Expression of maize zein genes in transformed sunflower cells
Peter B. Goldsbrough 1 *, Stanton B. Gelvin z, and Brian A. Larkins 1 Departments of 1Botany and Plant Pathology and 2Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
Summary. Genes encoding maize seed storage proteins of 19 and 15 kd have been inserted at various sites in the T-DNA of the Ti plasmid pTiA6. Agrobaeterium tumefa- ciens strains harboring these plasmids were used to incite tumors on sunflower stem sections. Some of the resulting tumors were found to contain mRNAs transcribed from the zein genes. The transcripts from both zein genes ini- tiated and terminated at precisely the same sites in both maize endosperm and sunflower tumors. The steady state level of zein mRNAs in tumors varied from undetectable to approximately 1% of the level in maize endosperm. This variation between tumors in zein mRNA levels did not cor- relate with either zein gene copy number or the level of octopine synthase mRNA. However, in six tumor lines con- taining a zein gene inserted at the same site in TR, the level of zein mRNA was directly proportional to the level of a TR encoded mRNA.
Key words: Crown gall tumors - Zein - Agrobacterium tumefaciens - Gene expression - Transgenic plants
Introduction
The major seed storage proteins of maize are the alcohol soluble zeins. These proteins are synthesized in the endo- sperm between 12 and 50 days after pollination, and are deposited in dense, membrane-bounded protein bodies (Larkins and Hurkman 1978). The zeins can be separated by size into groups having molecular weights of 27, 22, 19, 15 and 10 kilodaltons (Lee et al. 1976). Analysis of eDNA clones has shown that the genes encoding the 22 and 19 kd zeins are closely related (Marks and Larkins 1982). Both contain a sequence of some 20 amino acids that is repeated nine times within each polypeptide. Data on the physical properties of zein polypeptides indicate that this repeated sequence of amino acids may be important for folding the polypeptides into rod-shaped molecules and their assembly into protein bodies (Argos et al. 1982). Both the 22 and 19 kd zeins are encoded by multigene families (Hagen and Rubenstein 1981). In contrast, the 15 kd zein does not contain a repeated amino acid sequence, shares no homology with the 22 and 19 kd zeins (Marks and Lar-
* Present address: Department of Horticulture, Purdue University, West Lafayette, IN 47907, USA
Offprint requests to: S.B. Gelvin
kins 1982) and is encoded by only one to three genes in the maize genome (Wilson and Larkins 1984).
The expression of all zein genes is coordinated during endosperm development. Zein mRNAs appear at 10-12 days after pollination and reach their maximum level at 18-22 days postanthesis (Marks et al. 1985 a). Quantita- tion of the mRNAs for each class of zein has shown that, per gene, the genes for the 15 kd polypeptide account for a greater proportion of the zein mRNA in the endosperm (Marks et al. 1985a). A number of mutations have been characterised that affect the accumulation of particular classes of zein proteins (Salamini and Soave 1982).
One approach to studying the synthesis of these proteins is to transfer individual genes into a heterologous back- ground and examine their expression in the absence of the other members of these multigene families. To accomplish this, genes encoding a 19 kd and a 15 kd zein have been inserted into the T-DNA of the Ti plasmid of Agrobacter- ium tumefaciens. Bacteria containing these modified Ti plas- raids have been used to infect sunflower (Helianthus annuus) and the resulting tumors have been analyzed for expression of the zein genes.
Materials and methods
Insertion of zein genes into the Ti plasmid
The two zein genes that have been inserted into the Ti plasmid were originally isolated from a phage library of maize DNA fragments (Pedersen et al. 1982). Both genes and their flanking regions have been sequenced (Pedersen et al. 1982; Pedersen et al. 1986). Restriction fragments con- taining the coding region and 5' and 3' flanking DNA have been inserted into pBR322 prior to their introduction into A. tumefaeiens (Fig. 1). A 3 kb EeoRI--HindIII fragment, containing a gene for a 19 kd zein (gZI9ABI) was inserted into pBR322 to produce pgZ19AB1. A 1.1 kb HindIII frag- ment of the SV40 genome, containing the transcriptional enhancer sequences (Banerji et al. 1981), was inserted at the HindIII site of pgZI9ABI to produce pgZI9AB1SV. A 1.2 kb E c o R I - XhoI fragment of maize DNA, containing a gene for a 15 kd zein (gZ151), was similarly inserted be- tween the EeoRI and SalI sites of pBR322 to produce pgZ151. These three plasmids, containing zein genes in pBR322, carry a functional gene encoding resistance to am- picillin which can be used for selection in both E. colt and A. tumefaciens.
