Post on 11-Jan-2017
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
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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-
1 GGATCCTGTT ACAAGTATTG CACGTTTTAT AAATTCCATA TTAATGCMT CTTGATTTTC
61 AACAACGAAG CTAATGCCCT AAAAGAAAAA ATGTATGTTA TTCTATTGAT CTTTCATGAT
121 CTTGAAGCGT GCCATAATAT GATGATCTAT AATTAAAATA TTAACTGTCG CATTTTATTG
181 AAATGGCACT GTTATTTCAA CCATATCTTT GATTCTGTTA CATGACACGA CTGCAAGAAG
241 TAAATAATAG ACGCCGTTGT TAAAGAATTG CTATCATATG TGCCTAACTA GAGGGAATTT
301 GAGCGTCAGA CCTMTCAAA TATTACAAAA TATCTCACTC TGTCGCCAGC AATGGTGTM
361 TCAGCGCAGA CAAATGGCCT AAAGATCGCG GAAAAACCTC CCCGAGTGGC ATGATAGCTG
421 CCTCTGTATT GCTGATTTAG TCAGCCTTAT TTGACTTAAG GGTGCCCTCG TTAGTGACAA
4*1 ATTGCnTCA AGGAGACAGC CATGCCCCAC ACTTTGTTGA AAAACAAATT GCCTTTGGC;
541 AGACGGTAAA GCCAGTTGCT CTTCAATAAG GAATCTCGAG GAGGCAATAT AACCGCCTCT
601 GCTACTACAC TTCTCTAATC CAAAAATCAA TTTGTATTCA AGATACCGCA AAAAACTT
659 ATG GAT CTG CGT CTA ATT TTC GCT CCA ACT TGC ACA GGA AAG ACG TCGMET Asp Leu Arg Leu H e Phe Gly Pro Thr Cya Thr Gly Lyi Thr Ser
707 ACC GCG GTA GCT CTT GCC CAG CAG ACT GGG CTT CCA GTC CTT TCG CTCThr Ala V.I Ala Leu Ala Gin Gin Thr Gly Leu Pro Val Leu Ser Leu
755 GAT CGG GTC CAA TGT TGT CCT CAG CTG TCA ACC GGA AGC GGA CGA CCAAap Arg Val Gin Cya Cya Pro Gin Leu Ser Thr Gly Ser Gly Arg Pro
803 ACA GTC GAA GAA CTG AAA CGA ACG AGC CGT CTA TAC CTT GAT GAT CGGThr Val Glu Glu Leu Lya Gly Thr Ser Arg Leu Tyr Leu Aap Alp Arg
851 CCT CTG GTG AAG GGT ATC ATC GCA GCC AAG CAA GCT CAT GAA AGG CTGPro Leu Val Lya Gly lie lie Ala Ala Lya Gin Ala Hii Glu Arg Leu
899 ATG GGG GAG GTG TAT AAT TAT GAG GCC CAC GGC GGG CTT ATT CTT GAGMET Gly Glu Val Tyr Aan Tyr Glu Ala Uia Gly Gly Leu lie Leu Glu
947 GGA GGA TCT ATC TCG TTG CTC AAG TGC ATG GCG CAA AGC ACT TAT TGGGly Gly Ser H e Ser Leu Leu Lya Cya KET Ala Gin Ser Ser Tyr Trp
99S ACT GCG GAT TTT CGT TGG CAT ATT ATT CGC CAC GAG TTA GCA GAC GAASer Ala Asp Phe Arg Trp His H e H e Arg Hla Glu Leu Ala Aap Glu
1043 GAC ACC TTC ATC AAC GTG GCC AAG GCC AGA CTT AAG CAG ATG TTA CGCGlu Thr Phe MET Aan Val Ala Lys Ala Arg Val Lya Gin KET Leu Arg
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1091 CCT GCT GCA GGC CTT TCT ATT ATC CAA GAC TTG GTT GAT CTT TGG AAAPro Ala Al> Gly Leu Ser lie lie Gin Glu Leu Val Asp Leu Trp Lys
1139 GAG CCT CCG CTG ACC CCC ATA CTG AAA GAG ATC GAT GGA TAT CGA TATGlu Pro Arg Leu Arg Pro lie Leu Lys Glu lie Asp Gly Tyr Arg Tyr
1187 GCC ATG TTG TTT GCT AGC CAG AAC CAG ATC ACA TCC GAT ATG CTA TTGAla MET Leu Phe Ala Ser Gin A m Gin H e Thr Ser Aap MET Leu Leu
1235 CAG CTT GAC GCA GAT ATG GAG GAT AAG TTG ATT CAT GGG ATC GCT CAGGin Leu Aip Ala Asp HET Glu Asp Lys Leu H e His Gly lie Ala Gin
1283 GAG TAT CTC ATC CAT GCA CGC CGA CAA GAA CAG AAA TTC CCT CGA GTTGlu Tyr Leu H e His Ala Arg Arg Gin Glu Gin Lys Phe Pro Arg Vsl
