Retroviruses, including HIV‐1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV‐1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer‐binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3′ PBS for primer tRNAiMet annealing to the genomic RNA, and the 5′ PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA–tRNAiMet. Interestingly, HIV‐1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV‐1 and Ty3 RNA template–primer systems. Our findings impact on the understanding of non‐canonical reverse transcription as well as on the use of Ty3 systems to screen for anti‐NCp7 drugs.
Replication of retroviruses and retrotransposon elements proceeds by reverse transcription of genomic RNA followed by integration of the DNA copy into the host genome (Coffin, 1985; Varmus and Swanstrom, 1985; Boeke and Sandmeyer, 1991). Reverse transcriptase (RT), integrase, the RNA genome and primer tRNA that participate in this process are all packaged into a core nucleoprotein complex called a nucleocapsid (NC) (Chen et al., 1980; Hu and Temin, 1990; Katz and Skalka, 1994; Berkowitz et al., 1996; Mak and Kleiman, 1997). For human immunodeficiency virus type 1 (HIV‐1), nucleocapsid protein NCp7 is of one the major structural constituents of the virion core in which ∼2000 NCp7 molecules cover the dimeric genome (Darlix et al., 1990, 1995; Berkowitz et al., 1993). NCp7 is a small basic protein possessing two conserved ‘CCHC’ zinc fingers with key functions in virus structure and replication (Aldovini and Young, 1990; Summers et al., 1992; Dorfman et al., 1993; Morellet et al., 1994; Darlix et al., 1995; Berkowitz et al., 1996). NCp7 appears to function as a nucleic acid chaperone, with RNA–DNA binding and annealing activities implicated in the specificity of reverse transcription initiation, and in DNA strand transfers required for generation of the long terminal repeats (LTRs) and completion of proviral DNA synthesis (Barat et al., 1989; Allain et al., 1994; Dannul et al., 1994; Peliska et al., 1994; Rodriguez‐Rodriguez et al., 1995; Li et al., 1996; Berthoux et al., 1997; Lapadat‐Tapolsky et al., 1997). In addition, NCp7 acts as an essential co‐factor during dimerization, packaging, condensation and stability of genomic RNA (Housset et al., 1993; Berkowitz et al., 1995; Ottmann et al., 1995; Zhang and Barklis, 1995).
Nucleoprotein complexes resembling the virion NC can be formed in vitro in the presence of NCp7, viral RNA and primer tRNALys3, resulting in dimerization of viral RNA (provided the packaging E sequence is present) and annealing of primer tRNA to the primer‐binding site (PBS). Interestingly, the viral RNA is protected from nuclease degradation in these nucleoprotein complexes, yet initiation of cDNA synthesis by RT can take place at the PBS followed by elongation and strand transfer (Lapadat‐Tapolsky et al., 1993; Tanchou et al., 1995), thus reproducing the early phases of proviral DNA synthesis (Coffin, 1985; Varmus and Swanstrom, 1985).
In view of the ubiquitous nature of NC protein among retroviruses and of its numerous key roles in virus structure and replication, we set out to compare the functions of NC proteins from two distantly related retroelements, namely HIV‐1 and the yeast Ty3 retrotransposon. Ty3, an LTR‐containing retroelement, encodes the nucleocapsid protein NCp9 which possesses a single zinc finger and is required for Ty3 transposition in yeast (Kirschner and Sandmeyer, 1993; Orlinsky and Sandmeyer, 1994), implying functional homology with HIV‐1 NCp7. During an in vitro comparison of HIV‐1 NCp7 and Ty3 NCp9 functions in genomic RNA dimerization and reverse transcription, we discovered an unusual bipartite PBS organization for Ty3 with segments of complementarity to primer tRNAiMet located at opposite ends of the genome. Initiation of Ty3 cDNA synthesis requires the 3′ PBS for primer tRNAiMet annealing and the 5′ PBS as the transcriptional start site. Furthermore, Ty3 RNA dimerization, unlike HIV‐1 and other retroviral RNAs, appears to require and be mediated via dimeric tRNAiMet.
