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Discovery of a Zdel transposable element in Zea species as a consequence of a retrotransposon insertion
Gene 184 (1997) 257–261
Discovery of a Zdel transposable element in Zea species as a consequence of a retrotransposon insertion Carlos M. Vicient 1, Jose´ A. Martı´nez-Izquierdo * Departamento de Gene´tica Molecular, CID-CSIC, Jordi Girona Salgado 18–26, 08034 Barcelona, Spain Received 26 April 1996; revised 16 July 1996; accepted 20 July 1996; Received by A. Bernardi
Abstract Nucleotide sequences similar to del1 retrotransposon from Lilium henryi have been discovered in Zea diploperennis as a consequence of finding a Zea retrotransposon element inserted into one of them. These sequences named Zdel (Zea del1-like) elements are present in all the Zea species (about 100 copies per haploid genome) and in Tripsacum dactyloides and absent from closely related genera. Sequences corresponding to gag and protease domains from a Zdel element have been identified. The Zdel protease sequence shows a conserved active site motif (DT/SG) from aspartic proteases. The high level of DNA methylation found in Zdel elements may be related to the observed absence of transcriptional activity. Keywords: Maize; Methylation; Plant genome; Retrotransposon; Zea diploperennis
1. Introduction Retrotransposons are mobile DNA elements that transpose via an RNA intermediate and are flanked by long terminal repeats (LTRs). The LTRs provide cisregulatory sequences required for the transcription of the RNA intermediate ( Temin, 1989). The sequences between the two LTRs encode proteins necessary for reverse transcription and integration ( gag, protease, reverse transcriptase, RNAse H and integrase). Retrotransposons are widely distributed in eukaryotic genomes, including plants and animals. In addition, plant retrotransposons show typical features, namely, the majority of them are transcriptionally inactive and they have higher sequence heterogeneity and copy * Corresponding author. Tel. +34 3 4006127; Fax +34 3 2045904; e-mail: [email protected] 1 Present address: Laboratoire de Physiologie et Biologie Mole´culaire des Plantes, URA-CNRS 565, Faculte´ des Sciences, Universite´ de Perpignan, 52, avenue de Villeneuve, 66860 Perpignan Ce´dex, France. Abbreviations: aa, amino acid(s); BMS, Black Mexican Sweet; bp, base pair(s); dCTP, deoxycytidine triphosphate; GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase(s) or 1000 bp; LTR, long terminal repeat(s); ORF, open reading frame; PR, protease; rDNA, ribosomal gene DNA; SDS, sodium dodecyl sulfate; ss, single strand(ed); SSC, 0.15 M NaCl/0.001 M Na -EDTA/0.01 M sodium 2 phosphate pH 7.7; Zdel, Zea del1-like; ZLRS, Zea long repetitive sequence(s).
number than the animal ones (Grandbastien, 1992; Voytas et al., 1992; Flavell et al., 1994). Their high copy number could, in turn, facilitate plant retrotransposon sequence heterogeneity and divergence. We have recently reported the characterization of a family of dispersed repetitive sequences (Aledo et al., 1995; Monfort et al., 1995) that we have initially named ZLRS (Zea long repetitive sequences). ZLRS elements are present in all Zea species and their copy numbers have been estimated to be around 1500 in maize and Zea diploperennis (Aledo et al., 1995). The sequence of a full-length ZLRS element (clone 4) has been recently obtained and analyzed. The analysis revealed that ZLRS-4 has all the attributes that characterize normal retrotransposons. In addition, this new retrotransposon is unusually large ( larger than 13 kb) and has been named Grande1 ( Vicient, 1995). Flanking regions of the Grande1-4 element were sequenced. The analysis of these sequences reveals a new retrotransposon, whose molecular characterization is reported here. This is the first time that a plant retrotransposon is discovered as a consequence of the insertion of another transposable element.
