Nucleic Acids Research

RNA preparation

The YAC clones (vector pYAC4, host AB1380) were grown in 5 ml of a selective medium (6.7 g yeast nitrogen base with ammonium sulfate, without amino acids, 14 g casamino acids, 20 g glucose, 55 mg adenine, and 55 mg l-tyrosine per 1 l medium) overnight, then 15 ml of the rich medium YPD (1% yeast extract, 2% peptone, 2% glucose) were added, and the growth was continued to OD₆₀₀ = 1. Yeast RNA was prepared by extraction with hot acidic phenol (5), digested by RQ-RNase-free DNase (Promega, Mannheim, Germany), extracted with acidic phenol and isopropanol precipitated. The RNA integrity was checked on agarose.

RT-PCR

Five-hundred nanograms of yeast RNA were reverse transcribed using Superscript II (Gibco BRL, Life Technologies, GmBH, Eggenstein, Germany), according to manufacturer's instructions, with random, T_12-18, or gene-specific primers in 20 µl volume. Positive and negative control reactions were performed to verify the specificity of the results. The negative controls of RT included (i) treatment of 500 ng RNA with DNase-free RNase A, (ii) using 500 ng of RNA from an unrelated YAC clone and (iii) omitting RT of 1000 ng of RNA. Internal yeast controls included RT-PCR with two pairs of primers specific for two yeast genes, and with a pair of primers separated by an intergenic region. One microliter of each RT reaction and control mixtures was separately added to 40 µl of reactions prepared as master mixes containing a final concentration 0.15 nM of each of the four dNTPs, 50 mM KCl, 10 mM Tris-HCl pH 8.8, 1.6 mM MgCl₂, 0.08% Nonidet P40, 1 U of Taq polymerase (MBI Fermentas, Biogen sro, Prague, Czech Republic) and 15 pmol of each primer. The reactions were denatured on a thermal cycler (Hybaid, UK) at 94°C for 3 min, then cycled 37 times at 94°C for 30 s, at the corresponding annealing temperature (60°C, unless noted otherwise) for 30 s, and extension at 68°C for 2.5 min, and finally, extended at 70°C for 5 min. The PCR samples were run on 1-2.5% agarose gels containing ethidium bromide, and photographed under a UV illuminator to check the sizes of the products. Some gels were then blotted on Hybond N+ membranes (Amersham, MGP sro, Zlín, Czech Republic). The membranes were hybridized with corresponding DNA fragments of a known sequence labeled by hexamer priming, washed at a high stringency at 68°C, and exposed to a film. Some products were subcloned into pCR2.1 vector (Quiagen, Bioconsult sro, Prague, Czech Republic) according to supplier's instructions, and sequenced.

RPA

A DNA fragment [nt 1-411 of GenBank U64452 (6)], which covers 65 bp of the first exon of the Psmb1 gene and 346 bp of the Psmb1-Tbp intergenic region, was subcloned in pBluescript KS+ (Stratagene, Heidelberg, Germany). The plasmid was digested with EcoRI and transcribed by T3 RNA polymerase (Promega) in the presence of [[alpha]-³²P]CTP (300 Ci/mmol, Amersham). The total length of the probe including vector sequences was 516 bp. The template DNA was digested with RQ-RNase-free DNase (Promega) and the RNA probe was gel-purified. Aliquots of 8-25 × 10⁴ c.p.m. of the probe in elution buffer (0.25 M ammonium acetate, 1 mM EDTA, 0.1% sodium dodecylsulfate, 50% formamide) and the corresponding RNA in formamide were mixed, 20 µl of hybridization buffer (80% formamide, 40 mM PIPES pH 6.4, 400 mM NaCl, 1 mM EDTA) was added, the mixture was denatured at 85°C for 5 min and hybridized at 48°C for 16-18 h. The samples were diluted with 0.2 ml of RNase digestion buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 300 mM NaCl, RNases T1 and A) and incubated for 1 h at room temperature. Aliquots of 0.2 ml of stop solution (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7, 0.5% sarcosyl, 0.1 mg/ml yeast tRNA) and 0.4 ml isopropanol were then added, the RNA was precipitated for 30 min at -20°C, and spun for 15 min at 13 000 g, 4°C. RNA pellets were dissolved in 80% formamide, 1 mM EDTA pH 8, 0.1% bromphenol blue and 0.1% xylene cyanol FF, heated to 90°C for 3 min and loaded on a 6% polyacrylamide minigel containing urea. The minigel was then autoradiographed for 1-3 days.