Ty3 NCp9 promotes HIV‐1 RNA dimerization and annealing of primer tRNALys3
Ty3 NCp9 (Figure 1A) was synthesized by fmoc chemistry and purified by HPLC. Highly pure NCp9 thus obtained was found to complex an equimolar zinc cation with high affinity, as does NCp7 of HIV‐1 (Figure 1A, H.Deméné and D.Marion, unpublished data). In preliminary experiments, NCp9 exhibited strong nucleic acid binding and annealing activities, similar to HIV‐1 NCp7, and was able to form nucleoprotein complexes with both Ty3 and HIV‐1 RNA (data not shown; Tanchou et al., 1995).
The ability of Ty3 NCp9 to promote HIV‐1 RNA dimerization and annealing of HIV‐1 primer tRNALys3 to the PBS was examined using HIV‐1 5′ RNA corresponding to the leader and part of the 5′ GAG domain (position 1–415, Figure 1A), containing the packaging–dimerization (E/DLS) as well as dimerization initiation (DIS) sequences (Paillart et al., 1996). In accordance with published data (Barat et al., 1989; Lapadat‐Tapolsky et al., 1993; Tanchou et al., 1995), HIV‐1 NCp7 promoted dimerization of the 5′ RNA (Figure 1B, lanes 2–4) and annealing of primer tRNALys3 to the PBS (Figure 1C, lanes 2–4). Figure 1 shows that NCp9 of Ty3 promotes HIV‐1 RNA dimerization (Figure 1B, lanes 6–8) and tRNALys3 annealing (Figure 1C, lanes 6–8) as efficiently as does NCp7. Ty3 NCp9 was also found to function in place of HIV‐1 NCp7 during in vitro strong stop cDNA synthesis and its transfer to the 3′ end of HIV‐1 RNA (data not shown; for NCp7 see Barat et al., 1989; Tanchou et al., 1995).
A 5′–3′ bipartite PBS within Ty3 genomic RNA
The above data demonstrate functional homology of Ty3 NCp9 with HIV‐1 NCp7. To pursue this observation, we devised an in vitro Ty3 system equivalent to that used for HIV‐1 (Figure 1; Tanchou et al., 1995; Lapadat‐Tapolsky et al., 1997). However, the PBS of Ty3 RNA, deduced from sequence complementarity to the primer tRNA in the region analogous to the retroviral PBS, is only eight nucleotides long and located, as is usual for canonical PBS sequences, adjacent to the 5′ LTR (position 121–128; Hansen and Sandmeyer, 1990). Interestingly, Ty3 5′ RNA (Figure 2A, position 1–355) alone did not support binding of replication primer tRNAiMet (Figure 2C), dimerization (Figure 2B) or initiation of reverse transcription (Figure 3, lanes 1–3). This prompted us to perform a computer local alignment search for additional sequences within Ty3 RNA complementary to primer tRNAiMet. To our surprise, we found two adjacent sequences with high complementarity to tRNAiMet located near the 3′ end of the Ty3 genomic RNA. The first, at position 4947–4959 in U3 of the RNA, is complementary to 12 nucleotides of the TΨC‐arm of tRNAiMet while the second, at position 4964–4974 in U3, is complementary to 11 nucleotides of the D‐arm (Figure 2A). A Ty3 3′ RNA comprising nucleotides 4724–5011 was therefore generated in vitro (Figure 2A) and, as shown in Figure 2E, NCp9 efficiently promoted primer annealing to this RNA. Binding of tRNAiMet to the 3′ RNA also resulted in partial dimerization (Figure 2D, lanes 5–8); however, Moloney murine leukaemia virus (MoMuLV) RT was unable to extend primer tRNAiMet when annealed to this 3′ RNA by NCp9 (Figure 3A, lanes 4–6).
To examine whether the putative 3′ PBS sequences of Ty3 RNA might act in cis to present the 3′ acceptor stem of tRNAiMet to the 5′ PBS, thereby allowing initiation of reverse transcription, we generated a chimeric RNA containing both 5′ and 3′ sequences (Figure 2A). Ty3 NCp9 and HIV‐1 NCp7 efficiently promoted annealing of primer tRNAiMet to this 5′–3′ RNA (Figure 2G), resulting in high levels of RNA dimers (Figure 2F). Addition of MoMuLV RT and dNTPs to NCp9/5′–3′ RNA and NCp7/5′–3′ RNA complexes resulted in the synthesis of strong‐stop cDNA, the initial product of reverse transcription (Figure 3, lanes 10–13).
