Molecular identification and isolation of the Waxy locus in maize

Cell, Vol . 35, 225 -233, November 1983, Copyright ©1983 by MIT

0092-8674/83/110225-09 $02 .00/0

Molecular Identification and Isolation of the Waxy Locus in Maize M . Shure, S . Wessler,* and N . Fedoroff Department of Embryology Carnegie Institution of Washington 115 West University Parkway Baltimore, Maryland 21210

Summary The Waxy (Wx) locus in maize determines the amylose content of pollen and endosperm tissue . There are several mutant alleles of the locus caused by insertion of transposable controlling elements . In the present study, we have used the properties of controlling element alleles to identify the Wx locus and its gene product, with the subsequent objective of isolating the elements causing the mutations . We present evidence that the Wx locus encodes a starch granule-bound 58 kd polypeptide that is synthesized in vitro as a 65 kd precursor . We describe the isolation of recombinant plasmids containing cDNA inserts homologous to Wk mRNA and a recombinant A phage containing a genomic Eco RI fragment encompassing most or all of the Wk transcription unit . We show that a mutation caused by the controlling element Dissociation (Ds) is attributable to an insertion of approximately 2 .4 kb at the Wx locus. Introduction The Waxy (Wx) locus in maize determines the amylose content of endosperm tissue and pollen (Sprague et al ., 1943) . There is evidence that it encodes a starch granulebound UDP-glucose starch transferase (Nelson and Rines, 1962 ; Tsai, 1974) . Many mutations affecting expression of the locus have been identified, including unstable mutations caused by insertion of transposable controlling elements (McClintock, 1951, 1952, 1963, 1964 ; Nelson, 1968) . Among these are several alleles that arose by insertion of the autonomously transposing Activator (Ac) element, as well as numerous alleles that are attributable to insertion of elements incapable of autonomous transposition (McClintock, 1951, 1952, 1963, 1964 ; Nelson, 1968, 1976) . These include the Dissociation (Ds) element and an element belonging to the Suppressor-mutator (Spm) controlling element family . We undertook the isolation of the Wx locus with the eventual aim of isolating the controlling elements responsible for unstable mutations at the locus . In the present study, the properties of strains with controlling element mutations have been used to identify the Wx locus and its protein product . We present evidence that the Wx locus encodes a 58 kd starch granule-bound protein and that the 2 .3 kb Wx mRNA is translated in vitro into a 65 kd polypeptide which is probably an unprocessed precursor . We describe the construction and identification of cloned Present address : Department of Botany, University of Georgia, Athens, Georgia 30620 .

cDNAs homologous to the Wx mRNA sequence. We show that a cloned Wx cDNA hybridizes to a single genomic sequence that is longer by a 2 .4 kb insertion in a Ds allele of the locus than it is in either the progenitor Wx allele or in several newly derived Wx revertants, identifying the sequence as the Wx locus. We describe the construction, identification, and analysis of a recombinant A phage containing most or all of the transcription unit at the Wx locus . Results The Waxy Locus Encodes a 58 kd Starch Granulebound Protein Starch granules contain a tightly bound 58 kd protein whose abundance is proportional to the dosage of the Wx allele and correlates with the amount of starch granulebound glucosyl transferase (Tsai, 1974 ; Chaleff et al ., 1981 ; Echt and Schwartz, 1981 ; M . Shure and S . Wessler, unpublished data) . We unsuccessfully explored the ability of various agents to release the active enzyme from starch granules . These included weak acids and bases, high concentrations of salt, substrate analogs, amylase, detergents, and chaotropic agents . Compounds that disrupted starch granule structure released the 58 kd protein, but in no case was the UDP-glucosyl transferase activity recovered . We were also unable to reconstitute active enzyme . We therefore purified the inactive 58 kd protein and raised antiserum against it (see Experimental Procedures for details) . We obtained evidence that the 58 kd protein is encoded by the Wx locus from analyses of the proteins bound to endosperm starch granules in strains with controlling element mutations at the Wx locus . The mutant wx-m6 and wx-m9 alleles have Ds elements inserted at the Wx locus, while the wx-m8 allele is attributable to insertion of an element belonging to the Spm family of controlling elements (McClintock, 1952, 1961,' 1963 ; see Experimental Procedures for a note on the nomenclature used for the unstable alleles) . All three mutations are caused by insertion of elements that are incapable of autonomous transposition and the mutations give a stable recessive wx phenotype (wx-m6 and wx-m8) or a stable intermediate phenotype (wx-m9) in the absence of an autonomously transposing element . In the presence of Ac, the wx-m6 and wx-m9 alleles revert both somatically to give Wx endosperm tissue sectors and germinally to give Wx revertant alleles . The wx-m8 allele reverts in the presence of the Spm element, but is insensitive to the presence of an Ac element. Similarly the wx-m6 and wxm9 alleles are stable in the presence of an Spm element . We isolated starch granules from immature endosperm tissue of kernels carrying the wx-m6, wx-m9 and wx-m8' alleles and analyzed the starch granule-bound proteins on SDS-polyacrylamide gels (Figure 1) . When the kernels did not contain an autonomous controlling element (either Ac or Spm) and showed no somatic reversion of the wx-m6, wx-m9, or wx-m8 mutations, the 58 kd protein was either not detectable (Figure 1, lanes 5, 6, 9, and 10) or present

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wx- m6 I ll,

wx-m8

wx-m9 I

I

58K-

Figure 2. Immunological 56 kd Protein

,‘z?

