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BOR - Papers in Press, published online ahead of print January 17, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.057851
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BIOLOGY OF REPRODUCTION 76, 813–824 (2007)
DOI: 10.1095/biolreprod.106.057851
© 2007 by the Society for the Study of Reproduction, Inc.

The Cathepsin L First Intron Stimulates Gene Expression in Rat Sertoli Cells1

Martin Charron 2, Jing-Yi Chern , and William W. Wright 

Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205

ABSTRACT

Large amounts of cathepsin L (CTSL), a cysteine protease required for quantitatively normal spermatogenesis, are synthesized by mouse and rat Sertoli cells during stages VI to VII of the cycle of the seminiferous epithelium. We previously demonstrated that all of the regulatory elements required in vivo for both Sertoli cell- and stage-specific expression of the Ctsl gene are present within a ~3-kb genomic fragment that contains 2065 nucleotides upstream of the transcription start site and 977 nucleotides of downstream sequence. Most of the downstream region encodes the first intron. In this study, transient transfection assays using primary Sertoli cell cultures and the TM4 Sertoli cell line established that the Ctsl first intron increased reporter gene activity by ~5-fold. While the intron-mediated enhancement in reporter gene activity was not restricted to the Ctsl promoter, positioning the first intron upstream of the Ctsl promoter in either orientation abolished its stimulatory activity, suggesting that it does not contain a typical enhancer. Mutating the 5'-splice site of the Ctsl first intron or replacing the first intron by the Ctsl fourth intron abolished the stimulatory effect. Finally, the intron-dependent increase in reporter gene activity could be explained in part by an increase in the amounts of total RNA and transcript polyadenylation. Results from this study suggest that the stimulatory effect mediated by the Ctsl first intron may explain in part why Sertoli cells in seminiferous tubules at stages VI to VII produce high levels of CTSL.

gene regulation, Sertoli cells, testis

INTRODUCTION

As mammalian spermatogenic cells divide and differentiate into mature spermatozoa, they interact extensively with the adjacent somatic Sertoli cells (reviewed in Wright [1]). These dynamic interactions are obligatory for spermatogenesis. As male germ cells progress through the stages of the cycle of the seminiferous epithelium, the transcription of genes that influence the survival, replication, and development of these cells is regulated temporarily in Sertoli cells. Such genes encode cell adhesion proteins, growth factors, transport proteins, proteases, and protease inhibitors [2–4]. We are studying the consequences and the regulation of stage-specific gene expression in Sertoli cells using the rat cathepsin L (Ctsl) gene as a model. CTSL, a cysteine protease, is required for normal spermatogenesis, as mice that express enzymatically inactive CTSL exhibit increased seminiferous tubule atrophy, decreased production of preleptotene spermatocytes, and reduced maturation of these cells into pachytene spermatocytes [5]. In rat Sertoli cells, CTSL levels increase approximately 20-fold from their nadir during stages XII to I to their maxima during stages VI to VII [6, 7]. As a result, CTSL constitutes approximately 1% of the total proteins synthesized by seminiferous tubules at stages VI to VII [7]. The production of high CTSL levels is mediated predominantly by an increase in the transcription of this gene [8].

A requirement for defining how male germ cells modulate stage-specific gene expression in Sertoli cells is the identification of a promoter that confers accurate, stage-specific gene expression in vivo. We reported that a 3-kb genomic fragment located immediately upstream of the translation start site of the rat Ctsl gene directs in vivo expression of the reporter gene, β-galactosidase, only in Sertoli cells [9]. The expression pattern of the reporter gene recapitulated that of the endogenous gene (i.e., high levels of expression were observed in Sertoli cells located in seminiferous tubules at stages VI to VII). Thus, the 3-kb genomic fragment contained all of the regulatory elements required for accurate, stage-specific gene expression in Sertoli cells. To date, no other promoter has been shown to confer both Sertoli cell-specific and accurate stage-specific gene expression in transgenic mice. Thus, the Ctsl gene provides a unique model for elucidating the mechanisms that regulate gene expression in Sertoli cells.

The promoter region of the rat Ctsl gene tested in transgenic mice contained 2065 nucleotides upstream of the transcription start site, the first exon (74 nucleotides), the first intron (892 nucleotides), and the first 11 nucleotides of exon 2. Transient transfection assays and in vitro DNA-protein binding experiments allowed the identification of two functional domains in the region located upstream of the transcription start site. The first domain mediates the repressive effects of germ cells, whereas the second one contains the proximal promoter [8, 10].

The present study uncovers the presence of a third functional domain within the Ctsl promoter that possesses a stimulatory activity, which corresponds to the first intron. Introns are intervening, noncoding DNA sequences that must be precisely removed from the nascent pre-mRNA by a macromolecular complex termed the spliceosome (reviewed in Jurica and Moore [11] and Sanford and Caceres [12]). In mammals, the transcribed pre-mRNA contains several cis-elements that are essential for the splicing reaction. The 5'-splice site is defined by the sequence 5'-AGGURAGU-3', which contains the invariant GU dinucleotide at the start of the intron, whereas the 3' splice site is defined by the sequence 5'-YAGR-3', with the invariant AG dinucleotide at the end of the intron (reviewed in Proudfoot et al. [13]). The branchpoint, followed by a polypyrimidine-rich track, usually lies about 100 nucleotides upstream of the 3'-splice site [14, 15]. Intron-containing genes generally are expressed at a significantly higher level in eukaryotic cells than their intronless counterparts [16–20]. Results from several studies have demonstrated that this increase in gene expression can be mediated by the presence of a transcriptional enhancer located in the first intron [21–24]. Splicing also has been shown to directly stimulate the expression of several genes [25, 26]. Based on these previous observations, the current study explores whether the Ctsl first intron is capable of stimulating gene expression in Sertoli cells and, if so, how the stimulatory effect is achieved. We show that the Ctsl first intron does increase gene expression in Sertoli cell cultures and propose that this stimulatory effect is responsible in part for the high levels of CTSL produced by Sertoli cells within seminiferous tubules at stages VI to VII.

