Duplication-Dependent CG Suppression of the Seed Storage Protein Genes of Maize

Corresponding author: Gertrud Lund, Department of Plant Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark., gel{at}kvl.dk (E-mail)

Communicating editor: J. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study investigates the prevalence of CG and CNG suppression in single- vs. multicopy DNA regions of the maize genome. The analysis includes the single- and multicopy seed storage proteins (zeins), the miniature inverted-repeat transposable elements (MITEs), and long terminal repeat (LTR) retrotransposons. Zein genes are clustered on specific chromosomal regions, whereas MITEs and LTRs are dispersed in the genome. The multicopy zein genes are CG suppressed and exhibit large variations in CG suppression. The variation observed correlates with the extent of duplication each zein gene has undergone, indicating that gene duplication results in an increased turnover of cytosine residues. Alignment of individual zein genes confirms this observation and demonstrates that CG depletion results primarily from polarized C:T and G:A transition mutations from a less to a more extensively duplicated gene. In addition, transition mutations occur primarily in a CG or CNG context suggesting that CG suppression may result from deamination of methylated cytosine residues. Duplication-dependent CG depletion is likely to occur at other loci as duplicated MITEs and LTR elements, or elements inserted into duplicated gene regions, also exhibit CG depletion.

IN many organisms, nuclear DNA is methylated at cytosine residues, resulting in 5-methylcytosine (5mC). In plants, symmetrical 5'-CpG-3' (CG) and 5'-CpNpG-3' (CNG) are the most frequent targets of cytosine methylation, whereas in mammals 90% of methylation is restricted to the CG dinucleotide (GRUENBAUM et al. 1981 , GRUENBAUM et al. 1982 ). However, the degree and ratio of CG and CNG methylation can vary considerably between plant species (JEDDELOH and RICHARDS 1996 ; KOVARIK et al. 1997 ). For example, in maize, 28% of cytosine residues are methylated compared to only 6% in Arabidopsis (LEUTWILER et al. 1984 ; MATASSI et al. 1992 ; MONTERO et al. 1992 ). Furthermore, analysis of the rRNA genes from maize has shown that the external cytosine is twofold less methylated compared to the internal cytosine residue of the 5'-CCG-3' sequence (KOVARIK et al. 1997 ), indicating that CG methylation occurs more frequently than CNG methylation.

CG suppression, or depletion, refers to the underrepresentation of the CG dinucleotide compared to an estimated value based on the G + C content of the sequence investigated. CG suppression is especially evident in the mammalian genome where the frequency of the CG dinucleotide can be up to fivefold lower than the expected value (BIRD 1980 ). In contrast, both monocot and dicot plant genes are only slightly CG depleted (an average of 75–80% of expected values), and CNG suppression is lacking or less severe than CG depletion (MCCLELLAND 1983 ; GARDINER-GARDEN et al. 1992 ; ASHIKAWA 2001 ).

The most commonly explained mechanism of CG depletion relates to the tendency of 5mC to undergo spontaneous deamination to thymidine, resulting in C:T or G:A transition mutations (COULOUNDRE et al. 1978 ). Interestingly, the mutability of CG has been shown to be one of the most important causes of germline point mutations in human genetic diseases and is a frequent occurrence in somatic mutations leading to cancer (COOPER and KRAWCZAK 1989 ; JONES et al. 1992 ; HOLSTEIN et al. 1994 ). In addition to the mutability of 5mC, recent evidence has shown that cytosine deamination also contributes to CG suppression (FRYXELL and ZUCKERKANDL 2000 ).

Although the majority of methylation in plants is associated with repetitive DNA sequences such as transposons, duplicated gene regions can also be methylated (BIANCHI and VIOTTI 1988 ; FLAVELL et al. 1988 ; BENNETZEN et al. 1994 ; FLAVELL 1994 ; BENDER and FINK 1995 ; RONCHI et al. 1995 ; RABINOWICZ et al. 1999 ). In Neurospora crassa, duplicated sequences are efficiently targeted by methylation, and a large number of C:T transition mutations are introduced following duplication [hence the name repeat-induced point mutation (RIP; CAMBERERI et al. 1989 ; SELKER 1990 )]. Similarly, in Ascobolus immersus a process referred to as methylation induced premeotically (MIP) results in de novo methylation of a DNA sequence upon duplication (GOYON and FAUGERON 1989 ). The observed consequences of de novo methylation in RIP and MIP include gene inactivation and a reduction in the frequency of recombination (BARRY et al. 1993 ; ROUNTREE and SELKER 1997 ; MALOISEL and ROSSIGNOL 1998 ). Similar roles of duplication-induced DNA methylation have been proposed to occur in plants (FLAVELL 1994 ; BENDER 1998 ).

