-
1
-
-
0027398902
-
-
H. M. Robertson, Nature 362, 241 (1993); P. Capy et al., Genetica 93, 161 (1994).
-
(1993)
Nature
, vol.362
, pp. 241
-
-
Robertson, H.M.1
-
2
-
-
0028696781
-
-
H. M. Robertson, Nature 362, 241 (1993); P. Capy et al., Genetica 93, 161 (1994).
-
(1994)
Genetica
, vol.93
, pp. 161
-
-
Capy, P.1
-
5
-
-
0026322114
-
-
K. Maruyama and D. L. Hartl, J. Mol. Evol. 33, 514 (1991); P. Capy, D. Anxolabehere, T. Langin, Trends Genet. 10, 7 (1994); J. Garcia-Fernandez et al., Mol. Biol. Evol. 12, 421 (1995); A. R. Lohe, E. N. Moriyama, D. A. Lidholm, D. L. Hartl, ibid., 62.
-
(1991)
J. Mol. Evol.
, vol.33
, pp. 514
-
-
Maruyama, K.1
Hartl, D.L.2
-
6
-
-
0028058068
-
-
K. Maruyama and D. L. Hartl, J. Mol. Evol. 33, 514 (1991); P. Capy, D. Anxolabehere, T. Langin, Trends Genet. 10, 7 (1994); J. Garcia-Fernandez et al., Mol. Biol. Evol. 12, 421 (1995); A. R. Lohe, E. N. Moriyama, D. A. Lidholm, D. L. Hartl, ibid., 62.
-
(1994)
Trends Genet.
, vol.10
, pp. 7
-
-
Capy, P.1
Anxolabehere, D.2
Langin, T.3
-
7
-
-
0028950619
-
-
K. Maruyama and D. L. Hartl, J. Mol. Evol. 33, 514 (1991); P. Capy, D. Anxolabehere, T. Langin, Trends Genet. 10, 7 (1994); J. Garcia-Fernandez et al., Mol. Biol. Evol. 12, 421 (1995); A. R. Lohe, E. N. Moriyama, D. A. Lidholm, D. L. Hartl, ibid., 62.
-
(1995)
Mol. Biol. Evol.
, vol.12
, pp. 421
-
-
Garcia-Fernandez, J.1
-
8
-
-
0026322114
-
-
K. Maruyama and D. L. Hartl, J. Mol. Evol. 33, 514 (1991); P. Capy, D. Anxolabehere, T. Langin, Trends Genet. 10, 7 (1994); J. Garcia-Fernandez et al., Mol. Biol. Evol. 12, 421 (1995); A. R. Lohe, E. N. Moriyama, D. A. Lidholm, D. L. Hartl, ibid., 62.
-
Mol. Biol. Evol.
, pp. 62
-
-
Lohe, A.R.1
Moriyama, E.N.2
Lidholm, D.A.3
Hartl, D.L.4
-
9
-
-
0029818461
-
-
D. J. Lampe, M.E.A. Churchill, H. M. Robertson, EMBO J. 15, 5470 (1996); J. C. Vos, I. De Baere, R. H. Plasterk, Genes Dev. 10, 755 (1996).
-
(1996)
EMBO J.
, vol.15
, pp. 5470
-
-
Lampe, D.J.1
Churchill, M.E.A.2
Robertson, H.M.3
-
10
-
-
0029930255
-
-
D. J. Lampe, M.E.A. Churchill, H. M. Robertson, EMBO J. 15, 5470 (1996); J. C. Vos, I. De Baere, R. H. Plasterk, Genes Dev. 10, 755 (1996).
-
(1996)
Genes Dev.
, vol.10
, pp. 755
-
-
Vos, J.C.1
De Baere, I.2
Plasterk, R.H.3
-
13
-
-
0029549553
-
-
T. G. Loukeris, I. Livadaras, B. Area, S. Zabalou, C. Savakis, Science 270, 2002 (1995); T. G. Loukeris, B. Area, I. Livadaras, G. Dialektaki, C. Savakis, Proc. Natl. Acad. Sci. U.S.A. 92, 9485 (1995); C. J. Coates et al., Mol. Gen. Genet. 249, 246 (1995).
-
(1995)
Science
, vol.270
, pp. 2002
-
-
Loukeris, T.G.1
Livadaras, I.2
Area, B.3
Zabalou, S.4
Savakis, C.5
-
14
-
-
0028809850
-
-
T. G. Loukeris, I. Livadaras, B. Area, S. Zabalou, C. Savakis, Science 270, 2002 (1995); T. G. Loukeris, B. Area, I. Livadaras, G. Dialektaki, C. Savakis, Proc. Natl. Acad. Sci. U.S.A. 92, 9485 (1995); C. J. Coates et al., Mol. Gen. Genet. 249, 246 (1995).
-
(1995)
Proc. Natl. Acad. Sci. U.S.A.
