Transcriptional Analysis of the Adeno-Associated Virus Integration Site

293T (HEK_293T), RD, A673, and MCF7 cells were grown in Dulbecco's modified Eagle's medium (Mediatech Inc., Manassas, VA), 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA). HeLa cells were grown in minimal essential medium (Mediatech Inc.), 10% fetal bovine serum, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA). The human ES cell line H1 was grown on DR4 mouse embryonic feeder cells in Dulbecco's modified Eagle's medium-F12 supplemented with 20% (vol/vol) Knockout Serum Replacement (both from Invitrogen), basic fibroblast growth factor (20 ng/ml; R&D Systems), 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM l-glutamine, 0.1 mM nonessential amino acids (all from Invitrogen), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Prior to RNA isolation, H1 cells were grown under feeder-free conditions in matrigel-coated plates (Biocoat; Becton Dickinson, San Jose, CA).

Total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA). Ten micrograms of RNA was separated on a 1% formaldehyde-agarose gel and transferred onto a Magna nylon membrane (Osmonics, Minnetonka, MN). All membranes were hybridized with [α-³²P]dCTP-labeled probes (Prime-It RmT Random Primer Labeling Kit; Stratagene, Cedar Creek, TX). Northern blots were first hybridized to red fluorescent protein (RFP) or MBS85 (exons 18 to 22) probes; stripped by being boiled for 15 min in 0.05× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10 mM EDTA (pH 8), 0.1% sodium dodecyl sulfate (SDS); and rehybridized to a β-actin cDNA probe. The RFP, MBS85, and β-actin probes were generated by PCR performed on plasmids pRFP, pND83, and pβ-actin, respectively (see Tables S1 and S2 at http://www.kcl.ac.uk/linden ).

Transcription start sites (TSS) were identified using the 5′ rapid amplification of cDNA ends (5′ RACE) GeneRacer Kit (Invitrogen) according to the manufacturer's instructions. Total RNA was extracted from 293T cells (MBS85 TSS) and from 293T cells transfected with construct pND29 (antisense TSS) (see Table S1 at http://www.kcl.ac.uk/linden ). Briefly, DNase I-treated total RNA (5 μg) was dephosphorylated with calf intestinal phosphatase, and the cap structure was removed with tobacco acid pyrophosphatase. The decapped RNA was then ligated to the GeneRacer RNA primer. RNA was reverse transcribed for 1 h at 50°C using an oligo(dT) primer and SuperScriptIII reverse transcriptase (Invitrogen). The resulting cDNAs were amplified by PCR with the GC-rich PCR kit (Roche Applied Science, Indianapolis, IN) and GeneRacer 5′ and ND189 primers (MBS85 TSS) or GeneRacer 5′ and ND192 primers (antisense TSS) (see Table S2 at http://www.kcl.ac.uk/linden ). The PCR conditions were as follows: 95°C for 3 min; 10 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min 20 s (or 1 min for the antisense TSS); 25 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min 20 s with an elongation time of 5 s per cycle; and a final extension at 72°C for 7 min. The PCR products were cloned into the PCR2.1 Topo vector (Invitrogen), and the resulting clones were sequenced using a 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA).

Plasmids pDsRed2.1, pDsRed2-N1, and pIRES2-EGFP were purchased from Clontech (Mountain View, CA). EST R35625 (catalog number 363135) was purchased from the ATCC (Manassas, VA). Plasmid pDsRed2.1 was used to clone the MBS85 and p5 promoter regions. The p5 promoter used in this study is the region described by Chang et al. (reference 10 and Table S2 at http://www.kcl.ac.uk/linden ). All constructs have been sequenced.

Transfection experiments were performed in 60-mm plates. At 50% confluence, 293T cells were transfected with 6 μg of reporter construct using Lipofectamine Plus reagent (Invitrogen). HeLa cells were transfected at 80% confluence with 6 μg of reporter construct using Fugene 6 reagent (Roche Applied Science). 293T and HeLa cells were harvested 48 h and 72 h posttransfection, respectively, and assayed for plasmid DNA uptake, along with Northern blot, Western blot, and fluorescence-activated cell sorter (FACS) analyses. Transfection efficiencies were normalized by plasmid DNA uptake, as previously described (31). All transfection experiments were repeated four times using plasmids that were independently prepared at least twice.

