©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Polymorphic Sequences Encoding the First Open Reading Frame Protein from LINE-1 Ribonucleoprotein Particles (*)

(Received for publication, August 24, 1994; and in revised form, November 18, 1994)

Vladimir O. Kolosha Sandra L. Martin (§)

From the Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mouse LINE-1 (L1) retrotransposon contains two open reading frames (ORFs). Three classes of the protein encoded by the first open reading frame (ORF1) are expressed in the mouse embryonal carcinoma cell line, F9; the apparent molecular sizes of these proteins are 41.3, 43, and 43.5 kDa. Two of these three proteins (41.3 and 43 kDa) are translated in vitro from full-length, sense-strand L1 RNA isolated from ribonucleoprotein particles. A reverse transcription-polymerase chain reaction approach was used to clone the ORF1 region from RNA isolated from ribonucleoprotein particles, then the coding capacity of these clones was examined using in vitro transcription and translation. Multiple sequences that encode ORF1 were recovered by this approach, indicating that multiple loci of L1 in the mouse genome are expressed in F9 cells. In addition, L1 sequences with intact ORF1 regions appear to be selectively enriched in the ribonucleoprotein particles.


INTRODUCTION

The LINE-1, or L1, (^1)family of interspersed repeats comprises at least 10% of the mammalian genome. Like other interspersed repeated DNA families in genomes of other organisms, L1 is dispersed and amplified throughout the genome by a series of duplicative transposition events. The majority of L1 elements in the mouse genome are 5`-truncated and rearranged, leaving only 10,000 full-length copies(1) . Truncated copies originate from active, full-length versions of the element by retrotransposition (1, 2, 3) . It has been estimated that only about 60 copies of L1 in the mouse genome are competent or active for transposition(4) .

The biochemical details of the intermediate steps in L1 retrotransposition are unknown. Nevertheless, it is reasonable to hypothesize that L1 retrotransposition involves the expression of a full-length, sense-strand L1 transcript and the polypeptide products of ORF1 and ORF2(1, 5) . Full-length, sense-strand L1 transcripts and ORF1 protein have been detected in both human and mouse cell lines, primarily teratocarcinoma or embryonal carcinoma(6, 7, 8, 9) , and also in mouse testis(10) . In the mouse embryonal carcinoma cell line, F9, full-length, sense-strand L1 RNA is found in ribonucleoprotein particles (RNP), which also appear to include ORF1 protein(6) . In addition, human L1 RNA appears to be present in a high-molecular weight complex with reverse transcriptase activity in Ntera2D cells(11) .

Due to the high copy number of L1 sequences in the genome, L1 is abundantly represented in the RNA population of most cells. However, most of the transcripts that contain L1 are the result of fortuitous transcription and are not intermediates in L1 retrotransposition. This high background of L1-containing transcripts, many of which are truncated and rearranged, makes it difficult to distinguish the transcript encoded by an active L1 element(s). Two active human L1 elements revealed themselves by retrotransposing into the human factor VIII and dystrophin genes(2, 3, 12) . Both of these truncated, defective insertions provided sequence tags that allowed isolation of their active progenitors(2, 3) . Recently, the first potential example of a similar insertional mutagenesis event in the mouse genome has been reported(13) . However, the amount of sequence data that was reported is insufficient to discern whether the inserted element was derived from an active version of mouse L1.

There are two major subfamilies of mouse L1, A and F. The A subfamily is evolutionarily the youngest and is transcriptionally active(14) . Within the A subfamily, there are three length polymorphisms in the ORF1 coding region(14) . The shortest of these length variations defines subgroup 1, which is represented by L1MdA2(15) . Subgroup 2 contains an additional 42 bp in the 5` end of the ORF1 coding sequence and is represented by L1MdA9(16) . Although L1 elements with the subgroup 2 polymorphism are at least as abundant in the genome as subgroup 1 elements, they are not thought to be transcribed in F9 cells (14) .

In this study, the L1 sequences that are transcribed in F9 cells are characterized in more detail. In particular, we focused on RNA from L1-RNPs, which are enriched for 7.5-kb, full-length L1 transcripts. The ORF1 coding capacity of these transcripts was examined in order to identify the pool of L1 elements that express ORF1 protein in F9 cells and, therefore, are likely to represent active elements.


