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Abstract RNA interference is a powerful method for inhibition of gene expression in Trypanosoma brucei(Ngo, H., Tschudi, C., Gull, K., and Ullu, E. Here we describe a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably transformed cells. The dsRNA is synthesized from opposing T7 promoters. We tested the vector with genes involved in processes such as kinetoplast DNA replication, mitochondrial mRNA synthesis, glycosyl phosphatidylinositol biosynthesis, glycosome biogenesis, and polyamine biosynthesis. In most cases the induction of dsRNA caused specific and dramatic loss of the appropriate mRNA, and in many cases there was growth inhibition or cell death.
One striking phenotype was the loss of kinetoplast DNA after interference with expression of a topoisomerase II. The gene being analyzed by this procedure need not even be fully sequenced. In fact, many of the genes we tested were derived from partial sequences in the T. Brucei genome data base that were identified by homology with known proteins. It takes as little as 3 weeks from identification of a gene sequence in the data base to the appearance of a phenotype.
Trypanosoma brucei is the protozoan parasite that causes sleeping sickness, a fatal disease that currently affects several hundred thousand people in sub-Saharan Africa. Closely related parasites, Trypanosoma cruzi and Leishmania, are also significant pathogens in tropical countries.
In addition to their major impact on human health, these parasites have been important subjects for the study of a number of significant basic biological processes, including RNA editing, glycosyl phosphatidylinositol (GPI) anchoring, and antigenic variation. However, many questions concerning the virulence and general biology of these fascinating parasites remain to be answered. These problems are sometimes difficult to address because trypanosomes are diploid and the available genetic tools are labor intensive. RNA interference (RNAi) 1 is a recently discovered process in which the introduction of a double-stranded RNA into a cell causes specific degradation of an mRNA containing the same sequence (reviewed in Ref.
Specific loss of gene expression by RNAi has been shown to occur in a variety of organisms including Caenorhabditis elegans , T. Brucei , plants , Neurospora , Drosophila , planaria , mouse embryos , and zebrafish. Several genes involved in the RNAi mechanism have been described , and insights into the molecular mechanism of this process have recently been published (, ).
As suggested by Ullu and co-workers , RNAi could be a powerful tool to genetically manipulate trypanosomes. One advantage of RNAi over conventional gene knockout approaches is that only a few hundred base pairs of a gene sequence is required. This feature is noteworthy because the T. Brucei genome project is rapidly moving forward and currently there are at least partial sequences available for most of the genes of the parasite. Brucei was first achieved in transient fashion by electroporation of in vitro synthesized dsRNA. A more valuable approach utilizing inducible, stably maintained constructs that express stem-loop transcripts has recently been described (,). These integrated constructs are heritable and provide for prolonged expression of dsRNA, allowing interference of expression of genes of highly stable proteins.
We have now designed a vector (pZJM) in which a PCR-amplified gene fragment is ligated in a single step between opposing T7 promoters. PZJM also incorporates features of the powerful tetracycline-inducible promoters in vectors developed by Wirtz et al. Integration of pZJM into a nontranscribed region of the T. Brucei genome results in establishment of a stable cell line.
Tetracycline induction then leads to the production of dsRNA with concomitant degradation of the target mRNA. We have assessed the effectiveness of this vector in targeting a wide variety of T. Brucei genes. Given its relative ease of use and effectiveness, pZJM should prove to be a valuable tool in evaluating gene function in trypanosomes. Plasmid Constructs Two vectors for RNAi were constructed by modifying pLew100 (a generous gift from Drs.
Elizabeth Wirtz and George Cross, Rockefeller University ). The first vector is similar to that recently published by Shi et al. A) and produces dsRNA as a stem-loop structure.
The vector was constructed as follows. First, a ∼ 500-bp fragment of the gene of interest (target gene) was amplified by PCR from T.
Brucei 427 DNA using primers containing XbaI and HindIII linkers. This product was ligated into the NheI/ HindIII sites of pJM326 (a gift from Dr. Stephen Gould, Johns Hopkins University ). This plasmid carried the gene for an irrelevant Myc-tagged human protein (Pex 11 β), part of which will form the loop of the stem-loop transcript. This construct was then digested with HindIII and XbaI, liberating a ∼1050-bp fragment containing the target gene fused to the 3′ terminus of the Pex 11 β/Myc tag construct.
Figure 1 RNA interference vectors. A, the stem-loop vector. The 5′-3′ directions of the gene targeted for RNA interference are indicated with horizontal arrows. B, the pZJM vector.