MATERIALS AND METHODS

DNA Constructs

The different DNA constructs tested in this study were assembled using the plasmid pGL2-Basic (Promega Corp., Madison, WI) as the backbone and are described in their respective figures. A detailed description of the assembly of these constructs is available online as supplemental data (www.biolreprod.org). The size of the 5'-untranslated region (UTR) of the rat Ctsl gene is 85 nucleotides. The nucleotide sequence derived from the Ctsl gene in each of the constructs tested was verified by DNA sequencing. The reporter gene constructs used to transfect TM4 cells were purified using the EndoFree Plasmid Maxi Kit (Qiagen Inc., Valencia, CA). DNA constructs used to transfect Sertoli cells isolated from testes of sexually mature rat were further purified by CsCl2 centrifugation.

Animals, Cell Culture, and DNA Transfection

Sexually mature (60- to 70-day-old) Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). The use of animals in the experiments described in this manuscript was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University. Sertoli cells from sexually mature rats were isolated, cultured, and transfected as previously described [8]. The mouse TM4 cell line originated from primary cultures of Sertoli cell-enriched preparations from 11- to 13-day-old BALB/c mice [27] and was obtained from the American Type Cell Collection (Manassas, VA). TM4 cells were cultured in Dulbecco modified Eagle medium/F-12 medium supplemented with 10% fetal bovine serum and 10 µg/ml gentamycin. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA). TM4 cells were plated in six-well plates at a density of 2.5 x 105 cells per well the day prior to transfection. Transient transfections were performed using 15 µl lipofectamine (Invitrogen Corp.), 0.45 pmol firefly luciferase (Luc) reporter gene constructs that contained Ctsl promoter sequences, and 0.009 pmol plasmid pRL-CMV (Promega Corp.). This plasmid contained the Renilla Luc reporter gene and was used to correct for variations in transfection efficiency. In experiments in which RNA extraction and reverse transcription were performed, TM4 cells were plated in 100-mm dishes at a density of 1.2 x 106 cells per dish the day prior to transfection. Transient transfections were performed using 50 µl lipofectamine, 2.2 pmol promoter constructs, and 0.19 pmol plasmid pRL-CMV. Each transfection was performed at least three independent times.

LUC Assays

LUC activities were measured 24 h after transfection. Sertoli cells isolated from sexually mature rats and TM4 cells were lysed in Passive Lysis Buffer (Promega Corp.), frozen in dry ice, and stored at –80°C. Firefly and Renilla LUC activities were measured in cell extracts using the Dual-Luciferase Reporter Assay System (Promega Corp.) as previously described [8]. LUC activity was defined as the ratio between firefly and Renilla LUC activities. Titration of extracts from both mature Sertoli and TM4 cells demonstrated that firefly and Renilla LUC activities were within the linear range of the assays.

RNA Extraction and Reverse Transcription

Total RNA from transiently transfected TM4 cells was isolated using the RNeasy Mini Kit (Qiagen Inc.). To remove any traces of plasmid DNA, total RNA (1 µg) was treated twice with 3 units of TURBO DNA-free (Ambion, Austin, TX) for 30 min at 37°C, according to the manufacturer's instructions. TURBO-treated RNA (50 ng) then was reverse transcribed using the SuperScript III First-Strand Synthesis for RT-PCR (Invitrogen Corp.), according to the manufacturer's instructions. Cellular RNA was reverse transcribed using (2'-deoxy-thymidine)20 (oligo(dT)20) or random hexamers as primers. Random hexamers were used to prime reverse transcription of all RNA species regardless of their polyadenylation state, whereas oligo(dT)20 primers were used to initiate reverse transcription of polyadenylated RNA only.

Real-Time Quantitative PCR

Real-time PCR was performed using the ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA). The firefly and Renilla Luc primers and TaqMan probes were designed as gene expression assays from the Assays-in-Design service (Applied Biosystems), and their nucleotide sequences are listed in Table 1. TaqMan gene expression assays consisted of a 20x mix of unlabeled gene-specific primers and TaqMan probes, labeled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine at the 3' end. From a master mixture, 50-µl aliquots were placed in a 96-well optical tray. Wells contained 25 µl TaqMan Universal PCR master Mix (2x), 2.5 µl of each gene expression assay, and 12.5 µl H2O. In addition to 10 µl of the respective cDNA unknowns, each plate contained no template controls (DNA was replaced with 10 µl H2O) and known amounts of the plasmids pGL2-Basic (0.01 fg to 10 pg) and pRL-CMV (0.001 fg to 1 pg), which were used to generate two standard curves. All samples were prepared in duplicate. TaqMan PCR conditions were as follows: 50°C for 2 min; 95°C for 10 min; and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Data were analyzed using Sequence Detection Systems software version 1.7 (Applied Biosystems). The standard curves generated for every experiment were linear over the given standard range, with a linear correlation coefficient of >0.990. The amounts of firefly and Renilla Luc cDNA produced using oligo(dT)20 or random hexamer primers were calculated by plotting the Ct (cycle number above the threshold) against the respective standard curves. Firefly Luc cDNA levels in each sample were then normalized to that of Renilla Luc. Gel electrophoresis was performed to confirm the correct size of the amplified cDNA and the absence of nonspecific products. To exclude PCR amplification of contaminating plasmid DNA, controls with no RT (samples that contained RNA that was not reverse transcribed) were carried out and showed absence of amplification.


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  		TABLE 1. Nucleotide sequences of the primers used for TaqMan real-time PCR.

Assessment of Splicing

Following transient transfections of the construct C4 or C5 in TM4 cells, cellular RNA isolation and reverse transcription were performed as described above. PCR was performed with the Optimizer Buffer B kit (Invitrogen Corp.), according to manufacturer's instructions, using 2 µl of reverse transcribed RNA primed with oligo(dT)20. The forward primer SF (5'-ACCGTCGAGATCTAAGTAAG-3') and the reverse primer SR (5'-CCTTATGCAGTTGCTCTCCA-3') were used to monitor the splicing of the intron sequence. The construct C5 was used to generate PCR fragments that corresponded to the size of the unspliced transcripts. To exclude PCR amplifications of contaminating plasmid DNA, PCR was performed on RNA samples that were not reverse transcribed. Reactions were run in a Perkin-Elmer thermal cycler using the following conditions: 2 min at 94°C for 1 cycle, followed by 15 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C for 35 cycles. PCR products (1 µl of a 1:5 dilution) then were fractionated on DNA 1000 LabChips (Agilent Technologies Inc., Palo Alto, CA). Automated quantitation and size of each DNA fragment against internal standards were performed using the Agilent Bioanalyzer 2100 and the 2100 Expert software (Agilent Technologies Inc.).