In mammals, duplicated genes are more CG suppressed compared to single-copy genes. This observation has led to the suggestion that duplicated genes have a history of methylation and subsequent mutation of methylated residues (KRICKER et al. 1992 ). Likewise, in plants, the multigene families of 5S rRNA genes from Arabidopsis and rRNA genes from maize show elevated levels of transition mutations that are consistent with deamination of 5mC, in particular the nonfunctional members of these gene families (EDWARD et al. 1996 ; MATIEU et al. 2002 ). However, neither study can confirm if CG loss results from spontaneous deamination of methylated residues over time or whether depletion is the consequence of an active mechanism linked to duplication. We have analyzed the CG dinucleotide and CNG trinucleotide content of the large zein gene family, which encodes the seed storage proteins of maize. Due to differences in gene copy number of each subfamily, the zein genes provide an ideal model system to analyze the effect of gene duplication on CG suppression. In addition, the highly abundant LTR-retrotransposons and MITEs have also been analyzed for evidence of CG suppression.

The zeins constitute 50–60% of total endosperm protein and can be divided into two major fractions, zein-1 and zein-2, on the basis of solubility characteristics (ESEN 1986 ). The zein-1 fraction consists of the 19- and 22-kD polypeptides that are encoded by a large gene family, the -zeins. On the basis of DNA sequence identity and hybridization characteristics, this family can be further divided into four subfamilies, z1A, z1B, z1C, and z1D (HEINDECKER and MESSING 1986 ; RUBENSTEIN and GERAGHTY 1986 ). Genes belonging to the z1A, z1B, and z1D subfamilies encode the 19-kD zein genes, whereas the 22-kD zein genes are encoded mainly by the z1C subfamily. The 19-kD genes have an estimated copy number of 56 per haploid genome, whereas the 22-kD zeins are presumed to be present in 15 copies per haploid genome (HAGEN and RUBENSTEIN 1981 ; WILSON and LARKINS 1984 ). However, the exact copy number of the -zein genes can show considerable variation among different inbred lines (LLACA and MESSING 1998 ; SONG and MESSING 2002 ). In addition, within the 19-kD zein gene family, the z1A subfamily has the highest copy number, followed by z1B and z1C (WILSON and LARKINS 1984 ; HEINDECKER and MESSING 1986 ; SONG and MESSING 2002 ). In contrast, the 10-, 15-, 16-, and 27-kD proteins that represent the zein-2 fraction are encoded by one or two genes that show limited sequence similarity to the -zeins (PRAT et al. 1985 ; KIRIHARA et al. 1988 ; SWARUP et al. 1995 ).

The majority of 22-kD zein genes form a dense gene cluster, 168 kb in size, on chromosome 4 of maize (LLACA and MESSING 1998 ), whereas the 19-kD zein genes are distributed on five unlinked genomic locations on maize chromosomes 1, 4, and 7 (SOAVE et al. 1981 , SOAVE et al. 1982 ; WILSON et al. 1989 ; WOO et al. 2001 ; SONG and MESSING 2002 ). Phylogenetic analysis of the -zein genes has revealed that the 19- and 22-kD zein genes share a common ancestor (SONG and MESSING 2002 ). Given that only the 22-kD zein genes have been identified in Coix lacryma-jobi, an ancestor of maize (LEITE et al. 1990 ), it is probable that in maize the 19-kD zein genes have derived from the 22-kD zein genes. Interestingly, it has been estimated that the amplification of the -zein gene family in maize occurred within the last 3–4 million years (SONG et al. 2001 ; SONG and MESSING 2002 ).

In contrast to the clustered zein genes, MITEs and LTR elements are dispersed in the genome. MITEs are frequently associated with promoter and 3' regulatory regions of genes, whereas the larger LTR-transposons are typically found in intergenic regions (KUMAR and BENNETZEN 1999 ). The copy numbers of MITEs and LTR elements range from 3000 to 10,000 copies and a few to 50,000 copies, respectively (BUREAU and WESSLER 1992 , BUREAU and WESSLER 1994 ; SANMIGUEL et al. 1996 ; ZHANG et al. 2000 ). Similar to the amplification of the zein gene family, a majority of LTR-retrotransposons have colonized the maize genome during the last 5 million years (SANMIGUEL et al. 1998 ).

Our analysis shows that duplicated zein genes are CG suppressed and that the degree of suppression correlates with the copy of each subfamily. Likewise, within the 19- and 22-kD zein gene families the extent of CG depletion correlates with the number of duplications each individual gene has undergone. Despite their high copy number, most MITEs and LTR elements are not CG suppressed, except when duplicated or located in a duplicated DNA sequence. This suggests that the process leading to CG depletion is activated upon duplication or is a consequence of the duplication process itself. We discuss the possible role of duplication-dependent CG depletion in the evolution of the GC-poor isochores in which the zein genes are located.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sequences:
The di-and trinucleotide composition of the coding region of 32 zein genes belonging to the zein-1 fraction and 6 genes belonging to zein-2 fraction was analyzed. All 22-kD zein genes were derived from the inbred line BSSS53 (af090447), whereas the 19-kD gene sequences were derived from the B73 inbred line (af546187, af546188; af546189, af546190; SONG et al. 2001 ; SONG and MESSING 2002 ). Only full-length genomic and cDNA clones, including genes with in-frame stop codons, were analyzed. Clones in which the open reading frame was disrupted by insertions or deletions were omitted from the analysis. One exception was Z492M16-5, which was included as it is expressed despite a deletion in the open reading frame. The correct open reading frame was determined by the use of GenBank's annotations, and nucleotide compositions were generated using the Genetics Computer Group (GCG) analysis software package. Sequences of MITEs and LTR-retrotransposons were extracted from gene sequences according to the annotations of the authors (BUREAU and WESSLER 1992 , BUREAU and WESSLER 1994 ; SANMIGUEL et al. 1996 ; TIKHONOV et al. 1999 ; ZHANG et al. 2000 ). Only the LTR region and primer-binding site of the LTR-retrotransposon was analyzed.