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2. Experimental and discussion 2.1. Determination and analysis of Zdel sequence The sequence of approx. 1.5 kb of Grande1-4 flanking DNA was determined (GenBank/EMBL accession No. X87229). This DNA sequence was translated into putative peptides and compared with DNA databases using the TFASTA program of the GCG Package (Devereux et al., 1984). Relatively high similarities were found between aa sequences corresponding to the flanking sequences at both sides of Grande1-4, and the aa sequence of del1 retrotransposon from Lilium henryi (Sentry and Smyth, 1989; Smyth et al., 1989). Interestingly, the similarity at the left side continues exactly at the right side, that is more than 13 kb away (Fig. 1A). Moreover, there is a 5 bp duplication of a del1-like sequence (5∞-AAGAG-3∞) at both sides of Grande1-4 termini ( TG...CA) (Fig. 1A). Therefore, we conclude that Grande1-4 is inserted into a del1-like retrotransposable element that we have named Zdel (Zea del1-like) element. The aa sequences deduced from Zdel open reading frames (ORFs) show similarities to gag and protease domains of del1 retrotransposon. Those ORFs are interrupted prematurely in several places by stop codons, although no frameshift is necessary to have continuous aa sequences. The Zdel protease sequence can be aligned with the protease sequences corresponding to other retrotransposons, caulimoviruses and retroviruses (Fig. 1B). The highest similarity is observed with proteases from the Ty3/gypsy retrotransposon group around
a well conserved active site motif (DT/SG ) of aspartic proteases ( Katz and Skalka, 1994). The levels of similarity observed between Zdel and del1 are relatively high for gag (22%) (results not shown) and protease (29%) sequences if we consider the high diversity shown by both domains among retrotransposable elements (McClure et al., 1988). As the protease domain, in retrotransposable elements, is only present in retrotransposons and not, for example, in non-LTR retrotransposons, Zdel can be considered an LTR retrotransposon. The higher similarity observed between Zdel protease sequences and those belonging to the Ty3/gypsy group of retrotransposons compared to the proteases of other elements (Fig. 1) suggests that Zdel belongs to the Ty3/gypsy family of which del1 is a member. 2.2. Species distribution of Zdel retrotransposon To determine the species distribution and the relative abundance of Zdel elements, Southern hybridization analysis was performed with DNAs corresponding to different Zea and related species using the 4S3E3 fragment from the Zdel gag domain (Fig. 1A) as a probe. All Zea and Tripsacum species tested gave hybridization signals with the 4S3E3 probe and no detectable signals corresponding to sorghum or barley were found ( Fig. 2A). Estimation of the Zdel copy number by genomic reconstruction was done by slot-blot hybridizations with genomic DNA of Zea diploperennis, resulting in around 100 copies per haploid genome (data not shown). The band pattern is highly conserved among
Fig. 1. The Zdel element from Zea diploperennis. (A) Schematic representation of gag and protease (PR) Zdel domains. The Zdel gag domain is interrupted by Grande1-4 retrotransposon (expanded view), showing the TG..CA short inverted repeats (short arrows) typical of retrotransposon LTRs, flanked by two 5 bp direct repeats (AAGAG) duplication target ( long arrows). The filled box below the gag domain (4S3E3) indicates the probe used in hybridization experiments. (B) Alignments of the protease aa sequences of Zdel and other retroelements. Identical aa to Zdel are shadowed and the most conserved ones among the sequences shown are highlighted by bold letters. Dashes represent absence of aa at that position. At the bottom, alignment of sequences around the conserved active site motif (D T/S G) of retrotransposon proteases. CaMV, cauliflower mosaic virus; MoMLV, Moloney murine leukemia virus; HIV1, human immunodeficiency virus 1. All the sequences shown except del1 (Smyth et al., 1989) were obtained from McClure (1991).
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by the higher amount of DNA loaded into the gel in the case of the former species (results not shown). The Zdel elements are also present in Zea mexicana, Zea luxurians and other maize lines and varieties (A188, cateto and BMS ) but they are not present in rice (data not shown). We can conclude that Zdel elements are specific to the genera Zea and Tripsacum. The same distribution between genera has been observed for other retrotransposons as Bs1 (Fuerstenberg and Johns, 1990) and magellan (Purugganan and Wessler, 1994).
2.3. Preferential insertion of Grande1 elements into other transposable elements
Fig. 2. Zdel Southern hybridization analysis. Genomic DNA was isolated as described (Dellaporta et al., 1983). The DNA (10 mg per lane) was digested to completion with the appropriate restriction enzyme, fractionated by electrophoresis in 0.8% agarose gel and transferred to Nytran-N membranes (Schleicher and Schuell ) according to the manufacturer’s indications. Probes were labeled to high specific activity by random priming extension (Random Priming Kit, Boehringer) in the presence of radiolabeled dCTP. Hybridizations were performed in phosphate buffer (0.25 M pH 7.2, 7% SDS, 1 mM EDTA, 0.25 mg/ml ssDNA) at 65°C. Prehybridizations were performed during 30 min in the same conditions. After overnight hybridizations the filters were washed at 65°C in 0.5×SSC/0.1% SDS. After washing the filters were exposed to films (Agfa Curix RP2) for 48 h at −70°C with intensifying screens. The size of molecular weight markers in the middle of the figure are indicated in kb. (A) Southern hybridization of DNA from different species. Leaf DNAs were digested with EcoRI restriction enzyme and probed with the 0.6 kb 4S3E3 Zdel probe. W, W64A maize; D, Zea diploperennis; P, Z. perennis; Td, Tripsacum dactyloides dactyloides; Th, T. d. hispidum; Tm, T. d. mexicana; S, Sorghum bicolor; H, Hordeum vulgare. (B) DNA methylation status of Zdel. DNA from BMS maize cells was digested with MvaI (M ) or EcoRII ( E ) restriction enzymes and hybridized with the 4S3E3 fragment or a rDNA maize probe (McMullen et al., 1986).