27DAP I

1 I ‘2345678

Figure 1, SDS-Polyacrylamdie Gel Electrophoretic terns from En&sperms Containing wx-m Alleles

9 Analysis

IO

of Granule Pro-

The major band in lane 1 is the 58 kd granule-bound protein from Wx endosperms harvested 18-20 DAP. The other lanes show granule proteins for indrvidual endosperms harvested 27 DAP. The genotypes represented are: (2) homozygous wxm9, no AC; (3) Wx; (4) homozygous wx-m-8, Spm present; (5) homozygous wx-m8, no Spm in kernel; (6) homozygous wxm8, no Spm in plant; (7) heterozygous wx-mS/wx-m6, Spm present; (8) homozygous wx-m6, AC present; (9) homozygous wx-m6, no AC in kernel; (10) homozygous wx-m6, no AC in plant.

at a very low level (Figure 1, lane 2). When the Spm element was present with the wx-m8 allele, sectors of endosperm tissue showed the Wx phenotype and a 58 kd protein was present in isolated starch granules (Figure 1, lane 4). By contrast, when AC was present together with the wx-m6 allele, endosperm tissue showed sectors that were intermediate between the Wx and the wx phenotypes and a novel 60 kd protein was detectable on starch granules (Figure 1, lane 8). That the proteins detected in the presence of the AC and Spm elements are related to the purified 58 kd starch granule protein was demonstrated immunologically. A protein that cross-reacts with the purified 58 kd protein was detected among proteins derived from Wx starch granules (Figure 2, lanes 1 and 5) but not from starch granules purified from kernels carrying either a stable wx allele (Figure 2, lanes 2 and 8) or the wx-m6, wx-m9 or wx-m8 alleles, but lacking the AC and Spm elements (Figure 2, lanes 3, 6, and 10). When the starch granules were from kernels that carried an unstable allele of the Wx locus and the cognate autonomously transposing element (AC or Spm), polypeptides that cross-react

Detection

of Endosperm

Proteins

Related to the

Granule-bound proteins from individual endosperms analogous to those shown in Figure 2 were fractionated by SDS-polyacrylamide gel electrophoresrs and transferred to a diazotized cellulose filter. The filter was first incubated with antrserum directed against the 53 kd Wx polypeptide, then with purified 58 kd protein that had been labeled in vitro with ‘? The dried filter was autoradiographed. The labeled bands in lanes 1 and 5 comigrate with the 58 kd Wx protein, as judged by stainrng a strip of the gel with Coomassie blue. The Wx alleles in the kernels used as a source of starch granules, as well as the presence of controlling element, are indicated in the figure.

with the antiserum to the 58 kd protein were detected (Figure 2, lanes 4, 7, 9). Thus somatic reversion of the unstable wx-m6, wx-m9 and wx-m8 alleles correlates in each case with the appearance on starch granules of a polypeptide that is immunologically related to the purified 58 kd protein and has a similar mobility. Evidence that that Wx locus encodes the 58 kd protein is provided by two observations. First, the wx-m6 allele reverts somatically to give a phenotype intermediate between the Wx and wx phenotypes and reversion results in the appearance on starch granules of a 60 kd, rather than a 58 kd, protein. Hence reversion of the wx-m6 mutation gives a protein that is structurally abnormal and appears to have an altered enzymatic activity. Second, only the 58 kd protein is present in starch granules from kernels that are heterozygous for the wx-m6 and wx-m8 alleles, but contain only the Spm element (Figure 1, lane 7). Therefore, the structurally altered protein detected upon reversion of the wx-m6 allele in the presence of AC is not detected in the presence of Spm. Were the Wx locus a regulatory locus, both the 58 kd and the 60 kd proteins would be present in a kernel that is heterozygous for the wx-m6 and wx-m8 mutations and contains the Spm element. We conclude that the Wx locus is the structural locus for the 58 kd protein. We refer to it hereafter as the Wx protein.

ldentlflcation 227

and Isolation of the Wx Locus

The Wx Protein Is Synthesized as a Larger Polypeptide In Vitro Antiserum raised against the Wx protein was used to identify an antigenically related polypeptide translated in vitro from Wx mRNA. The labeled polypeptides obtained when poly(A)+ RNAs from immature wx and Wx endosperm tissue were translated in vitro are displayed in Figure 3 (lanes 2-5). No major 58 kd polypeptide is evident among the translation products. However, there is a major 65 kd polypeptide that is translated from Wx (Figure 3, lanes 4 and 5) but not from wx poly(A)’ RNA (Figure 3, lanes 2 and 3). The 65 kd polypeptide is specifically precipitated by antiserum directed against the 58 kd Wx protein (Figure 3, lanes 6 and IO). Antibodies that precipitate the labeled 65 kd protein can be adsorbed out by preincubation of the antiserum with excess unlabeled Wx protein (Figure 3, lane 11). By contrast, preincubation of sucrose synthetase antiserum with the Wx protein has no effect on the subsequent precipitation of the labeled 92 kd sucrose synthetase monomer encoded by the Shrunken locus (Figure 3, lanes 12 and 13). The 65 kd in vitro translation product is therefore related to the 58 kd Wx protein and may be a precursor that is synthesized, but not processed in the rabbit reticulocyte extract.