CG analysis:
To measure the extent of repression of a given di- or trinucleotide, a score was calculated by the formula = O/E, where O and E denote the observed and expected counts, respectively. Overall expected counts for di- and trinucleotides were calculated by multiplying the observed counts of each nucleotide and dividing the product by the total number of nucleotides found in the sequence. Position-dependent expected counts were calculated assuming the absence of any codon bias. The positions of the 5' and 3' di- or trinucleotides relative to codon triplets are indicated by roman numerals; e.g., I-II denotes a dinucleotide including the first two nucleotides of a codon triplet. For the CG dinucleotide, position-dependent expected counts were calculated as follows: I-II = 2/3 of arginine-specifying codons; II-III = the sum of 1/6 of serine-, 1/4 of proline-, threonine-, and alanine-specifying codons; III-I = NNC x GNN/T; N represents any nucleotide and T represents the total number of triplets in the sequence. In the case of CNG trinucleotide, position-dependent expected counts were calculated as follows: I-III = the sum of 1/6 of arginine- and leucine-, 1/4 of proline-, and 1/2 of glutamine-specifying codons; II-I = NCN x GNN/T; and III-II = NNC x NGN/T.

Alignment between members of the -zein gene family:
Pairwise alignments were conducted of individual expressed members of the 19- and 22-kD zein genes by GAP analysis (GCG analysis software package). For each alignment, the percentage of C:T and G:A transition mutations was compared to the total number of single-base-pair point mutations. To establish if transition mutations occur in a polarized fashion, i.e., from a younger to an older duplication, or from a gene that has undergone less to more duplications, transition mutations of each gene were counted in individual alignments. Importantly, only transition mutations occurring in a CG or CNG context were considered.

Statistical analysis:
Differences between O and E values were tested using the chi-square analysis. To test for differences in -values between groups, the Mann-Whitney U-test was employed. All statistical tests were performed using the STATISTICA software package for Macintosh (StatSoft, Tulsa, OK).

Bisulfite analysis:
Genomic DNA was extracted from young leaf tissue of the inbred lines W64A and W22 using the DNAeasy kit (QIAGEN, Valencia, CA). The W64 and W22 inbred lines contain the Tourist element located in the 5' flanking region of the single-copy or duplicated 27-kD zein gene, respectively (DAS and MESSING 1987 ). Between 1 and 2 µg of DNA were treated with bisulfite as described by ZESCHNIGK et al. 1997 . For PCR analysis 1/10 vol of bisulfite-treated DNA was employed in a standard PCR reaction. The primer pairs employed for amplification of the Tourist element from bisulfite-treated DNA were as follows: W64A, 5'-TAGGTATATGATTAGTGGTAATTTAATATT-3' and 5'-ATTCTTAAAACTTTACATACCAATACATAA-3'; W22, 5'-GGGTATATAATTAGTGTAATTTAATATATG-3' and 5'-ATTCTTAAAACTTTACATACCAATACATAA-3'. The resulting PCR products were cloned by TOPO cloning and 16 individual clones were sequenced at MWG Biotech (Ebersberg, Germany). To confirm the previously published MITE sequences, the Tourist element was also amplified from genomic DNA employing the following primer pairs: W64A, 5'-CCTTGGTTGTTGGCTCATAAT-3' and 5'-CAGATGAGTATGATCTCGGCA-3'; W22, 5'-ATAAGTGTTCTGGATATTGGTTGTT-3' and 5'-TCAGATGAGTATGATCTCGCA-3'. These primers were also tested on bisulfite-treated DNA and failed to give a product of the expected size. To ensure that the observed patterns of methylation did not result from incomplete strand separation during the bisulfite reaction, the Tourist element from the W64A inbred was cloned, bisulfite treated, and amplified with the bisulfite primers. This element contains only one cytosine that can be methylated by the Escherichia coli dcm methylase (i.e., the internal cytosine residue of the CCWGG sequence). As expected, sequence analysis of 10 independent clones showed that this cytosine remained unmodified. All the remaining cytosine residues of the Tourist element had undergone modification to thymidine.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