Zea species on the one hand, and among Tripsacum species on the other hand, but not between the two genera, indicating that Zdel sequence and their chromosomal distribution are conserved in both genera but not between them. Band intensities are similar among maize and Z. diploperennis, but they are lower in Zea perennis. Similar results have been observed for ZLRS/Grande1 (Aledo et al., 1995) and ZEAR elements (Raz et al., 1991). This variation in band intensities could not be explained by differences in the amounts of DNA loaded into the gel, but it can be attributed to the fact that Z. perennis is the only tetraploid species in the genus. The intensity of hybridization bands is lower for Tripsacum species compared to Zea species ( Fig. 2A) indicating either sequence divergence or lower copy number of Zdel in the former species. The higher intensity of bands seen in Tripsacum dactyloides mexicana compared to the two other Tripsacum species can be partially explained
Most plant retrotransposable elements have been found to be inserted in cellular genes or in their flanking regions ( White et al., 1994). In contrast, Zdel retrotransposon was discovered because a copy of another retrotransposon (Grande1-4) was inserted into it. Interestingly, we have also found that another copy of Grande1 ( ZLRS7/Grande1-7) (Monfort et al., 1995) is inserted into a sequence similar to maize Cin1 retroelement (Shepherd et al., 1984). Due to the fact that Grande1 (ZLRS ) is present in around 1500 copies in the teosinte and maize genomes (Aledo et al., 1995), it is reasonable to assume that a high number of elements could be deleterious for the organism if the majority of them are not integrated in non-essential genome regions of the plant cell. These non-essential regions could be represented by retrotransposable elements such as Zdel and Cin1 that are interrupted by Grande1 retrotransposons. Other plant retrotransposons with high copy number as barley BARE-1 ( Ty1/copia group, 5000 copies), IFG7 of Pinus radiata ( Ty3 group, 10 000 copies) or del1 ( Ty3 group, 13 000 copies) have not been associated yet either with plant genes or other transposable or repetitive elements (Sentry and Smyth, 1989; Grandbastien, 1992; Manninen and Schulman, 1993). Nevertheless, the finding of retrotransposable elements inserted into other transposable elements either from the same family or from a different type has been reported before (Sandmeyer et al., 1990). For example, Physarum polycephalum Tp1 retrotransposons have been found inserted in other Tp1 elements (Rothnie et al., 1990). Although the short inverted repeat element stowaway has been found as insertion into the inverted repeat tourist element (Bureau and Wessler, 1994), to our knowledge no retrotransposon has been discovered in plants as a consequence of another retrotransposon being inserted into it. However, more flanking sequences of plant retrotransposon are needed to know whether retrotransposons are located preferentially in or near plant genes or in other transposable elements or repetitive sequences in general.
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2.4. DNA methylation level of Zdel elements In order to know whether other copies of Zdel, besides the copy inactivated by the insertion of Grande1-4, are transcriptionally active, Northern blot analysis using RNAs from leaves of different Zea species and different maize tissues (7-day-old maize seedlings, styles, ovules and pollen) was performed. Hybridizations performed with the 4S3E3 fragment probe did not reveal any band in Northern blots (results not shown). Our failure to detect Zdel transcripts may be due either to the lack of functionality of Zdel elements at the present time, or to the inactivity of Zdel in the tissues and conditions tested. It is well known that transposable elements usually become active only in certain genomic stress conditions (McClintock, 1984), as has been observed, for example, during in vitro tissue culture. On the other hand, DNA methylation has been inversely correlated to transposon activity ( Fedoroff, 1989). Activation of some transposable elements has been observed after tissue culture, a fact that seems to be secondary to the demethylation of transposon DNA ( Finnegan et al., 1993). These observations prompted us to investigate the methylation levels of Zdel elements in normal plant tissues and during in vitro plant tissue culture. The endonuclease isoschizomers MvaI and EcoRII, recognizing the restriction site CCA( T )GG, were used to digest genomic DNA from cells in suspension culture and leaf tissues. MvaI digestion is insensitive to cytosine DNA methylation, whereas EcoRII does not cut when the internal cytosine of the recognition site is methylated. Southern blots of genomic DNA from maize BMS cell suspension cultures, digested with MvaI or EcoRII, were probed with 4S3E3 Zdel fragment (Fig. 2B, left). As a control for MvaI and EcoRII activity the same filter was hybridized with a rDNA probe ( Fig. 2B, right). Low molecular weight bands appeared in the case of MvaI digestion for both probes, but only high molecular weight DNA was present when genomic DNA was digested with EcoRII and probed with the 4S3E3 Zdel probe (Fig. 2B). Low molecular weight bands were observed when the same EcoRII digest was probed with the rDNA (Fig. 2B, right). The latter result indicates that in spite of the fact that the extent of CpG methylation is particularly high in plant rRNA genes (Flavell et al., 1988), EcoRII digestion allows to identify some copies of the ribosomal genes that are not methylated. These results indicate that Zdel elements are more extensively methylated than rDNA in cell suspensions of maize. The same results were obtained using DNA from maize leaves (data not shown).