Transfected cells were lysed in 0.2 M NaOH, 10 mM EDTA. Samples were boiled for 15 min at 90°C and loaded onto a Hybond XL nylon membrane (Amersham Biosciences, Piscataway, NJ) using a slot blot manifold (Bio-Rad). The membranes were hybridized to an RFP or green fluorescent protein (GFP) probe, generated by PCR, to determine the amount of reporter plasmid taken up by the cells.

Cells were solubilized in RIPA buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1× Complete protease inhibitor cocktail) (Roche Applied Science). Samples (10 μg) were loaded on a 15% SDS-polyacrylamide gel and transferred onto a Hybond C extra nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). The membranes were blocked in 5% fat-free milk and incubated with anti-DsRed polyclonal antibody at a dilution of 1:16,000 (catalog number 632397; Clontech) or anti-actin monoclonal antibody at a dilution of 1:10,000 (catalog number 612656; Becton Dickinson Biosciences). After being washed in Tris-buffered saline-Tween 20 buffer, the blots were incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of 1:10,000 (Jackson ImmunoResearch Laboratories, West Grove, PA). RFPs were visualized by the enhanced-chemiluminescence method using Pico and Femto detection kits (Pierce, Rockford, IL) for 293T and HeLa cell extracts, respectively. Actin proteins were visualized with the Pico detection kit. Membranes were first incubated with anti-DsRed antibody, stripped with the Restore Western Buffer Stripping Buffer (Pierce), and blotted with anti-actin antibody for normalization.

Protein quantification was performed using the Li-Cor Odyssey infrared imaging system (Li-Cor Biosciences UK Ltd., Cambridge, United Kingdom). Anti-mouse IRDye 680 (catalog number 926-32220; Li-Cor) or anti rabbit IRDye 800 (catalog number 926-32211; Li-Cor) fluorescent secondary antibody was used at a dilution of 1:5,000 in 1% milk, 0.5% Tween 20.

Cell suspensions from transfected 293T and HeLa cells were prepared and analyzed for RFP expression. The data were acquired using a FACScalibur (Becton Dickinson) and analyzed by Flowjo software (Tree Star, Inc., Ashland, OR). FACS data are presented as dot plots with linear axes for forward/side scatter and logarithmic axes for FL1 (empty; x axis) and FL2 (RFP; y axis). Gates were set to exclude dead cells based on forward/side scatter. This gated population was analyzed for RFP expression. The gate set to determine the percentage of RFP-expressing cells for each sample was obtained by the analysis of a negative population (cells transfected with a promoterless vector, pDsRed2.1) and was verified by a positive population (cells transfected with a cytomegalovirus [CMV]-controlled RFP vector, pDsRed2-N1). The geometric mean fluorescence intensity (Geo MFI) was calculated for this gated population of RFP-positive cells. FACS data collected from four experiments were used to generate graphs (see Fig. 3, 4, and 6).

Sorting experiments were performed on a Moflo cell sorter (Cytomation, Ft. Collins, CO).

DNase I-treated RNA was subjected to reverse transcription-PCR (RT-PCR) using SuperScriptIII reverse transcriptase (Invitrogen) and the KCL1 primer. The resulting cDNAs were amplified by PCR with GoTaq polymerase (Promega, Madison, WI) and the KCL2 and KCL4 primers. The PCR conditions were 94°C for 2 min; 35 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 40 s; and a final extension at 72°C for 10 min. The PCR products were cloned into the PCR2.1 Topo vector (Invitrogen), and the resulting clones were sequenced.

Transfected 293T cells were visualized for RFP and GFP expression 48 h posttransfection. Epifluoresecnce microscopy was carried out by using an inverted epifluorescence microscope (Leica; DM IRB) and an ×20 magnification lens. Images were acquired with a Mintron digital camera. Exposure times and camera gain values were kept strictly identical in all pictures. All images were assembled and processed identically (adjustment level correction and contrast enhancement were identical for each image file) with Adobe Photoshop 7.0 (Adobe Systems Inc., Mountain View, CA).