Xba I Xba |
• Xbal K nl ( - i :? ).,, ":':7'" Y-"''"' S a i l
E c o R ~ X / ~ hol/Salll
Fig. l. Plasmids containing zein genes that were inserted into pTiA6. Closed bars indicate the coding region of the zein genes, open bars indicate the flanking maize DNA sequences and hatched bars indicate DNA from SV40. Arrows indicate the direction of transcription of the zein genes
The method for introducing the zein genes into the T- DNA of the Ti plasmid is essentially that used by Garfinkel et al. (1981) and has been described previously (Golds- brough et al. 1983). Three intermediate vectors were con- structed that contained restriction endonuclease fragments of the Ti plasmid inserted in the broad host range plasmids pRK290 (Ditta et al. 1981) and pRK2501 (Kahn et al. 1979). These were used to make insertions of the zein genes in the T-DNA at the following sites (see Fig. 2): 1) replace- ment of the small HindIII fragment in EcoRI fragment 13 ; 2) the XbaI site also in EcoRI fragment 13; 3)the KpnI site in EeoRI fragment 7; 4) the SalI site in EcoRI fragment 7; 5) replacement of the small EcoRI fragment in BamHI fragment 8. DNA fragments containing a zein gene and ampicillin resistance marker were inserted into the interme- diate vectors at these sites. The orientation of the zein gene within the intermediate vector was determined, and the plasmids were subsequently transferred into A. tumefaciens strain A348 which contains the octopine-type Ti plasmid pTiA6 (Garfinkel et al. 1981). Integration of the zein gene and ampicillin resistance marker into the Ti plasmid re- sulted from a double recombination event between homolo- gous sequences on the intermediate vector and the Ti plas- mid. In order to select bacteria in which this had occurred, pPH1JI (Hirsh and Beringer 1984) was conjugated into A. tumefaciens strains containing the intermediate vectors. Bacteria were screened for the presence of the ampicillin resistance marker and the loss of the antibiotic resistance marker carried by the broad host range plasmid. The pre- dicted integration of the zein gene was confirmed by restric- tion endonuclease analysis of the Ti plasmid. The insertions that were made are shown in Fig. 2. The A. tumefaciens strains carrying these modified Ti plasmids will be referred to as pTintl, pTint2 etc., and the tumors resulting from infection by these bacteria will be termed T1, T2 etc. pTint32 contained gZ151; all other insertions contained gZI9AB1.
A. tumefaeiens strains carrying these modified Ti plas- mids were used to infect stem sections of sunflower (Barton
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Kb
T-DNA
Loci Transcripts
Born Hl" Eco RI Hind 1Tr
0 5 I0 15 20 25 I I i ~ i
I TL-DNA I I TR-DNA I
F--igr-1 E, N r ~
6 III I I I ~ 7 ill I 13 I
I I III II
12 V / / / / / / A - -~ i
2 ~ I I I I 4 - Y///////] 14 [ / / / / / / / / l ~ I 21 ~ Y / / / / / / A
19fl" " - - r / / / / . ; 2 ~ s l v / / / / / / , ~ - - i
201 --,- K / / / / / / A I 71 ~ - ' ' [ , ' / / / / / / . . 1 I
21 V / / / / / / ] ~ ~ 8 I V / / / / / / A " - - I
2 21 t ' / / / / / / / ] ~ I 9
Fig. 2. Sites of insertion of zein genes into pTiA6. A restriction endonuclease map of the T-DNA region of pTiA6 is shown with genetic loci and mRNA transcripts. The sites of insertion and orien- tation of the inserts are shown. Hatch bars are pBR322, open bars are maize DNA and closed bars are SV40 DNA. Arrows indicate the location and direction of transcription of the zein gene. pTintl, 2, 11 and 12 contain insertions in place of the small HindIII frag- ment in EeoRI fragment 13; pTint 6, 7, 8, 9 and 10 contain inser- tions at the XbaI site in EcoRI fragment 13; pTintt7 contains an insert at the KpnI site in EcoRI fragment 7; pTint 19, 20, 21 and 22 contain insertions at the SalI site in BamHI fragment 8; pTint 13, 14 and 32 contain insertions in place of the small EcoRI fragment in BamHI fragment 8
et al. 1983). The resulting tumors were removed from the stems and cultured on solid medium (Murashige and Skoog 1962) in the absence of phytohormones. Carbenicillin (1 mg/ml) and vancomycin (0.2 mg/ml) were added to the medium to kill any remaining bacteria.
