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Retand: a Novel Family of Gypsy-Like Retrotransposons Harboring an Ampliwed Tandem Repeat

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Retand: a Novel Family of Gypsy-Like Retrotransposons Harboring an Ampliwed Tandem Repeat Mol Gen Genomics DOI 10.1007/s00438-006-0140-x ORIGINAL PAPER Retand: a novel family of gypsy-like retrotransposons harboring an ampliWed tandem repeat Eduard Kejnovsky · Zdenek Kubat · Jiri Macas · Roman Hobza · Jaroslav Mracek · Boris Vyskot Received: 20 December 2005 / Accepted: 2 June 2006 © Springer-Verlag 2006 Abstract In this paper we describe a pair of novel Retand elements are also present in other Silene species, Ty3/gypsy retrotransposons isolated from the dioecious mostly in subtelomeric heterochromatin regions of all plant Silene latifolia, consisting of a non-autonomous chromosomes. The only exception is the subtelomere of element Retand-1 (3.7 kb) and its autonomous partner the short arm of the Y chromosome in S. latifolia which Retand-2 (11.1 kb). These two elements have highly is known to lack the terminal heterochromatin. An similar long terminal repeat (LTR) sequences but diVer interesting feature of the Retand elements is the pres- in the presence of the typical retroelement coding ence of a tandem repeat sequence, which is more ampli- regions (gag-pol genes), most of which are missing in Wed in the non-autonomous Retand-1. Retand-1. Moreover, Retand-2 contains two additional open reading frames in antisense orientation localized Keywords Pair of autonomous and non-autonomous between the pol gene and right LTR. Retand transcripts retrotransposons · Tandem repeat · Transcriptional were detected in all organs tested (leaves, Xower buds activity · Sex chromosomes · Silene latifolia and roots) which, together with the high sequence simi- larity of LTRs in individual elements, indicates their recent transpositional activity. The autonomous ele- Introduction ments are similarly abundant (2,700 copies) as non- autonomous ones (2,100 copies) in S. latifolia genome. Retrotransposons represent a class of mobile elements with duplicative mode of transposition via RNA inter- mediate. They are widespread among eukaryotes and often compose a signiWcant fraction of the nuclear Communicated by M.-A. Grandbastien genome (Bennetzen 1996; SanMiguel et al. 1996). In Nucleotide sequence data reported are available in the DDBJ/ plants, retrotransposons are found in all genomes that EMBL/GenBank databases under the accession numbers have been examined, but they seem to be highly abun- DQ023669 (Retand-2) and DQ023670 (Retand-1). dant only in species with large genomes, in some plants comprising more than 50% of nuclear DNA content E. Kejnovsky (&) · Z. Kubat · R. Hobza · J. Mracek · B. Vyskot (Pearce et al.1996a; SanMiguel et al. 1996; Suoniemi Laboratory of Plant Developmental Genetics, et al. 1998). The replication strategy of retrotranspo- Institute of Biophysics, sons oVers the potential for explosive increases in copy Academy of Sciences of the Czech Republic, numbers and thereby these elements increase the size Kralovopolska 135, 612 65 Brno, Czech Republic e-mail: [email protected] of the host genome (Kumar and Bennetzen 1999; San- Miguel and Bennetzen 1998). DiVerential ampliWcation J. Macas of retrotransposons is largely responsible for signiWcant Laboratory of Molecular Cytogenetics, diVerences in genome size even between closely related Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, plant species (SanMiguel 1998; Vicient et al. 1999; 37005 Ceske Budejovice, Czech Republic Kalendar et al. 2000; SanMiguel and Bennetzen 1998). 123 Mol Gen Genomics It has been found that plant genomes host diverse region, regions of gag and pol genes, ORF3 region and populations of retroelements which are classiWed into STR1 tandem repeat region with following primers, several basic classes (Hull 2001), each of them is repre- respectively: LTR-F1 (5Ј-TTCCGGGTGTAATTCC sented by various families including elements similar in AGAG-3Ј), LTR-R1 (5Ј-CATATTCTGCACCCGCT their nucleotide sequences and thus supposed to be of GAC-3Ј), GAG-F1 primer (5Ј-CCGACTCTCCAAA the same origin. However, even the retroelement GTCGAAC-3Ј) and GAG-R1 (5Ј-TTCTGCCCTTC sequences belonging to the same family may diVer con- ATTCTTGCT-3Ј), POL-F1 (5Ј-ACGGTCAGTGCC siderably in their length and structure, as they occur in GTAATCATC-3Ј), POL-R1 (5Ј-GTCGTCTCGGCT the genome as deletion derivatives of the full-length TCTATGTC-3Ј), STR1-F1 (5Ј-GAGGTGTCCAAG retroelements (Havecker et al. 2004). A typical exam- ACCGTTGT-3Ј), STR1-R1 (5Ј-GCCGTCGACTTCT ple is solo-LTRs derived from LTR-retrotransposons TTCTTGT-3Ј), ORF4-F1 (5Ј-TAACGTAACCCGGC by recombination-based excision and elimination of TCTGTC-3Ј) and ORF4-R1 (5Ј-TTTGGAGGATTTC internal element sequences between the two terminal GAGGATG-3Ј) with BAC4J4 clone containing an LTRs. In addition, non-autonomous elements lacking a autonomous Retand element as a template DNA. part or whole of the regions coding for retroelement After hybridization, detection was performed using proteins have been reported from several species. As CDP-Star detection reagent. BAC DNA was isolated these elements are not capable of encoding functional using