Construction and identification Homologous to Wx mRNA

of cDNA Clones

We enriched endosperm poly(A)’ mRNA for the species encoding the Wx protein by sucrose gradient fractionation prior to cDNA cloning. The size of the mRNA coding for the 65 kd Wx polypeptide was estimated to be 2 kb from its sedimentation rate. Since the Wx RNA is larger than the most abundant endosperm mRNAs, we were able to obtain an RNA preparation for cDNA cloning that was enriched for the Wx mRNA by a factor of 5-10 (see Experimental Procedures for details). Recombinant plasmids were initially screened by hybrid-selected translation (Ricciardi et al., 1979). The first chimeric plasmid identified that selectively hybridized the mRNA for the 65 kd Wx polypeptide had an inset of 0.35 kb and was therefore designated pcWxO.35. The insert from the pcWxO.35 plasmid was used to screen an additional 6000 transformants containing recombinant plasmids. Only two additional chimerit plasmids with homology to the insert in pcWxO.35 were identified. Thus despite a substantial enrichment of the mRNA used for cloning, very few Wx cDNA plasmids were identified. Moreover, the inserts were quite short (0.25-0.4 kb) in all three Wx cDNA plasmids, even though many of the randomly selected cDNA clones in the population had longer inserts (l-2 kb). The identity of the pcWx plasmids was verified both by hybrid selected translation (data not shown) and by hybrid ization to endosperm mRNAs. The pcWxO.35 plasmid is homologous to a 2.3 kb mRNA species that is abundantly represented in total, as well as in sucrose gradient-fractionated mRNA from Wx endosperm tissue (Figure 4, lanes 3 and 6). There is no RNA species of the same length and

Antiserum:

58K 58K + + IW pI 1w 1w 1w 1w 1s IS

None

Ei5K -5 i8~

I

I

2

3

4

5

6

7

8

9

IO II

12 13

I

Figure 3. Fluorograph of an SDS-Polyacrylamide Gel on Which %Methionine-labeled In Vitro Translation Products Were Fractionated mRNAs were translated in a rabbit reticulocyte lysate. The designations across the bottom of the figure are the genotypes of endosperms from which poly(A)+ RNA was purifled. The designations across the top of the figure Indicate the type of serum used when samples were lmmunoprecipitated. The proteins obtained without addition of exogenous RNA are shown in lane 1. Lanes 2-5 show the translation products of poly(A)+ RNA from Immature endosperms of the following genotypes; (2) Sh wx; (3) sh wx; (4) and (5) Sh Wx (Sh designates the Shrunken locus, encoding sucrose synthetase). Two different stable recessive wx alleles were used (lanes 2 and 3). Lanes 6-13 show the immunoprecipltable translation products obtained using: (6) Sh Wx RNA and Wx immune serum (Iw); (7) Sh Wx RNA and prelmmune serum (pl); (8) sh wx RNA and Wx immune serum; (9) Sh wx RNA and Wx immune serum; (10) Sh Wx RNA and Wx immune serum; (11) Sh Wx RNA and Wx immune serum preincubated with 5 ag of unlabeled 58 kd Wx protein; (12) Sh Wx RNA and sucrose synthetase antiserum (I’); and (13) Sh Wx RNA and sucrose synthetase antiserum preincubated with 5 pg of unlabeled 58 kd Wx protein. The position of the 56 kd Wx protein was determined by staining the gel with Coomassle blue prior to fluorography.

abundance with homology to the plasmid in poly(A)- RNA from Wx endosperm tissue (Figure 4, lane 2) or in poly(A)’ RNA from wx endosperm tissue, although a small amount of a slightly longer homologous mRNA is detectable in the latter (Figure 7, lanes 4 and 5).

The pcWx cDNA Plasmids Are Homologous to the wx Locus To determine whether the Wx cDNA plasmids were homologous to the genetically defined Wx locus, we used the pcWxO.35 insert to probe DNA derived from a maize strain with a well-characterized insertion mutation at the Wx locus. We chose the wx-m6 allele because both the progenitor Wx allele and several newly derived Wx revertant alleles were available (see Experimental Procedures). We used DNA from plants homozygous for the progenitor Wx allele, the mutant wx-m6 allele, and two germinally stable Wx revertant alleles. The insert in the pcWxO.35 plasmid is homologous to a single genomic DNA fragment in all of

Cell 228

poly A-

poly A+

III wx

wx wx wx wx

3256-

the restriction endonuclease digests analyzed (Figure 5). In four of the five d;$ests, the fragment detected in wx-m6 DNA is larger by 2.4 kb than it is in either the Wx progenitor DNA or in the Wx revertant DNAs. Since the wx-m6 mutation has been characterized as a Ds insertion mutation at the Wx locus (McClintock, 1951) we conclude that the cloned cDNA is indeed homologous to the Wx locus. We further conclude that the Ds element is approximately 2.4 kb in length and that reversion of the Ds mutation is associated with excision of the element (Figure 5).

- 2600 - 2300

l74614 33l106910-

Isolation and Structure of the Wx Locus Total Eco RI-digested DNA from a strain homozygous for the Wx allele that gave rise to the mutant wx-m6 allele was cloned in the Eco RI site of XEMBL4, and the recombinant phages were screened by plaque hybridization with a Wx cDNA clone (see Experimental Procedures). The 10.8 kb insert from one of the recombinant phages was subcloned into a plasmid, mapped with restriction endonucleases, and used to analyze the transcription unit. As shown in Figure 6, the Eco RI fragment contains most or all of the sequence encoding the Wx mRNA. The direction of transcription is as indicated (see Experimental Procedures for details). Although the ends of the transcription unit have not been located precisely, it appears to be quite long. Hybridization to the 2.3 kb Wx mRNA has been observed with probes extending beyond the Pst I site at 8.7 kb on

655520-

I Figure 4. Detectron

23456 of Endosperm

RNA Homologous

to a Wx cDNA Clone

RNA was fractionated on an agarose gel, transferred to a nitrocellulose filter, and hybridized with labeled pcWxO.35 DNA. (1) pBR322 DNA size standards; (2) 1 rg of poly(A)- RNA from Wx endosperms; (3) 1 pg of poly(A)+ RNA from Wx endosperms; (4) and (5) 1 pg and 2 pg, respectively, of poly(A)+ RNA from endosperms homozygous for a stable wx allele; and (6) 0.25 pg of poly(A)+ RNA from a sucrose gradient fractron enriched for Wx mRNA. The sizes of the marker DNAs are given In base pairs.

