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Nucleic Acids Research Pages 2339-2344  

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing
Introduction
Materials And Methods
   Plasmids
   Analysis of E.coli transformants
Results
Discussion
Acknowledgements
References

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing

Vera Holländer+ and Ulrich Kück*

Lehrstuhl für Allgemeine Botanik, Fakultät für Biologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany

Received February 4, 1999; Revised and Accepted April 19, 1999

ABSTRACT

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus' LSUrRNA gene, has been introduced into the lacZ gene encoding [beta]-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans-acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis-mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E.coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans-acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans-acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.

INTRODUCTION

Since group II introns were first discovered in fungal mitochondria, this intron type has been found in a wide range of plastid, mitochondrial and eubacterial genomes. In the latter case, organisms like cyanobacteria, proteobacteria and Gram-positive bacteria have all been shown to be sources of group II introns (1-6). The detection of group II introns in bacteria supports the hypothesis that group II introns entered eukaryotes together with prokaryotic endosymbionts (7). This invasion might have led to the evolution of discontinuous trans-spliced group II introns, and eventually to nuclear introns with a similar splicing mechanism (7-9).

Group II introns are characterized by a conserved secondary structure consisting of six stem-loop domains (10; Fig. 1). Splicing of group II introns proceeds via two transesterification steps, which release an intron lariat and the ligated exons. The splicing mechanism, which depends on the secondary and tertiary intron structure, has mainly been analyzed in in vitro self-splicing experiments using autocatalytic intron RNAs (reviewed in 11). In vivo, the few investigations using site-directed mutagenized introns include experiments with yeast in mitochondria, and with chloroplasts in the green alga Chlamydomonas reinhardtii chloroplasts (12-16). To date, as far as we are aware, there have been no in vivo analyses in prokaryotes of cis-acting group II intron sequences; although in vivo splicing, as well as its reverse reaction, has been demonstrated for the Lactococcus intron Ll.ltrB (4,6,17). In this latter example, the intron was able to splice itself out of its precursor RNA, and after transfer into Escherichia coli was also able to splice itself into the ligated exons (18,19). Ll.ltrB contains an open reading frame (ORF) encoding a protein with maturase, reverse transcriptase and endonuclease activities, which facilitate the intron's reactions. Only some of the group II introns characterized carry an ORF (10,11). Since all group II introns depend on trans-acting factors for in vivo splicing, cellular factors must facilitate splicing of those introns which do not encode their own maturase. Therefore, it remains questionable to what extent those introns without an ORF are able to move between different organisms. Intron transfer is thought to play a major role in intron evolution, and may have led to the wide distribution of group II introns amongst various organisms (7).

In order to test the proposed horizontal group II intron transfer, the mitochondrial group II intron rI1 from the green alga Scenedesmus obliquus (20) was inserted into the lacZ gene of E.coli, and subsequently analyzed with respect to its in vivo splicing activity. Intron rI1 does not contain an ORF, and as such processing of rI1 is dependent on cellular trans-acting factors provided by the host. Our analysis shows for the first time that a eukaryotic group II intron, which does not encode its own maturase, is correctly spliced in a prokaryotic host. In addition, our system also allows the mutational analyses of intron splicing in E.coli. From our data it becomes clear that intron processing in eukaryotes and prokaryotes shares identical features, i.e. both processes might depend on trans-acting factors with similar functions.

Table 1. List of rI1 mutations used for the splicing experiments in E.coli, and the function of the intron domains and nucleotides involved in vitro self-splicing
Transformant Mutation Function of cis-acting elements during in vitro splicing
[Delta]DV complete deletion of intron domain V formation of the catalytic core and catalysis (31,32)
[Delta]DVI complete deletion of intron domain VI lariat formation, 3[prime] splice site selection (33,78)
G1A substitution of the first intron nucleotide G1 to A 1. and 2. splicing step, lariat formation (34,12)
U608C single substitution of the [gamma] [prime] nucleotide 2. splicing step, 3[prime] splice site selection (35,16)
A398C U608 to C, or of the [gamma] nucleotide A398 to C
[gamma]G[gamma] [prime]C double mutations of the [gamma]-[gamma] [prime] base pair
[gamma]G[gamma] [prime]G carrying a substitution of [gamma] (A398) to
[gamma]U[gamma] [prime]A G and of [gamma] [prime] (U608) to C, of both [gamma] and [gamma] [prime] to G, and of [gamma] to U and of [gamma] [prime] to A, respectively
Numbers indicate references.