CG and CNG analysis of the zein genes:
Table 1 shows -values (observed/expected) of CG dinucleotides and CNG trinucleotides of genes belonging to the zein-1 and zein-2 fractions. All 19-kD zein genes analyzed were isolated from the B73 inbred line, whereas the 22-kD zein genes were derived from the BSSS53 inbred line (SONG et al. 2001 ; SONG and MESSING 2002 ). A -average was calculated of both zein fractions, of the 19- and 22-kD zein genes, and of each of the three 19-kD zein subfamilies, z1A, z1B, and z1D. The zein-1 fraction, representing the multicopy -zeins has a CG average of 0.40 (P < 0.001), whereas the -average of single-copy zein genes of zein-2 fraction is 0.75 (P < 0.001). Indeed, the zein-1 fraction is more suppressed than the zein-2 fraction (P < 0.001). Furthermore, the average GC content of the zein-1 fraction is 48% compared to 66% of the zein-2 fraction (results not shown), indicating that CG suppression is accompanied by a decrease in G + C (GC) content. In contrast, none of the zein fractions are suppressed at the CNG trinucleotide.

It can also be observed that the degree of suppression varies between subfamilies of the zein-1 fraction. The more abundant 19-kD zein genes are the more CG suppressed compared to the less abundant 22-kD zein genes (0.34 and 0.49, respectively; P < 0.001) and, likewise, within the 19-kD gene family the degree of suppression is associated with the copy number of each subfamily. The z1A subfamily, which has the highest copy number, is more suppressed compared to the less abundant z1B and z1C subfamilies (P < 0.009 and P < 0.027, respectively). We also analyzed 18 19-kD genes from different genetic backgrounds and found no differences in the average CG and CNG scores (results not shown).

The amino acid content of zein-1 and zein-2 fractions differs considerably, which could potentially influence overall CG and CNG scores. For example, the -zeins are particularly rich in glutamine, leucine, proline, and alanine, whereas the single-copy -zeins have a high content of methionine. To address this problem, CG and CNG frequencies were analyzed in a position-dependent context (Table 2). Position-dependent frequencies correct for differences in amino acid content but not for amino acid codon bias. Essentially, position-dependent frequencies of the zein genes largely reflect overall CG and CNG frequencies. The multicopy -zeins are suppressed at positions II-III and III-I (Table 2; = 0.41 and 0.55, respectively; P < 0.001); in contrast, the single-copy zein genes are not CG suppressed at any position. This implies that the low overall CG score observed for the 10-kD zein gene, m23537 (Table 1), is related to the amino acid content of this gene. Within the zein-1 fraction, the 19-kD zein genes are CG suppressed at positions II-III and III-I (P < 0.001). The z1A subfamily is more suppressed than the z1B subfamily at position III-I, whereas the opposite is true of position II-III (P < 0.002 and P < 0.025, respectively). The 22-kD zein genes are also CG suppressed at position II-III ( = 0.52; P < 0.001), but to a lesser degree than the 19-kD zein genes ( = 0.33; P < 0.001). The CNG trinucleotide is suppressed only at position I-III of the zein-1 fraction ( = 0.60; P < 0.009; results not shown) and, again, the 19-kD zein genes are more suppressed compared to the 22-kD zein genes at this position (P < 0.001).

A recent analysis of the B73 and BSSS53 inbred lines has shown that only a relatively small number of -zein genes are expressed (SONG et al. 2001 ; WOO et al. 2001 ). Most of the nonexpressed genes contain in-frame stop codons or insertion/deletions (SPENA et al. 1983 ; LIU and RUBENSTEIN 1992 ; LLACA and MESSING 1998 ; SONG et al. 2001 ). Analysis of the average overall CG content of seven expressed vs. seven nonexpressed 22-kD genes (marked with a superscript b in Table 1) showed no significant differences (0.51 vs. 0.47, respectively; P = 0.225). However, position-dependent frequencies indicated that the inactive genes are more CG suppressed at position II-III (P = 0.025). Due to small sample size, a similar calculation of the 19-kD genes was not undertaken. The relative contributions of the expressed -zein genes have been assessed in the B73 inbred line (WOO et al. 2001 ). The 19-kD zein genes are the most highly expressed, whereas the 22-kD zein genes are expressed at a threefold lower level. An estimated five genes encode most 19-kD zein gene transcripts, and four genes account for most 22-kD zein gene transcripts. Interestingly, the average CG values of these 19- and 22-kD zein genes correlate inversely with expression (P = 0.49; = 0.71). This is also true within the 22-kD zein gene family, and a similar tendency was observed for the 19-kD zein gene family (results not shown). This indicates that highly expressed genes are more CG suppressed compared to low expressing genes.

To understand if CG suppression is a general effect of a specific chromosomal region, the CG content of intergenic regions of the 22-kD zein gene cluster located on chromosome 4S was analyzed. The lengths of the 22-kD zein intergenic regions varied from 2517 to 14,438 bp. The -average CG of the intergenic region was 0.68, which is significantly higher than the -average of 0.50 of the 22-kD genes (P < 0.001).

