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Structure and Coding Properties of Bsl, a Maize Retrovirus-Like Transposon

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Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6235-6239, August 1989 Genetics Structure and coding properties of Bsl, a maize retrovirus-like transposon (transposable element/retrotransposon/reverse transcription/translation frameshift) YOUNG-KWAN JIN AND JEFFREY L. BENNETZEN Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Communicated by John D. Axtell, May 12, 1989 (receivedfor review July 11, 1988) ABSTRACT We have sequenced Bs), an insertion element do not integrate into chromosomal DNA (10). Recently, isolated from a null allele of the Adhi locus encoding alcohol Schwarz-Sommer et al. (11) have found that the Cin4 inser- dehydrogenase in maize. The Bs) element is 3203 base pairs tion element of maize could encode a reverse transcriptase (bp) in length, has 302-bp identical long terminal direct repeats activity and may fall into the subcategory of nonviral retro- (LTRs), and created a 5-bp flanking direct duplication oftarget transposons. AdhI DNA upon insertion. The 5' LTR is followed by a In 1985, Johns and coworkers (12) reported the isolation canonical primer binding site with homology to the plant and terminal repeat sequence of the Bs) insertion element initiator methionyl-tRNA, and the 3' LTR is directly preceded that they isolated from a null allele of the maize Adhl locus by a polypurine stretch like that observed in retroviruses and encoding alcohol dehydrogenase. The identical, direct ter- retrotransposons. Bs) encodes two overlapping open reading minal repeats of this approximately 3.3-kilobase pair (kb) frames specifying peptides of 740 and 168 amino acids. The insertion and its low genomic copy number led these inves- longer open reading frame specifies a peptide with amino acid tigators to describe Bs) as a "copia-like" transposable ele- homology to the protease and nucleic acid binding moiety of ment of maize. We report here the complete nucleotide retroviruses and retrotransposons. The deduced amino acid sequence* ofBs) and both structural and functional evidence sequence encoded by Bsl lacks convincing homology to the that show that BsJ is the DNA form of a retrovirus-like polymerase (reverse transcriptase) encoded by retroposons, transposon. despite the fact that this polymerase-encoding domain is rou- tinely the most conserved region of any such element. The MATERIALS AND METHODS sequence and relatively small size of Bs) suggest that this element is a deleted retrotransposon that inserted into AdhM Biologicals. Escherichia coli strain HB101 was used for all with the aid of a reverse transcriptase function provided in plasmid cloning, subcloning, and maintenance. Plasmids pJ1 trans. In vitro transcribed Bsl complementary RNA was trans- and pK18, containing overlapping portions of the Bs) ele- lated in vitro to produce both a protein of 81 kDa representing ment, were provided by M. A. Johns (Northern Illinois open reading frame 1 (ORF1) and one of the 95-kDa size University). Bs) contains a single HindIII site at base pairs predicted for the frame-shifted fusion of ORF1 and ORF2. As (bp) 1937-1942. Plasmids pJL-24, pJL+ 138, pJL+ 174, with many other retroposons, the efficiency of translational pJL+248, and pJL+267 were constructed by ligating a initiation at the AUG beginning ORF1 was not noticeably BamHI/HindIII fragment containing the 5' 60% of Bs) from affected by the presence of one or more upstream, unproduc- pK18 and a Xba I/HindIII fragment containing the 3' 40% of tive AUGs in the complementary RNA transcript. Bs) from pJ1 into a BamHI/Xba I-digested derivative of pGEM1 (Promega Biotec). BamHI and Xba I linkers were obtained from New England Biolabs. Restriction enzymes By both structural and mechanistic criteria, eukaryotic in- were purchased from Boehringer Mannheim, BRL, and New sertion sequences and transposable elements can be divided England Biolabs. Klenow DNA polymerase was obtained into two major categories. One class of elements is bounded from Boehringer Mannheim, and BAL-31 was supplied by by inverted terminal repeat sequences and transposes New England Biolabs. Radioactive triphosphates and wheat through excision (1, 2) and/or enhanced replication (3) ofthe germ translation extracts were purchased from Amersham. integrated element. Examples of this class of transposable [35S]Methionine and [3H]leucine were from New England elements are the controlling elements of maize (4) and the P Nuclear. Phage T7 RNA polymerase, RNasin, and rabbit elements of Drosophila (5). A second class of eukaryotic reticulocyte lysates were supplied by Promega Biotec. All insertion sequences, the "retroposons," integrate into the enzymes and extracts were used approximately as specified chromosome after reverse transcription of element-encoded by the manufacturer. RNA (6), are particularly abundant in animals, and are the DNA Sequencing. DNA was sequenced by the technique of only type