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The 3′ UTR elements of P. gallinaceum protein Pgs28 are functionally distinct from those of human cells
Molecular & Biochemical Parasitology 137 (2004) 355–359
Short communication
The 3 UTR elements of P. gallinaceum protein Pgs28 are functionally distinct from those of human cells夽 Peter Shuea,1 , Silvia V. Browna , Helen Canna,2 , Esme F. Singera , Susan Applebyb , Linnie M. Golightlya,b,∗ a
b
Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Anenue, New York, NY 10021, USA Received 21 February 2004; received in revised form 30 June 2004; accepted 6 July 2004 Available online 29 July 2004
Keywords: Plasmodium; Development; Pgs28; 3 UTR; Gene regulation; Sexual stages
Plasmodium parasites are major causes of morbidity and mortality worldwide. Despite this, their mechanisms of gene regulation have only recently begun to be elucidated [1,2]. Several studies have shown that Plasmodium species require parasite-specific 5 promoter and 3 gene regulatory elements for protein expression [3–5]. These findings suggest that understanding the differences between the gene regulatory mechanisms of Plasmodium and those of man could permit the development of novel parasite-specific chemotherapeutics targeting the parasite’s gene regulatory apparatus. While strides have been made in defining the 5 promoter elements of Plasmodium, the cis-acting elements in the 3 UTR elements remain poorly defined [1,5,6]. Similarities between the 3 gene regulatory domains of Plasmodium and those of metazoans, however, have been noted. In metazoans, cleavage and polyadenylation of mRNA occurs 10–30 nucleotides 3 of a hexamer motif (AATAAA/ATTAAAA) Abbreviations: P. gallinaceum, Plasmodium gallinaceum; P. falciparum, Plasmodium falciparum; LUC, firefly luciferase; GUS, -glucuronidase; CAT, chloroamphenicol acetyl transferase; UTR, untranslated region; CTD, carboxy-terminal domains 夽 Note: F148861. ∗ Corresponding author. Tel.: +1 212 746 6303; fax: +1 212 746 8675. E-mail address: [email protected] (L.M. Golightly). 1 Present address: New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. 2 Present address: Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA. 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.07.001
and upstream of G/U or U-rich sequences [7,8]. Similarly, polyadenylation of some Plasmodium mRNAs occurs downstream of an AATAA pentamer and upstream of G/U rich sequences [9,10]. In addition, the PfPuf1 P. falciparum RNAbinding protein is able to recognize and bind to 3 UTR Nanos-responsive element (NRE) sequences of Drosophila hunchback RNA, suggesting that there are conserved eukaryotic mechanisms of translational regulation [11]. Differences between Plasmodium and metazoans have also been noted. The capping apparatus of Plasmodium is yeast-like and unrelated to those of metazoans [12]. The signal for polyadenylation has been postulated to be the AATAA pentamer rather than a hexamer motif and many transcripts are minimally or unadenylated [9,10,13]. Previous studies analyzing the 3 UTR of the pgs28 gene, which encodes the major surface protein of mature zygotes and ookinetes in the chicken malaria P. gallinaceum, revealed 3 gene regulatory motifs similar to those used in metazoan development [6,14]. In metazoans, 3 gene flanking elements in association with eukaryotic polyadenylation consensus motifs (AATAAA/ATTAAA) mediate the timing of developmentally regulated protein expression [15–20]. Many of these elements are T-rich. In the pgs28 3 UTR, a 156 nucleotide 82% T-rich element and ATTAAA eukaryotic polyadenylation consensus motif occur 55 and 20 nucleotides upstream respectively from the polyadenylation site, which is 449 nucleotides downstream from the pgs28 stop codon [6]. Although the precise elements which mediate Pgs28
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P. Shue et al. / Molecular & Biochemical Parasitology 137 (2004) 355–359
expression are not known, previous studies suggested that the T-rich element and DNA sequences containing the ATTAAA eukaryotic polyadenylation consensus motif were necessary for optimal Pgs28 protein expression [6]. We postulated that studying the function of these elements in human cells would help in defining the differences and similarities in the species 3 gene regulatory machinery. We therefore studied the ability of pgs28 3 UTR elements, previously tested in P. gallinaceum cells, to mediate protein expression in human embryonic kidney cells (T293). T293 cells were transiently transfected with plasmid constructs containing SV40 promoter elements, CAT as a marker of transient gene expression and pgs28 3 gene flanking sequences. The test plasmids contained pgs28 3 gene flanking sequences capable of full expression in P. gallinaceum cells or deleted of putative gene regulatory domains. The CAT expression derived from constructs using the Plasmodium gene regulatory elements was compared to that derived from constructs containing SV40 gene regulatory elements. As shown in Fig. 1, cells transfected with the MLSISCAT pgs28, MLSISCAT dtl