Nucleotide sequence accession numbers were as follows: Homo sapiens MBS85 expressed sequence tag (EST), R35625; IMAGE clone identifier, 38310; H. sapiens MBS85 mRNA, AF312028; H. sapiens β-actin, NM_001101; AAV2 complete genome, AF043303; and upstream sequences from the MBS85 ATG start codon for H. sapiens (human) chromosome 19, NT_011109.15 from nucleotide (nt) 27897099 to nt 27898224, for Pan troglodytes (chimpanzee) chromosome 19, NW_001228247.1 from nt 1307249 to nt 1307515 and from nt 1305832 to nt 1306173 (there is a gap between the two contigs), for Bos taurus (cattle) chromosome 18, NW_001493632.2 from nt 1526744 to nt 1527802, for Equus caballus (horse) chromosome 10, NW_001867363.1 from nt 24348902 to nt 24349949, for Canis lupus familiaris (dog) chromosome 1, NW_876270.1 from nt 32959175 to nt 32960143, for Mus musculus (mouse) chromosome 7, NW_001030825.1 from nt 1246105 to nt 1247455, and for Rattus norvegicus (rat) chromosome 1, NW_047555.2 from nt 13365737 to nt 13367100.

In order to map the promoter region of the human MBS85 gene and identify potential regulatory elements, the monkey and mouse MBS85 genes (3, 16) were used to retrieve additional MBS85 orthologous genes from the NCBI database. The availability of genomic sequences from an increasing number of species indicated that MBS85 is evolutionarily conserved among higher eukaryotes. Analysis of the highly conserved regions from the human, monkey, cattle, horse, dog, mouse, and rat sequences upstream of the translation initiation site demonstrated that the MBS85 gene lacks the typical TATA and CAAT boxes, while several putative DNA motifs for transcription factors are conserved, i.e., Sp1, Staf, Brn, Egr-1, ATF-CREB, E4F, YY1, and zinc finger 5 (ZF5) (Fig. 1). Importantly, not only the sequence of the RBS is conserved among all these species, but also its position relative to the ATG codon (located 17 to 37 nt upstream from the ATG site), suggesting a possible role in MBS85 gene regulation.

In order to characterize the MBS85 TSS, we first investigated the expression of MBS85 by Northern blotting in six human cell lines. Total RNA was extracted from H1 (ES), 293T (embryonic kidney), HeLa (cervix epitheloid carcinoma), MCF7 (breast adenocarcinoma), and RD and A673 (rhabdomyosarcoma) cell lines. Northern blots hybridized to a probe containing MBS85 exons 18 to 22 revealed a unique transcript of 3 kb, indicating that MBS85 is expressed in all six cell lines (Fig. 2A). This result is consistent with the previously described findings of Tan et al., in which a unique transcript of 3 kb was detected in various human tissues. However, the possibility that additional transcripts (i.e., alternative splicing) expressed at low levels might be generated from the MBS85 promoter cannot be excluded. The relative expression level of MBS85 transcripts compared to that of the β-actin gene suggests that MBS85 is expressed at higher levels in 293T than in HeLa cells. These two cell lines were chosen for further experiments based on their endogenous MBS85 expression (Fig. 2A) and their relatively high transfection efficiencies.

We subsequently determined the MBS85 mRNA TSS by 5′ RACE on total RNA extracted from 293T cells. A single RT-PCR product was detected (Fig. 2B). Sequence analysis of several clones assigned the MBS85 TSS to an adenine located 94 bp upstream from the translation initiation site (Fig. 2B and Fig. S1 and S2 at http://www.kcl.ac.uk/linden ). This result also confirmed that the AAVS1 TRS-RBS motifs are located in the 5′ UTR of the MBS85 gene (Fig. 1 and Fig. S2 at the URL given above).

In order to identify regulatory regions involved in MBS85 promoter activity, a series of progressive 5′-truncated sequences were cloned in the sense orientation into the pDsRed2.1 promoterless RFP vector. These constructs contained MBS85 sequences all the way down to the ATG translation initiation site, including the TSS and 5′ UTR (Fig. 3A). To compare MBS85 and p5 promoter activities, the p5 promoter was also cloned into the pDsRed2.1 vector (Fig. 3A).

