Biochemical Genetics, Vol. 28, Nos. 1/2, 1990

The Effect of Insertion of the Maize Transposable Element Mutator Is Dependent on Genetic Background Daniel F. Ortiz, 1 Robert G. Gregerson, t and Judith S t r o m m e r 1

Received 19 June 1989--Final 3 Oct. 1989

A secondary mutant, derived from an allele o f maize alcohol dehydrogenase 1 ( A d h l ) carrying a Mutator transposable element ( M u l ) in its first intron, was reported to exhibit a threefold decrease in A D H enzymatic activity and steady-state R N A levels compared to the original mutant. The original mutant, A d h l - S 3 0 3 4 (abbreviated $3034), was previously characterized at the molecular level. The derivative, abbreviated $3034b, has now been cloned; at the DNA sequence level the insertion and surrounding A d h l sequences are indistinguishable from $3034. Furthermore, in our lines there is no difference in relative A D H activities between products o f the two putative alleles. A comparison o f gene expression in heterozygotes obtained by crossing to different tester lines reveals a correlation between the measured decrease in levels o f A D H polypeptide produced by the mutant allele and the background in which it is measured," this effect is distinct from any background-related variation in the expression o f the progenitor allele. It does not appear to be attributable to alternative patterns o f DNA modification. It appears to reflect a background-associated difference in the level o f normal Adh 1-RNA produced. Th us the previously reported distinction between $3034 and $ 3 0 3 4 b may be due to differences in the extent to which the mutant allele and a given genetic background interact to produce functional Adh I - R N A . KEY WORDS: mutator; transposable element; alcohol dehydrogenase; maize; gene expression.

This research was supported by United States Public Health Service Grant GM38616 and United States Department of Agriculture Grant 87-CRCR-1-2500 to J.S.D.O. was supported by an N IH predoctoral training grant to the Department of Genetics. Department of Genetics, University of Georgia, Athens, Georgia 30602. 9 0006-2928/90/0200-0009506.00/0

© 1990 Plenum Publishing Corporation

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Ortiz, Gregerson, and Strommer

INTRODUCTION Robertson's Mutator (Mu) is a transposable element family of maize, first identified by its capacity to induce mutation rates 30- to 50-fold above background levels. The phenotype does not segregate in Mendelian fashion; in general 90% of the progeny from a Mu outcross exhibit high mutability, and many of the mutant phenotypes are unstable (Robertson, 1978). Isolation and molecular analysis of the transposable element were originally undertaken using maize alcohol dehydrogenase 1 (Adhl), a locus with the advantage of detailed biochemical and genetic characterization. The A d h l gene is highly expressed in pollen and scutella, as well as in seedlings subjected to anaerobic stress (Scandalios and Felder, 1971; Sachs et al., 1979). Freeling and Cheng (1978) devised a scheme for positive selection of pollen impaired in A d h l function, relying on ADH-catalyzed conversion of allyl alcohol to acrolein, a toxic product. Plants exhibiting the Mutator phenotype were submitted to allyl alcohol selection, and a line was isolated that exhibited a 60% decrease in levels of ADH 1. The availability of a cDNA clone for maize A d h l permitted the detection of a 1.4-kb insertion in the gene (Strommer et al., 1982) and allowed for its isolation (Bennetzen et al., 1984). Mu elements of several lengths have since been found (Taylor and Walbot 1987); the 1.4-kb elements are commonly referred to as M u l . The mutant A d h l allele was designated A d h l - S 3 0 3 4 or $3034. R N A analyses demonstrated a comparable reduction in levels of ADH 1-S monomers assayed in scutella and A d h l message in anaerobically induced roots of mutants (Bennetzen et al., 1984; Rowland and Strommer, 1985). Characterization of gene expression by analysis of runoff transcripts indicated that the 60% drop in the level of hybridizing RNA could be attributed to reduced levels of transcription (Rowland and Strommer, 1985). Soon after isolating the original mutant line, Freeling and co-workers isolated derivatives by resubmitting $3034 pollen to allyl alcohol selection under conditions of increased stringency. Pollen was crossed to a tester line with a rare, electrophoretically distinguishable A d h l allele for quantification of ADH1-S polypeptide levels. The result was identification of two new mutants with further reductions in ADH1 (Freeling et al., 1982). A d h l $3034a (or S3034a) exhibited no detectable enzymatic activity and very low levels o f A d h l - R N A (Strommer et al., 1982). An explanation for the extreme phenotype of this mutant allele was subsequently provided by Taylor and Walbot (1985), who cloned an $3034a allele and demonstrated the presence of rearrangements immediately upstream of the M u element. These changes resulted in deletion of the 5' splice junction of the first intron of A d h l and part of the coding sequence of the first exon.