3[prime] RACE

RNA was reverse transcribed with the CDS poly(dT) anchored primer as described above, except that the reaction was prewarmed to 42°C for 2 min before adding the Superscript II. RT-PCR was carried out as above with one gene-specific primer and with the CDS primer. The sequence of the CDS primer is 5[prime]-TTCTAGAATTCAGCGGCCGCT₃₀VN-3[prime], where V is A, G or C. The Psmb1 gene-specific primer, C53, is: 5[prime]-CCCCTGCGGAAAGACTGACAT-3[prime].

Transcription of mouse genes in Saccharomyces cerevisiae

To confirm that the yeast cell is able to transcribe mammalian DNA and to verify the specificity of the process, RT-PCR was performed using randomly primed total yeast RNA from the previously characterized FFEH11 YAC clone (Fig. 1). The YAC clone, encompassing 580 kb of mouse chromosome 17 DNA, was mapped by restriction endonucleases and found to be stable and non-chimeric (7). Five genes are situated in the chromosomal region contained in the YAC clone: Tbp [or Gtf2d for TATA-binding protein (7)], Psmb1, encoding [beta]-subunit C5 of the proteasome (10), D17Ph4e, for a protein of an unknown function (10,11), Dll1 for Delta-like 1 membrane polypeptide (8; R.M.J.Hamvas, unpublished results), and D17Ph8e (Z.Trachtulec and J.Forejt, manuscript in preparation). The Tbp and Psmb1 genes are tightly linked and transcribed in the opposite orientation (6). In addition to the genes, a number of other loci, including four microsatellites [(D17)Mia6, Mia7, Ph6, and Ph7], were identified in this region (Fig. 1). The results of the transcription analysis are summarized in Table 1. It soon became obvious that RT-PCR detects not only transcripts from all the known mouse genes but also from intergenic regions and microsatellite repeats. To exclude a trivial explanation that the RT-PCR amplifies traces of genomic DNA, great care was taken to control the purity of the analyzed yeast RNA. All RNA was treated with RNase-free DNase, and controls included (i) PCR without previous RT; (ii) degradation of RNA with DNase-free RNase prior RT; (iii) RT-PCR of the RNA of a non-overlapping YAC clone; none of these controls yielded a PCR product, indicating that, indeed, only RNA sequences transcribed from the FFEH11 YAC served as templates in the main experiments; (iv) additional controls included two pairs of primers detecting yeast coding sequences (Table 1, exps 25 and 26) and one pair of primers spanning a yeast intergenic region (exp. 27). The yeast primer pairs amplified no product without reverse transcription. The specificity of the mouse products was established by Southern blotting and/or by sequencing.

Table 1. Identification of various mouse coding and non-coding sequences in the FFEH11 YAC clone RNA
*-, no product (a product out of the limit of PCR amplification); NA, not applicable; T_a, annealing temperature.