MATERIALS AND METHODS

Cell Culture and Cell Labeling

F9 embryonal carcinoma cells were obtained from Dr. E. Barry Pierce (Department of Pathology, University of Colorado Health Sciences Center) and maintained in modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone Laboratories), on gelatin-coated (0.1%), plastic tissue culture dishes. For metabolic labeling, cultures were incubated in methionine-free modified Eagle's medium containing dialyzed fetal bovine serum (1%) for 48 h, then 0.5 mCi/ml L-S in vitro cell labeling mix (Amersham Corp.) were added to the culture medium and growth was continued for 15 h.

Immunoprecipitation

Following metabolic labeling, F9 cells were washed twice in phosphate-buffered saline, lysed, and treated as described previously(7) . Cell extracts were diluted 20-fold (to 600 µl) in RIPB buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 0.25 mM EDTA, 0.05% Nonidet P-40, 0.1% Tween 20) with 1 mM phenylmethylsulfonyl fluoride and were centrifuged at 16,000 times g for 10 min. RNP complexes in the supernatant were disrupted by adding RNase A (10 µg/ml); proteins that interact nonspecifically with protein A-Sepharose were removed by preincubating with 20 µl of beads (7%, Sigma) for 20 min at 4 °C. After a brief centrifugation to sediment the protein A-Sepharose beads, the supernatant was removed to a new tube and incubated with antibody for 6 h at 4 °C in RIPB. 6 µg of affinity-purified, anti-ORF1 antibody (FP1) or preimmune IgG (described in (7) ) were used for each incubation. 80 µl of 7% protein A-Sepharose were added and incubated 2 h at 4 °C. Bound proteins were recovered after three washes in RIPB, one wash in RIPB containing 0.05% SDS, and one wash in TE (10 mM Tris-HCl, pH 7.5, and 0.1 mM EDTA). For each wash, beads were resuspended, then recovered by centrifugation at 2000 times g for 1 min. The final protein A-Sepharose pellet, containing washed immune complexes, was resuspended in SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), boiled for 5 min, then fractionated by SDS-PAGE (8-9% gel, bisacrylamide:acrylamide (1:30)).

Western Blot Analysis

50-300 µg (BCA assay, Pierce) of F9 cytoplasmic protein extract, or proteins following immunoprecipitation, were separated by SDS-PAGE (9%) and transferred to nitrocellulose. Blots were blocked in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) with 1% bovine serum albumin. For Western blots of immunoprecipitates, horseradish peroxidase-conjugated goat anti-rabbit IgG (Boehringer Mannheim) was also added to reduce later detection of IgG. After blocking, anti-L1 ORF1 antibodies were added (250 ng/ml) and allowed to bind for 16 h at 4 °C. Blots were washed three times 10 min each, in TBST with 0.05% Nonidet P-40 and 0.05% SDS, before adding alkaline phosphatase-conjugated goat anti-rabbit IgG (1:7500 dilution, Promega) or I-protein A (1:15,000; ICN). Following a 2-3-h incubation at room temperature, blots were washed four times as above, then developed (alkaline phosphatase, Promega) or exposed (I-protein A, Hyperfilm-MP, Amersham).

Plasmids, Probes, and Oligonucleotides

A 6050-bp region of CE102, extending from a SacII site at position 951 to an ApaI site at position 7001 of L1MdA2(15) , which includes all of ORF1 and ORF2, was subcloned into pBlueScript SK- (pVK15) and used for in vitro transcription and translation studies (Fig. 1). A 800-bp BamHI fragment from the 5` end of L1MdA9 was gel-purified for use as a hybridization probe (probe 1, Fig. 1; (6) and (16) ). The fragment was labeled with [alphaP]dCTP (Amersham) using a Random Primed DNA labeling kit (U.S. Biochemical Corp.).