Background Effects on Mutator Phenotype

11

The second derivative, Adhl-S3034b (or $3034b), exhibited 13% of the ADH activity produced by the non-Mu progenitor allele assayed in the scutellum of an Adhl heterozygote, threefold lower than the original mutant (Freeling et al., 1982). This quantitative change was reflected in decreased levels of hybridizing A d h I - R N A in induced roots of $3034b homozygotes (Strommer et al., 1982), but from Southern blots the derivative was indistinguishable from $3034. This allele seemed to provide a means of learning how dramatically different RNA levels could result from minor intragenic changes. To elucidate the molecular events underlying the decrease in gene expression we undertook detailed analysis of the $3034b allele and its expression.

MATERIALS A N D M E T H O D S

Corn Lines. The Berkeley Slow (BkS) line, homozygous for either Adhl-S, $3034, or $3034b, and the inbred line Funk G4343 (carrying an Adhl-F allele and designated FkF) were obtained from Michael Freeling, University of California at Berkeley. Boone County White (designated BCF) is a moderately inbred line carrying an Adh 1-F allele; it was the gift of Don Miles, University of Missouri. All lines were propagated in the field or greenhouse in Georgia. The cloned gene was from a plant grown from an ear obtained from M. Freeling. Generation and Screening of Genomic Library. Maize genomic DNA from young leaves of an $3034b homozygote was prepared by the method of Murray and Thompson (1980). The DNA was digested with BamHI and fractionated on sucrose gradients. Those fractions containing fragments 10-15 kb in length were pooled and cloned to generate a lambda EMBL 3 genomic library using BamHI arms purchased from Vector Cloning Systems (San Diego, CA) and in vitro packaging extracts prepared according to Maniatis et al. (1982). The library contained 5 x l05 plaque-forming units and was screened using nick-translated Adhl fragments derived from the genomic clone p428 (Dennis et al., 1984). Subcloning and Sequencing. Lambda phage-containing sequences homologous to Adhl were isolated and submitted to restriction analysis. A 13.5-kb BamHI insert was subcloned into pUC19; from this plasmid a 1.9-kb Pstl-BgllI fragment containing the Mu transposable element and 501 bp of flanking Adh 1 sequence was isolated and subcloned into pUC 19 to give rise to the plasmid designated pAL-4b. Various fragments from this plasmid were then subcloned into M 13 mp 18 and mp 19 vectors and sequenced following the chain termination procedure of Sanger et al. (1977). Both strands were