Analysis o f DNA and R N A f rom tumors
Total nucleic acids were isolated from tumors essentially as described by Parish and Kirby (1966). Nucleic acid prep- arations were fractionated in 2.5 M ammonium acetate. The insoluble RNA fraction was dissolved in water, re-extracted with a phenol-chloroform mixture and the RNA precipi- tated with ethanol. The salt-soluble DNA was precipitated with isopropanol and purified by banding on a CsC1 buoyant density gradient in the presence of ethidium bro- mide. Using these techniques, typical yields of 1-2 mg of RNA and 50-100 gg of DNA were obtained from 10 g fresh weight of tumor tissue.
The procedure of Southern (1975) was used to detect zein genes in tumors. Samples of DNA (3 gg) were digested with EcoRI, fractionated through a 1% agarose gel and transferred to a nitrocellulose filter. The hybridization probe was pgZ19AB1 radiolabelled with 32P-dCTP (Rigby et al. 1977). Specific activities of 1-5 x 1 0 7 dpm/gg were ob- tained. Filters were prehybridized (Denhardt 1966) in 3xSSC, 0.08% bovine serum albumin, 0.08% Ficoll, 0.08% polyvinylpyrollidone, 100 gg/ml denatured calf thy- mus DNA for 4 h at 68 ° C. Hybridization was under the
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same conditions in the presence of 1-2 x l0 s dpm/ml of ra- diolabelled DNA for 16 h. After hybridization, filters were washed extensively in 0.1 x SSC, 0.1% SDS at 68 ° C, dried and exposed to Kodak XAR5 film at - 70 ° C with an inten- sifying screen.
Specific RNAs were detected using an $1 nuclease pro- tection assay. DNA fragments were 5' end-labelled as de- scribed by Maxam and Gilbert (1980), or 3' end-labelled as described by James and Leffak (1984). To obtain a DNA fragment labelled at only one end, labelled DNA fragments were digested with an appropriate restriction endonuclease and the fragment of interest was isolated after electrophore- sis on a 5% polyacrylamide gel. Hybridizations and $1 nu- clease digestions were performed as described by Favaloro et al. (1980). The hybridization temperatures were as fol- lows: 49 ° C for the 19 kd zein mRNA; 58 ° C for the 15 kd zein mRNA; 50 ° C for the octopine synthase mRNA; and 51 ° C for transcript 25 of TR-DNA. $1 nuclease was used at a final concentration of 1000 units/ml. After digestion the products were analyzed on 5% polyacrylamide gels con- taining 8 M urea.
Results
Transcription of zein genes in sunflower tumors
Two genes encoding 19 kd and 15 kd zein polypeptides were introduced into the T-DNA of the octopine-type Ti plasmid pTiA6. In order to examine the effect that the site of inser- tion might have upon the transcription of a novel gene in the T-DNA, the 19 kd zein gene was inserted at a number of sites in both TL- and TR-DNA (Fig. 2). A. tumefaeiens strains carrying these modified Ti plasmids were used to infect sunflower stem sections. Sunflower was chosen as the host for these experiments because we have found that TR-DNA is more reliably transferred into this species than into tobacco. After propagation of the tumors in axenic culture, RNA was extracted and analyzed for the presence of zein mRNA transcripts.
S1 nuclease mapping was used to detect specific zein mRNAs and to determine the sites for the initiation and termination of transcription of these genes in both maize and sunflower (Fig. 3). The probe for the 5' end of tran- scripts from the 19 kd zein gene was 5' end-labelled at the BamH1 site within the coding region and extended to a HincII site 415 bp 5' of the site for the initiation of transla- tion. When this 595 bp DNA was hybridized to total RNA isolated from developing maize endosperm tissue, a major DNA fragment of 225 bases was protected from digestion by Sl nuclease (Fig. 3, lane 1). This places the 5' end of the mRNA approximately 30 nucleotides downstream of a putative TATA box (TATAAATA) (Pedersen et al. 1982). When RNA isolated from tumor line T1, containing gZ19AB1, was hybridized to the same probe, a DNA frag- ment was protected of the same size as that protected by maize endosperm RNA (Fig. 3, lane 2). No such fragment was observed when either RNA from a wild type sunflower tumor or tRNA were used in the hybridization (Fig. 3, lanes 3 and 4). This result shows that the 19 kd zein gene is being transcribed in this tumor and that the sites for initia- tion of transcription in a sunflower tumor and maize endo- sperm are identical.
