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Expression in Escherichia Coli by a Ribozyme

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Expression in Escherichia Coli by a Ribozyme Proc. Nail. Acad. Sci. USA Vol. 88, pp. 7303-7307, August 1991 Biochemistry Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme (intracediular/RNase HI) MOULDY SIOUD*t AND KARL DRLICA** *Public Health Research Institute, 455 First Avenue, New York, NY 10016; and tInstitute of Immunology and Rheumatology, Rikshospitalet, Fr. Qvamsgt. 1, 0172 Oslo, Norway Communicated by Thomas Cech, May 2, 1991 ABSTRACT Ribozymes are potentially very powerful pug/ml) and Casamino acids (0.5%), diluted 1:50, grown to an agents for perturbing intracellular gene expression. However, optical density of 0.4 at 600 nm, and induced with isopropyl pilot experiments in eukaryotes have met with mixed success. f3-D-thiogalactoside (IPTG) at 1 mM. Cells were harvested by We now report that a ribozyme designed to cleave the integrase centrifugation at 40C. In some experiments, integrase was gene of the human immunodeficiency virus (HIV), when tran- expressed as a fusion protein from p22KS6 obtained from the scribed from a plasmid in Escherichia coli, led to destruction of National Institutes of Health AIDS Research and Reference integrase RNA and complete blockage of integrase protein Program (contributed by S. Goff, Columbia University). This synthesis. These results indicate that ribozymes can be used to derivative ofpBR322 contains the 5' region ofthe E. coli trpE study intracellular gene expression in bacteria and that the gene fused to a short portion of the 3' end of the HIV-1 HIV-1 integrase gene may be a useful target for therapeutic reverse transcriptase gene plus the entire gene for HIV-1 ribozymes. integrase. For induction of this fusion, cells were grown overnight at 37°C in M9 medium supplemented with tryp- Ribozymes are RNA molecules that catalyze RNA cleavage tophan (20,g/ml), Casamino acids (0.5%), and ampicillin (50 (1-3). Those that act in trans first hybridize to a specific ,ug/ml), diluted 1:25 in M9 medium lacking tryptophan, sequence in a target RNA and then cleave the target within grown to an optical density of0.4 at 600 nm, and induced with that sequence. The hammerhead type refined by Haseloffand indoleacrylic acid (IAA) at 5 ,ug/ml. Gerlach (4) possesses a 22-nucleotide-long catalytic domain Plasmid pMPD48, expressing ribozymefB, was constructed that contains the consensus sequences responsible for cleav- by inserting a 118-base-pair synthetic DNA encoding a T7 age activity. This catalytic domain is embedded within flank- promoter and ribozyme ,B into the unique EcoRI site of ing sequences that are complementary to those surrounding pMPD51 (TetR), a derivative ofptrpl84 (10) lacking an EcoRI the cleavage site; they enable the ribozyme to interact with fragment. Plasmid pMPD52, which encoded ribozyme A3, a specific target sequence. The only sequence requirement was constructed in a similar manner. for the cleavage site is that it contain the trinucleotide GUC Nudeic Acid Analysis. Ribozyme activity was detected in (or a limited number of other triplets). Since appropriate vitro by incubating 2 ,ug of ribozyme with 20 ,ug of total target trinucleotides are common in RNAs, there are many cellular RNA extracted from strain MPD45 after induction of sites where ribozymes can be directed to cut. the trpE-integrase fusion gene (5 ,ug of IAA per ml at 30°C). To explore the possibility that bacterial cells could be used Mixtures oftarget RNA and ribozyme in 50 mM Tris-HCl (pH to refine ribozymes directed against viral RNA targets, we 8) were heated to 90°C for 1 min, rapidly cooled, and then examined the ability ofa ribozyme to cleave RNA containing incubated in 20 mM MgCl2 at 50°C or 37°C for 60 min. After the integrase gene ofthe human immunodeficiency virus type incubation, samples were treated with pancreatic DNase I 1 (HIV-1). The integrase gene, which is located within the 3' (ribonuclease free) for 15 min at 37°C. They were then domain ofthe HIV-1 pol gene, encodes a protein required for separated by electrophoresis through a 1% agarose gel con- the integration of viral DNA into the host chromosome (5, 6). taining 6.1% formaldehyde, transferred to a Hybond-N mem- Since integration is essential for productive infection, a brane (Amersham), and hybridized with a [32P]DNA probe ribozyme that interferes with the expression of integrase derived from the HIV-1 integrase gene [a 1631-base-pair Kpn should also block production of infectious HIV-1. The ribo- I/Sal I fragment of pNL4-3 (11)]. In some cases, the probe zyme that we designed completely blocked expression of was removed after hybridization by incubation in 10 mM integrase in Escherichia coli. Tris-HCI, pH 7.5/0.1% SDS/10 mM EDTA at 85°C for 2 hr. A second probe specific to trpE was then