C-l

Sh Bz

+Ac

WxDs,,

c

wx-m6

tAc

wx-m6r 1 z wx-m6r

EcoRI

Bgl II

Xmnl kb

EcoRV

2 Pvu

kb I,

Frgure 5. Hybridization

Analysis of Genomic

I ,‘y

” ;’

DNAs

Autoradiograph of filters, folIOwing hybridization with the 32P-labeled cDNA insert from pcWxO.35, contakring restnckon endonuctease-digested DNAs from plants homozygous for the WX-m6 allele, the progenitor Wx allele and two stable Wx revertants (wx-m6r7 and wx-m6Q). The diagram at the top of the figure outlines the relationship among these alleles. The chromosome in which the wx-m6 allele arose carried the C-l allele of the C lk~~s, Sh, 6z (&onze), and WX alleles and had Ds between the Wx locus and the centromere on the shod arm of chromosome 9. The restrictron endonucleases used, as welt as the sizes (In kb) of the fragments homologous to the cDNA insert, are indicated in the figure.



Identification and Isolation of the Wx Locus 2 29

3

-1 1 4

)) W 6

)Vf(( f , Y a

7

9

kt

Figure 6. A Restriction Endonuclease Cleavage Site Map of a Cloned Genomic Eco RI Fragment Containing Wx Locus Sequences The direction of transcription and the minimal extent of the transcription unit are indicated by the arrow above the map . The Wx cDNA plasmids hybridize to the 0 .7 kb Pst I fragment extending from 7 .8 to 8 .5 kb on the restriction site map.

the map, as well as beyond the 2 kb Eco RI-Bam HI fragment derived from the left end of the fragment, indicating a length of more than 7 kb for the mRNA coding region (M . Schwartz, unpublished data) . Discussion Characteristics of the Protein Encoded by the Wk Locus We have presented evidence that the Wx locus encodes a 58 kd protein that is a major constituent of starch particles . Echt and Schwartz (1981) identified a protein of similar mobility as the Wx gene product based on the study of alleles that give inactive mutant proteins with different isoelectric points . These authors also suggested that the wx-m6 insertion is likely to be within the protein-coding sequence at the Wx locus . We have observed that the wxm6 allele reverts somatically to a phenotype intermediate between the Wx and wx phenotypes, suggesting that reversion does not result in the full restoration of enzymatic activity. Moreover, revertant endosperm tissue contains a 60 kd, rather than a 58 kd, Wx protein . The intermediate phenotype may therefore result from the production of a structurally altered protein that is not fully active enzymatically . Schwartz and Echt (1982) also reported the synthesis of aberrant Wx polypeptides in endosperm tissue of plants carrying the Ac wx-m9 allele, suggesting that the insertion is within the transcription unit in this allele as well (Fedoroff et al ., 1983b) . Although the present experiments, taken together with those of Echt and Schwartz (1981) and Schwartz and Echt (1982), unambiguously identify the Wx locus as the structural locus for a major 58 kd starch granule-bound protein, they do not illuminate the relationship between the protein and the UDP-glucose starch transferase activity identified with the Wx locus by Nelson and Rines (1962) . Since our attempts to release active enzyme from starch granules were not successful, the identity of the 58 kd protein as the glucosyl transferase remains, at best, a reasonable conjecture . It is supported by the observation that both the activity and the Wx protein levels are proportional to the dosage of the Wx allele in endosperm tissue and that the structurally altered protein produced upon reversion of the wx-m6 allele gives a reduced level of amylose in endosperm tissue . We have reported that a 65 kd polypeptide immunologically related to the 58 kd Wx protein is synthesized in an in vitro rabbit reticulocyte protein-synthesizing extract . Be-