1331 AAC GCA GCC GCT TAC GAC GGA TTC GAA GGT CAT CCA TTC GGA ATG TATAsn Ala Ala Ala Tyr Asp Gly Phe Glu Gly His Pro Phe Gly MET Tyr
1379 TAG TTTGCACCAG CTCCGCGTCA CACCTGTCTT CATTTGAATA AGATGTTCGC
1432 AATTGTTTTT AGCTTTGTCT TGTTGTGGCA GGGCGGCAAG TGCTTCAGAC ATCATTCTGT
H92 TTTCAAATTT TATGCTGGAG AACAGCTTCT TAATTCCTTT GGAAATAATA GACTGCGTCT
1552 TAAAATTCAG ATGTCTGGAT ATAGATATGA TTGTAAAATA ACCTATTTAA GTGTCATTTA
1612 GAACATAAGT TTTATGAATG TTCTTCCATT TTCGTCATCG AACGAATAAG AGTAAATACA
1672 CCTTTTTTAA CATTATAAAT AAGTTCTTAT ACGTTGTTTA TACACCGGGA ATCATTTCCA
1732 TTATTTTCGC GCAAAAGTCA CGGATATTCG TGAAAGCGAC AAAAACTGCG AAATTTGCGG
1792 GGAGTGTCTT CAGTTTGCCT ATTAATATTT AGTTTGACAC TAATTGTTAC CATTGCAGCC
1852 AAGCTCAGCT G I H U I H C TTAAAAACGC AGGATCGAAA GAGCATGACT CGGCAAGGTT
1912 GGCTTGTACC ATGCCTTTCT CATGGCAAAG ATGATCAACT GCAGGATGAA CTCTCGGAGC
1972 TTTCAAAAGC TT
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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419 TCCCTCTGTA TTGCTGATTT ACTCAGCCTT ATTTCACTTA AGGCTGCCCT CCTTAGTCAC
450 TTCCTCTGCA TTGCCAATTT ATTCAGCTTT ATTTCACTTA GGTGTGCCTT CGTTAGCGAC
479 AAATTCCTTT CAAGGAGACA GCCATCCCCC ACACTTTGTT GAAAAACAAA TTGCCTTTGG
510 AAATTCCTTT CAAGGAGACA GCCATCCCCC ACACTTTGTT GAAAAACAAC TTGCCTTTTG
539 GGAGACGGTAAAGCCAGTTG CTCTTCAATA AGCAATCTCC AGCAGGCAAJ ATAACCOCCT
570 GCATAC^GTA AACC({AGTTG CACTTCAATA ATGAATTTCA AGCAGACAAfr ATAACCGCCT
599 CTGCTAGTAC ACTTCTCTAA TCCAAAAATC AATTTGTATT CAAGATACCC CAAAAAACTT ATG
630 CTGATAACAC AATTCTCTAA frATAAAAATC ACTTTGTATT CAATATACTC CAAAAAACTT ATG
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
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 eliminat-
ing this transcript of the pTiT37 tmr Iocu6. 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 ATC CAT CTG CCT CTA ATT TTC CGT CCA ACT TCC ACA CCA AAC ACG TCCMET Aap Leu Arg Leu H e Pbe Cly Pro Thr Cya Thr Gly Lyi Thr Ser
C A AAsp Hia Thr
707 ACC CCC CTA CCT CTT CCC CAC CAC ACT GGG CTT CCA GTC CTT TCC CTCThr Ala Val Ala Leu Ala Gin Gin Thr Cly Leu Pro Val Leu Ser Leu
A A Tlie Thr Leu
755 GAT CCG GTC CAA TGT TGT CCT CAG CTC TCA ACC CCA ACC GGA CCA CCAAap Arg Val Gin Cya Cye Pro Gin Leu Ser Thr Gly Ser Gly Arg Pro
C A ACya Gin Leu
803 ACA CTG GAA GAA CTG AAA GGA ACG ACC CGT CTA TAC CTT GAT GAT CGGThr Val Glu Glu Leu Lya Gly Thr Ser Arg Leu Tyr Leu Ajp Aap Arg
CLeu
851 CCT CTG GTG AAG GCT ATC ATC CCA CCC AAG CAA GCT CAT GAA AGG CTGPro Leu Val Lya Cly l i e H a Ala Ala Lya Gin Ala Eia Clu Arg Leu
G C TGlu Hia
899 ATG GGG GAG GTG TAT AAT TAT GAG CCC CAC GGC GGG CTT ATT CTT GAGMET Gly Glu Val Tyr Aan Tyr Clu Ala Hla Gly Gly Leu H e Leu Glu
C A C Al i e Glu Uia Aan
947 GGA GCA TCT ATC TCG TTC CTC AAG TGC ATG GCG CAA ACC ACT TAT TGGGly Cly Ser H e Ser Leu Leu Lye Cya MET Ala Gin Ser S«r Tyr Trp
C C C G A CSer Thr Aan Arg Aan Ser