The 19 kd zein gene that was inserted into the T-DNA
Fig. 3. $1 nuclease mapping of transcripts from gZ19AB1. The illustration below the autoradiograms shows a map of the gene and the fragments used for 5' and 3' mapping. 0.t lag of maize endosperm RNA (lane t), 100 lag of RNA from T1 (lane 2) and from a wild type tumor (lane 3) and/00 lag of tRNA (lane 4) were hybridized to the 5' end-labelled probe. 0.t lag of maize endosperm RNA (lane 5), 100 lag of RNA from Tt (lane 6) and from a wild type tumor (lane 7) and 100 ~tg of tRNA (lane 8) were hybridized to the 3' end-labelled probe. The sizes at the side of the autoradio- grams are nucleotides
contains a consensus polyadenylation signal sequence (AA- TAAA) 30 bp after the translational stop codon. There is a second putative polyadenylation signal (AATAAG) 40 bp downstream from this (Pedersen et al. 1982). $1 nuclease mapping of endosperm RNA and sequence analysis of cloned zein cDNAs have shown that both these sites are used for the polyadenylation of transcripts from this gene in maize endosperm (Marks et al. 1985b). To map the 3' end of transcripts from the 19 kd gene in sunflower cells, a similar $1 nuclease mapping experiment was performed (Fig. 3). The 920 bp probe, 3' end-labelled at the BamHI site within the coding sequence, extended to a HincII site 420 bp downstream from the translational stop codon. When hybridized to maize endosperm RNA, two DNA fragments were protected (Fig. 3, lane 5) as previously ob- served (Marks etal. 1985b). These fragments were esti- mated to be 760 and 720 nucleotides in size, placing the 3' ends of these transcripts approximately 120 nucleotides
downstream of either polydenylation signal sequence. How- ever, the estimate of the sizes of the two protected fragments is not precise, because the D N A fragments migrated outside the region of the gel where the relationship between mobi- lity and size was linear. It is likely that these two fragments resulted from protection by RNAs whose 3' ends terminate at the same sites observed by Marks et al. (1985b). When R N A from TI was hybridized to this probe two D N A frag- ments were protected from $1 nuclease digestion (Fig. 3, lane 6). These protected fragments were of the same size as those observed when maize endosperm R N A was hybrid- ized to this DNA. Again, no protection was afforded by R N A from a wild type tumor or t R N A (Fig. 3, lanes 7 and 8). Affinity chromatography on oligo-dT cellulose showed that the transcripts from gZ19AB1 were polyadeny- lated in sunflower tumors (data not shown). The relative intensities of the two protected D N A fragments differed between R N A isolated from endosperm and tumor tissues, indicating that the second signal sequence (AATAAG) was used more frequently in transformed cells. I t must be re- membered, however, that there are a number of closely related genes for 19 kd zeins that are expressed in maize. Individual genes may vary in their use of particular tran- scriptional termination signals.
We performed similar S1 nuclease mapping experiments to examine the expression of gZ151 in the tumor line T32. The results of 5' and 3' mapping of R N A transcripts from this gene, in both maize and sunflower cells, are shown in Fig. 4. The 5' mapping produced two major protected fragments of approximately 230 nucleotides in size. This was observed with both endosperm and T32 R N A prepara- tions (Fig. 4, lanes 1 and 2). Whether this result represents two sites for the initiation of transcription o f this gene or is an artifact of the experimental procedure is unknown. Based upon this analysis, the 5' end of the m R N A lies approximately 30 bp downstream of the sequence T A T A A (Pedersen et al. 1986). The mapping of the 3' end of the m R N A positions the 3' end of this transcript approximately 20 bp after the polyadenylation signal sequence A A T A A A (Fig. 4, lanes 5 and 6). As observed with gZ19AB1, pre- cisely the same sites are used for the initiation and termina- tion of transcription of the 15 kd zein gene whether it is present in a sunflower tumor or in maize endosperm.