Figure 1: Structure of mouse L1 and location of probes used for this study. The structure of L1MdA2 (15) is shown, including the location of relevant restriction endonuclease cleavage sites; A(n) indicates the A-rich tail at the 3` end of the element; the graylines on the extreme ends indicate sequence in which the A2 element was inserted (target sequence). The line labeled A2 represents the in vitro transcript from pVK15 linearized with Esp31, used for in vitro translation of ORF1. Locations and strand specificity of the oligonucleotides used for PCR and DNA sequencing are indicated by the arrows marking their 3` end.



Oligonucleotides were purchased from DNA Express (Colorado State University) for use as PCR or sequencing primers. Oligonucleotides, o21 (5`-GAAGAACAAGCTTTTAACAGTG-3`) and o22 (5`-GAGTTGGAATTCTGTTCTTGTGG-3`), were used for the PCR amplification. Purified plasmids were sequenced with the T7 and T3 promoter primers (Promega) and o2 (5`-TTAGTTCTAGTATGGTTT-3`).

RNA Preparation and Analysis

Total RNA was purified from cytoplasmic extracts of F9 cells, or from fractions enriched in L1 RNP following sucrose density gradient centrifugation, as described previously(6) . In order to obtain enough RNA for in vitro translation experiments, the L1 RNP fraction from several sucrose gradient fractionations of F9 cell extract were combined (approximately 3 times 10^9 cells). Poly(A) RNA was isolated using the PolyATtract® system (Promega). For Northern blots, RNA was fractionated in 1% agarose-2.2 M formaldehyde gels, then transferred to Hybond N (Amersham). Blots were hybridized in 1.0 M NaCl, 1% SDS, and 10% dextran sulfate overnight at 64 °C, then washed in 1 times SSC and 0.1% SDS once at room temperature, followed by two or three stringent washes (10-15 min) in 0.2 times SSC and 0.1% SDS at 63 °C.

RT-PCR, DNA Cloning, and Sequencing

cDNA was synthesized from poly(A) RNA using oligo(dT) as a primer and avian myeloblastosis virus reverse transcriptase (Promega). The resulting cDNA was amplified with the primers o21 and o22 (94 °C for 1 min, 56 °C for 1 min and 10 s, and 72 °C for 2 min and 20 s) using TaqI polymerase buffer containing 2.2 mM MgCl(2) (Promega). The amplified product was cleaved with EcoRI and HindIII, purified from a 1% agarose gel (GeneClean, Bio 101), and cloned into pBlueScript. Colonies containing L1 ORF1 fragments were identified by colony hybridization using probe 1 (Fig. 1). The plasmids were isolated and used directly for DNA sequence analysis(17) .

In Vitro Transcription and Translation

In vitro transcription utilized 1.0 µg of linearized DNA plasmid for template in the presence of m7G(5`)ppp(5`)G (Pharmacia Biotech Inc.) and T3 or T7 polymerase (Promega). Following transcription, the DNA template was digested with 10-20 units of RNase-free DNase (Boehringer Mannheim) for 30 min at 37 °C. The yield of RNA was measured by absorbance at 260 nm after purification of the product through a Sephadex G-50 (Pharmacia) spin column. The integrity of in vitro transcripts was monitored by their appearance(18) .

These transcripts were used for in vitro translation in rabbit reticulocyte lysates (Promega) in the presence of [S]methionine (Amersham). Proteins were analyzed by SDS-PAGE following immunoprecipitation. Gels were treated for fluorography in EN^3HANCE (DuPont), then dried and exposed to Hyperfilm-MP (Amersham).


RESULTS

The organization of a mouse L1 element and the location of probes used for these studies are shown in Fig. 1.