MATERIALS AND METHODS

Plasmids

All plasmids used for E.coli strain XL1Blue transformation (Stratagene; 21) are derivatives of plasmid prI1s-s and have previously been described (15,16). Plasmid prI1s-s was generated by inserting a 759 bp PCR fragment into vector pT3T7/EcoRV. This fragment carries the S.obliquus mitochondrial intron rI1, its intron binding site 1 (IBS1), as well as exon sequences from the C.reinhardtii tscA gene (46 bp from the 5[prime] exon, and 99 bp from the 3[prime] exon) (15). Plasmid designations indicate group II intron mutations derived from plasmid prI1s-s (Table 1; Fig. 1). After cloning, all PCR fragments were completely sequenced.


Figure 1. Secondary structure of the group II intron rI1. Deletion and substitution mutations used in this study are indicated. Intron sequences are shown as a solid line; 5[prime] and 3[prime] exon sequences are represented by white boxes. Roman numerals (I-VI) denote the six structurally conserved group II intron domains. Domain I is subdivided into sub-domains (A-D). EBS1-IBS1, EBS2-IBS2, [alpha]-[alpha] [prime], [gamma]-[gamma] [prime] and [epsis]-[epsis][prime] indicate three-dimensional base pairings. Nucleotides G1, A398 ([gamma]) and U608 ([gamma] [prime]), and domains V and VI were either substituted, or deleted by PCR-mediated mutagenesis, as described previously (16).

Analysis of E.coli transformants

In order to isolate nucleic acids, E.coli cultures were grown at 37 or 24°C to an optical density (OD590) of 0.8. The isolation of total RNA was performed according to Brosius et al. (22). Total RNA was used as a template for reverse transcription by AMV reverse transcriptase (Boehringer Mannheim) according to Krug and Berger (23) and Kennel and Pring (24). T7 primer and oligonucleotide no. 655 (ATT AAA ATC GGC ATT ACT TG) were used as primers. These oligonucleotides are complementary to the 3[prime] exon of pT3T7 and the 3[prime] exon sequence of the tscA insert, respectively. The derived cDNA was PCR amplified using T3 and T7 primers, as well as with oligonucleotides nos 654 (TAC CCA TTT ATT TGA AGG GC) and 655. PCR was conducted in incubation buffer [75 mM Tris-HCl pH 9.0, 1.5 mM MgCl2, 20 mM (NH4)2SO4, 0.1% Tween-20 and 50 µM of each deoxynucleotide] with 0.2 U of Goldstar DNA polymerase (Eurogentec). Amplification was undertaken using a GeneAmp PCR-System 9600 (Perkin Elmer-Cetus) for 40 cycles with the following profile `1 min 92°C, 1 min 50°C, 1 min 72°C' for the T3- and T7-primer combination, and with the profile `1 min 92°C, 1 min 45°C, 1 min 72°C' for oligonucleotides nos 654 and 655. Southern hybridizations of PCR products were carried out according to conventional procedures (25,26). Radioactively labeled oligonucleotide 607 (AAC TGG CTT TTA AGC CCT TC), which is complementary to the tscA 3[prime] exon, was used as a probe. For sequencing, the amplified cDNA products were eluted from an agarose gel and used as template in an asymmetric PCR using the reaction conditions described above. In order to produce a major single-stranded DNA product, 50 ng of the T3 primer were mixed with only 2 ng of the T7 primer. The obtained reaction products were purified with the QIAquick PCR purification kit (Qiagen) and directly used for sequencing.

RESULTS

In previous studies, we have shown that the S.obliquus mitochondrial group II intron rI1, can be successfully transferred into chloroplasts of the green alga C.reinhardtii (15,16,27). The heterologous intron is correctly spliced in vivo and the intron processing is supported by trans-acting factors provided by the heterologous host. Such data question the extent to which group II introns can be transferred between different organisms and organelles; and in order to investigate the possibility of a horizontal intron transfer, we analyzed processing of intron rI1 in a prokaryotic host.

Plasmid prI1s-s, carrying the S.obliquus rI1 intron together with its 6 bp intron binding site (IBS1), was used to construct a recombinant E.coli strain. Previously, we have shown that IBS1 is essential for splicing in a heterologous environment (15). As shown in Figure 2A, the intron is flanked by 46 bp of the tscA gene at its 5[prime] end and by 99 bp of the tscA sequence at its 3[prime] end. The tscA gene is a C.reinhardtii chloroplast gene which was used as a source of exonic sequence for the in vivo and in vitro splicing analyses mentioned above (15,16,27). Following its construction, the chimeric tscA-rI1 sequence was inserted into the EcoRV site of vector pT3T7BM (Fig. 2A). Transcription of the heterologous intron starts at the lacZ promoter of vector pT3T7BM, which leads to a precursor RNA containing the lacZ sequence, along with the multiple cloning site and the inserted tscA-rI1 construct. Total RNA isolated from transformants carrying the plasmid prI1s-s (Fig. 2) was analyzed by RT-PCR amplification, as a means of determining the splicing activity of intron rI1.


