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Volumn 283, Issue 5407, 1999, Pages 1530-1534

Exon shuffling by L1 retrotransposition

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; DNA; RNA DIRECTED DNA POLYMERASE;

EID: 0033525586     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5407.1530     Document Type: Article
Times cited : (507)

References (39)
  • 10
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    • J. V. Moran et al., Cell 87, 917 (1996).
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    • note
    • The mutant initiation codon and artificial splice acceptor sequences were introduced into pmneol by polymerase chain reaction (PCR). The oligonucleotide primers SJ1 (5′-AAAGAATTCTACTAACTCTCTTCTCTCCTGCAGATAGGATCGGCCATTGAAC), SJ1 + 1 (5′-AAAGAATTCTACTAACTCTCTTCTCTCCTGCAGCATAGGATCGGCCATTGAAC), or SJ1 + 2 (5′-AAAGAATTCTACTAACTCTCTTCTCTCCTGCAGCCATAGGATCGGCCATTGAAC) were used in conjunction with 1720NEOS (5′-TGCGCTGACAGCCGGAACACG) to generate a 166-bp product. Reactions (one cycle: 94°C for 10 min: 30 cycles: 94°C for 1 min. 50°C for 30 s, 72°C for 30 s; one cycle: 72°C for 10 min) were performed as described (10). PCR products were digested with Eag I and Eco Rl, and the 0.06-kb fragments were used to replace the SV40 promoter in pmneol. The mneolRF1 to RF3 cassettes were subcloned into an engineered Sma I site in the 3′ UTR of L1.2 (10), and 7.9-kb Not I-Bam Hl fragments containing the engineered L1s were subcloned into pCEP4 (InVitrogen) to create pJM101 RF1 to RF3. We replaced the L1.2 ORFs with a 5.9-kb Not I-Bst Z171 from JM101/L1.3 to create pJM101/L1.3 RF1 to RF3.
  • 13
    • 0344148920 scopus 로고    scopus 로고
    • note
    • R colonies were isolated with the Fast Track Kit (InVitrogen). cDNAs were synthesized from 5 μg of poly(A) RNA with a cDNA synthesis Kit (Stratagene). Products greater than 500 bp were isolated from a cDNA size fractionation column (Gibco-BRL), ligated into Uni-ZAP XR vector (Stratagene), and packaged with Gigapack III packaging extracts (Stratagene). The cDNA library was plated at a density of 10,000 to 30,000 plaques per plate, and ∼500,000 clones were screened with a 0.46-kb neo probe. Secondary and tertiary screens were used to purify positive clones. Neo-containing inserts were liberated from the positive plaques with the ExAssist/ SOLR in vivo excision protocol (Stratagene) and were sequenced with an Applied Biosystems DNA sequencer (ABI 377).
  • 14
    • 0029968488 scopus 로고    scopus 로고
    • H. M. Albertsen et al., Genomics 33, 207 (1996). The sequences upstream of neo in the cDNA dones pJET1 to pJET7 were used to search available databases in the Baylor College of Medicine search launcher (kiwi. imgen.bcm.tmc.edu:8088/search-launcher/launcher. html). We determined that L1.3 inserted into intron 7 of the DOC2 gene (accession number U41111). The neo cDNA sequence was fused to spliced exons 1 to 7 of the DOC2 gene. Notably, the exon 7-intron 7 junction in figure 1 of the cited paper is misplaced by one codon. The last triplet of the DOC2 exon 7 sequence is TCG. The fusion sequence is shown in Fig. 1C.
    • (1996) Genomics , vol.33 , pp. 207
    • Albertsen, H.M.1
  • 15
    • 0027245959 scopus 로고
    • J. G. Patton, E. B. Porro, J. Galceran, P. Tempst, B. Nidal-Ginard, Genes Dev. 7, 393 (1993). The cDNA sequence upstream of the neo gene in pJET2 was derived from the PSF gene (accession number A46302). The PSF gene undergoes alternative splicing to generate two different mRNAs. The mutated ATA of mneolRF1 was spliced to the fused upstream PSF exons precisely at the position in the cDNA that is known to undergo alternative splicing. Thus, we deduce that L1.3 inserted into the intron either immediately upstream or immediately downstream of the alternatively spliced exon.
    • (1993) Genes Dev. , vol.7 , pp. 393
    • Patton, J.G.1    Porro, E.B.2    Galceran, J.3    Tempst, P.4    Nidal-Ginard, B.5
  • 16
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    • note
    • -32) to a human EST (accession number AA004013).
  • 17
    • 0344148918 scopus 로고    scopus 로고
    • note
    • m (temperature at which 50% of double-stranded DNA or DNA/RNA hybrids is denatured) of the primers. The band-purified products were digested with Not I and Bam HI and then were subcloned into Bam HI-Not I-digested pCEP4 (In-Vitrogen). Transfections were carried out as described previously (70).
  • 18
    • 0344148919 scopus 로고    scopus 로고
    • note
    • + is also present in the L1 expression construct. cDNA from pJET13 was truncated within the neo sequence and was uninformative in this analysis.
  • 19
    • 0345011428 scopus 로고    scopus 로고
    • personal communication
    • M. Yandell and I. Korf, personal communication.
    • Yandell, M.1    Korf, I.2
  • 21
    • 0345442515 scopus 로고    scopus 로고
    • note
    • + to create pJCC130. An 8.2-kb Not I-Bsp 1201 fragment containing the tagged L1 was subcloned into the Not I site of pCEP4 to create pJM130. The L1.3 derivative was constructed as above. Constructs lacking pCMV were constructed by subcloning the engineered L1s into pCEP4ΔCMV.
  • 22
    • 0344148917 scopus 로고    scopus 로고
    • note
    • +.
  • 23
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    • note
    • +.
  • 25
  • 29
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    • S. L. Martin, Gene 153, 261 (1995).
    • (1995) Gene , vol.153 , pp. 261
    • Martin, S.L.1
  • 39
    • 0345011413 scopus 로고    scopus 로고
    • note
    • We thank M. Kimberland for excellent technical assistance; A. Lotia for assistance with computer graphics; and J. Boeke, D. Ginsburg, E. Luning Prak, J. Goodier, and E. Ostertag for critical reading of the manuscript. Supported in part by Damon Runyon Postdoctoral fellowship DRG-1332 (J.V.M.) and NIH grant GM45398 (H.H.K.).


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