Quantitation of Mature and Pre-mRNA

Following transient transfections of the construct C6 or C7 in TM4 cells, cellular RNA isolation and reverse transcription were performed as described above. PCR was performed with the Optimizer Buffer B kit (Invitrogen Corp.), according to manufacturer's instructions, using 2 µl reverse transcribed RNA primed with oligo(dT)20. Levels of mature Luc mRNA levels were compared to the levels of intron-containing Luc pre-mRNA by simultaneous amplification of both cDNA fragments in the same reaction. The forward primer SF and the reverse primer SR were used to measure the amounts of mature mRNAs derived from constructs C6 and C7. The forward primer S1 (5'-CTTGTGGGTATAATCCCTGC-3') and the reverse primer SR were used to quantify the amount of intron 1-containing pre-mRNA, whereas the forward primer S4 (5'-CAAGGCTGTCCCTGTATGTA-3') and the reverse primer SR were used to quantify the amount of intron 4-containing pre-mRNA. To exclude PCR amplifications of contaminating plasmid DNA, PCR was performed on RNA samples that were not reverse transcribed. Reactions were run in a Perkin-Elmer thermal cycler using the following conditions: 2 min at 94°C for 1 cycle, followed by 15 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C for 30 cycles. PCR products then were fractionated on DNA 1000 LabChips (Agilent Technologies Inc.), sized, and quantitated as described above.

Statistical Analysis

Data from transient transfection assays were analyzed by ANOVA, and differences between individual means were tested by Fisher test using StatView 5.0.1 (SAS Institute Inc., Cary, NC). Differences were defined as significant at P < 0.05.

RESULTS

The Rat Ctsl First Intron Enhances Reporter Gene Activity in Primary Cultures of Sertoli Cells and in TM4 Cells

Based upon the observation that the presence of an intron in the 5'-UTR of several genes leads to an increase in their expression, the ability of the rat Ctsl first intron to act in such a manner was addressed by testing two constructs. The construct C1 (Fig. 1A) contains 2065 nucleotides upstream of the transcription start site, the first exon (74 nucleotides), the first intron (892 nucleotides), and the first 11 nucleotides of exon 2 (up to the ATG initiation codon) of the rat Ctsl gene, whereas the construct C2 (Fig. 1A) is identical to the latter construct except that the first intron sequence has been removed. To address whether or not the Ctsl first intron was capable of stimulating gene expression, the two constructs described above were transiently transfected in Sertoli cells isolated from sexually mature (60- to 70-day-old) rats, and LUC activities were measured 24 h afterwards. As shown in Figure 1B, LUC activity was ~5-fold higher in cells transfected with the promoter construct that contained the first intron (C1) compared with that of cells transfected with the parent construct that lacked the intron (C2). Identical stimulation of reporter gene activity was observed when transfections were performed in Sertoli cells isolated from sexually immature (30-day-old) rats (data not shown). The ~5-fold increase in reporter gene activity mediated by the Ctsl first intron in primary cultures of Sertoli cells invites the speculation that the Ctsl first intron also contributes to enhancing the expression of the Ctsl gene in a similar manner in vivo in Sertoli cells. This increase may contribute to the high levels of CTSL observed in seminiferous tubules at stages VI to VIII.


Figure 01
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  		FIG. 1. The Ctsl first intron increases LUC activity in Sertoli cells. A) Schematic representation of the DNA constructs tested. In the construct C1, a DNA fragment that contains 2065 nucleotides upstream of the transcription start site of the rat Ctsl gene and 977 nucleotides of downstream sequence is linked to the firefly Luc gene. The construct C2 is identical to the above construct, except that the Ctsl first intron has been deleted. Box with vertical bars, DNA region located upstream of the transcription start site (denoted +1); hatched box, 5'-UTR; open box, intron sequence. Reporter gene activities were measured 24 h after transient transfections in Sertoli cells isolated from sexually mature rat testes (B) or in TM4 cells (C). The plasmid pRL-CMV was co-transfected to control for variation in transfection efficiencies. LUC activity was defined as the ratio between the firefly LUC activity and the Renilla LUC activity. The results in B (n = 8) and C (n = 4) are expressed as means ± SEM. In B, LUC activities in eight different preparations of Sertoli cells were analyzed.

To facilitate the analysis of the effect of the first intron on gene expression in Sertoli cells, constructs C1 and C2 were transiently transfected in the TM4 Sertoli cell line. Originated from primary cultures of Sertoli cell-enriched preparations from 11- to 13-day-old BALB/c mice [27], TM4 cells posses two main advantages over primary cultures of Sertoli cells: 1) they can be maintained in culture for long periods of time, allowing experiments to be conducted at any given time, and 2) for the purpose of the experiments conducted in the present study, they represent a cost-efficient alternative to the expensive isolation of Sertoli cells from rat testes. Following transient transfections into TM4 cells, LUC activity also was ~5-fold higher in cells transfected with the construct C1 compared with that of cells transfected with the construct C2 (Fig. 1C). These results indicate that TM4 cells could be used to study the quantitative effect of the Ctsl first intron on gene expression.

Effect of the Ctsl First Intron on two Heterologous Promoters

To determine whether the stimulatory effect of the first intron was specific to the Ctsl promoter, its ability to enhance the activity of a reporter gene driven by two heterologous promoters was examined. A DNA fragment that contained the Ctsl first intron was inserted between the firefly Luc coding sequence and either the SV40 or the TK promoter (Fig. 2A). The intron-containing promoter constructs SV1 and TK1 each were transiently transfected in TM4 cells. Constructs SV2 and TK2, which lacked the Ctsl first intron, served as controls. The presence of the first intron stimulated LUC activity by 3- and 9-fold when linked to the TK or SV40 promoter, respectively (Fig. 2B). These results indicate that the increase in reporter gene activity mediated by the first intron was not restricted to the Ctsl promoter, although the magnitude of the stimulation was not identical for the different promoters tested. Additional experiments demonstrated that the stimulatory effect of the first intron was not restricted to the Luc coding sequence, as TM4 cells transfected with a LacZ-based construct that contained the first intron showed a similar increase in reporter gene activity compared with cells transfected with the intronless counterpart (data not shown).