Figure 2. In vivo splicing of intron rI1 in E.coli. (A) Map of the recombinant plasmid prI1s-s, the derived pre-RNA and the spliced exon RNA. White boxes represent the tscA exons, the black box shows the mitochondrial intron rI1 of S.obliquus. The vector pT3T7BM's multiple cloning site and the portion of the lacZ gene in the vector is shown by thin lines. Arrows indicate the position and orientation of oligonucleotides nos 654 and 655 and of T3 and T7 primers, which were used for PCR amplifications. The lengths of the predicted PCR products are given. (B) RT-PCR analysis of splice products isolated from E.coli. Plasmid DNA from prI1s-s and cDNA from E.coli transformants grown at 37 and 24°C were used as templates for PCR amplification. T3 and T7 primers and oligonucleotides 654 and 655 were used as primers for RT-PCR. The molecular size marker (M) is given in base pairs.

Plasmid DNA of prI1s-s was used as a control for PCR experiments. The DNA amplification was carried out using the T3 and T7 primers which are complementary to the exonic sequences from the lacZ gene. PCR yielded a product of ~900 bp which carries the multiple cloning site together with the chimeric tscA-rI1 sequence (Fig. 2B). Consequently, amplification of the cDNA resulted in a 900 bp fragment containing the non-spliced intron. An additional fragment, of ~290 bp, was also obtained from amplifying the cDNA. This PCR product was not detected when control DNA was amplified, and most probably represents the spliced exon-exon molecule. These results were confirmed by RT-PCR, using another pair of oligonucleotides (nos 654 and 655) as primers. Both oligonucleotides are complementary to the tscA 5[prime] and 3[prime] exons, and in all experiments using either the DNA control, or the cDNA, as template (Fig. 2B), a 760 bp fragment from the chimeric tscA-rI1 sequence was generated. In addition, a 150 bp product was amplified only when the cDNA was used as a template for RT-PCR (Fig. 2B). Sequence analysis of the RT-PCR products representing the putative spliced exon-exon RNAs confirmed that they do not contain the intron rI1, rather the correctly spliced tscA exons from inside the lacZ gene's multiple cloning site (data not shown). Similar results were also obtained when using either plasmids containing rI1 inside short homologous S.obliquus exon sequences, or plasmids carrying rI1 integrated into different tscA sequences (data not shown). Detection of the ligated exon-exon RNAs clearly demonstrates that the mitochondrial intron rI1 is correctly spliced in E.coli.

Intron rI1 is one of only a few group II introns which show autocatalytic splicing in vitro. The autocatalytic activity of rI1 depends on the reaction conditions, and in particular is limited by the incubation temperature (28). In vitro splicing of rI1 is efficient at 45°C, somewhat reduced at 37°C, and at temperatures of [le]24°C there are no splicing products detected at all (data not shown). Escherichia coli is generally grown at a temperature of 37°C, which in vitro should support low levels of rI1 autocatalytic activity. In the next set of experiments we examined whether E.coli in vivo processing of rI1 is solely an autocatalytic activity. For this purpose, in vivo splicing of rI1 was conducted in E.coli cells grown at 24°C, as well as in cells grown at 37°C. If splicing of rI1 is mainly an autocatalytical process, without the support of any trans-acting factors from E.coli, then the reduced temperature should inhibit splicing, due to the strict temperature dependency of the self-splicing reaction. In contrast, in vivo splicing mediated by trans-acting factors is possible at low temperatures, while in vitro splicing is completely prevented. RT-PCR amplification of RNA from E.coli transformants grown at both 37 and 24°C was performed using the two primer combinations mentioned previously (Fig. 2). PCR products representing the unspliced precursor, as well as the spliced exons, were detected in equal amounts at each temperature. Sequencing of these PCR products confirmed correct in vivo splicing in E.coli cells grown at 375C as well as at 24°C (data not shown). Thus, splicing of rI1 does not show a dependency on growth temperature, and is therefore most probably supported by trans-acting factors.