All sense constructs were transiently transfected into 293T and HeLa cells. The expression of the RFP reporter gene was monitored by measuring the amounts of RFP mRNA and protein by Northern blot and Western blot analyses, respectively. Since a direct correlation between the relative mRNA and protein levels could be observed, the reporter protein activity directly reflects activity at the transcriptional level. As shown in Fig. 3B, a reporter construct containing sequences from nt −3302 to +94 exhibited moderate RFP expression in 293T and HeLa cells. Deletions of sequences spanning from −3.3 kb to −137 bp resulted in only minor changes in MBS85 promoter activities in the two cell lines. However, a further deletion from nt −137 to −99 almost completely abrogated MBS85 promoter activity (Fig. 3B). Together, these data indicate that the proximal region spanning from −137 bp to +94 bp is sufficient for constitutive MBS85 transcription in both 293T and HeLa cells. Sequence analysis revealed that several putative response elements for transcription factors are located within this region, e.g., Sp1, ATF-CREB, E4F, Staf, YY1, and ZF5 (see Fig. S2 at http://www.kcl.ac.uk/linden ). Of these potential cis-acting elements, the YY1 site at position −110 is of particular interest, since deletion of nt −137 to −99 almost completely abolished MBS85 promoter activity.

Since flow cytometry provides direct readout at the single-cell level, it was used to determine the percentage of RFP-expressing cells and to quantify the promoter activity in the RFP-expressing cell population. These data indicated that 20% to 30% of 293T cells and 10% of HeLa cells expressed the reporter gene (Fig. 3B). Comparison of the Geo MFI of RFP indicated that the MBS85 promoter activity was significantly stronger in 293T than in HeLa cells (Geo MFI, 50 in 293T cells compared to 20 in HeLa cells) (Fig. 3B). These data were correlated with the higher endogenous MBS85 mRNA levels detected in 293T cells in comparison to HeLa cells, as observed by Northern blotting (Fig. 2A).

We next compared the p5 and MBS85 promoter activities. As shown in Fig. 3B, the p5 promoter led to a significant increase in RFP expression compared to the MBS85 promoter in both 293T and HeLa cells. This increase in RFP expression is due not only to a higher number of expressing cells, but also to stronger promoter activity, as determined by FACS analysis (p5 Geo MFI, 150 and 40 in 293T and HeLa cells, respectively).

It has previously been reported that a reporter construct containing MBS85 sequences from nt −332 to +20 exhibited transcriptional activities in an orientation-independent manner, suggesting the presence of an enhancer element within this region (35, 36). However, our data clearly indicate that this putative enhancer-like sequence lies within the MBS85 minimal promoter region. It has recently become evident that bidirectional promoters might not be as uncommon as previously thought (1, 43), and a large number of transcripts are indeed arising from the opposite direction of protein-coding genes (13, 18, 26, 46, 52, 66). Therefore, we hypothesized that the MBS85 promoter might display bidirectional promoter activities rather than enhancer activities. In order to address this hypothesis, 5′-truncated sequences were cloned in the antisense orientation into the pDsRed2.1 promoterless vector, and the resulting antisense constructs were transiently transfected into 293T and HeLa cells (Fig. 4A). As shown in Fig. 4B, reporter constructs containing sequences from nt +94 to −332 exhibited transcriptional activities in both 293T and HeLa cells, whereas no promoter activity could be detected by additional sequences from nt −332 to −3302. Flow cytometry analyses further indicated weaker promoter activity for constructs +94/−137 and +94/−332 compared to constructs +94/−99 and +94/−236, suggesting possible negative regulatory elements located within the regions −332 to −236 and −137 to −99. Sequence analysis of these two regions revealed a putative YY1 binding site within each region (see Fig. S2 at http://www.kcl.ac.uk/linden ). Fluctuations of the percentage of cells expressing the RFP reporter construct could be observed between constructs, as was previously reported with different cellular promoters (17). Importantly, constructs exhibiting a lower number of RFP-positive cells also concomitantly resulted in a lower MFI. Even though the significance of these differences is not well understood, it may be that cells expressing the lowest RFP level cannot be detected under the conditions used in the assay.