Characterization of Proteins Recognized by L1 ORF1 Antibody from F9 Cells

Previous studies suggested that multiple forms of ORF1 protein are expressed in mouse cells of different types(6, 7, 10) . In extracts from F9 cells that have been metabolically labeled with [S]methionine, two polypeptides that immunoprecipitate with ORF1 antibody are detected (Fig. 2A, lane2, arrows). The largest of these two can be further resolved into two distinct bands by SDS-PAGE following immunoprecipitation of F9 extract and detection by Western blotting (Fig. 2B, lane2, arrows). Therefore, at least three forms of ORF1 protein are expressed in F9 cells, with apparent molecular sizes of 41.3, 43.0, and 43.5 kDa. None of these three proteins (41.3, 43, and 43.5 kDa) is immunoprecipitated with preimmune IgG (Fig. 2, panelA, lane3, and panelB, lane1). The smallest form of ORF1 protein from F9 cells (41.3 kDa) comigrates with the ORF1 protein translated in vitro from L1MdA2 (Fig. 2A, lane1). Results of potato acid phosphatase treatment of F9 cell extracts prior to immunoprecipitation and SDS-PAGE suggest that the 43.5-kDa ORF1 protein is likely to be a phosphorylated product of the 43-kDa protein (data not shown).


Figure 2: Multiple forms of L1 ORF1 protein in F9 cells. Arrows indicate ORF1 proteins. A, immunoprecipitation using ORF1 antibody following in vitro transcription and translation of L1MdA2 (lane1, A2 in Fig. 1) or from cytoplasmic extracts of F9 cells after metabolic labeling (lane2). Preimmune serum was used for the immunoprecipitation of the F9 cell extract in lane3. B, Western blot analysis of immunoprecipitated proteins detecting ORF1 antibody with alkaline phosphatase-conjugated goat anti-rabbit IgG. F9 cytoplasmic extract was immunoprecipitated with ORF1 antibody (lane 2) or a 5-fold excess of preimmune IgG (lane1). The stronger upper band, particularly evident in lane1, is the IgG used for immunoprecipitation. C, Western blot of F9 cytoplasmic extract using ORF1 antibody and I-protein A (lane 1). Lane 2 contains the 44.6-kDa ORF1 fusion protein expressed in bacteria(7) . A larger, 53-kDa protein (lane 1) was variably observed. p43 and p43.5 are not resolved on this gel.



Quantitative phosphorimage analysis of the ORF1 proteins that are immunoprecipitated from F9 cells and detected by Western blotting with I-protein A (Fig. 2C, lane1) reveals that the larger two forms (43 and 43.5 kDa) are approximately 5-fold more abundant than the smaller form. All three of these ORF1 polypeptides are rare in F9 cells; comparison of the signals obtained on Western blots for known amounts of F9 protein extract and bacterially expressed ORF1 fusion protein indicates that the three forms of ORF1 in F9 cells together account for less than 0.001% of the protein recovered in the cytoplasmic extract (Fig. 2C).

In Vitro Translation of L1 ORF1

In light of the fact that the majority of L1 cDNA clones described previously (14) would be expected to encode a 41.3-kDa ORF1 protein, it was surprising that three forms of ORF1 protein were present in F9 cells and that the 41.3-kDa form was the least abundant. In order to determine whether the 43.0- and 43.5-kDa ORF1 proteins result from posttranslational modification of the 41.3-kDa form, or if they are encoded by a novel polymorphic subset of L1, the ORF1 proteins obtained following in vitro translation of F9 RNA were examined.

Initial attempts to detect L1 ORF1 protein following in vitro translation of poly(A) RNA from F9 cells and immunoprecipitation with ORF1 antibody were not successful, in spite of an efficient in vitro translation reaction. This is most likely due to a combination of the relatively low abundance of L1 RNA in the RNA population and its poor translation efficiency. The presence of a C at -3 relative to the start of translation results in a weak Kozak consensus sequence (19) for the subgroup of L1 that has been reported to be predominantly transcribed in F9 cells (subgroup 1; (14) ).

To enrich the relative abundance of L1 sequences in the RNA for in vitro translation, poly(A) RNA from L1 RNP was used (Fig. 3). L1 RNP were prepared by sucrose gradient fractionation; this preparation is known to be enriched in full-length 7.5-kb L1 RNA, relative to truncated versions of L1 and the majority of other cellular mRNAs (e.g. actin; (6) ). A rabbit reticulocyte lysate programmed with this poly(A) RNA (isolated from about 10^9 cells) reproducibly yielded two proteins (p41 and p43) that could be immunoprecipitated from a more complex mixture of proteins with ORF1 antibody, but not with preimmune IgG (Fig. 4). Mixing experiments demonstrate that p41 comigrates with ORF1 protein translated in vitro from an L1MdA2 RNA template (data not shown). It seems unlikely that p41 is posttranslationally modified in the reticulocyte lysate after translation from L1 RNP RNA to give rise to p43, because there is no evidence for such a modification of the p41 translated from L1MdA2 RNA in a parallel reaction. Since p41 appears to be identical to ORF1 from L1MdA2, based on immunoreactivity and electrophoretic mobility, p41 and p43 must be encoded by distinct mRNAs.