Based on the above data, the distance between the 5′ and putative 3′ PBS sequences in the Ty3 genome is 4800 nucleotides (Hansen et al., 1988). In order to probe the requirement for a physical link between these PBS sequences for strong‐stop cDNA synthesis by RT, an experiment was devised in which 5′ RNA and 3′ RNA (Figure 2A) were used in trans in the presence of primer tRNAiMet, NC protein and MoMuLV RT. As reported (Figure 3A, lanes 7–9), high levels of strong‐stop cDNA were synthesized in the presence of Ty3 NCp9 despite the fact that the 5′ and the putative 3′ PBS sequences were on distinct RNAs. The level of strong‐stop cDNA synthesis was similar to that seen with the 5′–3′ chimeric RNA (Figure 3A, lanes 10–13; B lanes 1–5) in which both PBS segments were present in cis. To confirm the requirement for the 5′ PBS, we used 5′–3′ RNA with a 5′ PBS deletion (5′ΔPBS–3′ RNA) shown to completely abolish Ty3 transposition in vivo (S.Sandmeyer, unpublished results). The 5′ PBS deletion was found to completely preclude strong‐stop cDNA synthesis in the presence or absence of Ty3 NCp9 protein (Figure 3B, lanes 6–8).
We also examined the relative contribution of the two putative 3′ PBS sequences during primer tRNAiMet annealing, Ty3 RNA–tRNA dimerization and initiation of reverse transcription by replacing either the first or the second putative 3′ PBS (3′PBSa or b, see Figure 2A) by a heterologous sequence (see Materials and methods). Results in Figure 4 show that deletion of either sequence strongly reduced RNA dimer formation (Figure 4A, compare lanes 2–4 with 6–8 and 10–12) as well as primer tRNAiMet annealing (Figure 4B, compare lanes 3–4 with 7–8 and 11–12). Consistent with this, strong‐stop cDNA synthesis by RT in the presence of NC protein was decreased ∼10‐fold by the 3′ PBSa mutation and 4‐ to 5‐fold by the 3′ PBSb mutation (Figure 4C, lanes 4 and 5). Combining both the 3′ PBSa and b mutations precluded strong‐stop cDNA synthesis in vitro (data not shown).
The effect of mutating the 3′ PBSa and 3′ PBSb sequences was also investigated in yeast cells using a Ty3 His+ element (see Materials and methods). Mutating segments a or b of the 3′ PBS, or both a and b caused a 6‐ to 7‐fold, 4‐fold and >20‐fold decrease of transposition, respectively (see Materials and methods). These results clearly show that the 3′ PBS sequences are critical for transposition in vivo.
Mutations in primer tRNAiMet that impair transposition result in an attenuation of Ty3 RNA–tRNA dimerization in vitro
Mutations in primer tRNAiMet involving substitutions C3T and G70A in the acceptor stem previously were reported to inhibit Ty3 transposition in vivo (Keeney et al., 1995). We generated tRNAiMet with the same point mutations and used it in tRNA annealing and tRNA‐promoted dimerization, as well strong‐stop cDNA synthesis assays in vitro (Figure 5). Annealing of [32P]tRNAiMet containing the C3T and G70A mutations to Ty3 RNA was essentially retained, but the majority of Ty3 RNA–tRNA was in a monomeric form (Figure 5A, compare lanes 2–3 with 5–6, and lanes 8–9 with 11–12). Interestingly, strong‐stop cDNA synthesis was not affected by these mutations in primer tRNAiMet (Figure 5B, contrast lanes 2–6 and 8–11).
Another set of mutations previously reported to inhibit Ty3 transposition in vivo (A54T and A60T, Keeney et al., 1995) were introduced into the TΨC‐arm of tRNAiMet. These mutations attenuated Ty3 RNA–tRNA dimerization by NC protein while again strong‐stop cDNA synthesis was essentially unaffected (data not shown).