Splicing of mouse genes in S.cerevisiae

To investigate whether the yeast splicing apparatus is able to process introns of mammalian genes correctly, nine pairs of deoxyoligonucleotide primers whose sites are separated by an intron(s) were used to amplify reverse transcribed total yeast RNA from the YAC clone FFEH11 (Table 1, exps 2, 4, 6-10, 12 and 14). In three experiments, PCR primers spanning short introns (<0.4 kb) of the Dll1, D17Ph4e and D17Ph8e genes were used (Table 1, exps 2, 7, 14). The resulting PCR fragments were specific for the unspliced RNA species (Fig. 2A, C and D). Two of the PCR reactions (Table 1, exps 8 and 9), primed in different exons of the Psmb1 gene, yielded fragments corresponding to spliced RNAs (Fig. 2B). The correct splicing was confirmed by sequencing. Three primer pairs spanning large introns, two from the D17Ph4e and one from the Tbp genes (Table 1, exps 4, 6 and 12 ), did not amplify any product. Therefore, the control-nested primers were designed within a flanking exon (5F/31L, 77U/77O and TFF/TFR; Fig. 1) to test for the presence of particular transcripts (Table 1, exps 3, 5 and 11). All control primers produced specific bands upon RT-PCR amplification (data not shown) suggesting that their adjacent introns were not spliced. Alternative explanation could be either the presence of a yeast promoter-like sequence within the mouse intron or transcription from the non-template strand.

Figure 1. Map of the FFEH11 YAC clone and positions of primers used in this study. The YAC, 580 kb in length, is shown as a line with the loci given above it. The D17 prefix is omitted in the names of anonymous loci and genes. The gene names are underlined. The microsatellite loci are designated (D17)Mia6, Mia7, Ph6 and Ph7. Arrows indicate directions of gene transcription, if known. The exon-intron gene structure of three genes is expanded below the map. Exons are symbolized by boxes and the primers by arrows below them. The arrows above the boxes show the directions of gene transcription. The introns and intergenic regions are shown as lines and dashed lines, respectively, with their size given above the lines. The map is based on results of Trachtulec et al. (6). The Tbp gene organization is drawn according to Ohbayashi et al. (24) and Sumita et al. (25). The drawing is not to scale.

Table 2. Comparison of yeast (18) and vertebrate (17 and references therein) consensus splicing signals to signals of the mouse YAC genes spliced and not spliced (yes/no) in the yeast
*The exon sequences are in upper case. Y = C, T; R = A, G; M = A, C; N = A, C, G, T. The Tbp intron sequence was taken from Ref. (24).

Figure 2. Transcription and splicing of mouse genes by S.cerevisiae. (A) Dll1 locus (Table 1, exp. 2). Lane 1, DNA marker; lane 2, FFEH11 total yeast DNA PCR (genomic DNA control); lane 3, mouse cDNA PCR (spliced control); lane 4, FFEH11 random primed RT-PCR; lane 5, RT- control of lane 4; lane 6, the same as in lane 4, but RNA was degraded with RNase A prior RT (abbreviated hereupon RN-); lane 7, unrelated YAC clone RT-PCR (henceforth YAC-); lane 8, water control. The product in lane 4 was sequenced and found to correspond to the unspliced Dll1 gene. S, spliced; NS, not spliced product. (B) Splicing of the mouse Psmb1 gene by the yeast (exp. 8), stained agarose gel. Lane 1, DNA marker; lane 2, genomic DNA control; lane 3, mouse cDNA spliced control; lane 4, FFEH11 random primed RT-PCR; lane 5, RT- control; lane 6, RN- control. The product in lane 4 was sequenced and found to correspond to the correctly spliced transcript of the Psmb1 gene. (C) D17Ph4e locus is transcribed from both DNA strands (exp. 7). Lane 1, DNA marker; lane 2, genomic DNA control; lane 3, mouse cDNA spliced control; lane 4, FFEH11 oligo(dT) primed RT-PCR; lane 5, RT- control; lane 6, FFEH11 RT primed with a sense oligo (77B); lane 7, RT- control for lane 6; lane 8, FFEH11 RT primed with an antisense oligo (5F); lane 9, RT- control for lane 8; lane 10, RN- control; lane 11, YAC- control; lane 12, water control. The product in lane 4 was sequenced and found to correspond to the unspliced part of the D17Ph4e gene. (D) Autoradiograph of Southern blot of the agarose gel from (C) hybridized with a D17Ph4e probe.