CG analysis of MITEs and LTR-retrotransposons:
The observed copy number variation in CG suppression prompted us to investigate whether the high-copy-number LTR-retrotranposons and MITEs exhibited similar behaviors. Three MITE families (Tourist, Stowaway, and Heartbreaker) and three groups of LTR-retrotransposon (Ty1-copia, Ty3-gypsy, and an unclassified group), differing in element copy number between and within each group, were analyzed for evidence of CG suppression. The -value and G + C content was calculated for MITEs and LTR elements in different sequence contexts (Table 3 and Table 4, respectively). Despite the high copy number of MITEs and LTR elements, no association was found between the degree of suppression and element copy number. Most transposons were not, or were only slightly, CG suppressed. For example, the -average of Tourist and Heartbreaker families was 0.66 and 0.60 (P < 0.041 and P < 0.018), respectively, whereas no suppression was observed of Stowaway family or LTR elements. However, large differences in CG suppression were observed of both MITEs and LTR elements ( ranging from 0.16 to 1.46 and 0.26 to 1.12, respectively). We found that the copy number of the insertion site could largely explain the variation in ; i.e., elements inserted into multicopy gene regions were more CG suppressed than elements inserted into single-copy genes. This is nicely illustrated by the -values of Stowaway found 3' of the single-copy 10- and 27-kD zein genes and the multicopy 22-kD zein genes (0.66, 0.96, and 0.25, respectively; Table 3B; BUREAU and WESSLER 1994 ). Two -values of Tourist and Stowaway elements located 5' and 3', respectively, of the 27-kD zein genes are indicated (x53514 and x56118; Table 3A and Table 3B). x56118 represents an allele of a tandem duplication of a 27-kD zein gene (DAS et al. 1991 ), whereas x53514 (zc2) represents a single copy of a 27-kD zein gene (REINA et al. 1990 ). For both Tourist and Stowaway elements, CG suppression is more severe upon insertion in the duplicated allele. In addition, suppression of the Stowaway element is accompanied by a 2% decrease in G + C content. Severe CG suppression was also observed of tandemly duplicated MITEs. An example of this can be seen by comparing the -value of a single-copy or duplicated Tourist element located in the 3' region of the Adh1 locus (x17556 and x04049, respectively; Table 3A; BUREAU and WESSLER 1992 ). No suppression is observed of the single-copy Tourist element ( = 1.46), whereas the tandemly duplicated element is severely CG suppressed ( = 0.16). Again, CG suppression of the duplicated element is accompanied by a 4% decrease in G + C content compared to the single-copy insertion. LTR regions also exhibit severe CG suppression upon duplication or if located in a duplicated DNA region (Table 4). For example, CG suppression is observed of x58700, a Hopscotch-like transposon inserted in the promoter region of a multicopy 19-kD zein gene (WHITE et al. 1994 ), and of u68406, a tandem duplication of an element, Kake-1 (SANMIGUEL et al. 1996 ).

An average -value was calculated of elements inserted into single- vs. multicopy gene regions or tandemly duplicated elements of selected data points (Table 3 and Table 4; labeled with superscript a and b, respectively). The criterion for selection of data points was knowledge of copy number of both element and insertion sequence. In addition, only clustered multicopy genes were represented in the multicopy group. Therefore, the Gpc4 gene, which belongs to a small dispersed multigene family, was analyzed as a single-copy insertion (RUSSELL and SACHS 1991 ), and some elements inserted in multicopy sequences were not included as it is unknown whether these genes are clustered or dispersed (e.g., Gpa pseudogenes and oleosins). CG suppression was not observed of LTR elements and MITEs located in single-copy genic regions ( = 0.94), whereas tandemly duplicated elements or gene sequences were suppressed ( = 0.33; P < 0.020). Furthermore, the -average of MITEs and LTR elements inserted in single-copy gene regions was higher than the average value of tandemly duplicated elements and elements inserted into duplicated DNA regions (P < 0.004). Simply analyzing -values of MITEs and LTR elements located in single- vs. multi-copy gene regions produced results identical to the selected data set, the latter being less significant (P < 0.009).

CG depletion as a function of time or gene duplication:
If CG suppression results from passive deamination of methylated residues, the extent of depletion should reflect the number of years a sequence has been methylated. Table 5A shows a comparison between , the estimated time of insertion, and the number of duplications of the seven expressed 22-kD zein genes clustered on chromosome 4S. Similarly, the estimated times of retrotransposon insertion at the Adh1-F locus has been compared to CG frequencies (Table 5B). Neither CG nor CNG suppression correlated with time of insertion of the 22-kD zein genes or of the LTR-retrotransposons. However, the 22-kD zein genes showed an inverse correlation between CG content and the number of duplications each gene has undergone (r = 0.85; P < 0.014). This was found to be specific of the CG dinucleotide. Similarly, analysis of the expressed 19-kD zein genes isolated from the B73 cluster also showed that the degree of CG suppression correlated with the extent of duplication (r = 0.52; P < 0.05; n = 14; results not shown).