Figure 02
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  		FIG. 2. The effect of the rat Ctsl first intron on the activity of the SV40 and TK promoters. A) Schematic representation of the DNA constructs tested. In the constructs SV1 and TK1, the Ctsl first intron is positioned in the sense orientation between the promoter region and the firefly Luc coding sequence. The constructs SV2 and TK2 are identical to the latter constructs, except that they do not contain the Ctsl first intron. B) LUC activities were measured in TM4 cells transfected with constructs that harbor either the TK or SV40 promoter in the presence or absence of the first intron. Results (n = 4) are expressed as means ± SEM.

The First Intron Does Not Contain a Typical Enhancer

Work from numerous laboratories has established that introns located in the 5'-UTR of several genes contain typical enhancer elements, meaning that they exhibit a position- and orientation-independent effect on gene expression (reviewed in Khoury and Gruss [28] and Serfling et al. [29]). The ability of the Ctsl first intron to act in such a fashion was tested by placing it upstream of the Ctsl promoter in the construct C2 in both sense and antisense orientations (Fig. 3A). Results from transient transfections performed in TM4 cells indicate that the stimulatory effect of the first intron was not detected when this sequence was moved upstream of the transcription start site, positioned either in the sense or antisense orientation (Fig. 3B). The effect of the Ctsl first intron also was tested using a compact promoter, the 120-nucleotide proximal promoter of the rat Ctsl gene [10]. Again, the Ctsl first intron failed to stimulate LUC activity (data not shown). The inability to demonstrate any activity of the first intron when it was moved upstream of the promoter suggests that its function was dependent on both position and orientation and therefore did not contain a typical enhancer.


Figure 03
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  		FIG. 3. The Ctsl first intron does not contain a typical enhancer. A) Schematic representation of the DNA constructs tested. The Ctsl first intron sequence that spans nucleotides +75 to +966 was inserted upstream of the Ctsl promoter in the construct C2, either in the sense or antisense orientation (constructs C3F and C3R, respectively). The black arrow indicates the orientation of the first intron. B) Constructs C1, C2, C3F, and C3R were transiently transfected in TM4 cells, and LUC activities were measured 24 h afterwards. The results (n = 5) are expressed as means ± SEM.

Inclusion of the Ctsl First Intron Leads to an Increase in Both Polyadenylated and Total Luc RNA Levels

We reasoned that the ~5-fold increase in reporter gene activity observed in TM4 cells transfected with the construct C1 that contained the first intron of the rat Ctsl gene might be explained by a proportional increase in the amounts of firefly Luc mRNA. To test this possibility, the steady-state levels of polyadenylated firefly Luc RNA in TM4 cells transiently transfected with the construct C1 or C2 were analyzed by quantitative real-time PCR. Measurements of LUC activities showed the expected ~5-fold increase in reporter gene activity in aliquots of TM4 cells transfected with the intron-containing construct C1 (Fig. 4A). Cellular RNA was isolated from the remaining cells, and reverse transcription of polyadenylated RNA was performed using oligo(dT)20 as the primer. The amounts of firefly Luc cDNA produced were then quantitated and normalized to that of the internal control Renilla Luc. Results from this analysis (Fig. 4B) revealed that steady-state levels of polyadenylated firefly Luc RNA derived from the construct C1 were 3-fold higher than those from the construct C2. To directly compare LUC activities to the amount of polyadenylated Luc RNA, the translational yields (defined as the ratios between the reporter gene activity and the amount of firefly Luc polyadenylated RNA) derived from constructs C1 and C2 were calculated (Fig. 4C). Yields of 0.076 and 0.045 were obtained for constructs C1 and C2, respectively. It is interesting to note that the ratio between the translational yields derived from constructs C1 and C2 was 1.7, which means that TM4 cells transfected with the intron-containing construct yielded 70% more LUC protein per polyadenylated Luc RNA than cells transfected with the intronless construct (Fig. 4C).


Figure 04
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  		FIG. 4. The inclusion of the Ctsl first intron leads to an increase in the levels of polyadenylated and total RNA. A) Measurement of LUC activity in TM4 cells transiently transfected with the construct C1 or C2. Quantitative real-time PCR was used to compare the amounts of polyadenylated (B) and total (D) firefly Luc RNA in TM4 cells transfected with the construct C1 or C2. Cellular RNA was reverse transcribed using oligo(dT)20 (B) or random hexamers (D). Quantitative real-time PCR was performed using gene-specific primers (Table 1). The amounts of firefly and Renilla Luc cDNA produced were calculated by plotting the Ct (cycle number above the threshold) against the respective standard curves. Firefly Luc cDNA levels in each sample were then normalized to that of Renilla Luc. The results are from nine independent experiments. C) The translational yield derived from a given construct is defined as the ratio between the reporter gene activity (A) and the amount of firefly Luc polyadenylated RNA (B). The translational yields derived from constructs C1 and C2 were 0.076 and 0.045, respectively.

To establish whether the increase in polyadenylated Luc RNA levels was correlated with an increase in the levels of total Luc RNA, the amounts of total firefly Luc RNA were monitored by reverse transcribing cellular RNA with random hexamers as primers. Results from this analysis (Fig. 4D) revealed that steady-state levels of total Luc RNA derived from construct C1 were 2-fold higher than those from construct C2. Since measurements of total RNA levels reflect both the amounts of polyadenylated and nonpolyadenylated RNA, the results presented in Figure 4, B and D suggest that the ~5-fold increase in LUC activity mediated by the Ctsl first intron is due in part to a combined increase in the amounts of total RNA and in transcript polyadenylation.