Figure 3: Northern blot of L1 RNA from F9 cells. Lanes contain poly(A) RNA isolated from sucrose gradient fractions enriched in L1-RNP (lane1) or from total RNA from F9 cells (lane2). 180 pg of 6.0-kb RNA transcribed in vitro (A2 in Fig. 1) was loaded in lane3. The blot was hybridized to probe 1 (Fig. 1). Arrow indicates the 7.5-kb L1-RNA.




Figure 4: SDS-PAGE of proteins translated in vitro. The translation was programed with poly(A) RNA from L1 RNP (the same as shown in Fig. 3, lane1) and immunoprecipitated with preimmune IgG (lane1) or ORF1 antibody (lane2). The total translation reaction is shown in lane3. Arrows indicate the two proteins that reproducibly immunoprecipitated with ORF1 antibody, p41 and p43.



As was observed with proteins isolated from F9 cells, there is approximately 5-fold more 43-kDa ORF1 protein than 41-kDa ORF1 protein after in vitro translation ( Fig. 2and Fig. 4). Because of the different methods of protein detection, these quantitation results are tentative; nevertheless, the fact that there is the same ratio between p41 and p43 in two different types of experiment suggests that RNAs encoding both p41 and p43 are similarly enriched during RNP fractionation. This assumes that the relative efficiency of translation of the two messages is the same in reticulocyte lysates and in F9 cells.

RT-PCR, Cloning, and Analysis of ORF1 Clones by in Vitro Translation

In order to characterize the ORF1 coding capacity of 7.5-kb L1 RNP RNA in more detail, RT-PCR products obtained from that RNA were cloned and screened by in vitro transcription and translation. Oligo(dT) was used to prime cDNA synthesis from the same poly(A) RNP RNA (Fig. 3) that was used for in vitro translation (Fig. 4). The cDNA was subjected to PCR amplification using oligonucleotide primers o21 and o22, which bracket the ORF1 region of all previously characterized, A-type mouse L1 elements (Fig. 1). Two fragments were amplified reproducibly, with the smaller fragment being always far more abundant (Fig. 5, lane 2). No DNA amplification product was detected using identical reaction conditions except for omitting reverse transcriptase from the cDNA synthesis step (Fig. 5, lane3). Identical results were obtained using the same cDNA template that was diluted 5-fold, demonstrating reproducibility (data not shown). The two PCR amplification products differ in length by approximately 50 bp, roughly the difference expected for elements from subgroups 1 and 2 (see Introduction).


Figure 5: RT-PCR amplification of the ORF1 region of RNA isolated from L1 RNP. Ethidium bromide-stained agarose gel shows DNA digested with HindIII (lane1), the RT-PCR reaction (lane2), and the corresponding PCR reaction in the absence of reverse transcriptase (lane3).



The PCR-amplified fragments were cloned into pBluescript. 32 clones, known to contain L1 ORF1 by hybridization to probe 1 (Fig. 1), were pooled into six groups (four to six clones in each group) and characterized by in vitro transcription followed by in vitro translation. The resulting ORF1 proteins from three of the six groups contained clones encoding a larger form of ORF1 than the one encoded by L1MdA2 (41.3 kDa). Further analysis of the coding capacity of individual clones from two of these groups revealed that seven (cD33, cD36, cD37, cD40, cD42, cD46, and cD47) out 10 encode a protein that comigrates with ORF1 translated in vitro from L1MdA2 (Fig. 6A). Of the remaining three, two clones (cD35 and cD43) encode a 42.0-kDa protein, and the third, cD39, encodes a 42.5 kDa protein (Fig. 6A). To compare the mobility of this 42.5-kDa protein directly with p43, the two were mixed and examined by SDS-PAGE. The result of the mixing experiment demonstrates that these two proteins are distinct (Fig. 6B). Thus, the majority of ORF1-containing clones that were isolated by RT-PCR and studied by in vitro translation encode a protein whose mobility on SDS-PAGE is indistinguishable from the ORF1 encoded by L1MdA2, and none of them encode p43.