3. Conclusions (1) We have presented data on a retrotransposon ( Zdel )
similar to del1 retrotransposon from Lilium henryi. This type of retrotransposon is described for the first time in Gramineae (Zea and Tripsacum) species. (2) Zdel retrotransposons are transcriptionally inactive and their sequences are highly methylated in maize tissues. (3) Zdel is the first plant retrotransposon discovered as a consequence of another retrotransposable element being inserted into it.
Acknowledgement We thank Dr. R. Raz for obtaining the l genomic clone, Mrs. A. Pons (CID-Automatic Sequencing Service) for technical support in DNA sequencing and Dr. J. Doebley for kindly providing us with the maize rDNA probe. We are indebted to Dr. J.M. Casacuberta for critical reading of the manuscript and Miss A. Jessop and Dr. S. Jackson for carefully revising this manuscript. This work has been supported by grants from DGPC (PB90-0129 and PB93-0043). C.M.V. was the recipient of a FPI fellowship (CIRIT ).
References Aledo, R., Raz, R., Monfort, A., Vicient, C.M., Puigdome`nech, P. and Martı´nez-Izquierdo, J.A. (1995) Chromosome localization and characterization of a family of long repetitive DNA elements from the genus Zea. Theor. Appl. Genet. 90, 1094–1100. Bureau, T.E. and Wessler, S.R. (1994) Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. Plant Cell 6, 907–916. Dellaporta, S.L., Wood, J. and Hicks, J.B. (1983) A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1, 19–21. Devereux, J., Haeberti, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387–395. Fedoroff, N.V. (1989) About maize transposable elements and development. Cell 56, 181–191. Finnegan, E.J., Brettell, R.I.S. and Dennis, E.S. (1993) The role of DNA methylation in the regulation of plant gene expression. In: Jost, J.P. and Saluz, H.P. (Eds.), DNA Methylation: Molecular Biology and Biological Significance. Birkha¨user Verlag, Basel, pp. 218–261. Flavell, A.J., Pearce, S.R. and Kumar, A. (1994) Plant transposable elements and the genome. Curr. Opin. Genet. Dev. 4, 838–844. Flavell, R.B., O’Dell, M. and Thompson, W.F. (1988) Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat. J. Mol. Biol. 204, 523–534. Fuerstenberg, S.I. and Johns, M.A. (1990) Distribution of Bs1 retrotransposon in Zea and related genera. Theor. Appl. Genet. 80, 680–686. Grandbastien, M.A. (1992) Retroelements in higher plants. Trends Genet. 8, 103–108. Katz, R.A. and Skalka, A.M. (1994) The retroviral enzymes. Annu. Rev. Biochem. 63, 133–173. Manninen, I. and Schulman, A.H. (1993) BARE-1, a copia-like retroelement in barley (Hordeum vulgare L.). Plant Mol. Biol. 22, 829–846.