12

Ortiz, Gregerson, and Strommer

sequenced and the result compared to the previously published sequences for $3034 (Barker et al., 1984; Chandler et al., 1988). Measurement o f ADH1-S. ADH enzymatic activity from scutellar samples of S / F heterozygotes was measured by staining starch gels after electrophoresis (Yang and Scandalios, 1975). Quantification of activity was by densitometric scanning of photographic negatives on a Beckman DU-8 spectrophotometer. Relative levels of the S polypeptide were obtained from comparison of the staining intensity of the S/F heterodimer band with respect to the F / F homodimer. For comparison of samples, F activity was defined as 100%. Levels of S polypeptides from mutant alleles were corrected for the level of S product from the progenitor allele in the same genetic environment. Southern Hybridizations. DNA was isolated from the leaves of young plants by the method of Murray and Thompson (1980). Ten microgram samples were digested with EcoRII, BclI, HincII, or HinfI according to the supplier's recommendations (Bethesda Research Laboratories), electrophoresed, and transferred to MSI nylon membranes by the method of Southern (1975). Probe DNA was eluted from agarose gels and radioactively labeled by primer extension (Feinberg and Vogelstein, 1983). Hybridization and highstringency washes were as described by Strommer et al. (1982). Northern Hybridizations. RNA was isolated from roots of 5-day-old seedlings after 12 hr of anaerobic induction. RNA was isolated by the procedure of Longemann et al. (1987). Oligo(dT) selection, electrophoresis, and transfer were as described by Rowland and Strommer (1985). After several hours of prehybridization in 50% formamide, 6 x SSC, 0.1% sodium dodecyl sulfate (SDS) at 43°C, probe was added for overnight hybridization at the same temperature. Blots were washed in 0.2× SSC, 0.1% SDS at 65°C.

RESULTS DNA obtained from a plant homozygous for the mutant $3034b allele was used to generate a genomic library from which the A d h l gene was isolated and subcloned for sequencing. The sequenced region spans 51 bp upstream of M u l in the first intron of the A d h l gene, the M u l element, and 52 bp of downstream sequence (Fig. 1). We compared it with the published M u l sequences obtained from $3034 (Barker et al., 1984; Chandler et al., 1988) and found no discrepancy with the corrected sequence published by Chandler et al. The element in $3034b is thus indistinguishable from the M u I insertion in $3034. Restriction mapping with enzymes recognizing 4-bp sequences indicated that, within the limits of detection, rearrangements had not taken place in the A d h l gene (data not shown).

Background Effects on Mutator Phenotype ATG

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Fig. 1. Map of the Adhl-S3034 allele. Thicker black lines designate regions present in the mature message. The Mul transposable element is indicated by an open line in the first intervening sequences of the Adhl transcriptional unit. Hatched regions within Mul represent the 200 bp inverted terminal repeats of the element. The fragment spanned by Sau 3A sites, indicated by (S), was sequenced in its entirety, in both directions. In an effort to account for the unexpected identity of $3034 and $3034b, we considered the m e t h o d by which $3034b had been recovered. W e m e a s u r e d A D H a l l o z y m e ratios in several S / F heterozygotes, using kernels of different b a c k g r o u n d s c a r r y i n g the progenitor allele A d h l - S , $3034, or $3034b. Protein extracts were p r e p a r e d from scutella, s e p a r a t e d electrophoretically, and stained for A D H activity. P h o t o g r a p h i c negatives were scanned d e n s i t o m e t r i c a l l y to q u a n t i f y S and F m o n o m e r levels. M o r e than two-thirds of the available samples fell into two groups, those whose F allele was c o n t r i b u t e d from a F u n k line, G4343 ( F k F ) , and those resulting from a cross to Boone C o u n t y W h i t e ( B C F ) . F u r t h e r analysis was restricted to these heterozygotes; in all cases, the S allele was c a r r i e d in the BkS background. All these heterozygotes were produced the s a m e s u m m e r and represented a small n u m b e r of F u n k and Boone C o u n t y parents. A l l o z y m e p a t t e r n s from four representative gels are presented in Fig. 2. T h e relative contributions of A D H 1 - S m o n o m e r s in F1 kernels c a r r y i n g the n o n m u t a n t , progenitor A d h l - S allele are themselves different, approxim a t e l y 50% for S / F k F and 66% for S/BCF. This could be due to differences in the level of S, F, or both polypeptides. To t a k e this source of variation into

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Fig. 2. Samples from four starch gels stained for ADH activity. Orientation is with electrophoretic origin at the bottom. BCF, Boone County background; BkS, Berkeley background; FkF, Funk G4343 background. The two gels on the left contain samples from scutella carrying the progenitor Adhl-S allele; the third carries S-3034; and the fourth, S-3034b. Samples in each of the Adhl-S gels are from different kernels; samples in the third and fourth gels each are multiple loadings of samples from the same kernel to demonstrate that observed variations are not the result of random sampling or staining differences.