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Fig. 4. S 1 nuclease mapping of transcripts from gZl 51. The illustra- tion below the autoradiograms shows a map of the gene and the fragments used for 5' and 3' mapping. I lag of maize endosperm RNA (lane 1), 100 lag of RNA from T 32 (lane 2) and from a wild type tumor (lane 3) and 100 lag of tRNA (lane 4) were hybridized to the 5' end-labelled probe. 1 gg of maize endosperm RNA (lane 5) 100 lag of RNA from T32 (lane 6) and from a wild type tumor (lane 7) were hybridized to the 3' end-labelled probe
Effect of zein gene position in the T-DNA upon zein gene expression
A number of Ti plasmids were constructed that contained the 19 kd zein gene inserted at different sites within the T-DNA, and in opposite orientations at the same site (Fig. 2). Some of these constructions also had an enhancer element from SV40 (Banerji et al. 1981) inserted at the Hin- dIII site 3' to the zein gene to see if it had any effect on transcription o f the zein gene in sunflower tumors. Tumors incited by bacteria carrying these modified Ti plasmids were analyzed for the presence of R N A transcripts from gZ19AB1. The probe for the 5' end of this R N A was the same as that used for the experiments described in Fig. 3. An example of one such experiment is shown in Fig. 5. Of the ten tumor lines examined, four contained R N A that protected a fragment of the same size as that protected by maize endosperm RNA. Zein R N A transcripts were not detectable in the six other tumor lines. We have examined tumors incited by the 16 different strains shown in Fig. 2;
Fig. 5. Detection of gZ19ABI transcripts in different tumors. RNA isolated from tumors was hybridized to the 5' end-labelled probe shown in Fig. 3 and analyzed as described. Lane t : 1 gg of maize endosperm RNA. Lanes 2-12:100 gg of RNA isolated from a wild type tumor, T1, T2, T6, T7, T8, T9, T12, T19, T20 and T21. Lane 13:100 gg of tRNA
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Table 1. Expression of gZ19AB1 in different tumor lines
Tumor Number of tumor Number of tumor lines lines tested containing detectable
for some of these strains we analyzed a number of tumor lines arising from independent infections. R N A was isolated from these tumors and examined for the presence of R N A transcripts from gZ19ABI as described. The results of these experiments are given in Table 1. Of the 38 tumor lines that were examined, 22 contained detectable levels of zein mRNAs . We observed no consistent effect of posi t ion of the zein gene within the T - D N A upon the t ranscript ion of this gene. In cases where we examined more than one tumor line incited by the same bacterial strain, considerable variat ion in the steady state level of zein gene expression was also observed. F o r example, one tumor line incited by pTin t l contained the highest level of zein m R N A that we have detected in any tumor (approximately 1% of that found in maize endosperm), whereas a separate tumor in- cited by the same strain contained no detectable zein m R N A . The enhancer element from SV40 had no consistent effect on the level of zein m R N A in tumors.
In the absence of any obvious effect of locat ion of the zein gene within the T - D N A , we examined the copy number of the zein gene in these tumors to see if it correlated with zein m R N A abundance. D N A isolated from the tumors analyzed in Fig. 5 was digested with EcoRI, fract ionated on an agarose gel and transferred to a nitrocellulose filter. The filter was hybridized with 32p-labelled pgZ19AB1 (Fig. 6). The reconstruction lanes show the intensity of hy- bridizat ion expected if 2,1 and 0.5 copies of the zein gene were present per haploid genome (Fig. 6, lanes 1-3). Hy- bridizat ion was observed to many fragments in maize D N A which contains a number of genes that are related to gZI9AB1 (Fig. 6, lane 4). D N A isolated from tumors in- cited by bacteria containing the zein gene contained se- quences homologous to pgZ19AB1 (Fig. 6, lanes 6-15). In general the sizes of the fragments that hybridized to p g Z I g A B I were those expected from the structures of the Ti plasmids. However, in some cases other fragments also hybridized and these are presumably the result o f incom- plete digestion of the tumor D N A or rearrangement of the T -DNA. The number of copies of the zein genes in these tumors varied from 0.1 to approximate ly 5 copies per hap- loid sunflower genome. This var ia t ion is not unexpected