995 ACT GCG GAT TTT CGT TGG CAT ATT ATT CGC CAC GAG TTA GCA GAC GAASer Ala Aap Phe Arg Trp Hia l i e H e Arg Bla Glu Leu Ala Aap Glu
A A C C CAla Lya Pro Gin
1043 GAC ACC TTC ATG AAC GTG GCC AAG GCC AGA GTT AAC CAG ATG TTA CGCGlu Thr Phe HET Aan Val Ala Lya Ala Arg Val Lya Gin MET Leu Arg
A C G ALya.Ala Leu Hla
1091 CCT GCT GCA GGC CTT TCT ATT ATC CAA GAG TTG GTT GAT CTT TGG AAAPro Ala Ala Gly Leu Ser H e H e Gin Glu Leu Val Aap Leu Trp Lya
C A T T TPro Hia H e Tyr Aan
1139 GAC CCT CCG CTG AGO CCC ATA CTG AAA GAG ATC GAT GGA TAT CGA TATGlu Pro Arg Leu Arg Pro H e Leu Lya Glu H e Asp Gly Tyr Arg Tyr
A TGlu H e
1187 GCC ATG TTC TTT CCT AGC CAC AAC CAG ATC ACA TCC GAT ATG CTA TTGAla MET Leu Phe Ala Ser Gin Aan Gin H e Thr Ser Aap MET Leu Leu
G G AThr Ala
1235 CAG CTT GAC GCA GAT ATG GAG GAT AAG TTC ATT CAT GGG ATC CCT CAGGin Leu Aap Ala Aap HET Glu Aap Lya Leu H e Hia Gly H e Ala Gin
A A C AAan Glu Gly Aan
1283 GAG TAT CTC ATC CAT GCA CCC CGA CAA GAA CAG AAA TTC CCT CGA CTTGlu Tyr Leu H e Hia Ala Arg Arg Gin Glu Gin Lya Phe Pro Arg Val
T G A G C APhe Ala G I D Gin Pro Gin
1331 AAC GCA GCC GCT TAC GAC GGA TTC GAA GCT CAT CCA TTC GCA ATG TAT TAGAan Ala Ala Ala Tyr Aap Gly Phe Glu Gly Hia Pro Phe Cly MET Tyr .
T GPha Pro
Figure 4. Comparison of the nucleotide and derived anino acids sequences ofthe 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 Ms-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 tar proteins fulfills its intracellular role awaits identifi-
cation 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 tar 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 31 nontranslated regions of
different members of a gene family (33,36-37), a great amount of variability
exists between the two tmr 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
tmr 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 TTTCCACCAG CTCCGCCTCA CACCTCTCTT CATTTGAATA AGATGTTCCC AATTGTTTTT1413 GTTACCCCAG CCCTGCGTCG CACCTCTCTT CATCTCCATA ACATCTTCCT AATTGTTTTT
1447 AGCTTTCTCT TGTTGTGGCA GCGCGGCAAG TGCTTCAGAC ATCATTCTG TTTTCAAAT1473 GCCTTTGTCC TCTTGTGCCA GCGCGCCAAA TACTTCCGAC AATCCATCGT GTCTTCAAAC
1500 TTTATGCTGG AGAACAGCTT CTTAATTCCT TTGCAAATAA TAGACTGCGT CTTAAAATT1533 TTTATCCTGG TGAACAAGTC TTAGTTTCCA CGAAAGTA TTATCTTAAA TTTTAAAATT
1559 CAGATGTCTG GATATAGATA TGATTGTAAA ATAACCTATT TAAGTGTCA TTTAGAACAT1591 TCGATGTATA ATGTGGaAT AATTGTAAAA ATAAACTATC GTAAGTCTGC GTGTTATGTA
1618 AAGTTTTATG AATGTTCTTC CATTTTCGTC ATCGAACGAA TAAGAGTAAA TACACCTTTT1651 TAATTTGTCT AAATGTTTAA TATATATCAT AGAACGCAAT AAATATTAAA TATAGCGCTT
1678 TTAACAT TA TAAATAAGTT CTTATACGTT GTTTATACAC CGGGAATCAT TTCCATTATT1711 TTATGAAATA TAAATACATC ATTACAAGTT GTTTATATTT CGGGTACCTT TTCCATTATT
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.
*To whom correspondence should be addressed
ABBREVIATIONSnos nopaline synthasekb kilobases, 1000 bases
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