Group II introns are characterized by their complex secondary and tertiary structure (Fig. 1). Structural elements, conserved sequences and specific single nucleotides can all determine the nature of the splicing reaction, both in vitro as well as in vivo. In contrast to numerous in vitro splicing experiments already described (reviewed in 11) and few reports of in vivo investigations done with yeast mitochondria and C.reinhardtii (12-15,29,30), we have demonstrated an E.coli-based system which allows us to examine the relevance of various cis-acting elements in a prokaryotic host. Therefore, derivatives of plasmid prI1s-s, carrying different intron mutations, were transformed into E.coli (Fig. 1). Table 1 summarizes the intron mutants used for in vivo analyses, as well as the function of the corresponding cis-acting elements during autocatalytic in vitro self-splicing.


Figure 3. Analysis of in vivo splicing of mutated introns in E.coli. cDNA of E.coli transformants carrying various intron mutants as indicated, was used as template for RT-PCR. The amplification reactions were performed using oligonucleotides nos 654 and 655 (Fig. 2A). The PCR products obtained were blotted and hybridized with oligonucleotide no. 607, which is complementary to an internal tscA exon sequence.

The resulting pre-RNAs with deleted or substituted intron sequences were tested for their in vivo splicing activity by RT-PCR (using oligonucleotides nos 654 and 655 as primers; Fig. 2A). As shown in Figure 3, both the unspliced RT-PCR product (760 nt) and spliced 150 nt fragment were detected in E.coli transformants carrying the wild-type intron rI1. However, as expected, RT-PCR analyses of intron derivatives completely lacking domain V ([Delta]V) identified only unspliced precursor RNA (domain V is part of the catalytic center of group II introns and thus is essential for splicing; 31,32). Similarly, deletion of domain VI, which is involved in lariat formation during the first splicing step (33), leads to a complete loss of splicing activity ([Delta]VI). RT-PCR products from both mutants are smaller than the fragment representing the wild-type precursor due to the deletion of either domain V or VI, respectively. The first intron nucleotide G1, which is highly conserved and in group II introns, is involved in both splicing steps (34), was substituted by an A in intron derivative G1A. Amplification of cDNA from transformants carrying this derivative yielded only 760 nt products, representing the unspliced RNA. Intron nucleotides A398 and U608 form the so-called [gamma]-[gamma] [prime] tertiary interaction (Fig. 1), which is involved in the second step of group II intron splicing, both in vitro and in vivo (16,35). Again, the single substitution of either A398 (A398C) or U608 (U608C) results in a complete loss of any splicing activity, similar to the G1 mutant. In these cases, only the amplification product of the unspliced precursor RNA is visible (Fig. 3). Double mutants of the [gamma]-[gamma] [prime] base pairing result in different pattern of in vivo phenotypes. The double mutant [gamma]G[gamma] [prime]G, which does not form a Watson-Crick base pair between the [gamma] and [gamma] [prime] sites, shows a complete loss of spliced exons, as previously seen with the single substitutions of U608 ([gamma] [prime] ) and A398 ([gamma]). Southern hybridizations of the PCR products with an internal exon-specific oligonucleotide exclude the occurrence of low amounts of spliced RNAs, and confirm the lack of exon-exon molecules in the deletion mutants as well as in all of the point mutations mentioned above (Fig. 3). Only double mutants, which form a regular Watson-Crick base pair between the [gamma] and [gamma] [prime] positions, restore the splicing capacity of the heterologous intron. In transformants [gamma]G[gamma] [prime]C and [gamma]U[gamma] [prime]A, the spliced exons are detected as a RT-PCR product of ~150 nt (Fig. 3). Taken together, these data reveal that the identity of the first nucleotide and the tertiary base pairing between the [gamma] site and the [gamma] [prime] site are essential for successful splicing in E.coli. In conclusion, our data show that the mitochondrial intron rI1 is a useful tool for the mutational analyses of cis-acting elements in the prokaryote E.coli.

DISCUSSION

Group II introns are considered to be mobile genetic elements, and their transposition between loci and organisms is thought to have been a major determinant of intron evolution (36,37). Ongoing changes in intron distribution are dependent on recent horizontal intron transfers between different species. A horizontal intron transfer might be mediated by mobile genetic elements such as transposons and plasmids (2,4,5), or as postulated for group I introns, by parasites, symbionts, viruses and phages carrying intron sequences (38-40). Transposition of mobile group II introns into new loci results from the reverse splicing activity mediated by the intron RNA itself, and is facilitated by intron-encoded polypeptides (41-43). The hypothesis that these mechanisms help spread group II introns is supported by artificial intron transfers into new hosts, resulting in successful splicing of heterologous intron RNAs in vivo. This has recently been shown by intron transfer between eukaryotic organelles (from mitochondria of S.obliquus into chloroplasts of C.reinhardtii; 27) as well as between different prokaryotes (from Lactococcus lactis into E.coli; 18).