DTR, MLSISCAT dtl 110 or MLSISCAT dtl plasmid expressed 89%, 55%, 85% and 98% less CAT activity respectively than those transfected with the pMLSISCAT control plasmid. Therefore, with the exception of cells transfected with the MLSISCAT dtl DTR plasmid, CAT expression was minimal (Fig. 1). In P. gallinaceum cells, 3 gene flanking elements contained in the MLSISCAT pgs28 plasmid mediate maximal protein expression and deletion of the T-rich region leads to a 89% reduction [6]. The reasons for the four-fold increase in CAT expression seen in T293 cells are unclear. The Plasmodium genome, in contrast to that of humans, is AT-rich. It could be that the presence of the 82% U-rich element in the human cells interferes with transcription or pre-polyadenylation transcript stability. The MLSISCAT dtl 110 construct, however, is also deleted of the T-rich region and expresses only minimal CAT activity. It lacks, however, additional downstream DNA sequences, which are present in the MLSISCAT dtl DTR plasmid. This suggests that it is not just the deletion of the T-rich element, but perhaps the proximal positioning of these distal sequences, which enhances protein expression. In either case, our experiments, which measure CAT activity as a marker for protein expression, cannot distinguish between alterations in transcription, mRNA processing or transcript stability. These findings suggested that pgs28 3 gene regulatory domains were either not recognized or mediate different signals in human cells. To determine if transcript processing was different in the two species, the sites of transcript polyadenylation in human cells was determined using 3 RACE (Rapid Amplification of cDNA Ends, Invitrogen) (Fig. 2). Polyadenylation occurred predominantly downstream of the first two eukaryotic polyadenylation consensus motifs (ATTAAA/AATAAA) approximately 69 and 95 nucleotides, respectively, from the pgs28 stop codon (Fig. 2). The second AATAAA motif was favored, perhaps due to
proximal downstream T-rich sequences, which are a component of endogenous metazoan cis-acting signals [8,21] (Fig. 2). The site of polyadenylation did not correlate with the plasmid from which the transcript was derived (Fig. 2). No transcripts with polyadenylation at the site reported for the pgs28 endogenous gene transcript were detected. The failure to detect transcripts polyadenylated at the endogenous pgs28 transcript polyadenylation site, despite the presence of a proximal ATTAAA eukaryotic polyadenylation consensus motif, further suggests that the cis-acting signals recognized by P. gallinaceum and human cells are inherently different. It is unclear why the CAT expression derived from plasmids containing the pgs28 3 gene flanking region is low in comparison to that derived from the pMLSISCAT control plasmid (Fig. 1). In both cases the human cells appear to be using classic hexamer consensus motifs to signal polyadenylation. It is possible that there are parasite-specific signals in the pgs28 3 gene flanking region which suppress or inhibit 3 gene processing in human cells. The cis-acting 3 -end-processing signals of yeast, animals and plants are different, but share an overall pattern [8,22]. There are also similarities in the trans-acting proteins required for 3 -end-processing. Although studies initially suggested that a more complex set of trans-acting proteins were responsible for cleavage and polyadenylation in yeast, it now seems likely that most components of the yeast apparatus will have clear mammalian counterparts [23]. This suggests that overall similarities between the 3 gene processing mechanisms of Plasmodium and those of other eukaryotes will emerge. A recent functional analysis of the cis-acting 3 UTR elements necessary for Pgs28 expression suggests that they share similarities with those of yeast or plants [24]. There are few studies of the trans-acting proteins involved in Plasmodium 3 -end-processing. The RNA polymerase II (RNA Pol II) of Plasmodium falciparum and Plasmodium berghei were cloned and sequenced in the pregenomic era [25,26]. Analysis of their carboxy-terminal domains (CTD), which couple transcription to 3 -end formation, revealed that they contain the basic structure of most eukaryotic CTD with tandem heptatpeptide repeats [27]. Several 3 -end-processing trans-acting protein homologues have been annotated in the P. falciparum genome database [28] (http://plasmodb.org/). Studies to verify their function, however, have not been reported. The accumulating data suggests that additional attempts to discover systems in which the differences between the 3 -end-processing mechanisms of Plasmodium and those of other eukaryotes can be elucidated are merited. This, combined with functional studies of putative trans-acting factors now available in the Plasmodium genome database will permit a better understanding of the parasite’s 3 gene regulatory apparatus. As the current studies demonstrate, the gene regulatory apparatus of Plasmodium may be sufficiently different from that of its human host to permit the development of chemotherapeutics which target it [29].