14

Ortiz, Gregerson, and Strommer

account, subsequent measurements of m u t a n t phenotypes were normalized to the relative expression of the progenitor in the same background. In any case, our conclusions are unaffected by the normalization. Comparing allozyme ratios of $3034 and $3034b heterozygotes (carrying either BC or Fk A d h l - F alleles), we could not demonstrate any significant difference between $3034 and $3034b. Both sets of samples exhibited a large variance, %S = 23.4 _+ 10 (mean relative A D H activity + SD) for $3034 and 19.6 + 8 for $3034b; see Table I. However, analysis of the data array according to background contributed by the fast-migrating allele revealed a significant difference, whether or not values were normalized for expression of the progenitor allele in the same background. Levels of S monomers from the mutant alleles were consistently and significantly lower in B C / B k hybrids than in F k / B k hybrids: %S = 15.8 + 4.3 for B C F and 30.5 _+ 7.6 for FkF. W h e n the data were organized in this manner, moreover, the variance narrowed to that seen for the progenitor allele. Thus the mutant allele produced consistently less functional product in a BC-derived hybrid than in a Fk-derived hybrid. The methylation of cytosine residues in transcription units has been correlated with changes in gene expression (reviewed by Cedar, 1988) and M u elements have been demonstrated to be targets for plantwide, heritable D N A modification (Chandler and Walbot, 1986; Bennetzen, 1987). W e therefore examined the methylation state of the M u l element at A d h l in samples representing high and low expression of the m u t a n t allele. Scutellar tissue is an inadequate source of D N A for Southern blot analysis; we therefore isolated D N A from leaf tissue. It was digested with either H i n f l or EcoRII, methylation-sensitive restriction endonucleases whose recognition sites are

Table I. Scutellar ADHl-S ofS3034 and $3034b Heterozygotes Carrying Either BCF- or FkF-Derived Adh 1-F Alleles, Presented as Mean and Standard Deviationa Mean

SD

n

Adhl-S allele $3034 $3034b

23.4 19.6

_+10.2 _+8.3

11 47

Background of ADH1-F donor BC Fk

15.8" 30.5*

_+4.3 +7.6

39 19

°The number of individuals in each group is indicated as n. The same data set has been analyzed in two forms, grouping the values either according to allelic type ($3034 and $3034b) or according to genetic background contributed by the Adhl-F allele (BCF and FkF). *A significant difference (P < 0.025) is seen between the BCF and the FkF groupings.

Background Effects on Mutator Phenotype

15

modified in inactive M u e l e m e n t s (Chandler and Walbot, 1986; Bennetzen, 1987), and s u b m i t t e d to Southern blot analysis. Three to five sibling kernels for each of these plants were tested for their S / F allozyme ratios. The observed methylation patterns correlated with neither S polypeptide levels nor genetic background (Fig. 3). We next sought evidence for differences in poly(A +) A d h l - R N A levels, an experiment complicated by the need for homozygous A d h l - S plants. We

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Fig. 3. Methytation states of $3034 and $3034b alleles in Fkg and BCF genetic backgrounds. Genomic DNA blots were probed with a HindllI-HincI| fragment isolated from the progenitor AdhI-S allele and representing 700 nt just downstream from the site of Mu insertion. Bands present in Adhl-F homozygotes are indicated with arrows and can be disregarded. Genotype, background, and relative enzymatic activity measured in sibling kernels are indicated under each lane. (A) Genomic DNA digested with HinfI and HincII. (B) Genomic DNA digested with BclI, EcoRII, and HincII. (C) Map illustrating the digestion products found. Uncut, modified sites are marked by the letter