Sequence divergence of duplicated DNA sequences:
To identify the fate of CG dinucleotides, pairwise alignments of the seven expressed zein genes was conducted (Table 5). For each of the 21 alignments, the total number of C:T and G:A transition mutations and the total number of transition mutations occurring in a CG or CNG context were counted and compared to the total number of point mutations (results not shown). In addition, transition mutations occurring in a CG or CNG context were counted for each gene in the pairwise alignments. Transition mutations were the most common point mutations observed, representing on average 61% of total point mutations, and the vast majority occurred in a CG or CNG context (average 74%). In 7/21 alignments performed, it was possible to distinguish if CG depletion resulted from the time or extent of duplication, whereas the remaining alignments were noninformative. The informative alignments included zp22/D87 and azs22;8, azs22;10 and azs22;4, azs22;10 and azs22;14, azs22;12 and zp22/6, zp22/6 and azs22;14, azs22;12 and azs22;10, and zp22/6 and azs22;14. Four of these alignments showed that transition mutations occurring in CG context were consistent with CG suppression resulting from duplication, whereas none were consistent with the time of amplification. For example, 11 transition mutations (occurring in a CG context) were observed from azs22;12 to zp22/6, whereas only 7 were observed in the opposite direction. This is in agreement with duplication-dependent CG suppression as zp22/6 has undergone a larger number of duplications compared to azs22;12 (six vs. four, respectively). If CG suppression were the result of time-dependent deamination of cytosine residues, an equal number of polarized transition mutations would have been expected given the fact that these genes have both amplified 0.6 MYA. In contrast, no association between transition mutations occurring at the CNG trinucleotide and duplication was found for the informative alignments. Interestingly, 5 of the informative alignments did show a time-dependent decrease in CNG content. For the noninformative alignments, the majority of transition mutations observed at CG dinucleotides (12/14) also occurred in a polarized fashion from a less to a more duplicated gene (or from a younger to an older duplication). However, only 6/14 alignments showed a similar behavior at the CNG trinucleotide. These results support the prior observation that only CG suppression correlates with the extent of duplication.

Alignments were also performed between genes belonging to the 19-kD zein subfamilies, z1A, z1B, and z1D, which have undergone an average of eight, six, and four duplications, respectively. Two expressed genes representative of the z1A subfamily (Z448F14-3 and Z448F14-4) were aligned to all the expressed genes of z1B and z1C. In 16/18 alignments, a higher number of transition mutations at CG dinucleotides were observed from the less duplicated z1B and z1C genes to the more extensively duplicated z1A genes. The same could be concluded by alignments of two z1B genes (Z492M16-1 and Z492M16-4) to the two expressed z1D genes. Of the 18 alignments performed, transition mutations represented on average 57% of total single base pair mutations and, of these, 48% occurred in a CG or CNG context.

Similar conclusions were found by aligning the duplicated 27-kD zein gene, x56118, to the single-copy 27-kD zein gene, zc2 (x53514). These genes exhibit 97% sequence similarity. Only nine point mutations were observed, six being C:T or G:A transition mutations. Again, transition mutations occurred in a polarized fashion from the single copy to the duplicated gene (four vs. two, respectively). However, only transition mutations observed from the zc2 to the x56118 allele were in a CG context (3/4). This pattern of mutation was also mirrored by alignment of the Tourist and Stowaway elements located in the 5' and 3' flanking region of the single-copy and duplicated 27-kD zein gene, respectively. Finally, alignment of the single-copy and duplicated Tourist element 3'of the Adh1 locus confirmed that C:T and G:A transition mutations are polarized (i.e., occur from the single to the duplicated MITE sequence).

The degree of sequence identity between individual MITEs varies between 46 and 88% (BUREAU and WESSLER 1992 ). Interestingly, the average degree of similarity between duplicated MITEs and MITEs inserted in duplicated gene regions is higher than the average degree of similarity of MITEs inserted in single-copy regions (66 vs. 63%, respectively; P = 0.0431). This supports our observations that linked duplicated sequences evolve more similarly compared to single or dispersed, multicopy sequences.

Methylation status of single- vs. multicopy genic regions:
C:T and G:A transition mutations are the presumed products resulting from deamination of methylated cytosines. Therefore, a possible explanation of the enhanced turnover of CG dinucleotides might be that duplicated sequences exhibit qualitative or quantitative differences in methylation compared to single-copy genes. To this end, we analyzed the methylation status of the Tourist element located in the single-copy or tandem duplication of the 27-kD zein gene (zc2 and x56118) by bisulfite sequencing. These elements were chosen because they exhibit small differences in CG and G + C content (see Table 3). The results showed that in both sequence contexts the MITE was hypermethylated at all CG dinucleotides, in addition to a proportion of methylated cytosines in a nonsymmetrical sequence context (see Fig 1). All 29 cytosine residues of the MITE located 5' of the zc2 coding region were methylated compared to 25/30 cytosine residues of the element inserted in the tandem duplication. In addition, quantitative analysis of 16 independent clones indicated that the MITE located in the duplicated 27-kD zein gene showed 19 and 37% reduction in symmetrical and asymmetrical methylation, respectively, compared to the element located in the single-copy gene.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Methylation state of a Tourist element in the single-copy or duplicated 27-kD zein gene. Bisulfite sequencing of a Tourist element in the 5' region of the single or duplicated 27-kD zein gene. M, methylation of symmetrical CG and CNG sequences; M, methylation of nonsymmetrical sequences. The methylation state of 16 independent clones is indicated, and each letter represents two observations.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our results show that during a short evolutionary time span, individual -zein genes have accumulated large variations in CG content. In particular, within the 19- and 22-kD zein gene families, extensively duplicated genes are more CG depleted compared to less duplicated genes. In addition, the 19-kD zein genes are more suppressed than the 22-kD zein genes, indicating that the former have undergone a greater number of gene duplications. This is supported by the fact that the 19-kD genes are more abundant compared to the 22-kD genes (HAGEN and RUBENSTEIN 1981 ; WILSON and LARKINS 1984 ; HEINDECKER and MESSING 1986 ).