P. Shue et al. / Molecular & Biochemical Parasitology 137 (2004) 355–359
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Fig. 1. (A) Plasmids containing pgs28 3 gene regulatory elements. Plasmids containing pgs28 3 UTR elements for expression in a mammalian cell line were derived from the previously described pMLSISCAT plasmid, which contains the SV40 promoter, the coding region of the chloroamphenicol acetyl transferase (CAT) gene and the SV40 late polyadenylation signal [30]. The pMLSISCAT plasmid was deleted of its SV40 late polyadenylation signal by digestion with SalI and XhoI and religated to create MLSISCAT dtl. MLSISCAT dtl was used as the parent vector for insertion of pgs28 3 gene flanking sequences which were derived from the previously described pgs28.1LUC and pgs28.1LUC DTR plasmids [6]. MLSISCAT pgs28 contains 727 bp of the pgs28 3 gene flanking region of pgs28.1LUC, which includes seven eukaryotic polyadenylation consensus motifs and a T-rich element. The 3 gene flanking regions of the MLSISCAT dtl DTR and MLSISCAT dtl 110 plasmids were both derived from the Pgs28.1LUC DTR plasmid which lacks the T-rich element. MLSISCAT dtl DTR contains the 602 bp 3 gene flanking region of the pgs28.1LUC DTR plasmid, which includes all seven eukaryotic polyadenylation sites but lacks the T-rich element. MLSISCAT dtl 110 contains the proximal 210 bp of the pgs28 3 gene flanking region [6]. The expression cassette of the indicated plasmid is diagrammed. SV405 promoter elements and the CAT gene coding region are indicated by gray and hatched boxes, respectively. 3 gene flanking elements are indicated by white boxes. The letter P indicates eukaryotic polyadenylation consensus motifs (ATTAAA/AATAAA). The pgs28 T-rich element is indicated by a black labeled box. (B) CAT Expression in T293 cells. Human embryonic kidney cells (T293) were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% calf serum. At 70% confluency, cultures were co-transfected with 2 g pRSVLUC and 5 g of the specific MLSISCAT derived test plasmid in triplicate by the previously described Calcium Phosphate method [31]. The pRSVLUC plasmid, which contains a luciferase expression cassette, was used as an internal control as previously described [32]. The cells were incubated at 37 ◦ C for 48 h, washed two times with phosphate buffered saline (PBS), then incubated at room temperature 5 min with 1 ml TEN solution (0.04 M Tris–HCl [pH 7.4], 1 mM EDTA, 0.15 M NaCl). Pelleted cells were resuspended in 100 l of 0.25 M Tris–HCl pH 7.8, lysed by freeze-thawing and the cellular extracts assayed for LUC activity using the the Luciferase assay system (Promega) and for CAT using standard methods [31]. Levels of 14 C chloroamphenicol conversion were quantitated using the Storm Phosphoimager (Molecular Dynamics). In each experiment, LUC and CAT assays were performed in triplicate and the two most closely correlated values were averaged. Luciferase activity was used to normalize CAT values to a maximal value of 1. Data from three independent experiments are presented. Error bars represent the standard deviation from the mean.