hybridized to the MATERIALS AND METHODS RNA on the membrane. Ribozyme activity occurring in vivo was detected after Bacterial Strains, Plasmids, and Culture Conditions. The induction of expression of both ribozyme and target RNA. plasmids and bacterial strains used in this study are listed in For analysis of RNA, total RNA was prepared by an acid Table 1. Integrase was expressed from plasmid pLJS10 guanidinium thiocyanate procedure (12). In experiments in obtained from J. Groarke, J. Hughes, and L. Shaw (Sterling which both RNA and DNA were recovered, samples were Research Group). This derivative of pBR322 contains the collected by centrifugation at 4°C and suspended in 2 mM HIV-1 (BH10) int gene to which codons for methionine and EDTA/1% SDS/1% (vol/vol) glycerol/0.01% bromophenol glycine have been added at the 5' end; the gene is under blue. Each sample was mixed with 1/9th vol of 37% form- control of a bacteriophage T7 promoter. To express inte- aldehyde, placed in a boiling water bath for 2 min, and then grase, cells containing pLJS10 were grown overnight at 37°C analyzed by electrophoresis through agarose gels containing in M9 minimal medium (9) supplemented with ampicillin (50 6.1% formaldehyde. Integrase RNA and trpE-integrase fu- The publication costs of this article were defrayed in part by page charge Abbreviations: HIV-1, human immunodeficiency virus type 1; IPTG, payment. This article must therefore be hereby marked "advertisement" isopropyl ,-D-thiogalactoside; IAA, indoleacrylic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 7303 Downloaded by guest on September 29, 2021 7304 Biochemistry: Sioud and Drlica Proc. NatL Acad. Sci. USA 88 (1991) Table 1. Plasmids and bacterial strains promoter (14). Ribozyme f3, which was identical to ribozyme Plasmid gene a except that it possessed a bacteriophage 17 transcription Strain Plasmid expressed terminator (15) at its 3' end, was synthesized in vitro (16) by transcription from a double-stranded template cut from plas- MPD45 p22KS6 trpE-int mid pMPD48. Ribozyme (3 was also synthesized in vivo from MPD61 pATH1 trp pMPD48 introduced into E. coli strain BL21(DE3), which MPD77 pLS10 int contains a chromosomal copy of the T7 RNA polymerase MPD92 pMPD48 Ribozyme (3 gene under control of the lacUVS promoter (8). Thus, the MPD97 pMPD51 resulting strain could be induced to synthesize ribozyme 13 by MPD98 pMPD52 Ribozyme A3 adding IPTG to the growth medium. As a control for possible MPD99 pLJS10; pMPD48 int; ribozyme ( antisense effects, we constructed ribozyme AP, in which the MPD100 pLJS10; pMPD52 int; ribozyme A,& entire catalytic domain of ribozyme ,B was replaced by a MPD101 pLJS10; pMPD51 int single guanosine. Both ribozyme P and ribozyme A&P were MPD102 p22KS6 trpE-int detected in extracts from E. coli cells by Northern hybrid- MPD103 p22KS6; pMPD48 trpE-int; ribozyme (3 ization analyses after induction with IPTG (Fig. 1B). In some MPD104 p22KS6; pMPD52 trpE-4nt; ribozyme AP experiments, read-through transcripts were also present MPD105 p22KS6; pMPD51 trpE-int (data not shown). MPD146 pUS10; pMPD48 int; ribozyme (3 Cleavage of Integrase RNA by Ribozymes in Vifro. The MPD148 pWS10 int activities of ribozyme a and ribozyme 1 were examined in The host for MPD45 and MPD61 is HB101 (7); for all others it is vitro using as a target total cell RNA extracted from cells BL21(DE3) (8). Strains MPD146 and MPD148 are rnclOS glyA::TnS induced to transcribe a trpE-integrase fusion gene. Samples derivatives in which the rmc and gly alleles were transduced from were incubated with each ribozyme, RNA species were strain NC124, obtained from Donald Court (Frederick Cancer Re- separated by electrophoresis, and target and product RNAs search Facility, Frederick, MD), by selection for kanamycin resis- were detected by hybridization with a radioactive probe for tance and screening for accumulation of precursor rRNA. the HIV-1 integrase gene. Ribozyme a and ribozyme 1 (both prepared in vitro) cleaved the 2000-nucleotide-long target sion RNA were detected by Northern blot analysis (13), using RNA into fragments of 1500 and 500 nucleotides; cleavage the Kpn I/Sal I fragment encompassing the integrase gene as was much more extensive when incubation was at 50TC (Fig. a hybridization probe. 2A, lanes 2 and 3) than when at 37c (lanes 6 and 7). Two RESULTS observations confirmed that cleavage occurred at the ex- pected site within the integrase portion ofthe fusion gene, 500 Ribozymes Designed to Cleave HIV-1 Integrase. We pre- nucleotides from the 3' end of the RNA. First, the two pared several ribozymes directed at the GUC located at the cleavage products had the expected sizes. Second, a probe position corresponding to nucleotides 4027-4029 of HIV-1 specific for the E. coli trpE gene detected only the 1500- (ribozyme sequences are described in Fig.
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