Figure 6: In vitro translation of L1 ORF1 clones. A SDS-PAGE of ORF1 proteins encoded by the individual, cloned PCR-amplified fragments (indicated by their cD number). Lanes marked A2 or FP1 contain the products of in vitro transcription and translation from pVK15 or the construct used to make the ORF1 antibody(7) , respectively. B, SDS-PAGE of proteins translated in vitro from A2, cD39, and poly(A) RNA from RNP. All proteins were immunoprecipitated with ORF1 antibody prior to SDS-PAGE.



cDNA Sequences Encoding ORF1 Proteins Detected by the Screening Procedure

In order to characterize the ORF1 region of the clones isolated by this procedure in more detail, and to gain some appreciation for the amount of sequence polymorphism present in the L1 RNP population, the DNA sequences of 10 cDNA clones were determined in the NH(2)-terminal, most variable region of ORF1 (Fig. 7). Of the seven clones encoding p41 (i.e. their ORF1 comigrates with ORF1 from L1MdA2), only one, cD37, is identical to L1MdA2 throughout the sequenced region. Among the other six members of this category, two (cD33 and cD47) contain a single, silent nucleotide substitution and the remaining substitutions are replacements. Two cDNAs replace Lys-60 with Arg (cD36) or Glu (cD42), a third replaces Arg-6 with His (cD40), the fourth replaces Pro-30 with Ala (cD33), the fifth replaces Ser-68 with Gly (cD46), and the sixth replaces Leu-65 with Ser (cD47). The two cDNAs encoding the slightly larger, 42.0-kDa ORF1 protein are identical to one another throughout the sequenced region and contain a 42-nucleotide insertion, as well as several nucleotide substitutions relative to L1MdA2. The remaining clone, encoding the 42.5-kDa form of ORF1 (cD39), belongs to subgroup 1, but contains several amino acid substitutions relative to L1MdA2 that are scattered throughout its open reading frame. Only one of these replacements is found in the region shown in Fig. 7, replacing Pro-30 of L1MdA2 with Ser.


Figure 7: DNA sequence alignment of L1 ORF1 clones in the NH(2)-terminal variable domain. Numbering starts with the A in the first ATG (bold). The cDNAs, which represent cloned RT-PCR amplified fragments, are numbered between 33 and 47. Only the positions that differ from the ORF1 consensus sequence (14) are shown. Dottedline indicates the region of the 42-bp insertion that defines subgroup 2. Slash (/) denotes the end of the determined sequence. The sequences of cD35 between +1 and +130, and cD47 5` to +81, were not determined. The HindIII site that was introduced in the sequence during PCR amplification is underlined.



The sequence of 198 bp from the 3` end of seven of these cDNA clones was determined using the T3 primer. No amino acid replacements were detected in the carboxyl-terminal region (153 bp) of ORF1 between L1MdA2 and cD clones 33, 37, 40, 46, and 47. The only silent substitution is found 18 bp upstream of the termination codon in cD40. The two clones encoding larger forms of ORF1, cD39 and cD43, contain 2- and 3-bp substitutions, respectively, in this region.


DISCUSSION

Sucrose gradient fractionation of F9 cell extracts leads to an enrichment of full-length L1 RNA in a high molecular weight nucleoprotein complex, L1 RNP(6) . In vitro translation of poly(A) RNA isolated from these RNP yields two proteins that are recognized by ORF1 antibody and have identical mobilities by SDS-PAGE to the proteins from F9 cells (p41 and p43). A third form of ORF1 is detected in F9 cell extracts but not after in vitro translation of L1 RNP RNA (p43.5) and is likely to result from posttranslational modification of p43.