the Qiagen Plasmid Midi kit. BAC DNA was proteins required for their replication and/or transposi- digested with restriction enzymes and desired frag- tion, they can be propagated in the genome only at the ments containing parts of Retand elements were sub- presence of a functional intact element. However, the cloned into linearized and dephosphorylated plasmid molecular mechanisms producing these non-autono- pBluescript IISK+ and transformed into XL1-Blue mous elements, as well as the population dynamics of cells. Subcloned fragments were sequenced using autonomous and non-autonomous elements in plant GeneJumper Primer Insertion Kit for Sequencing genomes are only poorly understood. Moreover, (Invitrogen) based on transposon-mediated insertions except for a few extensively sequenced model species of primer binding sites. All parts of the Retand ele- like rice (Jiang et al. 2002a, b), only a little is known ments were read 2–4 times (by VBC-Genomics, about the occurrence of these elements in other plants. Vienna) and the clone sequences were assembled with Here we describe Retand, a novel family of Ty3/ Staden Package software (Staden et al. 1996). gypsy retrotransposons including the full-length and the corresponding non-autonomous elements occur- Fluorescence in situ hybridization ring in the genome of Silene latifolia. These elements are localized at subtelomeric heterochromatin regions In order to synchronize the germinating seeds of S. of a vast majority of chromosomes and are transcrip- latifolia and S. viscosa (seeds come from the seed col- tionally active. An interesting feature of these retro- lection of the Institute of Biophysics, Brno, Czech transposons is the presence of an array of tandem Republic), the DNA polymerase inhibitor aphidicolin repeats within their sequences which is more ampliWed was added for 12 h, and mitoses were then accumu- in the non-autonomous element, as well as the lated with oryzalin as described in Siroky et al. (2001). presence of two extra ORFs in antisense orientation Slides were treated as described in Lengerova et al. located upstream of right LTR of the autonomous (2004) with slight modiWcations. Slide denaturation element. was performed in 7:3 (v/v) formamide: 2£ SSC for 2 min at 72°C. Slides were immediately dehydrated through 50, 70, and 100% ethanol (¡20°C), and air Materials and methods dried. The probe was denatured at 70°C for 10 min, and 100 ng of the denatured probe was added at room Cloning and hybridization temperature and hybridized for 18 h at 37°C. Slides were analyzed using Olympus Provis microscope, and The construction of a sample BAC library of the male image analysis was performed using ISIS software S. latifolia and the preparation of a membrane with 960 (Metasystems). DNA was labeled with Fluorolink BAC clones were described in Lengerova et al. (2004). Cy3-dUTP (Amersham Pharmacia Biotech) (red Screening was performed by hybridization with Alk- labeling) in combination with the nick translation mix Phos Direct hybridization kit (Amersham Pharmacia (Roche) or with SpectrumGreen direct-labeled dUTP Biotech) according to the manufacturer’s protocol. (green labeling) and the Nick Translation kit (both Probes were prepared by PCR ampliWcation of LTR Vysis). 123 Mol Gen Genomics RNA isolation and RT-PCR hybridization (FISH) signals, we found a novel non- autonomous retroelement that we named Retand-1 Total RNA was isolated from S. latifolia male plant tis- because it contained a tandem repeat array instead of sues (leaves, Xower buds or roots) by extraction with genes (retroelement with tandem repeat). This ele- RNA Blue (TopBio) and chloroform. All RNA samples ment was 3,679 bp long and contained almost identical were treated with RNase free DNase (Gibco-BRL) to long terminal repeats (LTRs) diVering in only one base remove any contaminant DNA. Reverse transcription at 3Јend (99.8% identity) indicating a recent insertion was carried out with the SuperScriptIII First-Strand of the element. The LTRs began with TG and ended Synthesis System for RT-PCR (Gibco-BRL) by random with CA, similarly as the LTRs of most plant retro- priming method, with 1 g of the template RNA. RNase transposons. We propose that the sequence TATA- H was added to remove the RNA template from the AAT located in the center of left LTR could serve as cDNA:RNA hybrid molecule after Wrst-strand synthe- putative transcription initiation signal for Retand-1. At sis. The RT-PCR reaction mix (50 l) consisted of 1£ both ends of the element, we found 5 bp direct repeats PCR buVer, 0.2 mM dNTP, 0.2 M primers, 1.5 mM CTTTG, which probably represent the target site MgCl2, 1 U of Taq polymerase (TopBio) and 10–100 ng duplications (TSD) generated during the element inte- of reverse-transcribed RNA or equal amount of reverse gration. Several features necessary for retroelement transcriptase-untreated RNA as a negative control. The replication were also identiWed, including primer bind- following primers were used in the RT-PCR experi- ing site (PBS) located downstream of the left LTR and ments (sequences of primers see above): LTR-F1, LTR- polypurine tract (PPT) located upstream of the right R1, POL-F1, POL-R1, STR1-F1, STR1-R1, ORF3-F1 LTR (Fig.
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