Based on the data reported herein, the Ty3 retrotransposon possesses a bipartite PBS with sequences located at opposite ends of the Ty3 genomic RNA complementary to three domains of the primer tRNAiMet, namely the 3′ end, and TΨC‐ and D‐arms. Each of these complementary segments is essential for efficient initiation of Ty3 reverse transcription. In the yeast retrotransposon Ty1, the 3′ end, TΨC‐ and D‐arms of primer tRNAiMet make contact with three PBS sequences all located near the 5′ end of the genomic RNA (Friant et al., 1996, 1998), providing an interesting parallel with the bipartite PBS structure described herein for Ty3. Furthermore, mutations in Ty1 RNA that impair interactions with the TΨC and D‐arms of tRNAiMet severely attenuate transposition in vivo (Friant et al., 1996, 1998). This situation in Ty3, and to a lesser extent in Ty1, is different from that prevailing in retroviruses where a canonical 5′ 18 nucleotide PBS is commonly the major binding site of primer tRNA as well as the start site for reverse transcription (Coffin, 1985; Darlix et al., 1993, 1995; Berkowitz et al., 1996; reviewed in Mak and Kleiman, 1997).
Using 3′ PBS mutants found to attenuate transposition (see Results), we show that annealing of tRNAiMet to Ty3 RNA is essential for the formation of dimeric Ty3 RNA–tRNAiMet complexes (Figure 4). In agreement with this, mutations in the acceptor stem of tRNAiMet shown to impair transposition strongly (Keeney et al., 1995) were found to decrease the level of dimeric Ty3 RNA–tRNAiMet in vitro (Figure 5 and data not shown) (note tRNAiMet dimerization in Figures 2,3,4,5, whereas tRNALys3 remained monomeric in Figure 1). Moreover, preliminary experiments indicate that the genomic RNA of the yeast retrotransposon Ty1 also undergoes dimerization dependent upon binding of tRNAiMet to the PBS (data not shown; Friant et al., 1996). Dimerization of tRNAiMet probably occurs by interaction between the 5′ ends since this domain contains a 12 nucleotide palindrome (position 2–13, GCGCCGUGGCGC) (Keeney et al., 1995), and a DNA oligonucleotide complementary to the palindrome or a deletion of the 15 5′ nucleotides completely inhibits Ty3 RNA and tRNAiMet dimerization by NC protein (data not shown; see also Figure 5).
A plausible dimerization model for the Ty3 RNA–tRNAiMet complex is illustrated schematically in Figure 6. In this structure, the primer tRNA is key to both dimer formation and initiation of reverse transcription. Due to the proposed proximity of the 5′ and 3′ ends of Ty3 RNA imposed by dimerization, the tRNA would also indirectly play a role in the 5′ to 3′ transfer of strong stop DNA during minus strand DNA synthesis. Taken together, these data suggest that dimerization of the Ty3 RNA–tRNAiMet complex is coincident with positioning of the primer tRNA on the RNA template (see also Sandmeyer and Menees, 1996).
The fact that both Ty3 NCp9 and HIV‐1 NCp7 were shown to be functionally equivalent in these two distantly related systems favours the notion that Ty3 NCp9 is an ancestor of HIV‐1 NCp7. In support of this, preliminary experiments have shown that Ty3 reverse transcription can be inhibited by anti‐NCp7 compounds (Rice et al., 1993), raising the possibility that the Ty3 genetic system might be used to screen potential inhibitors of HIV replication.
Understanding the role of the NC protein, the detailed nature of the bipartite Ty3 PBS and the utilization of multiple molecular determinants of a primer tRNA for reverse transcription initiation are of importance for our comprehension of the mechanism by which non‐retroviral and cellular RNAs might be reverse transcribed in cells containing active retroelements (Klenerman et al., 1997). It is tempting to evoke a mechanism in which Ty3‐like RNA presents tRNA 3′ sequences (via a 3′ PBS ‘grip’) for interaction with non‐canonical RNAs in trans, resulting in encapsidation and reverse transcription of such RNAs. This hypothesis currently is under investigation.