As far as we are aware, this work is the first study showing splicing of a eukaryotic intron in bacteria. The mitochondrial intron rI1 from S.obliquus is correctly processed in E.coli, leaving the exonic intron binding site 1 from S.obliquus inside the ligated exons. In contrast to in vitro self-splicing, in vivo splicing in E.coli is independent of temperature, suggesting that trans-acting factors are supporting the processing of this heterologous intron.

Studies with group I introns have shown that the trans-acting factors which facilitate in vivo splicing, do so by supporting the folding of intron RNAs into specific secondary and tertiary structures (44-46). Certain splicing factors specifically recognize and stabilize the catalytic core of either individual introns, or intron groups (47,48). Alternatively, RNA chaperones can bind intron RNAs non-specifically and prevent formation of an inactive conformation. They can also resolve misfolded precursors and facilitate association of critical substructures, thus constraining them to a correct and catalytically active structure. In E.coli, two putative splicing factors with RNA chaperone activity, which promote splicing of heterologous group I introns in vitro and in vivo, have been identified (49-51). Nucleoid protein StpA and the ribosomal protein S12 both exhibit unspecific RNA binding and RNA chaperone activities in E.coli. These proteins, as well as any additional RNA chaperones, may also facilitate folding and splicing of other introns in E.coli, including the group II intron rI1. In vivo splicing of rI1 in E.coli is independent of temperature and proceeds in a similar fashion at both 37 and 24°C. The reduction of temperature leads to a cold-shock response in E.coli, which is characterized by both an alteration in the translational machinery and a dramatic induction of the major cold shock protein CspA (reviewed in 52,53). CspA and other cold shock proteins act as RNA chaperones which, at low temperatures, bind RNA molecules with low specificity to prevent `wrong' RNA secondary structures forming (54,55). We speculate that these RNA chaperones might also prevent the formation of inactive intron structures, which impede splicing at low temperatures in vitro. In addition, CspA can induce expression of several different genes, including hn-s, which encodes a nucleoid protein (56,57). This protein might act as a splicing factor at low temperatures like the homologous StpA nucleoid protein from E.coli (50).

All intron mutations analyzed in E.coli and in C.reinhardtii chloroplasts result in similar splicing defects in both systems (16). The deletion of domains V and VI, the substitution of the highly conserved first intron nucleotide G1 and the destruction of the tertiary [gamma]-[gamma] [prime] base pair all lead to a complete lack of exon-exon molecules in vivo. In sharp contrast, in vitro self-splicing of identical point mutants from rI1 and other group II introns results in the formation of ligated exons, suggesting the involvement of trans-acting factors in intron splicing and exon ligation in vivo (12,16,35). Since all rI1 mutations investigated cause similar splicing phenotypes in both heterologous organisms, either identical splicing factors, or trans-acting factors exhibiting corresponding functions, are involved in intron processing in both chloroplasts and bacteria.

Most of the known non-intron encoded splicing factors of group I and group II introns do not act in an intron-specific way, rather promoting either the splicing of several different introns, or different intron groups (58-61). In most cases, these trans-acting factors are bi-functional, and affect other functions beside intron splicing (49,62,63). Such trans-acting factors show general nucleic acid binding activity, and are involved in various stages of gene expression. For both RNA chaperones and RNA helicases, RNA binding proceeds unspecifically (49,50). Splicing factors such as tRNA synthetases specifically recognize a defined structure, which is shared by both tRNAs and group I intron RNA (46). Therefore, splicing factors may exhibit ancient functions, which have been adapted to facilitate intron splicing (64,65). Since most of the known bi-functional splicing factors are involved in gene expression, these proteins are often distributed ubiquitously and share high degrees of homology, particularly between prokaryotes and eukaryotic organelles (66). The conservation of splicing factors may allow intron sequences transferred horizontally to be correctly spliced. This idea is supported by several trans-factors which facilitate splicing of heterologous group I introns. The td intron from phage T4 can be processed by either the nucleoid StpA protein and protein S12 from E.coli, or by the CYT18 tRNA synthetase from Neurospora crassa (49,50,67). Splicing factor CYT18 also suppresses mutations in the group I intron from Tetrahymena thermophila (68), whilst the homologous yts1 gene from Podospora anserina can complement cyt18 mutants from N.crassa deficient in in vivo splicing activity (69).