A sequence from the targeted gene, in either orientation, replaces the α−tubulin sequence. Both vectors are shown in linearized form, after cleavage in the rDNA spacer. These vectors are based on previous constructs devised in the Ullu lab , the Cross lab , and the Fire lab. The tetracycline-inducible procyclin promoter ( procyclin arrow), the tetracycline operator ( Tet Op), dual T7 terminators ( ΩΩ), tetracycline-inducible T7 promoter ( T7 arrows), ribosomal DNA ( rDNA), actin poly(A) addition sequence ( ACT polyA), phleomycin resistance gene ( BLE), splice acceptor site ( SAS), aldolase poly(A) addition sequence ( ALD polyA), and “stuffer” DNA are indicated. Vectors are not drawn to scale.
The vector pLew100 was then digested with MluI and XbaI, releasing the luciferase reporter. The same region of the target gene as used above was amplified by PCR with MluI and XbaI linkers, followed by ligation into pLew100. Finally, the modified pLew100 was cut with XbaI and HindIII, and the ∼1050-bp target gene/Pex 11 β/myc tag fragment was ligated into the vector. The resultant plasmid contains two copies of the target gene fragment in opposite orientation separated by a ∼550-bp irrelevant stuffer region. Transcription from the tetracycline-inducible procyclin promoter yields an RNA that can fold into a stem-loop structure.
The pZJM vector (see Fig. B) was constructed as follows. Dual T7 terminators were amplified by PCR from pLew82 using primers containing KpnI and NheI linkers (the primers were GGTACCCCGGATATAGTTCCTCCTTTCA and GCTAGCCCGCTGAGCAATAACTAGCATAACC), and the 230-bp product was cloned into pCR-TOPO (Invitrogen, Carlsbad CA). A 71-bp fragment containing the T7 promoter/Tet operator was amplified from pLew82 using primers with NheI and XhoI linkers (the primers were GCTAGCACCTGATTAATACGACTCACT and CTCGAGGGGCTAGATCTCTATCACTG). This fragment was then ligated into the NheI and XhoI sites of the T7 terminator construct generated above. The T7 terminator/promoter cassette was then ligated into the KpnI and XhoI sites of pBluescript SK+ (Stratagene, La Jolla CA). A 650-bp fragment of the T.
Brucei α-tubulin gene was amplified by PCR with primers containing XhoI and HindIII linkers (the primers were CACCTCGAGATGCGTGAGGCTATCTGCATC and CACAAGCTTAGGTTGCGGCGAGTCAAATC). The product was then ligated into the XhoI and HindIII sites of the modified pBluescript SK+ described above. A second T7 terminator/promoter cassette was generated by PCR of the original cassette using primers with BamHI and PstI linkers (the primers were GGATCCCCGGATATAGTTCCTCCTTTCA and CTGCAGGGGCTAGATCTCTATCACTG). The product was then ligated into the modified pBluescript SK+ that contains the tubulin/T7 terminator/promoter cassette. This pBluescript SK+ construct contains two opposing T7 terminator/promoter cassettes separated by a 650-bp tubulin gene fragment. To generate pZJM, the KpnI- BamHI fragment was ligated into pLew100, replacing the luciferase gene.
The tubulin fragment was included to prevent the formation of cruciform structures. To insert a gene fragment into pZJM for transfection into the parasite, a 250–600-bp fragment of the coding region of the target was amplified by 30 cycles of PCR, with an annealing temperature of 55 °C using 50 ng of T. Brucei 427 genomic DNA as template. The PCR primers contained XhoI and HindIII linkers. The PCR products were then ligated into pZJM, replacing the tubulin stuffer. Trypanosome Growth and Transfection Procyclic T. Brucei strain 29–13 (also a gift from Drs.
Elizabeth Wirtz and George Cross), which harbors integrated genes for T7 RNA polymerase and the tetracycline repressor, were grown in SDM-79 supplemented with 15% fetal bovine serum. Parasites were cultured in the presence of G418 (15 μg/ml) and hygromycin (50 μg/ml) to maintain the T7 RNA polymerase and the tetracycline repressor constructs, respectively. For transfection, cells (1 × 10 8) were washed once in 5 ml of cytomix and resuspended in 0.5 ml of cytomix containing 10 μg of plasmid that had been linearized with EcoRV (stem-loop construct) or NotI (pZJM) so that it could target the rDNA spacer region. Transfections were carried out in 2-mm cuvettes using a BTX electroporator with peak discharge at 1.6 kV in resistance timing mode R2 (24 ohms).