Splicing Is Required for the Increase in Reporter Gene Activity Mediated by the Ctsl First Intron

Several reports have shown that splicing can modulate gene expression by affecting steady-state transcript levels and 3'-end processing (reviewed in Proudfoot et al. [13]; see also [30–34]). To determine whether splicing of the first intron of the rat Ctsl gene is required to enhance reporter gene activity, a strategy was devised to prevent the splicing of this intron by mutating one of its splice sites. Inspection of the nucleotide sequence of the Ctsl first intron revealed that only the authentic 5'-splice site (located at nucleotide positions +73 to +80) matched the consensus sequence (data not shown). While mutating the 5'-splice site in the construct C1 appeared to be the best strategy to test the effect of splicing on reporter gene activity, preliminary experiments indicated that retention of more than half of the first intron sequence resulted in LUC activity comparable to that of the promoterless vector pGL2-Basic (data not shown). To explain this result, one could speculate that translation was affected by the generation of a long and potentially highly structured 5'-UTR. To circumvent this problem, the construct C4 was assembled (Fig. 5A). The intronic sequence that spans nucleotides +121 to +765 was deleted because the inspection of this sequence indicated that the regulatory elements that are essential for the splicing reaction were not located inside this region. To simplify the assembly of the construct, the intron sequence was removed from its natural setting and introduced into a convenient restriction site in the construct C2. Using the construct C4, we set out to test whether or not the splicing of the truncated first intron was required to enhance reporter gene activity. The sequence of the 5'-splice site 5'-AGGTGAGT-3' in the construct C4 was altered to 5'-TTAATTCA-3' in the construct C5. When the intron construct that contains the mutated 5'-splice site was tested in transient transfection assays using TM4 cells, LUC activity was comparable to that of the construct C2 that lacked the first intron (Fig. 5B). In contrast, LUC activity measured in TM4 cells transfected with the construct C4 was 3-fold higher than that in cells transfected with the construct C2 or with the construct C5 that contained the mutated 5'-splice site. These results indicate that splicing was necessary for the increase in reporter gene activity mediated by the Ctsl first intron. It should be noted that moving the truncated first intron sequence outside of its natural setting reduced its stimulatory effect on reporter gene activity (compare C1 and C4 in Fig. 5B). A similar reduction in LUC activity also was observed when the full-length Ctsl first intron was moved (see below).


Figure 05
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  		FIG. 5. Splicing is required for the intron-dependent increase in LUC activity. A) Schematic representation of the DNA constructs tested. In the construct C4, a truncated Ctsl first intron that spans nucleotides +42 to +120 linked to nucleotides +766 to +977 is inserted immediately downstream of the 5'-UTR (nucleotide position +977). The construct C5 is identical to the construct C4, except that the sequence of the 5'-splice site 5'-AGGTGAGT-3' (nucleotide positions +73 to +80) is mutated to 5'-TTAATTCA-3'. B) Constructs C1, C2, C4, and C5 were transiently transfected in TM4 cells, and LUC activities were measured 24 h afterwards. Results (n = 5) are expressed as means ± SEM. C) The mutations introduced in the 5'-splice site of the construct C5 prevent splicing. Cellular RNA isolated from TM4 cells transiently transfected with the construct C4 or C5 was primed with oligo(dT)20 in the presence (lanes 2 and 4) or absence (lanes 3 and 5) of reverse transcriptase. PCR analyses then were performed using primers SF and SR, which bridge the intron sequence. The construct C5 was amplified in parallel to provide the size of the PCR products derived from unspliced transcripts (lane 6). The locations of primers SF and SR are shown in A. Sizes for PCR fragments derived from spliced intron transcripts: 181 nucleotides; and from unspliced transcripts: 426 nucleotides. Lane 1 shows molecular weight markers. The results presented here are representative of two independent transfections.

To establish that the mutations introduced into the 5'-splice site of the construct C5 did prevent splicing of the truncated intron 1, cellular RNA was extracted from TM4 cells transiently transfected with the construct C5 or C4 (the latter was used as a positive control for splicing). Following reverse transcription, PCR using the primers SF and SR (Fig. 5A) was performed to analyze the splicing patterns. Since primers SF and SR bridged the intron 1 sequence, spliced transcripts should have given rise to PCR fragments of 181 nucleotides, whereas unspliced transcripts should have given rise to PCR fragments of 426 nucleotides. RT-PCR analysis of RNA isolated from TM4 cells transiently transfected with the construct C4 gave rise to a predominant PCR product that was derived from spliced transcripts (Fig. 5C, lane 2). A minor PCR product that corresponded to the pre-mRNA also was detected. In contrast, RT-PCR analysis of RNA isolated from TM4 cells transiently transfected with the construct C5 gave rise to a predominant PCR product of 426 nucleotides that was derived from transcripts that retained the intron sequence (Fig. 5C, lane 4). A minor PCR product of ~250 nucleotides also was detected (denoted by the arrowhead in lane 4 of Fig. 5C). This minor product could reflect the usage of a cryptic splice site. However, judging by the intensity of the band, the usage of this cryptic splice site appeared very inefficient. The absence of PCR products in the negative RT controls confirmed that the PCR-amplified fragments were derived entirely from cDNA and not from plasmid DNA (Fig. 5C, lanes 3 and 5). Collectively, these results indicate that when splicing was prevented, the Ctsl first intron lost its ability to increase reporter gene activity.

Loss of the Stimulatory Effect When the First Intron is Replaced by the Fourth Intron of the Rat Ctsl Gene

To investigate whether the stimulatory effect of the Ctsl first intron on reporter gene activity in Sertoli cells could be explained solely by its splicing, the first intron was replaced by the fourth intron of the rat Ctsl gene. The size of the Ctsl intron 4 is 564 nucleotides and contains 5'- and 3'-splice sites similar to those of intron 1 (Fig. 6A). The constructs C6 and C7 (Fig. 6B) were engineered so that both intron sequences would be positioned in an identical manner downstream of the 5'-UTR of the rat Ctsl gene and upstream of the ATG initiation codon of the Luc coding sequence. Following transient transfections in TM4 cells, LUC activity was 3-fold higher in cells transfected with the construct C6 compared with cells transfected with the construct C2 (Fig. 6B). As noted above, moving the first intron sequence outside of its natural setting had an unexpected effect on reporter gene activity, the activity of construct C6 being reduced by 14% compared with construct C1. However, in contrast to the stimulatory effect of intron 1, intron 4 did not increase LUC activity either in TM4 cells (Fig. 6C) or in Sertoli cells isolated from sexually mature rats (data not shown).