One pair of primers, spanning the Tbp intron 1.1 kb in length (Table 1, exp. 10), produced bands corresponding to both spliced and unspliced RNA. The result can be interpreted either as partial splicing or as transcription of the Tbp gene from both DNA strands. The comparison of splicing signals of three available sequences of unspliced introns and one properly spliced intron (Table 2) did not indicate any differences that would correlate with successful splicing. Provided that the intron-exon organization of the mouse Psmb1 gene is the same as that of its human ortholog (12), five introns were correctly spliced in this gene. Altogether, 12 introns were assayed in this study, of which six were correctly processed.

Transcription by yeast of mouse DNA from coding and non-coding strand and from intergenic sequences

The next question concerned the specificity of yeast transcription from mouse DNA, in particular whether the mouse genes encoded by the YAC can be transcribed from one or both strands and whether the transcripts can be detected from non-coding, intergenic sequences. For this purpose, the RT of the yeast RNA was primed separately with specific sense and anti-sense primers for the D17Ph4e, Tbp and D17Ph8e genes. The subsequent PCR amplifications (pairs 5F/31L, 77F/77B, TFF/TFR and PH8F/PH8R) revealed specific bands from both sense and antisense strand primed reactions (Fig. 2C and D). In a control experiment, using primers for two yeast genes, RPG1 and ISW2, RT with the sense primers did not generate cDNA strands that could be PCR amplified, while random primed RT reactions produced PCR bands of the expected size.

To further inquire into the specificity of transcription of mammalian DNA in the yeast host, 10 primer pairs amplifying anonymous and repetitive loci in the FFEH11 YAC clone were used for RT-PCR of randomly primed total yeast RNA (Table 1, exps 15-24). Unexpectedly, all 10 combinations yielded specific PCR products, suggesting a high frequency of promiscuous transcription from the mouse YAC DNA. The loci studied included a pseudogene (exp. 16), four microsatellite markers (exps 17-18, 20-21) and two loci spanning repetitive regions (exps 23-24) which are unlikely to be transcribed in the mouse cells. None of these sequences was amplified by RT-PCR from the mouse testicular RNA (Table 1). Another pair of primers (TFO/C5R) was derived from the first exons of the head-to-head oriented Tbp and Psmb1 genes (Fig. 3A and B). The illegitimate transcription from the Tbp-Psmb1 intergenic region observed in this experiment was independently confirmed by RNase protection assay (Fig. 3C). The probe included 65 nt of the first Psmb1 exon and 346 nt of the Psmb1-Tbp intergenic region. All 411 nt of the sense probe were protected in the assay. In a control experiment, the intergenic region between two yeast genes, CPA1 and ISW2 on chromosome XV, was successfully amplified from the YAC clone genomic DNA, but not by RT-PCR from the YAC clone RNA (data not shown).

Figure 3. Promiscuous transcription of mouse non-coding DNA in the yeast (Table 1, exp. 14). RT-PCR detects an RNA transcript from the intergenic region between the first exons of the Tbp and Psmb1 genes: lane 1, DNA marker; lane 2, genomic DNA control; lane 3, FFEH11 random primed RT-PCR; lane 4, RT- control; lane 5, FFEH11 oligo(dT) primed RT-PCR; lane 6, RT- control for lane 5; lane 7, RN- control; lane 8, YAC- control; lane 9, water control. (A) stained agarose gel; (B) autoradiograph of the blotted gel hybridized with a sequenced probe from the Psmb1 promoter region. (C) RNase protection assay with an RNA probe for the intergenic region between the Tbp and Psmb1 genes. Lane 1, yeast RNA control (10 µg); lane 2, FFEH11 RNA (10 µg); lane 3, FFEH11 RNA (35 µg); lane 4, mouse RNA (2 µg); lane 5, no RNase; lane 6, elution control. The arrows mark the sizes of the eluted (516 bp) and fully protected (411 bp) RNA probe.