Immediately following transfection, cells were transferred into 10 ml of SDM-79 supplemented with G418 and hygromycin. After 1 day, selection was applied by culturing in the presence of 2.5 μg/ml phleomycin, and the cells were grown for 2 weeks to form stable lines.
The cells were not cloned. For induction of dsRNA, cells were cultured in the above medium containing 1.0 μg/ml tetracycline. Cells were diluted 10-fold when densities reached a minimum of 1 × 10 6 cells/ml.
They were not allowed to grow beyond 5 × 10 6 cells/ml before diluting again. Cell densities were determined using a Coulter counter model Z1 (Coulter Electronics) and plotted on the growth curves as the product of the cell density and the total dilution. Northern Blotting and Hybridization Total RNA was purified from 5 × 10 7 parasites (Purescript RNA Isolation Kit, Gentra Systems) and electrophoresed on formaldehyde-1.5% agarose gels. Ribosomal RNA levels were estimated by ethidium bromide staining to ensure equal amounts of total RNA were loaded in each lane. The RNA was then transferred to GeneScreen Plus transfer membrane (PerkinElmer Life Sciences).
32P-Labeled probes were made by random priming the same PCR products used as inserts in either the stem-loop or the pZJM vector. Hybridization was carried out overnight at 42 °C in 50% formamide, 5× SSC, 10% dextran sulfate, 1% SDS, 1× Denhardt's solution, 0.2 mg/ml salmon sperm DNA. Blots were washed twice for 5 min in 0.1× SSC, 0.1% SDS at 56 °C. The percentage of reduction of mRNA levels induced by RNAi was estimated by densitometry of appropriately exposed autoradiograms. Western Blotting Proteins from 2.5 × 10 6cell equivalents were separated by SDS-polyacrylamide gel electrophoresis (15% gel), transferred onto Immobilon P, and probed using mouse IgG polyclonal antibodies raised against Crithidia fasciculata universal minicircle sequence-binding protein (UMSBP).
The antibody was a generous gift of Dr. Joseph Shlomai (Hebrew University).
Primary antibodies were detected with secondary rabbit anti-mouse IgG conjugated to alkaline phosphatase and then developed with NBT/BCIP. RNA Interference Using the Stem-Loop Vector Using this vector (Fig. A), we tested sequences encoding parts of a topo II and a candidate mitochondrial RNA polymerase. The entire open reading frame of topo II had been cloned, whereas the RNA polymerase was identified in the TIGR database by searching for sequences homologous to a mammalian mitochondrial RNA polymerase. We selected stable transfectants, induced expression of dsRNA and then monitored the effects of dsRNA production on steady-state mRNA levels and cell growth. In the case of topo II (Fig.
), Northern analysis revealed a ∼90% reduction of its mRNA 24 h after induction with tetracycline. Cells stopped growing eight days after induction (Table and Fig. Interestingly, the kDNA of these cells, visualized by DAPI staining, became gradually smaller until the cells became dyskinetoplastic (not shown) and died (Table ). We are now conducting a detailed study of the mechanism of this loss of kDNA. Expression of the mitochondrial RNA polymerase dsRNA led to a ∼95% reduction of its mRNA after 24 h of induction, with growth inhibition 6 days after induction (Fig. This inhibition was followed by cell death (Table ).
Figure 2 Effect of stem-loop dsRNA on cell growth and mRNA level. The growth profiles of cells transfected with the stem-loop vector for topo II and mitochondrial RNA polymerase ( RNA Pol) were determined by growing the cells in the absence (○) or upon addition of 1 μg/ml tetracycline (▪).
See “Experimental Procedures” for a description of the calculation of cell density. Insets show Northern analysis of mRNA levels from uninduced (−) and induced (+) cells expressing stem-loop dsRNA for 24 h. The topo II mRNA was 5.4 kb, and that of RNA pol was 4.0 kb. Intact dsRNA and its fragments were detected on the Northern blots of RNA from the induced cells, but none was detected in RNA from uninduced cells. RNA Interference Using the pZJM Vector To avoid the time-consuming introduction of inserts into the stem-loop vector, we constructed pZJM, a vector that allows PCR-generated fragments of any target gene to be inserted between opposing tetracycline-inducible T7 promoters (Fig. In an initial characterization of this vector, we attempted to generate FAT cells, a swollen cell phenotype described previously for RNAi of the α-tubulin gene (, ). With a fragment of the α−tubulin gene in the cloning site of pZJM, we transfected procyclic forms of T.