Materials and methods
RNA substrates, NC proteins and enzymes
HIV‐1 5′ RNA corresponding to nucleotides 1–415 was generated in vitro as previously described (Barat et al., 1989). HIV‐1 3′ RNA (position 8583–9208 of the HIV‐1 genome) with a poly(A) tail was prepared by transcription as described (Darlix et al., 1993). DNA encoding Ty3 5′ RNA under T7 promoter control was generated by PCR using pEGTy3‐1 (Kirschner and Sandmeyer, 1993), a 5′ oligonucleotide containing T7 Po, an EcoRI site and 16 nucleotides of the 5′ end of Ty3 RNA, and a 3′ oligonucleotide complementary to position 1106–1121 of Ty3 RNA and containing a HindIII site. DNA was cloned into pSP64 and controlled by sequencing (pTy3‐CG1). Template DNA was linearized at position 355 (EcoRV) and 5′ RNA synthesized in vitro. DNA encoding Ty3 3′ RNA was generated after PCR using pETGY3‐1, a 5′ oligonucleotide containing a HindIII site and 16 nucleotides (position 4724–4738) of Ty3, a 3′ oligonucleotide complementary to position 4994–5011 and containing an NheI site, cloned into pBS and controlled by sequencing (pTy3‐CG2). Template DNA was linearized with SmaI for in vitro synthesis of 3′ RNA (T3 pol). For chimeric Ty3 5′–3′ RNA, pTy3‐CG1 was cut with EcoRV and NheI to remove sequences downstream of position 355, and treated with Taq DNA polymerase to add a 3′ dT (method described in Marchuk et al., 1991), allowing direct insertion of the 3′ DNA PCR fragment digested with NheI. The construct was verified by sequencing (pTy3‐CG3) and linearized by NheI to generate RNA in vitro.
5′ΔPBS–3′ RNA was generated the same way except that pKO271 (S.Sandmeyer, unpublished; nucleotides 77–172 deleted) was used as the template for the first round of PCR. The construct was verified by sequencing (pTy3‐CG4).
Ty3 sequence complementary to the TΨC‐arm (3′ PBSa) or D‐arm (3′ PBSb) of tRNAiMet was replaced by ACUAGU (a SpeI site) using a method based on two successive rounds of PCR (Mikaelian and Sergeant, 1992) and the pTy3‐CG3 clone (see above) as template. Mutations were verified by sequencing (pTy3‐CG5 and pTy‐CG6 for 3′ΔPBSa and 3′ΔPBSb, respectively) and mutated 5′–3′ RNAs synthesized in vitro (T7 pol).
Primer tRNALys3 (Barat et al., 1993) and tRNAiMet were kindly provided by G.Keith and B.Ehresmann (Strasbourg). Synthetic tRNALys3 was generated in vitro using T7 RNA polymerase (Barat et al., 1993). Plasmid DNA HG300 encoding tRNAiMet (Senger et al., 1992) and 5′ and 3′ mutated oligonucleotides (5′ containing the substitution C3→T and 3′ containing G70→A; see Keeney et al., 1995) were used for DNA amplification. Mutation of the TΨC‐arm of tRNAiMet (A54→C and A60→T; see Keeney et al., 1995) was performed using the PCR strategy outlined above. Mutant tRNAs were verified by sequencing (pΨMet,i‐CG2 and 3) and synthesized in vitro (T7 pol). All RNAs were purified by spin column chromatography (Pharmacia S‐300 HR) and dissolved at 1 mg/ml in sterile water. [32P]UMP‐labelled tRNALys3 and tRNAiMet were synthesized in vitro using T7 RNA polymerase, purified by polyacrylamide gel electrophoresis (PAGE) in 7 M urea, recovered and dissolved at 0.1 mg/ml in sterile water.
Highly pure NCp7 (72 amino acids, containing two Zn2+) was prepared by peptide synthesis as described previously (de Rocquigny et al., 1991). A Ty3 NCp9 variant (P23→A and R31 insertion, relative to Hansen et al., 1988) (Figure 1A) was synthesized by the fmoc/opfp chemical method and purified by HPLC in conditions previously described for HIV‐1 NCp7 (de Rocquigny et al., 1991, 1992); 50 mg of >98% pure Ty3 NCp9 was obtained. NCp9 and NCp7 stocks were at 1 mg/ml in 20 mM Tris–acetate pH 6.5, 30 mM NaCl, 5 mM dithiothreitol (DTT) and 1.5 equivalents of ZnCl2. HIV‐1 RT (p66/p51), purified from Escherichia coli (Le Grice and Grüninger‐Leitch, 1990), was provided by S.Le Grice. MoMuLV RT purified from E.coli was from Gibco‐BRL.