Duplication-dependent depletion of the -zein genes results largely from C:T and G:A transition mutations in a CG or CNG context. Similarly, elevated levels of transition mutations have also been identified in other multigene families in plants such as the GAPA and rDNA gene families of maize and the 5S RNA genes from Arabidopsis (QUIGLEY et al. 1989 ; EDWARD et al. 1996 ; MATIEU et al. 2002 ). For these gene families, a higher level of C:T (or G:A) transition mutations was observed of the nontranscribed genes, and it was argued that CG depletion resulted from relaxation of selective constraints at the transcriptional level (QUIGLEY et al. 1989 ; MATIEU et al. 2002 ). This explanation is, however, inadequate for the variation in CG suppression observed of the -zein gene family, as the most highly expressed genes are the most CG depleted. We speculate that the high expression levels of the most CG-suppressed -zeins are caused by the lack of CG dinucleotides available for methylation, thus relieving methylation-mediated transcriptional repression. Indeed, the fact that -zein genes are methylated in both the coding and noncoding regions and exhibit an inverse relation between CG methylation and expression lends support to this idea (BIANCHI and VIOTTI 1988 ; LUND et al. 1995 ; STURARO and VIOTTI 2001 ). In addition, differences in the extent of methylation could explain the large variations in expression levels of individual zein genes (WOO et al. 2001 ; SONG and MESSING 2002 ).

The in vivo rate of deamination of methylated cytosine residues in plants is not known, whereas in mammals the estimated half-life of a cytosine residue is between 24 and 60 million years (YANG et al. 1996 ). However, the average plant gene is less depleted in CG compared to the average mammalian gene (average CG score is 0.68 and 0.22, respectively) perhaps indicative of a decreased mutability of CG dinucleotides in plants (GARDINER-GARDEN and FROMMER 1987 ; GARDINER-GARDEN et al. 1992 ). On an evolutionary time scale, many transposons represent recent insertions in the maize genome. For example, a large number of LTR-retrotransposons that map to the Adh1-F locus have inserted during the last 5 million years (SANMIGUEL et al. 1998 ). Although these LTR regions exhibit a twofold increase in transition mutations compared to nonmethylated intronic regions (SANMIGUEL et al. 1998 ), we found no correlation between CG suppression and insertion time of these sequences. Given that spontaneous deamination of 5mC is a very slow process, this suggests that the observed low CG values of duplicated elements or of elements inserted into duplicated gene regions also result from enhanced turnover of CG as a result of gene duplication.

If duplicated sequences are methylated compared to their single-copy counterparts, an increase in methylation-related deaminations might be expected, subsequently resulting in a reduction in CG content. However, bisulfite analysis of a MITE inserted in the single-copy or duplicated 27-kD zein gene locus showed that the element was methylated in both sequence contexts. In addition, as most transposons are hypermethlyated (RABINOWICZ et al. 1999 ; TOMPA et al. 2002 ), it is probable that the majority of LTR regions analyzed in this study are methylated. Indeed, the observation that LTR regions of retrotransposons that map to the Adh-F locus exhibit a twofold increase in transition mutations compared to nonmethylated intronic regions strongly suggests that these single-copy insertions are methylated (SANMIGUEL et al. 1998 ). However, despite indirect evidence that these elements are methylated, most elements were not CG suppressed. Likewise, although both single- and multicopy zein genes have been shown to be hypermethylated in plant tissue (BIANCHI and VIOTTI 1988 ; LUND et al. 1995 ), only suppression of the multicopy zeins was observed. Taken together, the data suggest that methylation status per se is not sufficient to explain differences in CG suppression. However, duplicated sequences may exhibit a specific methylation pattern that alters the mutability of 5mC compared to a single-copy methylated sequence. The Tourist element inserted in the 5' region of the single-copy 27-kD zein gene showed a quantitative difference in methylation compared to the same element inserted in the duplicated 27-kD zein gene. The significance of these findings in relation to CG suppression can only be speculated. However, in rodents neither CG density nor the methylation status could explain the observed mutation frequencies of CG dinucleotides of a transgene (SKOPEK et al. 1998 ; MONROE et al. 2001 ). Likewise, the methylation status of the expressed and nonexpressed 5S RNA genes in Arabidopsis failed to account for the elevated levels of C:T and G:A transition mutations observed of the nonexpressed genes (MATIEU et al. 2002 ).