16

Ortiz, Gregerson, and Strommer

relied on available $3034/$3034 seedlings, in the original Bk background and from a low-expressing Bk/BC background. The result of hybridization to an Adhl probe is shown at the left in Fig. 4: there is more hybridization to R N A from the Bk line (lane 3) than to RNA from plants derived from Bk/BC heterozygotes (lanes 1 and 2), but in addition, the bands of hybridization from the mutant samples are wider--and somewhat higher--than the band seen in the lane carrying R N A from the progenitor (lane 4). When the same blot was probed with Mul sequences, significant hybridization was seen to two classes of R N A molecules. Strongest hybridization was to the same region recognized by the Adhl probe; a second band, representing R N A of approximately 1.2 kb, was also apparent. From the width and position of the longer class, we conclude that many discrete products longer than the normal Adhl transcript are generated in these lines. Subsequent hybridization to probes for two othel maize genes, Css and actin, were used to normalize for variation in the amount of R N A loaded per lane; results of hybridization to Css are included in Fig. 4. The Mul-hybridizing R N A is induced concomitant with A d h l - R N A (data not shown). These results were reproducible with R N A samples from other lines; in some cases there appeared to be variable proportions of aberrant R N A species (data not shown). DISCUSSION As part of a long-range effort to understand how intragenic sequences can affect levels of gene expression, we undertook the detailed analysis of a secondary mutant allele whose level of expression had fallen threefold below that of the original mutant. These mutant alleles of maize Adhl-S carry copies of the 1.4-kb Robertson's Mutator element (Mul) in their first introns. ADH 1-S monomer levels and steady-state A d h l - R N A levels of the mutants were originally determined to be 40% for the original mutant $3034 and 13% for the derivative $3034b (Freeling et al., 1982). Quantitative decreases in ADH1-S monomers in scutella of these mutants have been shown to be correlated with decreased transcription of these mutant alleles in anaerobic roots, determined from analysis of runoff transcription (Rowland and Strommer, 1985). A genomic DNA clone for the derivative mutant was isolated and characterized; its detailed restriction pattern was identical to that of the previously characterized $3034 allele (data not shown). When the newly isolated Mu element and adjacent Adhl-S sequences were determined, the insertion in $3034b was found to be indistinguishable from $3034, and surrounding sequences were found to be unchanged. The identity of the two elements, along with earlier results suggesting that some $3034b lines in our laboratory exhibited higher levels of ADH1-S

17

Background Effects on M u t a t o r Phenotype

i~

I

2

3

4

1

2

3

4

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Fig. 4. Northern blot hybridizations of Adhl, Mul, and Css probes to anaerobically induced poly(A +) R N A isolated from seedlings of segregating $3034 homozygotes, two expressing low levels of Adhl (lanes 1 and 2) and one expressing high levels (lane 3). Lane 4 contains DNA from an A d h l - S progenitor line. Hybridization of the blot to a probe for the sucrose synthase 2 gene (Css) provided assurance that roughly equal amounts of R N A (5 #g) were loaded into each well.

than expected (Rowland and Strommer, 1985; Gregerson, unpublished data), led us to examine the enzymatic activities associated with a larger population of $3034 and $3034b mutant alleles. The survey demonstrated that the two mutants could not be differentiated on the basis of mean ADH1-S activity levels, which varied widely in both populations. On the other hand, when the data were compared according to the inbred line contributing the A d h l - F allele, significant differences were seen. Both mutant alleles were expressed at higher levels in a Fk-derived than in a BC-derived hybrid (Fig. 2 and Table