cause the 58 kd protein is not produced in the extract, it appears likely that the 65 kd polypeptide constitutes an unprocessed precursor of the 58 kd protein . The existence of such a precursor is not unexpected in view of the fact that the Wx protein is encoded in the nucleus, but functions on starch granules within amyloplasts . The transport of other plant proteins encoded by nuclear genes into plastids has been shown to result in the removal of a transit peptide from a precursor protein (Chua and Schmidt, 1978 ; Grossman et al ., 1982) . The putative Wx precursor protein is 7 kd larger than the mature protein, well within the 4-8 kd range of transit polypeptide sizes identified in other proteins . Molecular Cloning of DNA Complementary to Wk mRNA Poly(A)+ mRNA fractions substantially enriched for Wx mRNA were used to construct cDNA clones . Despite the use of a cloning method designed to produce long cDNAs and our observations that the resulting endosperm cDNA inserts were generally long, all of the Wx cDNA clones isolated had very short cDNA inserts and the frequency with which Wx cDNA clones were recovered (0 .05%) was much less than expected from the abundance of the corresponding mRNA . These anomalies suggest that the Wx mRNA has a secondary structure inimical to reverse transcription . Plasmids were initially identified as containing Wx cDNA inserts by selective hybridization to the mRNA sequence encoding the immunoprecipitable 65 kd Wx polypeptide, as well as by cross-homology . Further testing showed that the Wx cDNA hybridized to an abundant 2 .3 kb poly(A)+ RNA present in immature endosperm of plants homozygous for the Wx allele, but absent from immature endosperm tissue of a plant homozygous for a stable recessive wx allele . Finally, the Wx cDNA was shown to hybridize to a unique sequence in maize genomic DNA . That the unique sequence corresponds to the Wx locus was deduced from the results of hybridization analyses of DNA isolated from maize strains with and without a Ds insertion mutation at the Wx locus . The presence of the insertion at the Wx locus is correlated with the presence of a 2 .4 kb insertion in the sequence homologous to the cloned Wx cDNA, unequivocally establishing the homology of the cDNA to the Wx locus . Isolation of Genomic Wx Clones We isolated an Eco RI fragment of the Wx locus from genomic DNA of plants homozygous for the Wx allele that gave rise to the wx-m6 allele . The transcription unit occupies most of the 10 .8 kb fragment and may extend beyond the Eco RI sites bounding it . At present we know only that the 3' end of the sequence encoding the mRNA extends beyond a Pst I site located roughly 2 kb from the right end of the fragment and that its 5' end extends beyond a Bam HI site located 2 kb from the left end of the fragment . Because two of the cDNA clones isolated contain Pst I sites, as well as poly(A) sequences (S . Wessler and M .

Cell 230

Shure, unpublished data), it appears likely that the 3' end of the mRNA coding sequence lies near a Pst I site . Since the cDNA clones hybridize to the 0 .7 kb Pst I fragment extending from 7 .8 kb to 8 .5 kb on the restriction map, the Pst I site at 8 .5 kb probably falls within an intervening sequence and the mRNA coding sequence is likely to end in the vicinity of the Pst I site at 8 .7 kb . The wx-m6 Allele Has a 2 .4 kb Insertion Near the 3' End of the mRNA Coding Sequence The results of filter hybridization experiments indicate that the wx-m6 mutation is attributable to a 2 .4 kb insertion . Because the difference in size of Wx restriction endonuclease fragments between Wx and wx-m6 alleles is evident in Eco RV, Eco RI, Pvu I, and Bgl II digests, the insertion is likely to be near the 3' end of the transcription unit . That this is indeed the case is shown in the accompanying paper (Fedoroff et al ., 1983b) . Reversion of the mutation results in the excision of the element . However, the observation that an abnormal protein is made when the wx-m6 allele reverts suggests that excision of the element is not precise and does not completely restore the coding sequence . Peacock and his coworkers (personal communication) have recently observed that a Ds-like insertion at the Adh I locus of maize is excised imprecisely, leaving a small duplication at the site of insertion . We do not yet know whether the Ds element is inserted in a coding or an intervening sequence . Thus the production of an altered Wx protein upon reversion of the wx-m6 allele may result either from alterations in the processing of the Wx mRNA precursor or from a change in the protein-coding sequence itself . Experimental Procedures Maize Strains The F2 generation of the W23 X K55 hybrid strain was used throughout for the preparation of Wx protein and Wx RNA populations ; inbred strains carrying two different stable wx alleles were also used . These maize strains were kindly provided by Drs . E . H . Coe and G . M . Neuffer. Strains carrying the wx-m6, wx-m9, and wx-m8 alleles, as well as the strain carrying the Wx allele from which the wx-m6 allele was derived, were obtained from Dr . B. McClintock . It should be noted that we have used the designation wxm9 for an allele that behaves genetically as a Ds mutation. McClintock (1963) initially designated the Ac insertion mutation from which it was derived wx-m9, but later renamed the allele Ac wx-m9 in her personal records to distinguish it from the wx-m9 derivative . We have used these designations here and in Fedoroff et al., 1983b because they facilitate distinguishing between mutations caused by the autonomous Ac element and the nonautonomous Ds element, yet preserve the genetic relationship between the alleles in the nomenclature . Various combinations of wx-m alleles with and without the autonomous Ac and Spm elements were constructed for this study . The presence of an autonomous element was verified both by visualizing the somatic reversion phenotype in endosperms and by test-crossing plants to stocks carrying an independent genetic marker which could be destabilized by the appropriate autonomous controlling element. Wx revertants were detected visually or by iodine staining of kernels. Wx kernels appearing on ears that were homozygous for an unstable allele of the Wx locus were selected, grown, and the plants were tested for stability of the Wx phenotype, as well as the presence of the controlling element at the Wx locus . Maize plants were propagated either in the field or in a growth chamber . Immature ears were harvested 18-22 days after pollination (DAP) or 27 DAP . The ears were frozen in liquid nitrogen and stored at -70°C.