As observed for the MBS85 sense promoter activity, the antisense promoter activity was also stronger in 293T cells than in HeLa cells (Geo MFI, 40 and 20 in 293T and HeLa cells, respectively). Interestingly, weak p5 antisense promoter activity could be detected in both 293T cells and HeLa cells.

These results demonstrate that the MBS85 region spanning from nt +94 to −332 is able to drive the expression of a reporter gene from the antisense orientation. Our data further indicate that the MBS85 promoter might have bidirectional transcriptional activities, as detected from reporter constructs in which the same fragments were individually cloned in opposite directions.

We next investigated whether the MBS85 antisense promoter would function in a truly bidirectional manner. Fragment −236/+94, which exhibited sense promoter activity together with the strongest antisense promoter activity, was cloned in both orientations into a dual reporter construct carrying the RFP and enhanced GFP (EGFP) genes in opposite directions (see Table S1 at http://www.kcl.ac.uk/linden ). To exclude any promiscuous transcription occurring from the bidirectional vector, a 0.3-kb fragment originating from the first intron of the human MBS85 gene was cloned in both directions into the bidirectional vector. All bidirectional constructs were transiently transfected into 293T cells, and reporter gene expression was detected by Western blot analysis. As shown in Fig. 4C, expression of both RFP and EGFP genes was detected from each MBS85 promoter construct, whereas no EGFP or RFP expression could be detected with all three negative controls. Two constructs driving the expression of only one reporter gene (RFP or EGFP) were also transfected to assess the specificity of each antibody. These results show that the MBS85 promoter can drive the expression of two inversely oriented reporter genes. Taken together, our data provide strong evidence that the MBS85 promoter displays bidirectional promoter activities.

We next mapped the antisense TSS by 5′ RACE in 293T cells transfected with the +20/−332 antisense construct. The lengths of the RT-PCR products were in the range from 200 bp to 400 bp, suggesting the existence of multiple antisense start sites (Fig. 4D). Sequence analysis of several RT-PCR products revealed four antisense start sites spread over 70 bp (see Fig. S2 at http://www.kcl.ac.uk/linden ). However, since antisense promoter activity could be detected with shorter promoter fragments (+94/−99, +94/−137, and +94/−236), it is more likely that additional start sites will be identified and it is therefore difficult to ascertain which site is the predominant antisense TSS. Furthermore, it remains unclear whether the MBS85 and antisense transcripts overlap.

In order to identify potential genes that are transcribed in the opposite direction from MBS85, we searched the NCBI human EST database for the genomic sequences that are located between the MBS85 and TNNT1 genes (15). No full-length EST was found to perfectly match the intergenic region between these two genes. However, three short sequences upstream from the MBS85 TSS are highly homologous to the 3′ end of the nuclear transport factor 2 (NUTF2) mRNA, suggesting the presence of a NUTF2 pseudogene (ψNUTF2) (Fig. 5A). To first assess whether NUTF2 pseudogene-derived antisense transcripts could be generated by the MBS85 antisense promoter, we performed an RT reaction on total RNA extracted from 293T cells using a primer that could anneal only to the NUTF2 pseudogene antisense transcripts. As shown in Fig. 5B, a product of 650 bp was detected by RT-PCR. Nucleotide sequence analysis of this 650-bp RT-PCR product showed 100% homology with the NUTF2 pseudogene present upstream of the MBS85 gene. This result further suggests that ψNUTF2 is transcribed in the antisense orientation with respect to the NUTF2 gene. We next determined whether the ψNUTF2 antisense transcripts could hybridize to NUTF2 sense transcripts and lead to suppression of NUTF2 protein synthesis through an antisense repression mechanism. Constructs containing different lengths of the intergenic region between MBS85 and TNNT1 were transfected in 293T cells (Fig. 5C). All constructs contained the antisense promoter driving ψNUTF2 antisense transcription and the RFP gene under the control of the MBS85 promoter to selectively enrich for ψNUTF2 antisense transcripts. Total protein was extracted from the RFP-positive cell population, and NUTF2 expression was assessed by Western blot analysis. As shown in Fig. 5D, overexpression of ψNUTF2 antisense transcripts did not significantly decrease NUTF2 protein expression compared to endogenous NUTF2 expression. These results indicate that under the conditions used in this assay, detectable ψNUTF2 antisense transcripts do not affect NUTF2 expression.