I). The apparent difference between the original and the derivative mutant alleles, in reality the same allele, may be explained by the methodology of mutant recovery and identification. Expression of A d h l - S is traditionally measured as an allozyme ratio, in relation to an electrophoretically distinct A d h l - F allele, which provides an internal standard for quantification. In this case the "derivative" mutant alleles had been passed through a line different from that in which the original mutant had been identified and characterized (Freeling et al., 1982). The activity of the mutant allele was normalized to that of the progenitor in the same background, but the possibility of background variations in response to the presence of M u was not considered. This effect is presented numerically in Table I. The presence of a M u l element in A d h l S in the heterozygous BC background depressed the level of

18

Ortiz, Gregerson, and Strommer

ADH1-S twofold compared to the level in a heterozygous Fk background. A few $3034 and $3034b heterozygotes in other backgrounds were available and also fell into distinct groups irrespective of mutant designation (data not shown). For the original mutant recovery and analysis, a Mutator parent served as pollen donor to an A d h l - F maternal plant. In our experiments, the A d h l - F parents served primarily as pollen donors. Thus it appears that whatever components of the cell respond to the presence of Mul in the mutant allele, they are not determined solely by the maternal parent. The effect, moreover, is not unique to the $3034 mutants. Two additional sets of A d h l - S mutants, designated $4477 and $4478, also carry Mu elements in their first introns, a few hundred base pairs downstream from the site of $3034 insertion (Strommer and Freeling, unpublished). These mutant alleles exhibited the same bimodal distribution of activities, the peak of higher expression associated with the Fk background (data not shown). Methylation of DNA has been shown to be capable of depressing levels of gene expression in both plants and animals (Cedar, 1988), and inactivationlinked methylation of Mu elements is a common occurrence (Chandler and Walbot, 1986; Bennetzen, 1987). Our results might, therefore, reflect a background-dependent difference in methylation patterns associated with observed variations in the level of gene expression. Neither the level of gene expression for the mutant allele nor the genetic background correlated with the level of DNA modification measurable in leaves with methylationsensitive restriction endonucleases. Our tentative conclusion, limited by the inability to analyze scutellar DNA directly, is that variations in the expression of Mu-Adh I alleles do not correlate with differences in M u l modification. We did find that, in the lines assayed, higher levels of functional ADH 1 monomers were associated with higher levels of A d h l - R N A . To some extent levels of aberrant A d h l - R N A species may also vary with background. The overall decrease in A d h l - R N A could be the result of decreased transcription or increased turnover of some A d h l - R N A species; it is not possible from these data to determine the extent to which the background-associated variation is transcriptional or posttranscriptional. Aberrant poly(A +) R N A species may reflect alternative processing of normally initiated transcripts, and they may represent transcription from novel initiation sites. To characterize the aberrant transcripts we have isolated several cloned cDNAs carrying both AdhI and M u l sequences; DNA sequence analysis should permit us to determine how these transcripts have been generated. Neither the Fk nor the BC line used as pollen donor had any obvious distinguishing trait to suggest that transcription or processing was affected in a general way, and yet expression of the mutant Adhl allele varied in a significant way between hybrids constructed from the two. This variability

Background Effects on Mutator Phenotype

19

suggests that a significant degree of v a r i a b i l i t y in basic cellular m a c h i n e r y m a y be tolerated. In s u m m a r y , we conclude t h a t the presence of a M u l element in the first intervening sequence of m a i z e A d h l has different consequences on the expression of t h a t gene, depending on the genetic b a c k g r o u n d of the plant h a r b o r i n g the gene. T h e effect a p p e a r s to be i n d e p e n d e n t of M u - a s s o c i a t e d m e t h y l a t i o n but, rather, due to variations in some factor or factors acting at an early stage in gene expression to d e t e r m i n e the a m o u n t of functional t r a n s c r i p t produced from a m u t a n t template. W e are c u r r e n t l y p r e p a r i n g inbred lines c a r r y i n g the relevant A d h l - S alleles in Bk, BC and F k backgrounds. A n a l y s i s o f A d h l expression in specific b a c k g r o u n d s should facilitate the m e a s u r e m e n t of b a c k g r o u n d effects and the identification of factors responding differentially to the presence of M u l within a s t r u c t u r a l gene.