tRNA annealing assay
Reactions with HIV‐1 or Ty3 RNA, in vitro synthesized 32P‐labelled tRNA, or natural primer 5′ [32P]tRNA and NC protein were for 10 min at 37°C (HIV‐1) or 28°C (Ty3) in 10 μl containing 20 mM Tris–HCl pH 7.5, 30 mM NaCl, 0.2 mM MgCl2, 5 mM DTT, 0.01 mM ZnCl2, 5 U of RNasin (Promega), 1.5 pmol of RNA, 3 pmol of in vitro synthesized tRNA (or natural tRNA) and NCp7 or NCp9 at the indicated molar protein to nucleotide ratios. Reactions were stopped by SDS/EDTA (0.5%/5 mM), treated with proteinase K (2 μg) for 10 min at room temperature, phenol–chloroform extracted and RNA analysed by 1.3% agarose gel electrophoresis in 50 mM Tris–borate pH 8.3 and visualized by ethidium bromide staining followed by gel fixation in 5% trichloroacetic acid (TCA), drying and autoradiography. A 0.16–1.77 kb RNA ladder was used for size determination. The percentage of primer tRNA in HIV‐1 or Ty3 RNA annealing was determined by densitometric scanning of the autoradiograph.
Reverse transcription assays
The reactions were performed basically as described previously (Barat et al., 1989; Darlix et al., 1993; Allain et al., 1994). After 5 min at 30°C for the nucleic acid‐binding assay in 10 μl (see above), the reaction volume was increased to 25 μl by addition of 2 pmol of HIV‐1 RT or MoMuLV RT (Gibco‐BRL), 0.25 mM each dNTP, 60 mM NaCl and 2.5 mM MgCl2. Incubation was for 20 min at 30°C (or 37°C with HIV‐1 RT), and they were stopped and processed as for the analysis of tRNA annealing (see above), except that after phenol extraction nucleic acid was ethanol precipitated, recovered by centrifugation, dissolved in formamide, denatured at 95°C for 2 min and analysed by 8% PAGE in 7 M urea and 50 mM Tris–borate pH 8.3. 5′ 32P‐labelled FX174 DNA Hinf markers (Promega) were used for size determination (not shown). The levels of cDNA synthesized by RT were quantified by scanning densitometry.
The effect of 3′ PBS mutations on Ty3 transposition was examined using a low‐copy helper plasmid and a high‐copy Ty3 plasmid encoding HIS (Kirschner and Sandmeyer, 1993). The yeast strain yTM443 (derivative of TMy18: MATa, trp1‐H3, ura3‐52, his3‐200, ade2‐101, lys2‐1, leu1‐12, can1‐100, bar1::his G, GAL3+, ΔTy3) was transformed with one HIS3‐marked high‐copy donor plasmid (pKO254 for the wild‐type plasmid) (3′ PBS sequences of pKO254 were replaced by a SpeI site, ACTAGT, to generate plasmids pCG100 with 3′ΔPBSa, PCG101 with 3′ΔPBSb and pCG102 with 3′ΔPBSab) and with one unmarked low‐copy helper plasmid to supply the necessary Ty3 proteins in trans (pJK312). The transformation was done with a lithium acetate protocol at 30°C. Transformants were selected on synthetic medium minus uracil and tryptophan at 30°C.
Two transformants of each type were streaked onto selective medium containing either 2% glucose, to repress, or 2% galactose, to induce, transcription of the Ty3 elements once at 24°C and other times at 30°C. After 3 days, five colonies from each transformant were patched onto YPD (+glucose) to allow for loss of the plasmids at 30°C. Three days later, these patches were replicated onto minus‐histidine, 5‐fluoroorotic acid (5‐FOA)‐containing medium (+glucose) to select for cells which lost the URA3‐marked donor plasmid but retained the HIS3‐marked Ty3 element (at 30°C). To distinguish growth which was due to transposition of the marked Ty3 element from background recombinant events, the cells grown on galactose were compared with the cells grown on glucose. Experiments were repeated four times, and HIS+ colonies were counted. Galactose‐induced HIS+ colonies obtained with the wild‐type HIS+ plasmid were found to be 10–20 times more abundant than background colonies and much bigger.
We thank F.Fasiolo (Strasbourg, France) and H.Grosjean (Paris, France) for the HG300 plasmid, and S.Le Grice (Case Western, USA) for HIV‐1 RT. Thanks are due to H.Déméné and D.Marion (IBS, Grenoble) for communicating unpublished data on Ty3 NCp9. This work was supported by grants from the French National AIDS Programme (ANRS), SIDACTION and the Mutuelle Générale de l'Education Nationale (MGEN). S.S. is supported by US Public Health Service Grant GM33281.
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