Figure 06
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  		FIG. 6. Loss of the stimulatory effect when the Ctsl first intron is replaced by the fourth intron. A) Comparison between the nucleotide sequences of the 5'- and 3'-splice sites of introns 1 and 4 of the rat Ctsl gene is shown. The last 30 nucleotides of both intron sequences that contain the polypyrimidine-rich track also are included. Upper case, intron sequence; bold upper case, exon sequence; R, purine; Y, pyrimidine. B) Schematic representation of the DNA constructs tested. The constructs C6 and C7 were engineered so that both introns were positioned in an identical manner after the 5'-UTR of the rat Ctsl gene. In contrast, in the construct C1, the natural setting of the Ctsl first intron has been conserved. In the construct C6, a 972-nucleotide DNA fragment that corresponds to the last 33 nucleotides of exon 1, intron 1 (892 nucleotides), and the first 11 nucleotides of exon 2 of the rat Ctsl gene is inserted immediately downstream of the 5'-UTR (nucleotide position +977). In the construct C7, a 605-nucleotide DNA fragment that corresponds to the last 33 nucleotides of exon 4, intron 4 (564 nucleotides), and the first 11 nucleotides of exon 5 of the rat Ctsl gene is inserted immediately downstream of the 5'-UTR. The fourth intron of the rat Ctsl gene contains 5'- and 3'-end splicing sites similar to those of the first intron (A). C) Constructs C1, C2, C6, and C7 were transiently transfected in TM4 cells, and LUC activities were measured 24 h afterwards. Results (n = 4) are expressed as means ± SEM. D) Quantitation of mature and pre-mRNAs in TM4 cells transiently transfected with constructs that contain the Ctsl intron 1 or 4. Cellular RNA isolated from TM4 cells transiently transfected with the construct C6 or C7 was primed with oligo(dT)20 in the presence (lanes 2 and 4) or absence (lanes 3 and 5) of reverse transcriptase. PCR analyses then were performed using two sets of primers (SF, S1, and SR for intron 1; SF, S4, and SR for intron 4). The locations of primers SF, S1, S4, and SR are shown in B. Sizes for PCR fragments derived from spliced intron 1 or 4 transcripts: 181 nucleotides; from unspliced intron 1 transcripts: 258 nucleotides; and from unspliced intron 4 transcripts: 278 nucleotides. Lane 1 shows molecular weight markers. E) The PCR products that corresponded to the mature and pre-mRNAs in TM4 cells transfected with the construct C6 or C7 were sized and quantified using the Agilent Bioanalyzer 2100. Data are expressed as a ratio between mature and pre-mRNAs (means ± SEM). The results presented here are representative of three RNA isolations.

To verify that transcripts derived from the construct C7 were spliced as efficiently as those derived from the construct C7, the amounts of mature and pre-mRNAs were quantitated. Cellular RNA from TM4 cells transfected with the construct C6 or C7 was isolated and reverse transcribed using oligo(dT)20 as a primer. Levels of mature Luc mRNA were compared to the levels of intron-containing Luc pre-mRNA by simultaneous amplification of both cDNA fragments in the same reaction. The locations of the primers are indicated in Figure 6B. Since primers SF and SR bridged the intron 1 and 4 sequences, spliced mRNA should have given rise to DNA fragments of 181 nucleotides. Primers S1 and S4 are located 134 and 154 nucleotides upstream of the 3'-splice site of Ctsl introns 1 and 4, respectively. Amplication from primer sets S1-SR and S4-SR targeted specifically pre-mRNA that contained either intron 1 or 4, respectively. Unspliced intron 1 transcripts should have given rise to PCR fragments of 258 nucleotides, whereas unspliced intron 4 transcripts should have given rise to PCR fragments of 278 nucleotides. To ensure that the amplification was within the linear range, PCR was performed using different numbers of cycles and different initial inputs of RNA (data not shown). The PCR products were sized and quantified using the Agilent Bioanalyzer 2100. Using 10 ng total RNA isolated from TM4 cells transiently transfected with the construct C6 or C7, the predominant PCR products generated were derived from spliced transcripts (Fig. 6D, lanes 2 and 4). A minor product that corresponded to intron 1 or 4 pre-mRNA (unspliced) also was detected. The absence of PCR products in the negative RT controls confirmed that the PCR-amplified fragments were derived entirely from cDNA and not from plasmid DNA (Fig. 6D, lanes 3 and 5). The quantitation of the PCR products revealed that the ratios between the mature and pre-mRNAs from TM4 cells transfected with the construct C6 or C7 were not statistically different (Fig. 6E). Identical results were obtained when PCR quantitation of both Luc mature and pre-mRNAs was performed using serial 2-fold dilutions of total RNA (ranging from 80 to 2.5 ng) isolated from TM4 cells transfected with the construct C6 or C7 (data not shown). Taken together, these data suggest that the enhancement effect mediated by the Ctsl first intron was dependent on both splicing and the presence of specific cis-elements located within this intron.

Steady-State Levels of Polyadenylated and Total Luc RNA Derived from Constructs That Contained Either the Ctsl Intron 1 or 4

Total and polyadenylated firefly Luc RNA levels from TM4 cells transiently transfected with the construct C6 or C7 were analyzed by quantitative real-time PCR. As shown before, LUC activity was 3-fold higher in aliquots of TM4 cells transfected with the construct C6 compared with cells transfected with the construct C7 (Fig. 7A). Following LUC activity measurements, cellular RNA was isolated from the remaining cells and reverse transcribed using either oligo(dT)20 or random hexamers as primers. The amounts of firefly Luc cDNA produced by both primers then were quantitated and normalized to that of the internal control Renilla Luc. Results from this analysis revealed that the steady-state levels of polyadenylated Luc RNA derived from the construct C6 were 2-fold higher than those from the construct C7 (Fig. 7B). However, contrary to what was observed previously when comparing constructs C1 and C2, the fold difference between the levels of polyadenylated and total RNA derived from constructs C6 and C7 were identical (both 2-fold, compare Fig. 7, B and C).


Figure 07
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  		FIG. 7. Steady-state levels of polyadenylated and total Luc RNA derived from constructs that contain the Ctsl intron 1 or 4. A) Measurement of LUC activities in TM4 cells transiently transfected with the construct C6 or C7. Quantitative real-time PCR was used to compare the amounts of polyadenylated (B) and total (C) firefly Luc RNAs in TM4 cells transfected with the construct C6 or C7. Cellular RNA was reverse transcribed using oligo(dT)20 (B) or random hexamers (C). Quantitative real-time PCR was performed using gene-specific primers. The amounts of firefly and Renilla Luc cDNAs produced were calculated by plotting the Ct (cycle number above the threshold) against the respective standard curves. Firefly Luc cDNA levels in each sample then were normalized to that of Renilla Luc. The results (n = 4) are expressed as means ± SEM.