However, the horizontal transfer of introns is limited, for several different reasons. Introns which depend on a specific host-encoded splicing factor are unlikely to be transferred successfully. In contrast to rI1, which belongs to intron sub-group IIB, a plastid group IIA intron from tobacco is not spliced in C.reinhardtii (70), since processing of plastid group IIA introns depends on a chloroplast encoded factor (71). In addition, it appears that introns dependent on further RNA modifications, such as RNA editing (72,73) or trans-splicing (74,75), cannot be transferred successfully. Moreover, the post-splicing metabolism of excised intron RNAs may actually prevent successful intron transmission. The Tetrahymena group I intron is spliced in yeast and E.coli; however, the insertion of this heterologous intron reduces viability of both hosts (51,76). The stability of excised intron RNA is most probably not regulated correctly in E.coli and yeast, resulting in intron RNA interfering with cellular proteins or nucleic acids. Free intron RNA may well be toxic, since it can cleave both ligated exons and homologous sequences (77). Therefore, although the spreading of introns is generally limited, the relatively ancient group II introns which only need unspecific cellular factors (e.g. rI1; 27), or introns which encode their own maturase (e.g. Ll.ltrB; 18), can actually be transferred into new loci and hosts and thus may contribute to intron evolution.

ACKNOWLEDGEMENTS

We thank Ms Andrea Heinzl and Ms Beate Hübner for technical assistance, and Mr Hans-Jürgen Rathke for the artwork. This research was supported by the `Deutsche Forschungsgemeinschaft' SFB 480. V.H. received a stipend from the Graduiertenkolleg `Biogenese und Mechanismen komplexer Zellfunktionen' at the Ruhr-University Bochum.

REFERENCES

1. Ferat,J. and Michel,F. (1993) Nature, 364, 358-361. MEDLINE Abstract

2. Knoop,V. and Brennicke,A. (1994) Nucleic Acids Res., 22, 1167-1171. MEDLINE Abstract

3. Ferat,J., Le Gouar,M. and Michel,F. (1994) CR Acad. Sci. III, 317, 141-148.

4. Mills,D.A., McKay,L.L. and Dunny,G.M. (1996) J. Bacteriol., 178, 3531-3538. MEDLINE Abstract

5. Mullany,P., Pallen,M., Wilks,M., Stephen,J.R. and Tabaqchali,S. (1996) Gene, 174, 145-150. MEDLINE Abstract

6. Shearman,C., Godon,J.-J. and Gasson,M. (1996) Mol. Microbiol., 21, 45-53. MEDLINE Abstract

7. Wittop Koning,T.H. and Schümperli,D. (1994) Eur. J. Biochem., 219, 25-42.

8. Sharp,P.A. (1991) Science, 254, 663.

9. Copertino,D.W. and Hallick,R.B. (1993) Trends Biochem. Sci., 18, 467-471. MEDLINE Abstract

10. Michel,F., Umesono,K. and Ozeki,H. (1989) Gene, 82, 5-30. MEDLINE Abstract

11. Michel,F. and Ferat,J.-L. (1995) Annu. Rev. Biochem., 64, 435-461. MEDLINE Abstract

12. Peebles,C.L., Belcher,S.M., Zhang,M., Dietrich,R.C. and Perlman,P.S. (1993) J. Biol. Chem., 268, 11929-11938. MEDLINE Abstract

13. Boulanger,S.C., Belcher,S.M., Schmidt,U., Dib-Hajj,S., Schmidt,T. and Perlman,P.S. (1995) Mol. Cell. Biol., 15, 4479-4488. MEDLINE Abstract

14. Schmidt,U., Podar,M., Stahl,U. and Perlman,P.S. (1996) RNA, 2, 1161-1172. MEDLINE Abstract

15. Holländer,V. and Kück,U. (1998) Curr. Genet., 33, 117-123. MEDLINE Abstract

16. Holländer,V. and Kück,U. (1999) Nucleic Acids Res., 27, 2345-2353. MEDLINE Abstract

17. Mills,D.A., Manias,D.A., McKay,L.L. and Dunny,G.M. (1997) J. Bacteriol., 179, 6107-6111. MEDLINE Abstract

18. Matsuura,M., Saldanha,R., Ma,H., Wank,H., Yang,J., Mohr,G., Cavanagh,S., Dunny,G.M., Belfort,M. and Lambowitz,A.M. (1997) Genes Dev., 11, 2910-2924. MEDLINE Abstract

19. Cousineau,B., Smith,D., Lawrence-Cavanagh,S., Mueller,J.E., Yang,J., Mills,D., Manias,D., Dunny,G., Lambowitz,A.M. and Belfort,M. (1998) Cell, 94, 451-462. MEDLINE Abstract