of insertion sequence yet discovered in fungi. Both Maxam and Gilbert (13) on restriction fragments end-labeled the retroviruses (6) and the LI elements (7) ofvertebrates and with Klenow DNA polymerase or phage T4 polynucleotide the "retrotransposons" Ty of yeast (8) and copia of Dro- kinase. DNA sequencing products were resolved on 43- sophila (9) fall into this latter category. cm-long 0.4-mm-thick 6%, 8%, 12%, and/or 20%o denaturing Despite the early discovery and characterization of numer- DNA ous distinct transposable elements in maize (1, 4), a canonical acrylamide gels. Between 200 and 350 nucleotides of retrovirus or retrotransposon has not been described in sequence were read from a given labeled end. plants. The DNA caulimoviruses ofplants have the structural In Vitro RNA Synthesis. Complementary RNA (cRNA) was properties and encode the reverse transcriptase activity separately synthesized off restriction enzyme-digested definitional of a retrovirus or retrotransposon but apparently Abbreviations: BSMV, barley stripe mosaic virus; CaMV, cauli- flower mosaic virus; LTR, long terminal repeat; ORF, open reading The publication costs of this article were defrayed in part by page charge frame; cRNA, complementary RNA. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M25397). 6235 Downloaded by guest on September 29, 2021 6236 Genetics: Jin and Bennetzen Proc. Natl. Acad. Sci. USA 86 (1989) pJL-24, pJL+138, pJL+175, pJL+248, and pJL+267 by duced amino acid sequence of Bs] does contain some ho- following the protocol advised by Promega Biotec and in mology with the most conserved consensus sequence of reactions containing added 7-methylguanosine(5')triphos- reverse transcriptase, it also specifies two nearby arginines pho(5')guanosine (New England Biolabs) to provide a 5' cap and a cysteine, which would be incompatible with all other (14). The quantity and integrity of in vitro transcribed Bsl reverse transcriptase sequences (Fig. 2). RNA were confirmed by agarose slab gel electrophoresis. The reverse transcription of DNA off retroviral or retro- In Vitro Translation and Analysis. Approximately equiva- transposon RNA is generally primed by the 3' end of a host lent quantities of Bsl cRNAs were added to either wheat cellular tRNA (6). This priming event often is initiated 2 bp germ or rabbit reticulocyte lysate translation extracts. Re- downstream from the 5' LTR. The sequence 5'-TGGTAT- actions were run for 1 hr, following the manufacturers' CAAAGGTCACCGATCCTGG-3', beginning 2 bp down- specifications. Translation products were resolved on 10%o or stream from the BsJ 5' LTR, shares 21 bp of homology with 12.5% polyacrylamide/NaDodSO4 gels containinga5% acryl- the 3' end of the only initiator methionyl-tRNAs sequenced amide stacking phase. Gels were immersed in Amplify (Am- in plants, those from the monocot wheat and the dicots ersham), dried, and fluorographed by standard procedures. Lupinous luteus and Phaseolus vulgaris (20, 21). The homol- ogy observed is found in three interrupted blocks of 8, 4, and 9 bp (Fig. 3), thereby providing a theoretical overall binding RESULTS energy equivalent to approximately 13 or 14 bp ofcontinuous Both strands ofthe BsJ element were DNA-sequenced by the homology. Initiator methionyl-tRNA sequences are highly technique of Maxam and Gilbert (13). Bs] is 3203 bp in length conserved in nature (20, 21), and it is likely that the maize and terminates in identical 302-bp direct repeats (Figs. 1 and initiator methionyl-tRNA has an identical 3' sequence. Re- 2). The BsJ element encodes two long, overlapping open verse transcriptions of Ty, copia, and CaMV (22) RNAs are reading frames (ORFs) that could specify peptides of 740 primed by initiator methionyl-tRNAs. amino acids and 168 amino acids (Figs. 1 and 2). Within the To compare the translational competence ofBsl RNA with sequence domain that encodes ORFi are two nonoverlapping the ORFs predicted by the Bsl DNA sequence, we engi- ORFs specifying peptides of 181 and 133 amino acids in a neered the Bs] element for expression off bacterial SP6 and second reading frame (Fig. 1). As observed for most inte- T7 promoters in vitro. The progressive exonuclease BAL-31 grated DNA forms of retroviruses and retrotransposons (6, was used to digest both strands of DNA toward and, in some 16), the long terminal repeats (LTRs) start with TG and end cases, into the 5' and 3' Bs] LTRs. Oligonucleotides encod- with CA, the 5' LTR is followed after 2 bp by the sequence ingBamHI (5') orXba I (3') restriction sites were ligated onto 5'-TGG-3', and the 3' LTR is directly preceded by a poly- the ends of the digestion products. These fragments were purine tract (Fig. 2). It is these consistent and strong retro- then inserted between the opposing T7 and SP6 promoters in viral homologies that permit the identification ofthe 5' and 3' the plasmid pGEM1, and the endpoints of the BAL-31 LTRs and orient the expected direction of expression ofBsJ digestions were identified by DNA sequencing.
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