biology of Plant Tumors, Kahl, G. and Schell, J. Eds.,
Academic Press, New York.
5. Bevan, M. W. and Chilton, M.-D. (1982) Ann. Rev. Genet. 16, 357-384.
6. Braun, A. C. (1958) Proc. Natl. Acad. Sci. USA 44, 344-349.
7. Braun, A. C. (1978) Biochim. Biophys. Acta. 516, 167-191.
8. Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M.
P. and Nester, E. W. (1981) Cell 27, 143-153.
9. Leemans, J., Deblaere, R., Willmitzer, L., De Greve, H., Hernalsteens,
J.-P., Van Montagu, M. and Schell, J. (1982) The EMBO Journal 1,
147-152.
10. Joos, H., Inze, D., Caplan, A., Sormann, M., Van Montagu, M. and Schell,
J. (1983) Cell 32, 1057-1067.
11. Willmitzer, L., Dhaese, P., Schreier, P. H., Schmalenbach, W., Van
Montagu, M. and Schell, J. (1983) Cell 32, 1045-1056.
12. Bevan, M. W. and Chilton, M.-D. (1982) J. Mol. Appl. Genet. 1, 539-546.
13. Akiyoshi, D. E., Morris, R. O., Hinz, R., Mischke, B. S., Kosuge, T.,
Garfinkel, D. J., Gordon, M. P. and Nester, E. W. (1983) Proc. Natl.
Acad. Sci. USA 80, 407-411.
14. Ooms, G., Klapwijk, P. M., Poulis, J. A. and Schilperoort, R. A. (1980)
J. Bacteriology 144, 82-91.
15. Heidekamp, F., Dirkse, W. G., Hille, J. and van Ormondt, H. (1983)
Nucleic Acids Res. 11, 6211-6223.
16. Engler, G., Depicker, A., Maenhaut, R., Villarroel, R., Van Montagu, M.
and Schell, J. (1981) J. Mol. Biol. 152, 183-208.
17. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) Molecular Cloning,
A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York.
18. Messing, J., Crea, R. and Seeburg, P. H. (1981) Nucleic Acids Res. 9,
309-321.
19. Roberts, R. J. (1982) Nucleic Acids Res. 10, 1830 (r117-rI44).
20. Murray, N. E., Bruce, S. A. and Murray, K. (1979) J. Mol. Biol. 132,
4.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
4676
493-505.
Davis, R. W., Botstein, D. and Roth, J. R. (1980) Advanced Bacterial
Genetics. Cold Spring Harbor Labortory, Cold Spring Harbor, New York.
Anderson, S. (1981) Nucleic Acids Res. 9, 3015-3027.
Taylor, A. F., Silicano, P. G. and Weiss, B. (1980) Gene 9, 321-336.
Hernalsteens, J.-P., Van Vliet, F., De Beuckeleer, M., DePicker, A.,
Engler, G., Lemmers, M., Holsters, M., Van Montagu, M. and Schell, J.
(1980) Nature 287, 654-656.
Soberon, X., Covarrubias, L. and Bolivar F. (1980) Gene 9, 287-305.
Hepburn, A. G., Clarke, L. E., Blundy, K. S. and White, J. (1983) J.
Mol. Appl. Genet. 2, 211-224.
Matzke, A. J. M. and Chilton, M.-D. (1981) J. Mol. Appl. Genet. 1,
39-49.
Barton, K. A., Binns, A. N., Matzke, A. J. M. and Chilton, M.-D. (1983)
Cell 32, 1033-1043.
Schr6der, G., Klipp, W., Hillebrand, A., Ehring, R., Koncz, C. and
Schroder, J. (1983) The EMBO J. 2, 403-409.
Schroder, G. and Schroder, J. (1982) Mol. Gen. Genet. 185, 51-55.
Breathnach, R. and Chambon, P. (1981) Ann. Rev. Biochem. 50, 349-383.
Cashmore, A. R. (1983) in Genetic Engineering of Plants, an Agricultural
Perspective, Kosuge, T., Meredith, C. P. and Hollaender, A. Eds., pp.
29-38, Plenum Press, New York.
Broglie, R., Coruzzi, G., Lamppa, G., Keith, B. and Chua, N.-H. (1983)
Biotechnology 1, 55-61.
Nucleic Acids Research
De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and
Schell, J. (1982) J. Mol. Appl. Genet. 1, 499-512.
35. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. and Goodman, H. M.
(1982) J. Mol. Appl. Genet. 1, 561-574.
36. Berry-Lowe, S. L., McKnight, T. D., Shah, D. M. and Meagher, R. B.
(1982) J. Mol. Appl. Genet. 1, 483-498.
37. Coruzzi, G., Broglie, R., Edwards, C. and Chua, N.-H. (1984) Cell,
submitted for publication.
38. Messing, J., Geraghty, D., Heidecker, G., Hu, N.-T., Kridl, J. and
Rubenstein, I. (1983) in Genetic Engineering of Plants, an Agricultural
Perspective, Kosuge, T., Meredith, C. P. and Hollaender, A. Eds., pp.
211-227, Plenum Press, New York.
39. Herrera-Estrella, L., De Block, M., Hernalsteens, J.-P., Van Montagu, M.
and Schell, J. (1983) The EMBO Journal 2, 987-995.
40. Bevan, M. W., Flavell, R. and Chilton, M.-D. (1983) Nature 304, 184-187.
41. Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J.
S., Adams, S. P., Bittner, M. L., Brand, L. B., Fink, C. L., Fry, J. S.,
Galluppi, G. R., Goldberg, S. B., Hoffmann, N. L. and Woo, S. C. (1983)
Proc. Natl. Acad. Sci. USA 80, 4803-4807.
42. Morris, R. 0., Akiyoshi, D. E., MacDonald, E. M. S., Morris, J. W.,
Regier, D. A. and Zaerr, J. B. (1982) in Plant Growth Substances,
Waring, P. F., Ed., Acadmic Press, London.
34.
4677