DISCUSSION

The Rat Ctsl First Intron Increases Reporter Gene Activity in Sertoli Cells

CTSL constitutes approximately 1% of all of the proteins synthesized in the tubules at stages VI to VII of the cycle of the seminiferous epithelium of the rat [7]. We have previously showed that this increase in protein levels is mediated by the stage-specific transcription of the Ctsl gene via a 3-kb promoter, spanning nucleotides –2065 to +977, relative to the transcription start site [9]. As noted in the Introduction, this is the only promoter described to date that confers both Sertoli cell-specific and accurate stage-specific gene expression in transgenic mice. A major finding of the present study is that the first intron, located between nucleotide positions +75 and +966, increased reporter gene activity by ~5-fold in both primary Sertoli cell cultures and in the TM4 Sertoli cell line (Fig. 1, B and C). While the Ctsl first intron had a stimulatory effect on reporter gene activity, it is important to point out that the presence of an intron in the 5'-UTR does not always result in the stimulation of gene expression [35–38], and that a variety of genes that are naturally devoided of introns are expressed efficiently in higher eukaryotes [39–41]. The stimulatory effect of the Ctsl first intron was observed when it was linked to different promoters and reporter genes (Fig. 2 and data not shown). While the increase in the expression of several genes that contain an intron in their respective 5'-UTR is often mediated by typical enhancers, the intron-mediated enhancement observed here could not be explained by such a mechanism, since it was both position dependent and orientation dependent (Fig. 3).

Ctsl First Intron Increases the Levels of Polyadenylated and Total RNA of the Reporter Gene

Published reports have indicated that splicing can affect mRNA biogenesis in higher eukaryotes by increasing both cytoplasmic and nuclear mRNA levels and by enhancing transcript polyadenylation [26, 33, 42]. It is important to point out that based upon data currently available, splicing is thought to have little or no effect on cytoplasmic mRNA accumulation in mammalian and insect cells [16, 26, 42, 43].

While transcription and splicing have long been considered to be two independent cellular processes taking place in a sequential fashion, recent experimental evidence suggests that they are intimately connected. Furger and colleagues established that transcription was reduced when the promoter proximal intron from two yeast genes or the promoter proximal splicing signals from a mammalian gene were removed [44]. In addition, Fong and Zhou have shown that splicing factors increased transcriptional elongation by RNA polymerase II [45], and Kwek and colleagues have demonstrated that in addition to its role during splicing, the factor U1 snRNA associates with the general transcription factor TFIIH, leading to a stimulation of the rate of initiation by RNA polymerase II [46]. While the exact mechanism that underlies the stimulatory effect of splicing on transcription remains unknown, available data clearly indicate that factors that are required during the removal of introns also are able to associate with components of the transcription machinery and are important determinants of the levels of transcripts produced.

Introns also have been shown to stimulate mRNA polyadenylation. The stimulatory effect of introns on the accumulation of polyadenylated RNA has been observed using different introns [30, 31, 33]. In addition, Nott and colleagues noted that a strong correlation existed between polyadenylation efficiency and intron position, with the 5'-proximal intron increasing polyadenylation and RNA accumulation more efficiently than a 3'-proximal intron [42]. It is noteworthy that the coupling established between splicing and transcription also exists between splicing and polyadenylation (reviewed in Proudfoot et al. [13], Minvielle-Sebastia and Keller [47], and Le Hir et al. [48]). For example, the splicing factor U1 snRNP-A interacts with the 160-kDa subunit of cleavage-polyadenylation specificity factor [32, 49], whereas the poly(A) polymerase interacts with the splicing factor U2AF 65 [34].

In the present study, measurements of steady-state RNA levels (Fig. 4) revealed that the presence of the Ctsl first intron increased polyadenylated Luc RNA levels by 3-fold and total Luc RNA levels by 2-fold. Since 1) polyadenylated Luc RNA levels were 1.5-fold higher than total Luc RNA levels and 2) measurements of total RNA levels reflect both the amounts of polyadenylated and nonpolyadenylated RNA, these results suggest that the presence of the Ctsl first intron leads to a combined increase in the amounts of total RNA and in transcript polyadenylation.

Higher Translational Yields Are Observed in the Presence of the Ctsl First Intron

Aside from stimulating transcription and polyadenylation, splicing also can enhance the translational efficiency of the mature transcript (reviewed in Le Hir et al. [48]; see also [42, 50]). Data presented in Figure 4C indicate that the first intron of the rat Ctsl gene increased the translational yield for a given amount of polyadenylated RNA. The ratio between the translational yields derived from constructs C1 and C2 was 1.7, which meant that spliced Luc mRNAs yielded ~70% more protein per mRNA molecule than unspliced Luc mRNAs. Based on published reports, one can speculate that this effect on translation is mediated by the ~335-kDa protein complex termed the exon junction complex (EJC) [51–53]. Upon the removal of introns from the pre-mRNA, EJCs are deposited ~20–24 nucleotides upstream of the exon-exon junctions [53]. Several proteins that form part of the EJC remain associated with the mRNA during its transport from the nucleus to the cytoplasm [51, 54, 55]. Nott and colleagues showed that spliced transcripts exhibited higher translational yields than their intronless counterparts (two to four times more protein per mRNA molecule) [42], and that EJC proteins play a direct role in targeting spliced mRNAs more efficiently to polysomes than their intronless counterparts [50]. Results from the present study suggest that in addition to increasing the levels of polyadenylated and total RNA, the Ctsl first intron also stimulates mRNA translational efficiency. To definitively address this point, the analysis of the mRNA distribution between monosomes and polysomes will need to be performed. It is predicted that mRNA derived from DNA constructs that contain the Ctsl first intron will be more efficiently recruited to polysomes than mRNA derived from DNA constructs that lack the first intron.

Location of the Ctsl First Intron Is Conserved in Several Species

In view of the stimulatory effect of the Ctsl first intron on gene expression, it may be informative to compare its location in different species. While the first intron of the mouse gene is longer than its rat counterpart (1071 vs. 892 nucleotides), the distance between the ATG initiation codon and the end of the first intron is exactly 11 nucleotides in both species [56]. The 5'-UTR of the human Ctsl gene also contains an intron, which is located 10 nucleotides upstream of the translation start site [57]. It should be noted that the first intron of the human Ctsl gene might have an additional and unique function, as it contains a promoter responsible for the generation of a second Ctsl transcript [58]. The first introns of the cow and dog orthologs also are located 11 nucleotides upstream of the ATG initiation codon [59, 60]. An intron also is present in the 5'-UTR of the Drosophila melanogaster ortholog [61], which is located 13 nucleotides upstream of the translation start site. Since the location of the Ctsl first intron within the 5'-UTR is highly conserved in several species, this common feature invites the speculation that the Ctsl first intron plays a general role in the upregulation of this gene. It also is noteworthy that the location of the Ctsl first intron, ~10 nucleotides upstream of the ATG initiation codon, may promote the recruitment of the EJC close to the translational start site and enhance translation [50].