Following the generation of stable transfectants, we induced with tetracycline and after 1 day FAT cells were evident, comparable with previous results. To further characterize pZJM, we tested RNA interference of a number of other genes, including one homologous with UMSBP, a C.
Fasciculata protein that binds to the replication origin of kDNA minicircles. After 40 h of tetracycline induction, UMSBP mRNA was reduced by ∼80%.
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In contrast, probing the same blot for mitochondrial pol β mRNA revealed no change in level after tetracycline induction (Fig. A, bottom panel), confirming the specificity of RNAi.
Although a substantial amount of UMSBP-specific dsRNA of the predicted size is detected in the tetracycline-induced lane, a small amount of the same species was detected in the uninduced culture (indicated by an asteriskin Fig. Therefore, there is a low level of expression of dsRNA even in the absence of added tetracycline. Induction of dsRNA led to loss of detectable UMSBP protein after 10 days (Fig. In another experiment, UMSBP protein levels were reduced by ∼32% after 1 day of induction and ∼75% after 3 days of induction (data not shown). Growth was slowed in the tetracycline-induced cells starting at about day 7. After 12 days of induction, 10–15% of the cells lacked nuclei, whereas less than 0.1% of the uninduced cells were anucleate. Figure 3 RNA interference of UMSBP expression using the pZJM vector.
A, Northern analysis of total RNA from uninduced (−) and induced (+) cells harboring pZJM(UMSBP). Cells with stable integration of the linearized vector were grown in the absence or presence of 1 μg/ml tetracycline. Northern analysis of RNA from cells induced for 40 h was performed using a UMSBP probe. As a loading control, the same blot was probed for pol β mRNA (2.8 kb).
UMSBP mRNA ( arrow) and dsRNA ( asterisk) are indicated. It is likely that a large fraction of the dsRNA was cleaved to small fragments. B, Western analysis of whole cell lysates from T. Brucei harboring pZJM(UMSBP). Proteins from 2.5 × 10 6 induced (+ tetracycline ( Tet), after 10 days) and uninduced cells were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot using an antibody against UMSBP from C. The arrow indicates the cross-reacting species of the predicted molecular weight for T.
Brucei UMSBP (14.6 kDa). C, cell growth after expression of UMSBP dsRNA. Cultures were grown in the absence (○) or presence of 1 μg/ml tetracycline (▪), and cell density was plotted against days of growth. We then tested the pZJM system on genes involved in a number of biological processes, including kDNA replication (topo II ), SSE1 (homologous to C. Fasciculata SSE1 ), mitochondrial pol β (homologous to C. Fasciculata mitochondrial pol β )), GPI biosynthesis (GPI10 ), nuclear replication (CDC47 (homologous to Saccharomyces cerevisiae CDC47 ), glycosome biogenesis (Pex 11 (30)), polyamine biosynthesis (ornithine decarboxylase (ODC) ), RNA metabolism (RNase H1 (homologous to C. Fasciculata RNase H1 ), and p34/p37 ).
The RNAi effect on growth kinetics and mRNA levels for all of these proteins is shown in Fig., and the results are summarized in Table. Using pZJM(topo II) to interfere with topo II expression, we were able to reduce its mRNA level by ∼80% at 40 h (Fig.
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The growth rate of a culture expressing topo II dsRNA was similar to that with the stem-loop vector, with cell growth inhibited about 8 days after induction. Furthermore, expression of dsRNA from pZJM(topo II) led to dyskinetoplasty and cell death, as seen with the stem-loop construct (not shown). As was the case with UMSBP, a small amount of dsRNA was synthesized in uninduced cells (not shown), but this had little impact on cell growth. The doubling time of the uninduced cell line was 17.8 h compared with the doubling time of the parental cell line 29–13, which was 17.4 h (not shown). Furthermore, cells harboring pZJM(topo II) cultured for 2 months in the presence of phleomycin (to maintain the integration construct) grew normally. These cells retained the ability to express dsRNA upon induction and became dyskinetoplastic and died at rates similar to those seen with the original transfectants (not shown).
These results indicate that the low level of topo II-specific dsRNA in uninduced cells does not have detectable deleterious effects. Figure 4 Analysis of a wide range of genes using the pZJM vector. The growth curves show cells uninduced (○) or induced (with 1 μg/ml tetracycline) (▪).