Purification of Starch Granules Starch granules were prepared by powdering frozen dissected endosperms in a mortar and pestle, suspending the powder in 30% (w/v) sucrose containing 50 mM Tricine (pH 8 .0), 0 .1 M KCI, 5 mM MgCl2 , 1 mM EDTA, 1 mM D1T, (3 ml buffer/gram of endosperm tissue), followed by homogenization with a Bounce homogenizer or in a Sorvall Omni-mix homogenizer for 1 min . The homogenate was centrifuged at 75 X g for 5-10 min and the resulting pellet was washed twice in the same buffer containing 30% sucrose. To remove zein-containing protein bodies the granule pellet was suspended in the 30% sucrose buffer and layered on top of a sucrose cushion (2 vol of suspended granules per volume of cushion) containing 75% (w/v) sucrose in the same buffer as the 30% sucrose solution (Burr and Burr, 1976). Centrifugation was in an HB4 rotor at 10,000 rpm for 15 min . After removal of the supernatant, including the protein bodies which remain trapped at the top of the cushion, the granule pellet was washed twice with H2 O, lyophilized, and stored at -20°C. All of the steps were performed at 0°C-4°C. UDP-glucose: starch glucosyltransferase activity was assayed as described by Nelson et al . (1978) . Analysis of Starch Granule-bound Proteins For the analysis of starch granule-bound proteins from wx-m strains, starch granules were purified from individual endosperms by the procedure described above, using 1 ml of the homogenization buffer per endosperm . Granules (1-4 mg) were boiled for 2-5 min in 20 Al of a solution containing 20% glycerol, 2% SDS, 20 mM DTT, 0 .05% bromophenol blue . The insoluble starch was pelleted in a microfuge, and the supernatant was immediately removed . The entire supernatant was applied to one gel slot if the gel was to be stained with Coomassie blue and 1-5 Al of the supernatant was used if the gel was to be stained with silver . Purification of the Wk Protein Starch granules were prepared from 100 grams of dissected endosperms from strain W23 X K55 (harvested 20 DAP) by a modification of the protocol outlined above . Prior to the lyophilization step, the granule pellets were resuspended in the Tricine-30% sucrose buffer containing 1% Triton X100, incubated at 4°C for 10 min, pelleted and washed with H 2O. This procedure yielded 16 g of dried granules containing approximately 40 mg of protein, as determined by the method of Bradford (1976) . To further purify the Wx protein and prepare a soluble antigen, 5 g of granules were incubated in 50 ml of a solution containing 8 M urea, 50 mM Tris-HCI (pH 7 .5), and 2 mM DTT at 37°C for 15 min . Following centrifugation in an HB4 rotor at 9000 rpm for 10 min, the aqueous supernatant was adjusted to 4 M urea and applied to a DE-52 column . After extensive washing, the protein was eluted with a 0 to 0 .1 M NaCl gradient in 4 M urea, 50 mM Tris-HCI (pH 7 .5) . Fractions containing the Wx protein (determined by SDS-polyacrylamide gel electrophoresis) were pooled . Following dialysis against 50 mM Tris-HCI (pH 7 .5), 2 mM EDTA, and concentration by ultrafiltration (Amicon), the Wx protein was further purified by preparative gel electrophoresis. Acrylamide gels were incubated in cold KCI to detect the protein and the protein was then eluted as described by Hager and Burgess (1980) . From 5 g of granules purified from approximately 150 immature endosperms, we obtained 10 mg of Wx protein, as determined by the method of Bradford (1976). This represents approximately 0 .75% of the total endosperm protein at 20 DAP . SDS-Polyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis was performed as described by O'Farrell (1975) . Gels containing samples of granule-bound proteins were stained with Coomassie brilliant blue R-250 or with silver as described by Oakley et al . (1980) . Gels containing samples of in vitro translation reactions were either stained with Coomassie brilliant blue and then fluorographed (Laskey and Mills, 1975) or were fluorographed directly using either DMSOPPO as desribed by Bonner and Laskey (1974) or Enhance (New England Nuclear) . Preparation of Antiserum to the Wx Protein One milligram of Wx protein in a buffer containing 0 .15 M NaCl, 20 mM Na phosphate (pH 7 .0) was mixed with an equal volume of complete Freund's adjuvant and injected subcutaneously into 3 kg female New Zealand rabbits . After 5 weeks, the rabbits were given a booster injection containing