20. Kück,U., Godehardt,I. and Schmidt,U. (1990) Nucleic Acids Res., 18, 2691-2697. MEDLINE Abstract

21. Bullock,W.O., Fernandez,J.M. and Short,J.M. (1987) BioTechniques, 5, 376-379.

22. Brosius,J., Cate,R.L. and Perlmutter,A.P. (1982) J. Biol. Chem., 257, 9205-9210. MEDLINE Abstract

23. Krug,M.S. and Berger,S.L. (1987) Methods Enzymol., 152, 316-323. MEDLINE Abstract

24. Kennell,J.C. and Pring,D.R. (1989) Mol. Gen. Genet., 216, 16-24.

25. Southern,E.M. (1975) J. Mol. Biol., 98, 503-517. MEDLINE Abstract

26. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

27. Herdenberger,F., Holländer,V. and Kück,U. (1994) Nucleic Acids Res., 22, 2869-2875. MEDLINE Abstract

28. Winkler,M. and Kück,U. (1991) Curr. Genet., 20, 495-502. MEDLINE Abstract

29. Boulanger,S.C., Faix,P.H., Yang,H., Zhuo,J., Franzen,J.S., Peebles,C.L. and Perlman,P.S. (1996) Mol. Cell. Biol., 16, 5896-5904. MEDLINE Abstract

30. Robineau,S., Bergantino,E., Carignani,G., Michel,F. and Netter,P. (1997) J. Mol. Biol., 267, 537-547. MEDLINE Abstract

31. Koch,J.L., Boulanger,S.C., Dib-Hajj,S.D., Hebbar,S.K. and Perlman,P.S. (1992) Mol. Cell. Biol., 12, 1950-1958. MEDLINE Abstract

32. Peebles,C.L., Zhang,M., Perlman,P.S. and Franzen,J.S. (1995) Proc. Natl Acad. Sci. USA, 92, 4422-4426. MEDLINE Abstract

33. Schmelzer,C. and Schweyen,R.J. (1986) Cell, 46, 557-565. MEDLINE Abstract

34. Chanfreau,G. and Jacquier,A. (1993) EMBO J., 12, 5173-5180. MEDLINE Abstract

35. Jacquier,A. and Michel,F. (1990) J. Mol. Biol., 213, 437-447. MEDLINE Abstract

36. Lambowitz,A.M. and Belfort,M. (1993) Annu. Rev. Biochem., 62, 587-622. MEDLINE Abstract

37. Belfort,M. and Perlman,P.S. (1995) J. Biol. Chem., 270, 30237-30240. MEDLINE Abstract

38. Belfort,M. (1990) Annu. Rev. Genet., 24, 363-385. MEDLINE Abstract

39. Yamada,T., Tamura,K., Aimi,T. and Songsri,P. (1994) Nucleic Acids Res., 22, 2532-2537. MEDLINE Abstract

40. Vaughn,J.C., Mason,M.T., Sper-Whitis,G.L., Kuhlman,P. and Palmer,J.D. (1995) J. Mol. Evol., 41, 563-572. MEDLINE Abstract