ACKNOWLEDGMENTS W e gratefully acknowledge M i c h a e l Freeling and Don Miles for contributing maize stocks and t h a n k Kenneth Buckley, M i c h a e l M c L e a n , and helpful reviewers for critical r e a d i n g of the manuscript.

REFERENCES

Barker, R. F., Thompson, D. V., Talbot, D. R., Swanson, J., and Bennetzen, J. L. (1984). Nucleotide sequence of the maize transposable element Mul. Nucl. Acids Res. 12:5955. Bennetzen, J. L. (1987). Covalent DNA modification and the regulation of Mutator element transposition in maize. 31ol. Gen. Genet. 208:45. Bennetzen, J. L., Swanson, J., Taylor, W. C., and Freeling, M. (1984). DNA insertion in the first intron of maize Adhl affects message levels: Cloning of progenitor and mutant alleles. Proc. Natl. Acad. Sci. USA 81:1767. Cedar, H. (1988). DNA methylation and gene activity. Minireview. Cell 53:3. Chandler, V. L., and Walbot, V. (1986). DNA modification of a maize transposable element correlates with loss of activity. Proc. Natl. Acad. Sci. USA 83:1767. Chandler, V. L., Talbert, L. E., and Raymond, F. L. (1988). DNA modification ofa Mul element in non-mutator maize stocks. Genetics 119:951. Dennis, E. S., Gerlach, W. L., Pryor, A. V., Bennetzen, J. L., and Ingels, A. (1984). Molecular analyses of the alcohol dehydrogenase (Adhl) gene of maize. Nucl. Acids Res. 12:3983. Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6. Freeling, M., and Cheng, D. S.-K. (1978). Radiation induced alcohol dehydrogenase mutants in maize following allyl alcohol selection of pollen. Genet. Res. 31:107. Freeling, M., Cheng, D. S.-K., and Alleman, M. (1982). Mutant alleles that are altered in quantitative organ specific behavior. Dev. Genet. 3:179. Longemann, J., Schell, J., and Weillnitzer, L. (1987). Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163:16. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning." A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

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Murray, M. G., and Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res. 8:4321. Robertson, D. S. (1978). Characterization of a mutator system in maize. Mutat. Res. 51:21. Rowland, L. J., and Strommer, J. N. (1985). Insertion of an unstable element in an intervening sequence of maize Adhl affects transcription but not processing. Proe. Natl. Acad. Sci. USA 82:2875. Sachs, M. M., Freeling, M., and Okimoto, M. (1979). The anaerobic proteins of maize. Cell 20:761. Sanger, R., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain terminator inhibitors. Proc. Natl. Acad. Sci. USA 74:5463. Scandalios, J. G., and Felder, M. R. (1971). Developmental expression of alcohol dehydrogenase in maize. Dev. Bio125:641. Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 91:315. Strommer, J. N., Hake, S., Bennetzen, J. L., Taylor, W. C., and Freeling, M. (1982). Regulatory mutants of the maize Adhl gene caused by DNA insertions. Nature 300:542. Taylor, L. P., and Walbot, V. (1985). A deletion adjacent to the maize transposable element Mul accompanies loss of Adhl expression. EMBO 4:869. Taylor, L. P., and Walbot, V. (1987). Isolation and characterization of a 1.7 kb transposable element from a Mutator line of maize. Genetics 117:297. Yang, N. S., and Scandalios, J. G. (1975). De novo synthesis and developmental control of the multiple gene-controlled malate dehydrogenase isozymes of maize scutella. Biochem. Biophys. Acta 384:293.

The effect of insertion of the maize transposable element mutator is dependent on genetic background.

A secondary mutant, derived from an allele of maize alcohol dehydrogenase 1 (Adh1) carrying a Mutator transposable element (Mu1) in its first intron, ...
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