More detailed characterization of the L1 RNA present in RNP by in vitro translation, RT-PCR, and DNA sequence analysis demonstrates that it is a heterogeneous population of L1 sequences, rather than a single RNA species. Clearly, the RNP RNA contains distinct transcripts that specify translation of the 41- and 43-kDa forms of ORF1, yet clones that encode p43 were not isolated in this study. The most likely explanation for the absence of clones encoding p43 after RT-PCR is that those cDNA(s) were not amplified with one or both of the primers that were used for the PCR. p43 could be a retrotransposed, truncated L1 (but retaining enough of ORF1 to be recognized by the antibody) that has acquired a new 5` sequence that includes a promotor for transcription in F9 cells. Alternatively, p43 could be encoded by either an entirely new family of L1 that is represented by small number of copies in the genome, a rearranged element, or an unrelated protein that fortuitously shares an epitope with L1. The latter possibility is unlikely because the mRNAs encoding p41 and p43 cofractionate as RNP through sucrose gradients in conditions where polysomes are known to be disrupted. Additional experiments to isolate and characterize the sequence(s) encoding p43 are in progress.

L1 RNP transcripts that encode a 41-kDa form of ORF1 protein are heterogeneous, since six out of seven of them contain at least one amino acid substitution relative to each other and to L1MdA2. This high frequency of replacement substitutions (6 replacement/2 silent) is expected from a coding sequence evolving in the absence of selective constraint (20) and is also consistent with the high variability observed between species in the NH(2)-terminal region of L1 ORF1(21) . Thus, it seems likely that this part of the protein has little impact on its functional role.

ORF1 was intact in all 10 clones that were tested individually in this study using in vitro translation. This result contrasts with the results of a previous study, where 2 out of 10 ORF1 sequences contained a single nucleotide deletion that disrupted the coding sequence of ORF1(14) . Seven of the remaining sequences were open throughout the region studied, but this region was too small to conclude that ORF1 is intact, and coding capacity was not evaluated by translation(14) . The relatively higher proportion of clones with an intact ORF1 in cDNAs derived from poly(A) RNA derived from L1 RNP compared to F9 suggests that there is an enrichment for transcripts that encode intact ORF1 in RNP over a more heterogeneous population of L1 transcripts present in F9 cells.

A similar story emerges from studies of human L1. A protein corresponding to ORF1 could not be translated in vitro from two of five cDNA clones that were derived from full-length RNA from NTera2D cells(8, 9) . The ORF1 proteins encoded by the remaining three cDNA clones differed in mobility on SDS-PAGE from endogenous ORF1 protein isolated from the same cells, yet the ORF1 encoded by a known, active human L1 had the same mobility on SDS-PAGE as the endogenous ORF1 in NTera2D cells(22) . This result again suggests some type of selection for a subset of the transcribed sequences leading to production of ORF1 protein and active transposition. The two elements of human L1 that are known to be active for transposition contain intact ORF1(2, 3) .

These observations, together with sequence analysis which supports evolutionary arguments that active elements dominate the retrotransposition process(1) , suggest that retrotransposition is most likely to occur when L1 ORF proteins are supplied in cis from the L1 transcript. This leads to the hypothesis that L1 RNP, likely intermediates in L1 transposition, are assembled co-translationally, and makes the prediction that only L1 transcripts encoding functional ORF1 can be assembled into RNP complexes. This prediction is supported by the results of this study that demonstrate an enrichment for intact ORF1s in the full-length L1 transcripts found in RNPs. Thus, it appears likely that selection of full-length transcripts with intact ORF1 coding capacity from L1 RNP provides a useful handle for isolation of an active version of mouse L1.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM40367 (to S. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16662[GenBank]-U16672[GenBank].

§
To whom correspondence should be addressed: Dept. of Cellular and Structural Biology, Box B111, University of Colorado School of Medicine, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-270-6284; Fax: 303-270-4729.

(^1)
The abbreviations used are: L1, LINE-1; bp, base pair(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; RNP, ribonucleoprotein particle(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank H. Srere and S. Trelogan for a critical review of the manuscript.


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