Fig. 6. Detection of gZ19AB1 DNA sequences in tumors. DNA isolated from tumors was digested with EeoRI and analyzed as described. The probe was pgZ19AB1. Lanes 1,2 and 3:2,1 and 0.5 copy reconstructions, respectively, of pgZ19AB1 digested with EcoRI. Lane 4 : 3 gg of maize DNA digested with EcoRI. Lanes 5-15:3 gg of DNA from a wild type tumor, T1, T2, T6, T7, T8, T9, T12, T19, T20 and T21 digested with EcoRI. The sizes at the side of the autoradiogram are in kilobases
Fig. 7. Detection of octopine synthase mRNA in tumors. The illus- tration below the autoradiogram shows a map of the gene and the 5' end-labelled fragment used for $1 nuclease mapping as de- scribed. Lane 1 :10 gg of maize endosperm RNA. Lanes 2-12: 10 gg of RNA from a wild type tumor, TI, T2, T6, T7, T8, Tg, T12, T19, T20 and T21. Lane 13:10 gg of tRNA
because the tumors were uncloned and consisted of a chi- mera of t ransformed and untransformed cells. However, no correlat ion was observed between gene copy number and the abundance of zein R N A transcripts. Fo r example, the T1 tumor line examined in this experiment contains less than 0.5 copies of the zein gene (Fig. 6, lane 6), yet the highest level of zein transcripts was detected in this part icular tumor line (Fig. 5, lane 3). In contrast , T20 con- tains approximate ly 2 copies o f the gene (Fig. 6, lane 14) but produces no detectable zein m R N A (Fig. 5, lane 12).
There is an alternative explanat ion for the variat ion in
Fig. 8. Variation in the level of gZ19AB1 transcripts in different T1 tumor lines. RNA isolated from six independent TI tumor lines was analyzed for the presence of transcripts from gZ19AB1 as described in Fig. 4. Lane 1 : 0.1 lag of maize endosperm RNA. Lanes 2-8:100 lag of RNA from tumors Tla, Tlb, Tic, Tld, Tie, Tlf and a wild type tumor
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zein gene expression between tumor lines. Because the zein gene was within the T-DNA, its transcription might be gov- erned by some mechanism that controls all genes in the T-DNA. If this were so, variation in mRNA levels similar to that observed for zein would be expected for transcripts of other T-DNA genes. To test this hypothesis, we analyzed RNA isolated from tumors for the presence of octopine synthase mRNA, again using an $1 nuclease protection assay (Fig. 7). The probe was 5' end labelled at the EcoRI site, 270 bp downstream of the translational initiation co- don, and extended 412 bp to the BamHI site (Barker et al. 1983). RNA from the same tumors analyzed in Fig. 5 and 6 was hybridized to this DNA. All of these RNA prepara- tions protected a fragment of 300 nucleotides as expected (Fig. 7). However, little variation in the abundance of the octopine synthase mRNA was observed. This is in contrast to the result obtained for the levels of zein mRNA in these tumors, and suggests that there is no process that regulates the expression of all genes within the T-DNA.
We also examined the variation in expression of gZ19ABI between independent tumor lines incited by the same strain of A. turnefaciens. Tumors from six individual sunflower stem sections innoculated with pTintl were ana- lyzed for the presence of transcripts from gZI9AB1 (Fig. 8). All six contained zein transcripts but the levels of these transcripts ranged between approximately 0.05% and 1.0% that observed in maize endosperm tissue. Again no correla- tion was observed between the level of mRNA and gene copy number (data not shown). In these tumors the zein gene was inserted in TR-DNA, which is normally trans- ferred independently of TL-DNA, and variation in the level of TR-DNA encoded mRNAs has been reported (Karcher et al. 1984). To determine if the level of zein mRNA in these tumors was related to the level of other TR-encoded mRNAs, we analyzed these six T1 tumors for the presence of the internal 1600 base transcript of TR-DNA [open read- ing frame 25 in Barker et al. (1983)]. The probe for the 5' end of this mRNA is shown in Fig. 9. RNA from tumors protected a fragment of 500 nucleotides as expected (Fig. 9, lanes 2-7). There was substantial variation, however, in the level of transcript 25 between these six T1 tumor lines. Interestingly, the abundance of transcript 25 in a particular tumor line was directly proportional to the level of zein mRNA in that tumor. This result suggests that there may be a mechanism that regulates the expression of closely linked genes within these tumors.