Identification and Isolation of the Wx Locus 231

0 .6 mg of Wx protein . Two weeks later, they were given a final booster injection of 0 .5 mg each . Serum was collected after one additional week and used directly as described. Immunological Analysis of Starch Granule-bound Proteins Proteins fractionated on an SDS-polyarylamide gel were transferred to DBM paper (BRL), following which the filter was incubated with Wx immune serum as described by Symington et al . (1981) . The filters were then incubated with purified Wx protein that had been labeled in vitro with ' 251 (Hunter and Greenwood, 1962) to a specific activity of 4 .5 X 10° cpm/µg . The incubation was performed using 2 x 105 cpm/ml, following which the filters were washed and autoradiographed. Isolation of Endosperm mRNA RNA was purified from Wx endosperms as described by Fedoroff et al . (1983a) . Using this protocol, the hydrophilic starch from wx mutants dissolved in the aqueous phase and resulted in a gel from which RNA could not be extracted . The gelation of wx starch was prevented by substituting 0 .35 M Na2SO 4 for 0 .35 M NaCl in the lysis buffer and by equilibrating the phenol with 0.35 M Na2 SO 4. Because Na2SO4 has limited solubility in ethanol, the RNA was dialyzed prior to ethanol precipitation . In Vitro Translation and Immunoprecipitation Poly(A)' RNA was translated in a rabbit reticulocyte in vitro translation system, obtained from BRL, in the presence of 35 S-methionine (600-1200 Ci/mmole, Amersham) . Immunoprecipitation from in vitro translation reactions was performed as described by Fedoroff et al . (1983a), except that no detergents were used in the binding reactions of antigen with antibody or of antibody to protein A-sepharose (Pharmacia) . Enrichment for Wk RNA Sequences Poly(A)' RNA (50-60 µg) from immature Wx endosperms was boiled for 30 sec in 0 .2% SDS, 10 mM Tris-HCI (pH 7 .5), and 2 mM Na EDTA, quickcooled on ice, and layered on an 11 .8 ml linear 10%-30% sucrose gradient in 0 .1% SDS, 10 mM Tris-HCI (pH 7.5), 1 mM Na EDTA, 0.1 M NaCl . Centrifugation was at 28,000 rpm, 15°C, for 16 hr in an SW41 rotor . Fractions were collected and the RNA in each was precipitated twice with ethanol, resuspended in H20 and stored at -70°C . The position of Wx RNA was determined by translating an aliquot of each fraction in vitro . Construction and Screening of cDNA Clones An RNA preparation that had been enriched 5- to 10-fold for Wx sequences was used as a template for the synthesis of double-stranded cDNA according to the procedure of Land et al . (1981) . Following addition of homopolymeric stretches of dC residues to both 3' termini of the doublestranded cDNA, the cDNA was annealed to dG residues that had been added to the 3' termini of the cleaved Pst I site of the plasmid vector, pBR322 (Bolivar et al ., 1977) . Initially, a small fraction of the annealed mixture was used to transform E . coli strain HB101 (Boyer and RoullandDussoix, 1969), yielding 187 TetR, Amp s colonies . The transformants were grown individually in 5 ml cultures, amplified with chloramphenicol, and then pooled into groups of six . Cleared lysates were made from the pools, and the plasmid DNAs were linearized or nicked, denatured and bound to nitrocellulose filters as described by Kafatos et al . (1979) . The filters were screened by a modification of the method for hybrid-selected translation described by Ricciardi et al . (1974), using poly(A)' RNA from immature Wx endosperms . Hybridized RNA was eluted from the filters and translated in vitro as described above . The RNA eluted from one pool programmed the synthesis of a 65 kd polypeptide that was specifically immunoprecipitable by Wx antiserum. DNA from each of the six individuals in this pool was then screened, and one of these chimeric plasmids was found to selectively hybridize Wx mRNA . An additional 6000 transformants were generated in E . coli MCI1061 (M. Casadaban, personal communication) and screened by colony hybridization (Grunstein and Hogness, 1975; Hanahan and Meselson, 1980) using the 32P-labeled cDNA insert from pcWx0 .35 (O'Farrell, 1981) . Two additional Wx cDNA plasmids were isolated . These had inserts of 0.25 and 0 .4 kb and were designated pcWx0 .25 and pcWx0 .4, respectively . Filter Hybridization Analysis of Fractionated RNA RNA was fractionated by electrophoresis through a formaldehyde-containing 1 .2% agarose gel as described by Rave et al . (1979). After electropho-

resis, the gel was soaked for 60 min in several changes of 10x SSC, 1% (w/v) glycine, and the RNA was transferred to nitrocellulose filters (Thomas, 1980) using 10x SSC as the transfer buffer . The prehybridization, hybridization, and wash conditions used were the same as those used for Southern hybridization analysis of maize DNA . The filters were probed with DNAs internally labeled with 32P by nick translation or end-labeled with T4 DNA polymerase. The direction of transcription was deduced from the ability of fragments labeled at the 3' end at the Eco RV site and extending to the right (Figure 6) to hybridize to the 2 .3 kb Wx mRNA . Southern Hybridization Analysis of Maize DNA Maize DNA was prepared either by the procedure described by Murray and Thompson (1980) or as described below for the preparation of genomic DNA for cloning . Hybridization analysis of restriction endonuclease digests of maize genomic DNA (Southern, 1975) was done essentially as described by Fedoroff et al. (1 983a) . The probe used was the cDNA insert from pcWx0.35, labeled with 32P by nick translation (Maniatis et al ., 1975 ; Rigby et al ., 1977) to a specific activity of 3 x 10 8-6 x 1011 cpm/µg, using a -32P dATP and dCTP (specific activity, 400 Ci/mmole, Amersham) . Isolation of Maize DNA for Cloning DNA was isolated from 5-day etiolated seedlings, 3-week-old plants, or the inner leaves and immature tassels of 6-week-old plants . Frozen plant tissue was pulverized in a mortar with crushed glass in the presence of liquid nitrogen . The powdered plant tissue was taken up in 8 mug of a cold lysis buffer containing 8 .0 M urea, 0.35 M NaCl, 0 .05 M Tris-HCI (pH 7 .5), 0.02 M EDTA, 2% sarcosyl and 5% phenol . The mixture was stirred gently with a glass rod until homogeneous and then added to 1 vol of a 3 :1 mixture of phenol and chloroform containing 5% isoamyl alcohol . Sodium dodecyl sulfate was added to 0 .5%, the mixture was warmed to room temperature and gently shaken for 10 min . The phases were separated by low speed centrifugation and the phenol-chloroform extraction was repeated twice . The salt concentration was increased by the addition of '/2o vol of 3 M sodium acetate and the DNA was spooled out after addition of 2 vol of cold ethanol . The DNA was immediately and gently redissolved in approximately '/s of the original lysis volume of 10 mM Tris-HCI (pH 7 .5), 10 mM EDTA . The DNA was banded twice in a CsCI-ethidium bromide gradient . The ethidium bromide was removed by extraction with CsCI-saturated isopropanol and the DNA was dialyzed against 10 M Tris-HCI (pH 7 .5), 1 mM EDTA (TE) . Cloning of Maize DNA Fragments in XEMBL4 The AEMBL4 phage (Frischauf, Lehrach, Poustka, and Murray, unpublished data) was grown in strains 0358 (Karn et al ., 1980) or K803 (LE392, Maniatis et al ., 1982) in NZCYM medium (Maniatis et al,, 1982) . Phages and phage DNA were purified by modification of the procedure described by Yamamoto et al. (1970) . Phage DNA was self-annealed at 42°C in 132 mM Tris-HCI (pH 7 .5), 13 mM MgCI2 , 0 .8 mM rATP and 20 mM DTT for 1 hr. The solution was diluted 2-fold, T4 DNA ligase was added (BRL, 1 .5 U/ 100µg DNA) and the mixture was incubated at 12°C overnight . The ligated vector was cleaved with 4 U/µg of Bam HI (BRL) or Eco RI (BRL) for 3 hr, followed by digestion with Sal I (4 U/µg, BRL) under conditions specified by the enzyme supplier. The digests were phenol- and ether-extracted, dialyzed against TE and centrifuged through a 10%-40% sucrose gradient, as described by Maniatis et al . (1978). Fractions containing the ligated vector arms were detected by agarose gel electrophoresis (Maniatis et al ., 1978), pooled, ethanol precipitated, redissolved in TE at a concentration of 0 .5 mg/ml and stored frozen. Maize DNA was digested with 4-5 U of Eco RI (BRL) or Bg1 11 (BRL) for 1-3 hr under conditions specified by the enzyme supplier. The DNA was phenol- and ether-extracted, ethanol precipitated, redissolved in TE at a concentration of 0 .5 mg/ml and stored frozen. Maize DNA fragments were ligated to purified XEMBL4 arms in the ligation buffer described above . The 5-µl ligation reactions contained a total of 1 µg of DNA and the ratio of arms to insert varied between 4 :1 and 1 :1 . In vitro packaging extracts and in vitro packaging were done by as described by Hohn (1979) . The recombinant phages were plated on E . coli strain K803. As described by Fedoroff (1983), phages containing maize DNA inserts plated less efficiently by a factor of about 15 on the E . coli 0364 or 0359 strains generally used as selective hosts for propagation of recombinant phages prepared in the XEMBL derivatives of the A1059 vector (Karn et al ., 1980), than on E . coli