C.M. Vicient, Jose´ A. Martı´nez-Izquierdo/Gene 184 (1997) 257–261 McClintock, B. (1984) The significance of responses of the genome to challenge. Science 226, 792–780. McClure, M.A. (1991) Evolution of retroposons by acquisition or deletion of retrovirus-like genes. Mol. Biol. Evol. 8, 835–856. McClure, M.A., Johnson, M.A., Feng, D.F. and Doolittle, R.F. (1988) Sequence comparisons of retroviral proteins: relative rates of change and general phylogeny. Proc. Natl. Acad. Sci. USA 85, 2469–2473. McMullen, M., Hunter, B., Phillips, R.L. and Rubenstein, I. (1986) The structure of the maize ribosomal DNA spacer region. Nucleic Acids Res. 14, 4953–4968. Monfort, A., Vicient, C.M., Raz, R., Puigdome`nech, P. and Martı´nezIzquierdo, J.A. (1995) Molecular analysis of a putative transposable retroelement from the Zea genus with internal clusters of tandem repeats. DNA Res. 2, 255–261. Purugganan, M. and Wessler, S.R. (1994) Molecular evolution of magellan, a maize Ty3/gypsy-like retrotransposon. Proc. Natl. Acad. Sci. USA 91, 11674–11678. Raz, R., Puigdome`nech, P. and Martı´nez-Izquierdo, J.A. (1991) A new family of repetitive nucleotide sequences is restricted to the genus Zea. Gene 105, 151–158. Rothnie, H.M., McCurrach, K.J., Glover, L.A. and Hardman, N. (1990) Retrotransposon-like nature of Tp1 elements: implications for the organisation of highly repetitive, hypermethylated DNA in the genome of Physarum polycephalum. Nucleic Acids Res. 19, 279–286.
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Sandmeyer, S.B., Hansen, L.J. and Chalker, D.L. (1990) Integration specificity of retrotransposons and retroviruses. Annu. Rev. Genet. 24, 491–518. Sentry, J.W. and Smyth, D.R. (1989) An element with long terminal repeats and its variant arrangements in the genome of Lilium henryi. Mol. Gen. Genet. 215, 349–354. Shepherd, N.S., Schwarz-Sommer, Z., Blumnerg, J., Gupta, M., Wienand, U. and Saedler, H. (1984) Similarity of the Cin1 repetitive family of Zea mays to eukaryotic transposable elements. Nature 307, 185–187. Smyth, D.R., Kalitsis, P., Jospeh, J.L. and Sentry, J.W. (1989) Plant retrotransposon from Lilium henryi is related to Ty3 of yeast and the gypsy group of Drosophila. Proc. Natl. Acad. Sci. USA 86, 5015–5019. Temin, H.M. (1989) Retrons in bacteria. Nature 339, 254–255. Vicient, C.M. (1995) Caracterizacio´n Molecular de Grande1, un Nuevo Retrotransposo´n del Ge´nero Zea. Ph.D. Thesis, University of Barcelona, Barcelona. Voytas, D.F., Cummings, M.P., Konieczny, A., Ausubel, F.M. and Rodermel, S.R. (1992) copia-like retrotransposons are ubiquitous among plants. Proc. Natl. Acad. Sci. USA 89, 7124–7128. White, S.E., Habera, L.F. and Wessler, S.R. (1994) Retrotransposons in the flanking regions of normal plant genes: a role for copia-like elements in the evolution of gene structure and expression. Proc. Natl. Acad. Sci. USA 91, 11792–11796.
Discovery of a Zdel transposable element in Zea species as a consequence of a retrotransposon insertion Carlos M. Vicient 1, Jose´ A. Martı´nez-Izquierdo * Departamento de Gene´tica Molecular, CID-CSIC, Jordi Girona Salgado 18–26, 08034 Barcelona, Spain Received 26 April 1996; revised 16 July 1996; accepted 20 July 1996; Received by A. Bernardi
Abstract Nucleotide sequences similar to del1 retrotransposon from Lilium henryi have been discovered in Zea diploperennis as a consequence of finding a Zea retrotransposon element inserted into one of them. These sequences named Zdel (Zea del1-like) elements are present in all the Zea species (about 100 copies per haploid genome) and in Tripsacum dactyloides and absent from closely related genera. Sequences corresponding to gag and protease domains from a Zdel element have been identified. The Zdel protease sequence shows a conserved active site motif (DT/SG) from aspartic proteases. The high level of DNA methylation found in Zdel elements may be related to the observed absence of transcriptional activity. Keywords: Maize; Methylation; Plant genome; Retrotransposon; Zea diploperennis
1. Introduction Retrotransposons are mobile DNA elements that transpose via an RNA intermediate and are flanked by long terminal repeats (LTRs). The LTRs provide cisregulatory sequences required for the transcription of the RNA intermediate ( Temin, 1989). The sequences between the two LTRs encode proteins necessary for reverse transcription and integration ( gag, protease, reverse transcriptase, RNAse H and integrase). Retrotransposons are widely distributed in eukaryotic genomes, including plants and animals. In addition, plant retrotransposons show typical features, namely, the majority of them are transcriptionally inactive and they have higher sequence heterogeneity and copy * Corresponding author. Tel. +34 3 4006127; Fax +34 3 2045904; e-mail: [email protected] 1 Present address: Laboratoire de Physiologie et Biologie Mole´culaire des Plantes, URA-CNRS 565, Faculte´ des Sciences, Universite´ de Perpignan, 52, avenue de Villeneuve, 66860 Perpignan Ce´dex, France. Abbreviations: aa, amino acid(s); BMS, Black Mexican Sweet; bp, base pair(s); dCTP, deoxycytidine triphosphate; GCG, Genetics Computer Group (Madison, WI, USA); kb, kilobase(s) or 1000 bp; LTR, long terminal repeat(s); ORF, open reading frame; PR, protease; rDNA, ribosomal gene DNA; SDS, sodium dodecyl sulfate; ss, single strand(ed); SSC, 0.15 M NaCl/0.001 M Na -EDTA/0.01 M sodium 2 phosphate pH 7.7; Zdel, Zea del1-like; ZLRS, Zea long repetitive sequence(s).