Polyadenylation of mouse mRNAs in S.cerevisiae

To examine whether the transcripts from the FFEH11 YAC are polyadenylated in the yeast the same way as in the mouse, RT-PCR reactions were primed using oligodeoxythymidine. Thirteen primer pairs amplifying fragments of genes, anonymous loci and a sequence of the Psmb1-Tbp intergenic region produced RT-PCR specific fragments (Figs 2 and 3). As this assay does not allow recognition of the location of polyA, a 3[prime] RACE experiment was performed with four gene-specific primers. The products were of different sizes than would be expected if the mouse polyA signals were used by the yeast (data not shown). One of the 3[prime] RACE products was therefore subcloned and sequenced (GenBank accession no. AF090314). The sequence of the product, amplified with the Psmb1 gene-specific primer C53, covers 84 bp of the previously characterized 3[prime] untranslated region including the putative polyA signal ATTAAA of the Psmb1 gene mRNA and 290 bp of what was at first considered to be the 3[prime] intergenic region. However, examination of the Expressed Sequence Tag database using BLAST2 revealed that this sequence is closely related to several single read mouse and rat cDNA sequences (99 and 87% similarity, respectively) over 316 bp. The conservation of the sequence, its polyA signals, and cleavage sites indicate that the YAC RT-PCR sequence spans only 58 bp of the 3[prime] intergenic region of the Psmb1 gene, and that the rodent Psmb1 gene transcript carries two alternative polyA signals. The second mouse positioning signal (AATAAA) is surrounded by three hexanucleotides (two of them overlapping) similar to the yeast efficiency polyA signal (TATDTA). While both the yeast and mouse polyA signals are putative, detected by computer searching for consensus sequences downstream to polyA sites, the polyA sites were detected experimentally by cDNA sequencing. Neither mouse polyA signal is used by the yeast cell, as the 3[prime] RACE sequence exceeds the length of any mouse Psmb1 mRNA, and the putative yeast polyadenylation site is located downstream to two overlapping TATDTA yeast signals.

DISCUSSION

Previous studies on yeast and mammalian gene expression revealed common features as well as differences in their transcription, splicing and polyadenylation mechanisms. It has been documented that yeast can recognize mammalian promoters (13-15), but yeast and mammals vary in their requirements for the sequence context of the TATA boxes (16). Also, the intron splicing signals show several differences in spite of the common GT-AG rule, shared across the eukaryotic kingdom. For example, the yeast 3[prime] splicing signal consensus does not have a polypyrimidine stretch preceding the AG sequence, and the branching site is more conserved in the yeast than in mammals (17,18).

The mammalian polyadenylation signals are not generally recognized in S.cerevisiae (19,20). Although the yeast and mammalian signals are both composed of at least three elements, the yeast efficiency elements are different and the other two elements are more degenerate than the mammalian signals (reviewed in 21). The yeast efficiency signal optimal sequence is TATATA and hexanucleotides TATDTA have some activity, but several non-optimal elements are usually located downstream to the polyA site (22).

In spite of all these differences, Still et al. (4) reported successful screening for human genes in yeast RNA from the yeast clones carrying overlapping human YACs. Of 27 differentially expressed RNA fragments tested, the expression of four clones in human tissues (15%) was proven by PCR screening of five human cDNA libraries or by their match to expressed sequence tags. The specificity of the processing of the human genes by the yeast was not tested.

In the present report, we took advantage of a well characterized mouse YAC clone (7,10,11) to determine how efficient and specific is the yeast processing of the mouse DNA. Transcripts from five tested mouse genes encoded within the YAC clone were all found in the total yeast RNA. Of 12 mouse introns assayed, six were correctly spliced by the yeast. Besides the transcripts of exon sequences, `yeast-specific' transcription of the YAC DNA was observed. At least three genes were transcribed from their sense and antisense strands. Microsatellite, inter-repetitive, and anonymous mouse loci were detected in random- and oligo(dT)-primed YAC RNA. A pair of primers derived from the first exons of two head-to-head oriented mouse genes yielded an RT-PCR product. An RNA probe, derived from this intergenic region, was wholly protected by the YAC RNA in an RNase protection assay. This finding indicates that the steady state levels of transcribed RNA from the non-coding mouse sequences are high enough to be detected by a technique other than RT-PCR. The sequence analysis of a 3[prime] RACE product has shown that, in agreement with expectation (19,20), the mouse polyadenylation signals are not used by the yeast cell.