Splicing and the Intron-Mediated Enhancement of Gene Expression

As noted above, results from the present study have demonstrated the necessary role of splicing in the increase of reporter gene activity mediated by the rat Ctsl first intron. Mutating the 5'-splice site abolished the intron-mediated enhancement of reporter gene activity (Fig. 5B). While splicing is clearly required for the Ctsl first intron to stimulate gene expression, splicing per se may not be sufficient to achieve this stimulation. As shown in Figure 6C, the Ctsl fourth intron was unable to recapitulate the stimulatory effect of the first intron on reporter gene activity when it was positioned in the same location in the 5'-UTR, even though both introns 1 and 4 were properly removed from the pre-mRNA (Fig. 6, D and E). If splicing is a key step in the intron-mediated enhancement of gene expression, why did intron 4 fail to augment gene expression?

Korb and colleagues showed that the fourth intron of the mouse thymidylate synthase gene was unable to stimulate gene expression [62]. The failure of this intron to augment gene expression was explained by the fact that it was spliced very inefficiently, due to a weak 5'-splice site (only five of eight nucleotides matched the consensus sequence). Altering the 5'-splice to the consensus sequence resulted in a ~6-fold increase in gene expression. This scenario is unlikely in the case of the Ctsl intron 4, since it is as efficiently spliced as intron 1 (Fig. 6, D and E). Wiegand and colleagues demonstrated that intron splicing did not enhance gene expression if the EJC formation was blocked [63]. They showed that EJC formation was compromised if the intron sequence was located too close to the 5'-end of the mRNA, even though splicing still occurred. This scenario also appears unlikely to explain our results, since both introns 1 and 4 are located exactly at the same distance from the transcription start site in constructs C6 and C7, respectively (Fig. 6B). Using transgenic Arabidopsis plants, Rose and Beliakoff demonstrated that either intron 1 or 2 of the tryptophan pathway gene PAT1 was capable of increasing mRNA levels. However, neither specific intron sequences nor splicing were required for this effect [64]. To explain this observation, the authors speculated that redundant elements were located within the intron. These results argue that splicing per se is not always required to give rise to the intron-mediated enhancement of gene expression.

The differences in the activities of the rat Ctsl introns 1 and 4, which are both spliced, suggest that the splicing of the first intron is required but is not sufficient to explain the stimulatory effect on reporter gene activity. Additional regulatory elements present in the Ctsl first intron but absent in the intron 4 sequence may be required to trigger the intron-mediated stimulatory effect. Preliminary experiments were conducted to address this possibility. The truncated Ctsl first intron was inserted downstream of the 5'-splice site of the intron 4 sequence in the construct C7, in a region predicted not be important for splicing. We assumed that if the Ctsl first intron contained crucial regulatory elements required for the enhancement effect, its insertion into the intron 4 sequence might result in the stimulation of reporter gene activity. However, LUC activity in TM4 cells transfected with this construct was identical to that of the parent construct C7 (Charron and Wright, unpublished results). The outcome of this experiment suggests that the action of these putative regulatory elements within the Ctsl first intron is dependent on their position relative to the transcription and/or translation start sites. Nonetheless, while the exact mechanism remains elusive, results from the present study indicate that the first intron plays an important role in increasing the levels of CTSL synthesized by TM4 cells and by isolated Sertoli cells and, by extrapolation, by Sertoli cells within tubules at stages VI to VII of the cycle of the seminiferous epithelium.

Possible Role of the Ctsl First Intron in Sertoli Cells During Stages VI to VII of the Cycle of the Seminiferous Epithelium, When High Levels of CTSL Are Produced

In rat Sertoli cells, CTSL exhibits one of the largest stage-specific changes in transcript levels, protein synthesis, and secretion that has been reported, as they increase approximately 20-fold from their nadir during stages XII to I to their maxima during stages VI to VII [6, 7]. As a result, CTSL constitutes approximately 1% of the total proteins synthesized by seminiferous tubules at stages VI to VII [7]. We had previously shown that these high levels of CTSL during stages VI to VII of the cycle of the seminiferous epithelium are achieved by the action of a stage-specific promoter [9]. Under the control of this promoter, the transcription of the Ctsl gene is markedly increased in Sertoli cells in tubules at stages VI to VII. The results of the present study have provided new insights on the regulation of the Ctsl gene in Sertoli cells. The splicing of the first intron of the Ctsl gene can further elevate the levels of transcripts produced by its promoter. The results presented in this study invite the speculation that the first intron stimulates Ctsl gene expression in Sertoli cells in vivo and that a mechanism that relies on the combined action of the promoter and of the first intron ensures that high CTSL protein levels are produced in mouse and rat Sertoli cells during stages VI to VII of the cycle of the seminiferous epithelium.

ACKNOWLEDGMENTS

We are grateful to Mr. Thomas Visone and Drs. Terry Brown, David Levin, Mike Matunis, Joel Shaper, and Barry Zirkin for helpful comments on the manuscript. We would like to thank Joyce C.Y. Cheung for performing the initial experiments presented in Figure 3. Real-time PCR was performed using the ABI PRISM 7000 sequence detection system at the Gene Array Core Facility, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health. We are grateful to Anne E. Jedlicka, Meg Mintz, Jeff Strain, and Dr. Ernesto Marques for their help with the real-time PCR. Additional thanks go to Anne E. Jedlicka for performing DNA electrophoresis using the DNA 1000 LabChips.

FOOTNOTES

1Supported by National Institutes of Health grant RO1-HD-044183. Back

Correspondence: 2Martin Charron, Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room W3606, 615 North Wolfe St., Baltimore, MD 21205. FAX: 410 614 2356; e-mail: mcharron{at}jhmi.edu

Received: 2 October 2006.

First decision: 27 October 2006.

Accepted: 15 January 2007.

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