number than the animal ones (Grandbastien, 1992; Voytas et al., 1992; Flavell et al., 1994). Their high copy number could, in turn, facilitate plant retrotransposon sequence heterogeneity and divergence. We have recently reported the characterization of a family of dispersed repetitive sequences (Aledo et al., 1995; Monfort et al., 1995) that we have initially named ZLRS (Zea long repetitive sequences). ZLRS elements are present in all Zea species and their copy numbers have been estimated to be around 1500 in maize and Zea diploperennis (Aledo et al., 1995). The sequence of a full-length ZLRS element (clone 4) has been recently obtained and analyzed. The analysis revealed that ZLRS-4 has all the attributes that characterize normal retrotransposons. In addition, this new retrotransposon is unusually large ( larger than 13 kb) and has been named Grande1 ( Vicient, 1995). Flanking regions of the Grande1-4 element were sequenced. The analysis of these sequences reveals a new retrotransposon, whose molecular characterization is reported here. This is the first time that a plant retrotransposon is discovered as a consequence of the insertion of another transposable element.
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2. Experimental and discussion 2.1. Determination and analysis of Zdel sequence The sequence of approx. 1.5 kb of Grande1-4 flanking DNA was determined (GenBank/EMBL accession No. X87229). This DNA sequence was translated into putative peptides and compared with DNA databases using the TFASTA program of the GCG Package (Devereux et al., 1984). Relatively high similarities were found between aa sequences corresponding to the flanking sequences at both sides of Grande1-4, and the aa sequence of del1 retrotransposon from Lilium henryi (Sentry and Smyth, 1989; Smyth et al., 1989). Interestingly, the similarity at the left side continues exactly at the right side, that is more than 13 kb away (Fig. 1A). Moreover, there is a 5 bp duplication of a del1-like sequence (5∞-AAGAG-3∞) at both sides of Grande1-4 termini ( TG...CA) (Fig. 1A). Therefore, we conclude that Grande1-4 is inserted into a del1-like retrotransposable element that we have named Zdel (Zea del1-like) element. The aa sequences deduced from Zdel open reading frames (ORFs) show similarities to gag and protease domains of del1 retrotransposon. Those ORFs are interrupted prematurely in several places by stop codons, although no frameshift is necessary to have continuous aa sequences. The Zdel protease sequence can be aligned with the protease sequences corresponding to other retrotransposons, caulimoviruses and retroviruses (Fig. 1B). The highest similarity is observed with proteases from the Ty3/gypsy retrotransposon group around
a well conserved active site motif (DT/SG ) of aspartic proteases ( Katz and Skalka, 1994). The levels of similarity observed between Zdel and del1 are relatively high for gag (22%) (results not shown) and protease (29%) sequences if we consider the high diversity shown by both domains among retrotransposable elements (McClure et al., 1988). As the protease domain, in retrotransposable elements, is only present in retrotransposons and not, for example, in non-LTR retrotransposons, Zdel can be considered an LTR retrotransposon. The higher similarity observed between Zdel protease sequences and those belonging to the Ty3/gypsy group of retrotransposons compared to the proteases of other elements (Fig. 1) suggests that Zdel belongs to the Ty3/gypsy family of which del1 is a member. 2.2. Species distribution of Zdel retrotransposon To determine the species distribution and the relative abundance of Zdel elements, Southern hybridization analysis was performed with DNAs corresponding to different Zea and related species using the 4S3E3 fragment from the Zdel gag domain (Fig. 1A) as a probe. All Zea and Tripsacum species tested gave hybridization signals with the 4S3E3 probe and no detectable signals corresponding to sorghum or barley were found ( Fig. 2A). Estimation of the Zdel copy number by genomic reconstruction was done by slot-blot hybridizations with genomic DNA of Zea diploperennis, resulting in around 100 copies per haploid genome (data not shown). The band pattern is highly conserved among
Fig. 1. The Zdel element from Zea diploperennis. (A) Schematic representation of gag and protease (PR) Zdel domains. The Zdel gag domain is interrupted by Grande1-4 retrotransposon (expanded view), showing the TG..CA short inverted repeats (short arrows) typical of retrotransposon LTRs, flanked by two 5 bp direct repeats (AAGAG) duplication target ( long arrows). The filled box below the gag domain (4S3E3) indicates the probe used in hybridization experiments. (B) Alignments of the protease aa sequences of Zdel and other retroelements. Identical aa to Zdel are shadowed and the most conserved ones among the sequences shown are highlighted by bold letters. Dashes represent absence of aa at that position. At the bottom, alignment of sequences around the conserved active site motif (D T/S G) of retrotransposon proteases. CaMV, cauliflower mosaic virus; MoMLV, Moloney murine leukemia virus; HIV1, human immunodeficiency virus 1. All the sequences shown except del1 (Smyth et al., 1989) were obtained from McClure (1991).