How to explain the observed high frequency of illegitimate transcription of mouse DNA in the yeast? The presence of unspliced introns, transcripts from both DNA strands or from microsatellite repeats could be easily understood, if genomic DNA rather than reverse transcribed cDNA acted as a template for PCR amplification. However, we have effectively ruled out this possibility not only by including the RNase-free DNase treatment and RT minus controls, but also by abrogation of the PCR signal by RNA treatment with DNase-free RNase before reverse transcription. Moreover, when the primers for intergenic endogenous yeast region were used, the RT-PCR did not yield any signal. No products were obtained also when two yeast genes were amplified from sense-oligo primed RT reactions.

Yeast transcription initiation complex recognizes the TATA box consensus, but it is not sensitive to the TATA box sequence context that is preferred by the mammalian transcription complex (16). Thus, due to a high ratio of non-coding to coding DNA in mammals, some random mouse sequences could serve as promoters in the yeast and explain the presence of transcripts from YAC non-coding regions or a non-template DNA strand. The same argument may hold true for TATA-less transcription initiation.

The splicing of six mouse introns (out of 12 tested) described in this report provides the first piece of evidence for a successful splicing of mammalian introns by yeast. The failure to splice all introns could be explained by the fact that the branching site sequence is more conserved in the yeast compared with mammals, and its variation can abolish splicing in the yeast (18). Admittedly, splicing signals of four analyzed introns did not show any apparent differences that could distinguish three non-spliced introns from one that was spliced. None of the examined mouse introns displayed a branching site identical to the yeast consensus. One possible explanation could be that these introns are spliced only partially and the non-spliced product was not detectable by our method. It may be of some interest that though the yeast introns are generally short, at least one of the successfully spliced mouse introns spanned >5 kb. Our study has suggested that although mammalian polyA signals are not used by yeast, the YAC RNAs can be polyadenylated, apparently due to the redundant occurrence of the degenerate yeast polyA signal sequences.

The present report shows that the yeast transcription apparatus transcribes mouse coding and non-coding sequences with comparable efficiency. The enrichment with mammalian mRNA in YAC RNA seems rather low and a method based on a selection of mammalian mRNA in the YAC clone RNA would be expected to produce a high background. For example, a hypothetical mouse gene of 25 and a mRNA of 2 kb in size would be transcribed and spliced by the yeast into an RNA >12 kb, provided that half of its introns were properly spliced. The enrichment would be even lower if the gene was transcribed from a random sequence recognized by the yeast transcription apparatus on a non-template DNA strand that cannot be spliced at all. The YAC clone under study covers a GC-rich region, as judged by the frequency of rare-cutter restriction sites (10). Such regions are gene-rich and under-represented in mammals (23). Although it cannot be a priori excluded that the YAC transcription and RNA processing would be more faithful in GC-poor YAC clones, the expected occurrence of TATA and TATDTA sequences, and thereby of possible transcription initiation and RNA 3[prime] end formation sites is higher.

The results presented here indicate that YAC clones can serve as in vivo test tubes for further analysis of the conservation of gene processing sequences. An intriguing possibility emerges to develop new yeast host strains capable of recognizing some mammalian DNA and/or RNA processing signals, and thus enriching RNA of YAC clones with mammalian exons.

ACKNOWLEDGEMENTS

We wish to thank Dr R. M. J. Hamvas for sharing physical mapping data on Dll1, Drs T. Vogt and P. Jansa for helpful comments, Drs P. Vavrickova and J. Hasek for yeast control primers, Dr J. Felsberg for sequencing, and Dr S. Takacova for editing the manuscript. This work was supported by grant nos 204/98/P13 and 204/98/KO15 from the Grant Agency of the Czech Republic, and A5052709/1997 from the Grant Agency of the Academy of Sciences of the Czech Republic. J.F. is an International Research Scholar of the Howard Hughes Medical Institute.

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*To whom correspondence should be addressed. Tel: +4202 475 2256; Fax: +4202 471 3445; Email: trachtul@biomed.cas.cz

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