An alternative explanation for the observed differences in CG content is that selective pressures differ between chromosomal regions, which could result in different mutation rates of CG dinucleotides. For example, this might explain why the 22-kD zein genes, which map to chromosome 4S, are suppressed, whereas no suppression of DNA sequences that map to the Adh1-F chromosomal region was observed. However, analysis of intergenic and LTR regions of retrotransposons, located in the 340-kb 22-kD zein gene cluster, showed that CG suppression was localized to zein gene regions and was not a common feature of this chromosomal region. That CG suppression is independent of chromosomal content is also supported by the fact that many sequences analyzed exhibit large variations in CG despite being located in identical genomic regions, e.g., Tourist and Stowaway elements located in the noncoding regions of the single-copy or duplicated 27-kD zein gene or the single or duplicated Tourist element located in the 3' region of the Adh1 locus.

A study of 101 maize genes has shown that 40% of codon-usage variation is due to a bias toward G or C vs. A or U ending codons (FENNOY and BAILEY-SERRES 1993 ). The bias toward C or G in the third position (GC3) is larger for the single-copy zeins, whereas the -zeins have low GC3 values. We found that CG dinucleotides of the -zeins were suppressed at positions II-III and III-I, whereas the single-copy genes showed an excess of CG at these positions. This is interesting as CG suppression at position III-I seems to be specific of methylating species, whereas nonmethylating species show an excess of CG at this position (SCHORDERET and GARTLER 1992 ). Therefore, we argue that the differences in the GC3 bias between the single- and multicopy -zein genes reflect the fact that the -zein genes have undergone extensive gene duplication and are not due to a bias in codon usage between the single- and multicopy zein genes.

Mammalian and plant genomes are made up of large regions of relatively homogeneous base composition known as isochores (BERNARDI 2000 ), and the debate is ongoing of whether this mosaic structure is caused by mutation bias, natural selection, or biased gene conversion (reviewed by EYRE-WALKER and HURST 2001 ). Of particular interest in this context is the finding that cytosine deamination plays a primary role in the evolution of isochores (FRYXELL and ZUCKERKANDL 2000 ). In maize, most genes are confined to isochores with a narrow GC range, with the exception of the -zeins and ribosomal genes that are located in GC-poor and GC-rich fractions, respectively (CARELS et al. 1995 ). We have previously argued that the low GC3 content of the -zeins could be explained by duplication-dependent CG depletion. Given that the GC content and, in particular, the GC3 content of a gene is highly correlated to the overall GC content of the isochore in which it is located (BERNARDI et al. 1985 ; CLAY et al. 1996 ; EYRE-WALKER and HURST 2001 ), duplication-dependent CG loss may, in part, explain the evolution of this particular GC-poor isochore.

We have shown that duplicated zein genes, LTR elements, and MITEs undergo specific changes in nucleotide sequence. These changes have been observed in CG dinucleotides and result in C:T and G:A transition mutations and a net reduction in GC content. Such a process is reminiscent of RIP in N. crassa, where duplications are de novo methylated and riddled with point mutations (SELKER 1990 ). Unfortunately, this study cannot discern if transition mutations have occurred immediately upon duplication or result from subsequent mitoses. This question has been addressed in Arabidopsis by sequence analysis of multicopy insertions of transgenes after three sexual generations (MITTELSTEIN-SCHEID et al. 1994 ). None of the transition mutations characteristic of RIP were found, and it was argued that if RIP occurs in plants it occurs at a much lower frequency. However, this experiment does not necessarily exclude the possibility of a RIP-like mechanism in plants. Indeed, if transition mutations are linked to the duplication process, merely inserting a multicopy locus into the Arabidopsis genome would, expectedly, fail to recover any transition mutations.

From the detailed analysis of the 22-kD zein genes it is clear that the rate of CG depletion of duplicated sequences is enhanced compared to the average deamination of methylated residues of dispersed multicopy sequences. Further analysis will reveal if this mechanism is common to other duplicated gene families. Suppression of MITEs and LTR elements inserted in duplicated gene regions indicates that this might be the case. Interestingly, rapid responses to genome-wide duplication of genomes have been shown to occur in wheat and Arabidopsis allotetraploids. These changes include nonrandom alterations in methylation and gene silencing caused by methylation or gene loss (KASHKUSH et al. 2002 ; MADLUNG et al. 2002 ). Although it is unclear whether the observed effects result from chromosome doubling or from the hybridization of different genomes, it suggests that specific mechanisms are activated in plants in response to DNA amplification that presumably function to maintain genome stability.

	ACKNOWLEDGMENTS

The authors thank Mik Noordeweir for assisting in part of the sequence analysis, Angelo Viotti and Vincenzo Rossi for critical reading of the manuscript, and E. Linton for estimates of insertion time of the 22-kD zein genes. This work was supported by a grant from the Danish National Research Foundation.

Manuscript received February 28, 2003; Accepted for publication June 13, 2003.

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