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Fig. 2. (A) Determination of polyadenylation sites. 3 RACE analysis was performed using total RNA purified from transfected T293 cells using the TRI reagent (Molecular Research Center Inc.). RNA samples were DNase treated (RQ1 Rnase-Free Dnase, Promega). First strand cDNA synthesis was performed on 2 g of total RNA using the AP primer (5 -GGC CACGCG TCG ACT AGT AC(T)17 -3 ) according to the manufacturer’s instructions (Invitrogen Life Technologies). PCR reactions were incubated at 94 ◦ C for 3 min, followed by 35 cycles at 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 60 ◦ C for 1 min. Reactions were then incubated at 60 ◦ C for 1 min. In the first round of PCR amplification 5 l of the cDNA reaction mixture was used with the CAT gene specific 5 291 primer (5 -GTT ATT GGT GCC CTT AAA CGC CTG-3 ) and the 3 UAP primer (5 -CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3 ) (Invitrogen Life Technologies). A second round of nested PCR was performed using a gel plug extract of a 300 bp agarose gel electrophoresis band or 2 l of the total PCR reaction with the CAT gene specific 5 291-int primer (5 -CGC CTG AAT AAG TGA TAA TAA GCG G-3 ) and the 3 primer AUAP (5 -GGC CAC GCG TCG ACT AGT AC-3 ) (Invitrogen Life Technologies). PCR products of approximately 250 bp and 300 bp derived from MLSISCAT pgs28 series plasmids and pMLCISCAT transcripts, respectively, were resolved in a 2% agarose gel. Purified PCR products were cloned then sequenced. The sequences are shown with the corresponding pgs28 DNA sequence given in italics beginning with the indicated eukaryotic polyadenylation consensus motif downstream of the stop codon. The eukaryotic polyadenylation consensus motif is underlined. Variations in the site of polyadenylation may be due to degradation of RNA prior to reverse transcription or artifacts introduced during PCR amplification. (B) Schematic of pgs28 polyadenylation sites. The pgs28 DNA sequence downstream of the stop codon is diagrammed. Eukaryotic polyadenylation consensus motifs (AATAAA/ATTAAA) are represented as P-labeled boxes numbered from the stop codon. AATAAA and ATTAAA motifs are represented as black and stippled boxes, respectively. The T-rich element is indicated by a hatched box and the stop codon by a labeled arrowhead. Major and minor polyadenylation sites of MLSISCAT plasmid-derived transcripts are indicated by arrows or arrowheads with asterisks, respectively. The site of pgs28 polyadenylation in P. gallinaceum cells in indicated by a Pg*-labeled arrow.
Acknowledgements This work is supported by grant #AI 46494 from the National Institute of Health awarded to Linnie M. Golightly which includes a Minority Supplement to Silvia V. Brown. Raphael Oguariri is supported by a grant from the Fogarty Foundation. The authors wish to thank Raphael Oguariri, Laura Pologe, and Erik Falck-Pedersen for critically reviewing this manuscript.
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Short communication
The 3 UTR elements of P. gallinaceum protein Pgs28 are functionally distinct from those of human cells夽 Peter Shuea,1 , Silvia V. Browna , Helen Canna,2 , Esme F. Singera , Susan Applebyb , Linnie M. Golightlya,b,∗ a
b
Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Anenue, New York, NY 10021, USA Received 21 February 2004; received in revised form 30 June 2004; accepted 6 July 2004 Available online 29 July 2004
Keywords: Plasmodium; Development; Pgs28; 3 UTR; Gene regulation; Sexual stages
Plasmodium parasites are major causes of morbidity and mortality worldwide. Despite this, their mechanisms of gene regulation have only recently begun to be elucidated [1,2]. Several studies have shown that Plasmodium species require parasite-specific 5 promoter and 3 gene regulatory elements for protein expression [3–5]. These findings suggest that understanding the differences between the gene regulatory mechanisms of Plasmodium and those of man could permit the development of novel parasite-specific chemotherapeutics targeting the parasite’s gene regulatory apparatus. While strides have been made in defining the 5 promoter elements of Plasmodium, the cis-acting elements in the 3 UTR elements remain poorly defined [1,5,6]. Similarities between the 3 gene regulatory domains of Plasmodium and those of metazoans, however, have been noted. In metazoans, cleavage and polyadenylation of mRNA occurs 10–30 nucleotides 3 of a hexamer motif (AATAAA/ATTAAAA) Abbreviations: P. gallinaceum, Plasmodium gallinaceum; P. falciparum, Plasmodium falciparum; LUC, firefly luciferase; GUS, -glucuronidase; CAT, chloroamphenicol acetyl transferase; UTR, untranslated region; CTD, carboxy-terminal domains 夽 Note: F148861. ∗ Corresponding author. Tel.: +1 212 746 6303; fax: +1 212 746 8675. E-mail address: [email protected] (L.M. Golightly). 1 Present address: New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. 2 Present address: Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA. 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.07.001