The insets show Northern analyses of mRNA levels in these uninduced (−) or induced (+) cells lines. RNA was collected after 40 h of induction, except in the case of pol β mRNA, which was collected after 33 h. The sizes of the mRNAs are: topo II, 5.4 kb; SSE1, 1.9 kb; p34/p37, 1.2 kb; Pex 11, 1.5 kb; ODC, 1.9 kb; CDC47, 3.3 kb, RNase H1, 1.6 kb; GPI10, 2.2 kb; pol β, 2.8 kb.
Table summarizes the effects of dsRNA expression on cell growth and mRNA levels for the genes examined. RNA interference of p34/p37 (mRNA reduced ∼95%), SSE1 (mRNA reduced ∼35%), ODC (mRNA reduced ∼98%), and Pex 11 (mRNA reduced ∼93%) led to aberrant cell growth after 5–10 days (Fig. Following induction of p34/p37 dsRNA, 20% of the parasites became multinucleate, whereas uninduced cells did not show this effect. In the case of SSE1, there appeared to be an enlargement of the kinetoplast after expression of dsRNA. On the other hand, RNA interference of other genes, including GPI10 (mRNA reduced ∼50%), CDC47 (mRNA reduced ∼80%), and pol β (mRNA reduced ∼55%), had little or no effect on trypanosome growth. PZJM(RNase H1)-transfected cells expressed dsRNA (not shown) which, in contrast to all the other genes studied, did not lead to a reduction of the targeted mRNA (Fig. RNA Interference of Ornithine Decarboxylase Yields Putrescinedependent Mutants ODC converts ornithine to putrescine, a reaction in the polyamine biosynthetic pathway that is required for parasite growth.
Induction of ODC dsRNA led to a 98% reduction of mRNA level and inhibition of cell growth after 3 days (Table and Fig. Parasites lacking ODC because of conventional gene knockouts could not grow but persisted in an elongated, slender form for up to 8 weeks. We found the same morphology in cells expressing ODC dsRNA (not shown). Furthermore, like cells with the ODC genes knocked out , the RNAi parasites could be rescued if putrescine was added to the growth medium (Fig.). Finally, cells that had been expressing ODC dsRNA for 13 days were rescued by removal of tetracycline, with recovery occurring after 2 days of growth in tetracycline-deficient medium (Fig. The fact that cells deficient in ODC expression because of RNAi behave similarly to ODC knockout cells provides confidence in the reliability of RNAi for studying T. Brucei gene expression.
Figure 6 RNAi of multiple genes in a single pZJM construct (pZJM(topo II/pol β)). A, Northern analysis of total RNA from uninduced (−) and induced (+) cells expressing chimeric dsRNA probed for either topo II mRNA ( rightward arrow) or the pol β mRNA ( leftward arrow). The asterisk indicates dsRNA. The blot was also probed for UMSBP mRNA as a loading control.
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B, growth curves of cells expressing dsRNA of both mitochondrial topo II and pol β. Cultures were grown in the absence (○) or presence of 1 μg/ml tetracycline (▪). DISCUSSION Traditional methods for generating genetic knockouts in T. Brucei are time-consuming. In contrast, RNAi has been shown to be a much simpler and faster method for selective interference of gene expression (, ).
Unlike gene knockouts in which each locus must be individually disrupted, RNAi can inhibit expression of multiple copies of a target gene. Furthermore, RNAi does not even require fully cloned genes, whereas traditional knockouts generally need at least the 5′- and 3′-flanking regions. This feature makes RNAi a powerful tool to assess the function of partially sequenced genes available in the rapidly expanding T.
Brucei genome data base. It was our objective to make a vector that could be stably integrated and easily used for the analysis of a variety of genes. PZJM uses opposing tetracycline-inducible T7 promoters flanking a partial sequence of the target gene, and it is integrated into a nontranscribed region of the genome of procyclic trypanosomes that express both the T7 polymerase and the tetracycline repressor. PZJM is simple to use because it requires only a single cloning step in which the target gene fragment is inserted between the two T7 promoters. The convenience of the pZJM method is emphasized by the fact that after identification of a candidate gene sequence in the data base it takes as little as 3 weeks to obtain results from induction of RNAi in a stable cell line.
Both the stem-loop and pZJM vectors were effective in inducing the selective reduction of mRNA levels 24–40 h after induction with tetracycline. In many cases induction of RNAi resulted in a slowing or termination of cell growth, often followed by cell death. There was some variation in the effectiveness of interference of gene expression. For example, in the case of topo II with the stem-loop vector (Fig. ) or with pZJM(topo II/pol β) (Fig. ) reduction of the mRNA level was at least 90%.