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by the higher amount of DNA loaded into the gel in the case of the former species (results not shown). The Zdel elements are also present in Zea mexicana, Zea luxurians and other maize lines and varieties (A188, cateto and BMS ) but they are not present in rice (data not shown). We can conclude that Zdel elements are specific to the genera Zea and Tripsacum. The same distribution between genera has been observed for other retrotransposons as Bs1 (Fuerstenberg and Johns, 1990) and magellan (Purugganan and Wessler, 1994).
2.3. Preferential insertion of Grande1 elements into other transposable elements
Fig. 2. Zdel Southern hybridization analysis. Genomic DNA was isolated as described (Dellaporta et al., 1983). The DNA (10 mg per lane) was digested to completion with the appropriate restriction enzyme, fractionated by electrophoresis in 0.8% agarose gel and transferred to Nytran-N membranes (Schleicher and Schuell ) according to the manufacturer’s indications. Probes were labeled to high specific activity by random priming extension (Random Priming Kit, Boehringer) in the presence of radiolabeled dCTP. Hybridizations were performed in phosphate buffer (0.25 M pH 7.2, 7% SDS, 1 mM EDTA, 0.25 mg/ml ssDNA) at 65°C. Prehybridizations were performed during 30 min in the same conditions. After overnight hybridizations the filters were washed at 65°C in 0.5×SSC/0.1% SDS. After washing the filters were exposed to films (Agfa Curix RP2) for 48 h at −70°C with intensifying screens. The size of molecular weight markers in the middle of the figure are indicated in kb. (A) Southern hybridization of DNA from different species. Leaf DNAs were digested with EcoRI restriction enzyme and probed with the 0.6 kb 4S3E3 Zdel probe. W, W64A maize; D, Zea diploperennis; P, Z. perennis; Td, Tripsacum dactyloides dactyloides; Th, T. d. hispidum; Tm, T. d. mexicana; S, Sorghum bicolor; H, Hordeum vulgare. (B) DNA methylation status of Zdel. DNA from BMS maize cells was digested with MvaI (M ) or EcoRII ( E ) restriction enzymes and hybridized with the 4S3E3 fragment or a rDNA maize probe (McMullen et al., 1986).
Zea species on the one hand, and among Tripsacum species on the other hand, but not between the two genera, indicating that Zdel sequence and their chromosomal distribution are conserved in both genera but not between them. Band intensities are similar among maize and Z. diploperennis, but they are lower in Zea perennis. Similar results have been observed for ZLRS/Grande1 (Aledo et al., 1995) and ZEAR elements (Raz et al., 1991). This variation in band intensities could not be explained by differences in the amounts of DNA loaded into the gel, but it can be attributed to the fact that Z. perennis is the only tetraploid species in the genus. The intensity of hybridization bands is lower for Tripsacum species compared to Zea species ( Fig. 2A) indicating either sequence divergence or lower copy number of Zdel in the former species. The higher intensity of bands seen in Tripsacum dactyloides mexicana compared to the two other Tripsacum species can be partially explained
Most plant retrotransposable elements have been found to be inserted in cellular genes or in their flanking regions ( White et al., 1994). In contrast, Zdel retrotransposon was discovered because a copy of another retrotransposon (Grande1-4) was inserted into it. Interestingly, we have also found that another copy of Grande1 ( ZLRS7/Grande1-7) (Monfort et al., 1995) is inserted into a sequence similar to maize Cin1 retroelement (Shepherd et al., 1984). Due to the fact that Grande1 (ZLRS ) is present in around 1500 copies in the teosinte and maize genomes (Aledo et al., 1995), it is reasonable to assume that a high number of elements could be deleterious for the organism if the majority of them are not integrated in non-essential genome regions of the plant cell. These non-essential regions could be represented by retrotransposable elements such as Zdel and Cin1 that are interrupted by Grande1 retrotransposons. Other plant retrotransposons with high copy number as barley BARE-1 ( Ty1/copia group, 5000 copies), IFG7 of Pinus radiata ( Ty3 group, 10 000 copies) or del1 ( Ty3 group, 13 000 copies) have not been associated yet either with plant genes or other transposable or repetitive elements (Sentry and Smyth, 1989; Grandbastien, 1992; Manninen and Schulman, 1993). Nevertheless, the finding of retrotransposable elements inserted into other transposable elements either from the same family or from a different type has been reported before (Sandmeyer et al., 1990). For example, Physarum polycephalum Tp1 retrotransposons have been found inserted in other Tp1 elements (Rothnie et al., 1990). Although the short inverted repeat element stowaway has been found as insertion into the inverted repeat tourist element (Bureau and Wessler, 1994), to our knowledge no retrotransposon has been discovered in plants as a consequence of another retrotransposon being inserted into it. However, more flanking sequences of plant retrotransposon are needed to know whether retrotransposons are located preferentially in or near plant genes or in other transposable elements or repetitive sequences in general.