, vol.92
, pp. 9485
-
-
Loukeris, T.G.1
Area, B.2
Livadaras, I.3
Dialektaki, G.4
Savakis, C.5
-
15
-
-
0028867621
-
-
T. G. Loukeris, I. Livadaras, B. Area, S. Zabalou, C. Savakis, Science 270, 2002 (1995); T. G. Loukeris, B. Area, I. Livadaras, G. Dialektaki, C. Savakis, Proc. Natl. Acad. Sci. U.S.A. 92, 9485 (1995); C. J. Coates et al., Mol. Gen. Genet. 249, 246 (1995).
-
(1995)
Mol. Gen. Genet.
, vol.249
, pp. 246
-
-
Coates, C.J.1
-
16
-
-
0030048276
-
-
E. R. Lozovskaya, D. I. Nurminsky, D. L. Hartl, D. T. Sullivan, Genetics 142, 173 (1996); D. A. O'Brochta, W. D. Warren, K. J. Seville, P. W. Atkinson, ibid., p. 907.
-
(1996)
Genetics
, vol.142
, pp. 173
-
-
Lozovskaya, E.R.1
Nurminsky, D.I.2
Hartl, D.L.3
Sullivan, D.T.4
-
17
-
-
0030048276
-
-
E. R. Lozovskaya, D. I. Nurminsky, D. L. Hartl, D. T. Sullivan, Genetics 142, 173 (1996); D. A. O'Brochta, W. D. Warren, K. J. Seville, P. W. Atkinson, ibid., p. 907.
-
Genetics
, pp. 907
-
-
O'Brochta, D.A.1
Warren, W.D.2
Seville, K.J.3
Atkinson, P.W.4
-
19
-
-
0025049856
-
-
A. Cruz and S. M. Beverley, Nature 348, 171 (1990); M. A. Curotto de Lafaille and D. F. Wirth, J. Biol. Chem. 267, 23839 (1992).
-
(1990)
Nature
, vol.348
, pp. 171
-
-
Cruz, A.1
Beverley, S.M.2
-
22
-
-
1842310606
-
-
note
-
pX63TKNEO (strain B2179) and pX63PAC (B1129) are Leishmania expression vectors derived from pX63NEO (28) by replacing the NEO-containing Spe I fragment with the Bgl II-Sma I 2.2-kb fragment of pTNFUS69Py (TKNEO) (29) or the Not I-Nco I 0.7-kb fragment of pVN3.1 (PAC) (30). pX63TKNEO-TPASE (B3078) contains the 1.4-kb Cla I-Hind III fragment from pBluescribe M13+/Mos1 (12) inserted into the Sma I expression site of pX63TKNEO. This fragment starts 69 nucleotides before the transposase ATG and extends beyond the stop codon, including the 3′ inverted repeat and nearly 200 bp of Drosophila flanking DNA. pX63PAC-Mos1 (B3079) contains the 2.2-kb Xho I-Hind III fragment of pBluescribe M13+/ Mos1, inserted into the Bgl II site of pX63PAC. pX63PAC-MosHYG (B3081) was constructed by blunt-end cloning of a 1.1-kb Spe I-Bam HI hygromycin phosphotransferase cassette from pX63HYG (31) into the Cla I site of Mos1. The sequence of the Drosophila DNA flanking Mos1 has been deposited in GenBank (U96147, U96148).
-
-
-
-
23
-
-
0025339650
-
-
N. Agabian, Cell 61, 1157 (1990).
-
(1990)
Cell
, vol.61
, pp. 1157
-
-
Agabian, N.1
-
24
-
-
1842379942
-
-
note
-
- line C1 (21) by transfection with a wild-type DHFR-TS fragment [9-kb Cla I fragment of pK300 (16)] and selection for thymidine prototrophy. The +/Δ1 strain of L. major was initially transfected with pX63TKNEO-TPASE to give rise to line TPASE1. TPASE1 was subsequently transfected with pX63PAC-Mos1 in the presence of both G418 and puromycin. Selection against DHFR-TS expression was accomplished by plating cells on semisolid M199 medium containing TdR (10 μg/m|) and MTX (100 μM) (27) but lacking G418 or puromycin.
-
-
-
-
26
-
-
1842312529
-
-
note
-
Leishmania DNAs were prepared by the LiCl mini-prep method (33) and used in standard Southern blot analysis (34) with probes prepared by the random priming method (35).
-
-
-
-
27
-
-
1842307717
-
-
note
-
Several of these colonies did show higher molecular weight bands; however, their mobility was the same as higher molecular weight bands arising from the input transfecting DNA, and we attribute this to the presence of partial digestion of monomer or multimeric pX63PAC-Mos1, which is expected to occur in both Escherichia coli and Leishmania (16, 36).