Cell 232

K803 . Hence the background of nonrecombinant phages was minimized

Grunstein, M ., and Hogness, D. S . (1975) . Colony hybridization : a method

by purification of vector arms, rather than by genetic selection, Recombinant

for the isolation of cloned DNAs that contain a specific gene . Proc . Nat .

phages were screened by plaque hybridization (Benton and Davis, 1977) to the pWxO .4 plasmid labeled with 32P by nick translation (Maniatis et al .,

Acad . So . USA 72, 3961-3965 .

1975 ; Rigby et al ., 1977) . The cloning efficiency of total Eco RI or Bgl II-

dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate,

digested maize DNA was somewhat lower (2-4 x 10 5 pfu/µg maize DNA)

Hager, D . A ., and Burgess, R . R . (1980) . Elution of proteins from sodium

and renaturation of enzymatic activity : results with sigma subunit of Esch-

than of maize DNA enriched for fragments in the correct size range for

erichia coli RNA

cloning (1-3 x 10 6 pfu/µg maize DNA) . Nonetheless, the resulting recom-

enzymes . Anal . Biochem . 109, 76-86 .

binant phage were enriched for large maize fragments and yielded two or more Wx clones/105 recombinant phages .

polymerase, wheat germ DNA topoisomerase, and other

Hanahan, D ., and Meselson, M . (1980) Plasmid screening at high colony density . Gene 10, 63-67 . Hohn, B . (1979) . In vitro packaging of A and cosmid DNA . Meth . Enzymol .

Acknowledgments

68,299-309 . We thank Drs . E . H . Coe, B . McClintock, and G . M . Neuffer for their generosity in providing us with maize strains, Dr . L. Mets and J . Mauvais

Hunter, W. M ., and Greenwood, F . C . (1962) . Preparation of Iodine-131

for helpful discussions, Dr . D . Frambrough for assistance in the iodination

496 .

of the purified Wx protein, S . Downing for technical help and Ms . Pat

Kafatos, F . C ., Jones, C . W ., and Efstratiadis, A. (1979) . Determination of

Schmidt for preparation of the manuscript . M. S . was a fellow of the Damon

nucleic acid sequence homologies and relative concentrations by a dot

Runyon-Walter Winchell Cancer Fund (DRG-397-F [-FT]) . S . W . was sup-

hybridization procedure . Nucl . Acids Res . 7, 1541-1552 .

ported by a postdoctoral fellowship from the American Cancer Society (PF1837) . This work was funded by NSF grant PCM-8307708 and USDA grant

Karn, J ., Brenner, S., Barnett, L ., and Cesareni, G . (1980). Novel bacterio-

labelled human growth hormone of high specific activity . Nature 194, 495-

phage A cloning vector. Proc . Nat . Acad . Sci . USA 77, 5172-5176.

82-CRCR-1-1065 . The costs of publication of this article were defrayed

in part by the

payment of page charges . This article must therefore be hereby marked

"advertisement"

in accordance with 18 U .S .C. Section 1734 solely to

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indicate this fact .

Laskey, R . A ., and Mills, A. D . (1975). Quantitative film detection of 3H and

Received July 8, 1983 ; revised August 19, 1983

Maniatis, T., Jeffrey, A ., Kleid, D . G . (1975) . Nucleotide sequence of the

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