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2.4. DNA methylation level of Zdel elements In order to know whether other copies of Zdel, besides the copy inactivated by the insertion of Grande1-4, are transcriptionally active, Northern blot analysis using RNAs from leaves of different Zea species and different maize tissues (7-day-old maize seedlings, styles, ovules and pollen) was performed. Hybridizations performed with the 4S3E3 fragment probe did not reveal any band in Northern blots (results not shown). Our failure to detect Zdel transcripts may be due either to the lack of functionality of Zdel elements at the present time, or to the inactivity of Zdel in the tissues and conditions tested. It is well known that transposable elements usually become active only in certain genomic stress conditions (McClintock, 1984), as has been observed, for example, during in vitro tissue culture. On the other hand, DNA methylation has been inversely correlated to transposon activity ( Fedoroff, 1989). Activation of some transposable elements has been observed after tissue culture, a fact that seems to be secondary to the demethylation of transposon DNA ( Finnegan et al., 1993). These observations prompted us to investigate the methylation levels of Zdel elements in normal plant tissues and during in vitro plant tissue culture. The endonuclease isoschizomers MvaI and EcoRII, recognizing the restriction site CCA( T )GG, were used to digest genomic DNA from cells in suspension culture and leaf tissues. MvaI digestion is insensitive to cytosine DNA methylation, whereas EcoRII does not cut when the internal cytosine of the recognition site is methylated. Southern blots of genomic DNA from maize BMS cell suspension cultures, digested with MvaI or EcoRII, were probed with 4S3E3 Zdel fragment (Fig. 2B, left). As a control for MvaI and EcoRII activity the same filter was hybridized with a rDNA probe ( Fig. 2B, right). Low molecular weight bands appeared in the case of MvaI digestion for both probes, but only high molecular weight DNA was present when genomic DNA was digested with EcoRII and probed with the 4S3E3 Zdel probe (Fig. 2B). Low molecular weight bands were observed when the same EcoRII digest was probed with the rDNA (Fig. 2B, right). The latter result indicates that in spite of the fact that the extent of CpG methylation is particularly high in plant rRNA genes (Flavell et al., 1988), EcoRII digestion allows to identify some copies of the ribosomal genes that are not methylated. These results indicate that Zdel elements are more extensively methylated than rDNA in cell suspensions of maize. The same results were obtained using DNA from maize leaves (data not shown).
3. Conclusions (1) We have presented data on a retrotransposon ( Zdel )
similar to del1 retrotransposon from Lilium henryi. This type of retrotransposon is described for the first time in Gramineae (Zea and Tripsacum) species. (2) Zdel retrotransposons are transcriptionally inactive and their sequences are highly methylated in maize tissues. (3) Zdel is the first plant retrotransposon discovered as a consequence of another retrotransposable element being inserted into it.
Acknowledgement We thank Dr. R. Raz for obtaining the l genomic clone, Mrs. A. Pons (CID-Automatic Sequencing Service) for technical support in DNA sequencing and Dr. J. Doebley for kindly providing us with the maize rDNA probe. We are indebted to Dr. J.M. Casacuberta for critical reading of the manuscript and Miss A. Jessop and Dr. S. Jackson for carefully revising this manuscript. This work has been supported by grants from DGPC (PB90-0129 and PB93-0043). C.M.V. was the recipient of a FPI fellowship (CIRIT ).
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