-
-
-
-
28
-
-
1842313460
-
-
note
-
We obtained the sequences shown in Fig. 1C by inverse PCR, using different pairs of primers for each side of the transposon. The 5′ side primers (as defined by the transposase ORF within the Mos1 element) were B440 (5′-GCCGAACTGCAAGCATTATTGG; sense) and B441 (5′-TGAAGCGTTGAAACCACCGTTC; antisense); the 3′ side primers were B442 (5′-TCCACAAATTGCCCGAGAGATG; sense) and B443 (5′-ATGTGATGGAGCGTTGTCATGG; antisense). First, genomic DNAs were separated on agarose gels after restriction digestion, and DNA fragments in the size range for the new mariner-hybridizing band were isolated. These were then digested with Sau 3A, self-ligated under dilute conditions, and then used in standard PCR reactions (37). Products differing in size from those arising from the parental plasmids were identified by gel electrophoresis, inserted into pGEM-T (Promega), and sequenced with ΔTaq 2.0 polymerase and the Taquenase kit (United States Biochemical). Sequences were deposited in GenBank under the accession numbers B07506 through B07511.
-
-
-
-
29
-
-
1842359729
-
-
F. J. Gueiros-Filho and S. M. Beverley, data not shown
-
F. J. Gueiros-Filho and S. M. Beverley, data not shown.
-
-
-
-
30
-
-
0029831106
-
-
_, Mol. Cell. Biol. 16, 5655 (1996).
-
(1996)
Mol. Cell. Biol.
, vol.16
, pp. 5655
-
-
-
32
-
-
1842304842
-
-
note
-
-4.
-
-
-
-
33
-
-
1842383755
-
-
note
-
RNAs were prepared with Tri-reagent (Sigma) (38), following the procedures of the manufacturer. cDNA was synthesized from ∼1 μg of total RNA, with a random hexamer priming protocol (39). A nested RT-PCR protocol was used to amplify chimeric Leishmania-MosHYG mRNAs (39). For the first PCR, the anchor primer was B466 (5′-AACGCTATATAAGTATCAG), corresponding to nucleotides 1 through 19 of the mini-exon, and the HYG-specific primer was B467 (5′-AAAGCACGAGATTCTTCG). Primers for the second round of PCR were B145 (5′-ATCAGTTTCTGTACTTTA; nucleotides 15 through 32 of the mini-exon) and the HVG-specific primer B456 (5′-ATCAGAAACTTCTCGACAG). RT-PCR products were cloned into pGEM-T and sequenced. Sequences were deposited in GenBank under the accession numbers AA195776 through AA195783.
-
-
-
-
34
-
-
0025650360
-
-
J. H. LeBowitz, C. M. Coburn, D. McMahon-Pratt, S. M. Beverley, Proc. Natl. Acad. Sci. U.S.A. 87, 9736 (1990).
-
(1990)
Proc. Natl. Acad. Sci. U.S.A.
, vol.87
, pp. 9736
-
-
Lebowitz, J.H.1
Coburn, C.M.2
McMahon-Pratt, D.3
Beverley, S.M.4
-
39
-
-
0024403778
-
-
R. A. Lacalle, D. Pulido, J. Vara, M. Zalacain, A. Jimenez, Gene 79, 375 (1989).
-
(1989)
Gene
, vol.79
, pp. 375
-
-
Lacalle, R.A.1
Pulido, D.2
Vara, J.3
Zalacain, M.4
Jimenez, A.5
-
43
-
-
0004136246
-
-
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
-
J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
-
(1989)
Molecular Cloning: A Laboratory Manual
-
-
Sambrook, J.1
Fritsch, E.F.2
Maniatis, T.3
-
45
-
-
0026499725
-
-
L. Martin-Parras, P. Hernandez, M. L. Martinez-Robles, J. B. Schvartzman, J. Biol. Chem. 267, 22496 (1992).
-
(1992)
J. Biol. Chem.
, vol.267
, pp. 22496
-
-
Martin-Parras, L.1
Hernandez, P.2
Martinez-Robles, M.L.3
Schvartzman, J.B.4
-
46
-
-
0023850178
-
-
R. K. Saiki et al., Science 239, 487 (1988).
-
(1988)
Science
, vol.239
, pp. 487
-
-
Saiki, R.K.1
-
48
-
-
0003505215
-
-
M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. Academic Press, San Diego, CA
-
E. Kawasaki, in PCR Protocols. A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, San Diego, CA, 1990).
-
(1990)
PCR Protocols. A Guide to Methods and Applications
-
-
Kawasaki, E.1
-
49
-
-
1842362703
-
-
note
-
Northern blots were performed with glyoxal-denatured RNAs (34). The probes used in Northern analysis (Fig. 3B) were prepared by inverse PCR, using +/Δ 1 DNA digested with Sau 3A or Hpa II, followed by self-ligation under dilute conditions, as template; for sequence T3.5B, the primers were B523 (5′-CGGCTGCTCTGCTCTTCC; sense) and B524 (5′-TCTCTCGTGGCTCTGTGTACC); for sequence T3.6B, the primers were B525 (5′-CGCTTACATGCACGGTGC; sense) and B526 (5′-AACGGTGTCCACGACGGC).
-
-
-
-
50
-
-
1842320160
-
-
note
-
We thank D. Hartl for the gift of Mos1 DNA, S. Chatterjee and M. Lee for helpful discussions, and D. E. Dobson, L. A. Garraway, L. K. Garrity, A. Hubel, and L. R. O. Tosi for comments on the manuscript. Supported by NIH grant AI2964.
-
-
-
|