Fig. 9. Variation in the level of "transcript 25" in different TI tumor lines. The illustration below the autoradiogram shows a map of the gene and the 5' end-labelled fragment used for SI nuclease mapping as described. Lane 1:10 lag of maize endosperm RNA. Lanes 2-7:10 lag of RNA from tumors Tta, Tlb, Tlc, Tld, Tle and Tlf
Discussion
These experiments show that zein genes integrated in T- DNA can be accurately transcribed when these genes are introduced into sunflower cells, and that the 5' and 3' ends of zein gene transcripts are identical in sunflower and maize tissues. Matzke et al. (1984) have reported similar results on the transcription in sunflower tumors of a zein gene that is closely related to gZIgABI. There is sufficient infor- mation within the 5' flanking regions of these genes to ac- curately direct transcription. Deletion mapping will be nec- essary to determine the minimum amount of upstream se- quences required to promote transcription of these genes in sunflower cells. Signal sequences at the 3' ends of both zein genes are correctly recognized as sites for polyadenyla- tion. For gZI9AB1 in sunflower cells, the second signal, AATAAG, was used preferentially over the proximal se-
380
quence AATAAA. This has been noted for other zein genes (Heideker and Messing 1983; Marks et al. 1985b). Further analysis of individual zein genes in transformed cells may lead to a better understanding of the importance of these transcriptional regulatory sequences.
The level of zein transcripts in tumors varied from being undetectable to approximately 1% of that present in maize endosperm R N A at 18 days after pollination. We have used a variety of techniques to detect zein polypeptides in these tumors. None of these methods has shown that zein pro- teins are present in transformed sunflower cells. I f the zein m R N A in sunflower cells was translated with an efficiency similar to that in maize endosperm, we would have been able to detect the protein. We do not know if the absence of zein proteins in sunflower cells results from a failure in the translation of mRNA, or from degradation o f the protein, as has been observed for phaseolin in transgenic tobacco plants (Sengup ta - Gopalan et al. 1985). Zein pro- teins were also not detected in sunflower tumors analyzed by Matzke et al. (1984).
Sunflower tumors containing the zein gene at different sites within the T - D N A show great variation in the abun- dance of zein transcripts. This variation does not appear to be related either to the site of insertion of the zein gene within the T-DNA, or to the number of copies of the zein gene that are integrated. This apparently random variation makes it impossible to compare the relative activities of different zein gene promoters using this type of analysis. This difficulty may be overcome by examining the develop- mental expression of zein genes in plants regenerated from cells transformed with a non-tumorigenic Ti plasmid (Bevan 1984). Alternative approaches include comparing the activi- ty of different zein gene promoters in an in vitro transcrip- tion system (Manley et al. 1980) and the analysis of runoff transcripts in isolated nuclei (Gallagher and Ellis 1982).
The abundance of zein m R N A in tumors incited by pTintl was directly proportional to the level of the TR encoded transcript 25. Gene 25 is involved in the biosynthe- sis of mannopine and mannopinic acid (Komro et al. 1985). In these tumors the promoters of gZ19ABI and gene 25 were separated by approximately 2.5 kb. One explanation for the similar levels of expression of these two closely linked genes is that the site of integration of TR within the sunflower genome in some way regulates the activity of all genes within TR-DNA. We have not yet examined the abundance of other TR transcripts in these tumors to see if the levels of all transcripts from TR are similarly affected. Karcher et al. (1984) have examined the steady state levels of TR m R N A s in a number of tumor lines. Their data showed variation in the steady state levels of these transcripts between tumor lines. Their results, how- ever, are not consistent with the hypothesis that position effect within the host genome is an important factor in regulating the abundance of all TR mRNAs. The coordi- nate variation of zein m R N A and transcript 25 in TI tu- mors may result from a phenomenon that is restricted to a relatively small region of TR DNA, and may be caused by the insertion of the zein gene itself.
Our results show that both of the maize seed storage protein genes used in these experiments contain sufficient information for their correct transcription in sunflower cells. At least some of the signals that regulate transcription are therefore conserved between monocots and dicots. Ex- periments are now in progress to determine if these genes
contain the necessary information for their developmental expression in the seeds o f dicotyledonous plants.
Acknowledgements. The authors are indebted to Marlaya Wyncott, Judy Lindell and Evelyn Hatch for their excellent technical assis- tance, to Jane Carter and Karen Lewis for preparation of this manuscript and to Caroline Logan for photography. This work was funded by Agrigenetics Research Associates. This is Journal Paper Number 10,548 of the Purdue Agricultural Experiment Station.
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