41. Zimmerly,S., Guo,H., Eskes,R., Yang,J., Perlman,P.S. and Lambowitz,A.M. (1995) Cell, 83, 529-538. MEDLINE Abstract

42. Zimmerly,S., Guo,H., Perlman,P.S. and Lambowitz,A.M. (1995) Cell, 82, 545-554. MEDLINE Abstract

43. Yang,J., Zimmerly,S., Perlman,P.S. and Lambowitz,A.M. (1996) Nature, 381, 332-335. MEDLINE Abstract

44. Shaw,L.C. and Lewin,A.S. (1995) J. Biol. Chem., 270, 21552-21562. MEDLINE Abstract

45. Weeks,K.M. and Cech,T.R. (1995) Cell, 82, 221-230. MEDLINE Abstract

46. Caprara,M.G., Lehnert,V., Lambowitz,A.M. and Westhof,E. (1996) Cell, 87, 1135-1145. MEDLINE Abstract

47. Guo,Q. and Lambowitz,A.M. (1992) Genes Dev., 6, 1357-1372. MEDLINE Abstract

48. Caprara,M.G., Mohr,G. and Lambowitz,A.M. (1996) J. Mol. Biol., 257, 512-531. MEDLINE Abstract

49. Coetzee,T., Herschlag,D. and Belfort,M. (1994) Genes Dev., 8, 1575-1588. MEDLINE Abstract

50. Zhang,A., Derbyshire,V., Galloway Salvo,J.L. and Belfort,M. (1995) RNA, 1, 783-793. MEDLINE Abstract

51. Zhang,F., Ramsay,E.S. and Woodson,S.A. (1995) RNA, 1, 284-292. MEDLINE Abstract

52. Jones,P.G. and Inouye,M. (1994) Mol. Microbiol., 11, 811-818. MEDLINE Abstract

53. Thieringer,H.A. and Jones,P.G. (1998) BioEssays, 20, 49-57. MEDLINE Abstract

54. Jiang,W., Hou,Y. and Inouye,M. (1997) J. Biol. Chem., 272, 196-202. MEDLINE Abstract

55. Graumann,P.L. and Marahiel,M.A. (1998) Trends Biochem. Sci., 23, 286-290. MEDLINE Abstract

56. La Teana,A., Brandi,A., Falconi,M., Spurio,R., Pon,C.L. and Gualerzi,C.O. (1991) Proc. Natl Acad. Sci. USA, 88, 10907-10911. MEDLINE Abstract

57. Brandi,A., Pon,C.L. and Gualerzi,C.O. (1994) Biochimie, 76, 1090-1098. MEDLINE Abstract

58. Séraphin,B., Simon,M., Boulet,A. and Faye,G. (1989) Nature, 337, 84-87. MEDLINE Abstract

59. Labouesse,M. (1990) Mol. Gen. Genet., 224, 209-221. MEDLINE Abstract

60. Altamura,N., Groudinsky,O., Dujardin,G. and Slonimski,P.P. (1992) J. Mol. Biol., 224, 575-587. MEDLINE Abstract

61. Wiesenberger,G., Waldherr,M. and Schweyen,R.J. (1992) J. Biol. Chem., 267, 6963-6969. MEDLINE Abstract

62. Akins,R.A. and Lambowitz,A.M. (1987) Cell, 5, 331-345. MEDLINE Abstract

63. Wiesenberger,G., Link,T.A., von Ahsen,U., Waldherr,M. and Schweyen,R.J. (1991) J. Mol. Biol., 217, 23-37. MEDLINE Abstract

64. Lambowitz,A.M. and Perlman,P.S. (1990) Trends Biochem. Sci, 15, 440-444. MEDLINE Abstract

65. Saldanha,R., Mohr,G., Belfort,M. and Lambowitz,A.M. (1993) FASEB J., 7, 15-24. MEDLINE Abstract

66. Watson,J.C. and Surzycki,S.J. (1983) Curr. Genet., 7, 201-210.

67. Mohr,G., Zhang,A., Gianelos,J.A., Belfort,M. and Lambowitz,A.M. (1992) Cell, 69, 483-494. MEDLINE Abstract

68. Mohr,G., Caprara,M.G., Guo,Q. and Lambowitz,A.M. (1994) Nature, 370, 147-150. MEDLINE Abstract

69. Kämper,U., Kück,U., Cherniack,A.D. and Lambowitz,A.M. (1992) Mol. Cell. Biol., 12, 499-511. MEDLINE Abstract

70. Deshpande,N.N., Hollingsworth,M. and Herrin,D.L. (1995) Curr. Genet., 28, 122-127. MEDLINE Abstract

71. Hübschmann,T., Hess,W.R. and Börner,T. (1996) Plant Mol. Biol., 30, 109-123. MEDLINE Abstract

72. Börner,G.V., Mörl,M., Wissinger,B., Brennicke,A. and Schmelzer,C. (1995) Mol. Gen. Genet., 246, 739-744. MEDLINE Abstract

73. Zanlungo,S., Quiñones,V., Moenne,A., Holigue,L. and Jordana,X. (1995) Curr. Genet., 27, 565-571. MEDLINE Abstract

74. Kück,U., Choquet,Y., Schneider,M., Dron,M. and Bennoun,P. (1987) EMBO J., 6, 2185-2195.

75. Knoop,V. and Brennicke,A. (1993) In Brennicke,A. and Kück,U. (eds), Plant Mitochondria. Group II Introns in Plant Mitochondria-Trans-Splicing, RNA Editing, Evolution and Promiscuity. VCH Verlagsgesellschaft Weinheim, New York, Basel, Cambridge, Tokyo, pp. 221-232.

76. Good,L., Abou Elela,S. and Nazar,R.N. (1994) J. Biol. Chem., 269, 22169-22172. MEDLINE Abstract

77. Margossian,S.P. and Butow,R.A. (1996) Trends Biochem. Sci., 21, 392-396. MEDLINE Abstract

78. Jacquier,A. and Jacquesson-Breuleux,N. (1991) J. Mol. Biol., 219, 415-428. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +49 234 700 6212; Fax: +49 234 709 4184; Email: ulrich.kueck@ruhr-uni-bochum.de
+Present address: QIAGEN GmbH, Max-Volmer-Straße 4, D-40724 Hilden, Germany

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