However, with pZJM(topo II) (Fig. ) RNAi was less effective, with only about 80% reduction of mRNA level. In a different induction of the same pZJM(topo II) cell line, there was only a 70% reduction of topo II mRNA. In all cases, however, cell growth reduction and the timing of appearance of dyskinetoplasty were similar.
With pZJM, different genes varied in their sensitivity to RNAi as judged by Northern blots (Fig. In the cases of p34/p37, Pex 11, and ODC, the depletion of mRNA was nearly complete, and in all cases there was a striking effect on cell growth. In contrast, interference of expression of topo II, GPI10, and pol β had less severe effects on mRNA levels (Fig.
In the latter two cases there was no effect on growth, either because the genes are not essential under our experimental conditions or because there is sufficient mRNA present after tetracycline induction to sustain normal cell growth. Again, pol β interference showed variation with different constructs.
Using pZJM(pol β), mRNA reduction was 55% and using pZJM(topo II/pol β), it was 93%. It is worth noting that in one experiment using the stem-loop construct (not shown), pol β mRNA was depleted by 98%, and in that case the growth of the parasites was affected.
One reason for the differences could be that the cells are not clonal. RNase H1 mRNA was not reduced by expression of dsRNA (Fig.
This insensitivity to RNAi could be a consequence of rapid turnover of its mRNA. Alternatively, RNase H1 might play a heretofore unrecognized role in the RNAi mechanism so that its reduction would diminish the process of RNAi. As for phenotypes induced by RNAi, there is considerable variation with different genes in the rate of appearance of the growth effects, and this variation is probably due to differences in the turnover kinetics of both the mRNA and the protein product. DsRNA is detected several hours after the addition of tetracycline , and in most cases a large fraction of the mRNA is degraded within 40 h (Table and Fig. After depletion of mRNA, the appearance of a phenotype must depend on the abundance and the stability of the protein. For example, UMSBP mRNA was depleted by 80% after 40 h of dsRNA induction, and after 3 days protein levels dropped 75%.
The growth phenotype only became convincing after 10 days of induction, when the protein was undetectable by Western blotting (Fig. Some phenotypes, such as the appearance of anucleate cells after interference with UMSBP expression and multinucleate cells after blocking p34/p37 expression, are complex and have not yet been studied further. We consistently detected a small amount of dsRNA expressed from the pZJM vector in the absence of added tetracycline.
This did not appear to be the case with the stem-loop vector, which uses the procyclin promoter (Fig. ), because even with long exposures of the autoradiograms there was no detectable dsRNA in any of the uninduced cells. In all of the genes tested, this uninduced transcription had little or no detectable effect on the growth of the cells. In the case of pZJM(topo II), uninduced cells grew normally for over 2 months and, upon induction, exhibited the same phenotype as that of newly transfected strains.
We have shown that pZJM can be used to analyze a large number of genes quickly (Fig. ), and it can also be used to study two genes simultaneously (Fig. We are currently generating a pZJM-based library of genomic fragments that should allow the use of “forward genetics” to discover genes responsible for interesting phenotypes. Footnotes. This work was supported by Grants GM27608 and AI21334 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “ advertisement” in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. ‡ These authors contributed equally to this work. § To whom correspondence should be addressed: Dept. Of Biological Chemistry, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3790; Fax: 410-955-7810; E-mail: [email protected]. Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M008405200.
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2 Z. Englund, unpublished observations.
Blagnac Coordinates:: Country Government. Mayor (2014–2020) Bernard Keller Area 1 16.88 km 2 (6.52 sq mi) Population (2014) 2 23,416. Density 1,400/km 2 (3,600/sq mi). Summer /Postal code /31700 Elevation 119–153 m (390–502 ft) (avg. 135 m or 443 ft) 1 French Land Register data, which excludes lakes, ponds, glaciers 1 km² (0.386 sq mi or 247 acres) and river estuaries. 2: residents of multiple communes (e.g., students and military personnel) only counted once. Blagnac (: Blanhac) is a in the in southwestern.
It is the third-largest of the city of, although governed by a separate council, and is adjacent to it on the northwest side. It is a member of the. Archived from on 2009-10-31. Retrieved 2009-10-12. 27 April 2005.
Updated 28 April 2005. Retrieved on 12 February 2010. ' January 10, 2010, at the.'
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