Since the RBS is located within the 5′ UTR of the MBS85 gene and is conserved among all the species analyzed, we investigated whether the TRS-RBS region is involved in the regulation of MBS85 expression. Constructs with the TRS-RBS sequence deleted were transiently transfected into 293T and HeLa cells (Fig. 6A). As shown in Fig. 6B, promoter constructs in which the TRS-RBS sequence was deleted exhibited higher RFP expression than control promoters harboring the TRS-RBS sequence in both 293T and HeLa cells. In order to further confirm the effect of the TRS-RBS region on gene expression, we performed quantitative Western blots using a Li-Cor Odyssey system. Deletion of the TRS-RBS sequence resulted in a 4- to 5.8-fold increase in promoter activity in 293T cells (Fig. S3A and S3B at http://www.kcl.ac.uk/linden ). This higher RFP expression is directly correlated with a higher number of RFP-expressing cells and stronger promoter activity, as shown by FACS analysis (Fig. 6B and Fig. S3C and S3D at http://www.kcl.ac.uk/linden ). Despite an increase in the amount of reporter protein, Northern blot analysis showed constant mRNA levels (Fig. 6B). Together, these results indicate that the TRS-RBS region present in the 5′ UTR of MBS85 contains an inhibitory element and that this inhibition likely occurs at the posttranscriptional level.

We next tested whether the TRS-RBS region could also regulate the expression of a reporter gene construct under the control of a heterologous promoter. A construct containing the TRS-RBS sequence cloned between the CMV promoter and the RFP reporter gene was transiently transfected into 293T and HeLa cells (Fig. 6A). Insertion of the TRS-RBS sequence decreased the expression of the RFP reporter gene compared to the CMV promoter control in both 293T and HeLa cells (Fig. 6C). These results were further confirmed by quantitative Western blot and FACS analyses (see Fig. S4 at http://www.kcl.ac.uk/linden ). The TRS-RBS sequence significantly reduced CMV promoter activity by 3.7-fold and 16-fold in 293T and HeLa cells, respectively. However, the 16-fold decrease in promoter activity might be overestimated, since in HeLa cells, the signal is near the limit of detection. When placed between the promoter and the reporter gene, the TRS-RBS sequence led to a significant decrease in protein levels whereas the mRNA levels were unchanged or even slightly increased (Fig. 6C). These data strongly suggest that the TRS-RBS region present in the 5′ UTR of MBS85 is able to regulate the expression of a reporter gene at a posttranscriptional level, regardless of the promoter used.

We further tested whether the TRS-RBS region can interfere with the expression of a reporter gene in the context of the bidirectional promoter vector. The TRS-RBS sequence was therefore removed from the bidirectional promoter constructs used previously. The bidirectional promoter constructs were transiently transfected in 293T cells, and reporter gene expression was visualized by epifluorescence microscopy. As shown in Fig. 7A, higher gene expression was observed in 293T cells with a reporter construct with the TRS-RBS region deleted than with the control construct containing the TRS-RBS region (compare subpanels b and c [RFP expression] and subpanels i and j [GFP expression]). However, the TRS-RBS sequence decreased the expression level of only the sense reporter gene (compare subpanels b and c to i and j) without affecting the expression level of the antisense reporter gene (compare subpanels g and h [GFP expression] to d and e [RFP expression]). To confirm the effect of the TRS-RBS sequence on gene expression, quantitative Western blot analyses were performed using the Li-Cor Odyssey system (Fig. 7B and C). Deletion of the TRS-RBS sequence resulted in 5.4-fold (RFP expression) and 3-fold (GFP expression) increases in the promoter activity of only the sense reporter gene. The images generated by epifluorescence microscopy, furthermore, confirm the bidirectional promoter activity of the MBS85 promoter, since simultaneous expression of both RFP and GFP genes can be detected with all constructs. Together, these results indicate that the TRS-RBS region can inhibit MBS85 expression in an orientation-dependent manner, consistent with a possible role in posttranscriptional regulation. It remains to be determined whether a secondary structure or cis-acting elements are involved in the TRS-RBS-mediated inhibition of MBS85 expression.