and upstream of G/U or U-rich sequences [7,8]. Similarly, polyadenylation of some Plasmodium mRNAs occurs downstream of an AATAA pentamer and upstream of G/U rich sequences [9,10]. In addition, the PfPuf1 P. falciparum RNAbinding protein is able to recognize and bind to 3 UTR Nanos-responsive element (NRE) sequences of Drosophila hunchback RNA, suggesting that there are conserved eukaryotic mechanisms of translational regulation [11]. Differences between Plasmodium and metazoans have also been noted. The capping apparatus of Plasmodium is yeast-like and unrelated to those of metazoans [12]. The signal for polyadenylation has been postulated to be the AATAA pentamer rather than a hexamer motif and many transcripts are minimally or unadenylated [9,10,13]. Previous studies analyzing the 3 UTR of the pgs28 gene, which encodes the major surface protein of mature zygotes and ookinetes in the chicken malaria P. gallinaceum, revealed 3 gene regulatory motifs similar to those used in metazoan development [6,14]. In metazoans, 3 gene flanking elements in association with eukaryotic polyadenylation consensus motifs (AATAAA/ATTAAA) mediate the timing of developmentally regulated protein expression [15–20]. Many of these elements are T-rich. In the pgs28 3 UTR, a 156 nucleotide 82% T-rich element and ATTAAA eukaryotic polyadenylation consensus motif occur 55 and 20 nucleotides upstream respectively from the polyadenylation site, which is 449 nucleotides downstream from the pgs28 stop codon [6]. Although the precise elements which mediate Pgs28
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P. Shue et al. / Molecular & Biochemical Parasitology 137 (2004) 355–359
expression are not known, previous studies suggested that the T-rich element and DNA sequences containing the ATTAAA eukaryotic polyadenylation consensus motif were necessary for optimal Pgs28 protein expression [6]. We postulated that studying the function of these elements in human cells would help in defining the differences and similarities in the species 3 gene regulatory machinery. We therefore studied the ability of pgs28 3 UTR elements, previously tested in P. gallinaceum cells, to mediate protein expression in human embryonic kidney cells (T293). T293 cells were transiently transfected with plasmid constructs containing SV40 promoter elements, CAT as a marker of transient gene expression and pgs28 3 gene flanking sequences. The test plasmids contained pgs28 3 gene flanking sequences capable of full expression in P. gallinaceum cells or deleted of putative gene regulatory domains. The CAT expression derived from constructs using the Plasmodium gene regulatory elements was compared to that derived from constructs containing SV40 gene regulatory elements. As shown in Fig. 1, cells transfected with the MLSISCAT pgs28, MLSISCAT dtl DTR, MLSISCAT dtl 110 or MLSISCAT dtl plasmid expressed 89%, 55%, 85% and 98% less CAT activity respectively than those transfected with the pMLSISCAT control plasmid. Therefore, with the exception of cells transfected with the MLSISCAT dtl DTR plasmid, CAT expression was minimal (Fig. 1). In P. gallinaceum cells, 3 gene flanking elements contained in the MLSISCAT pgs28 plasmid mediate maximal protein expression and deletion of the T-rich region leads to a 89% reduction [6]. The reasons for the four-fold increase in CAT expression seen in T293 cells are unclear. The Plasmodium genome, in contrast to that of humans, is AT-rich. It could be that the presence of the 82% U-rich element in the human cells interferes with transcription or pre-polyadenylation transcript stability. The MLSISCAT dtl 110 construct, however, is also deleted of the T-rich region and expresses only minimal CAT activity. It lacks, however, additional downstream DNA sequences, which are present in the MLSISCAT dtl DTR plasmid. This suggests that it is not just the deletion of the T-rich element, but perhaps the proximal positioning of these distal sequences, which enhances protein expression. In either case, our experiments, which measure CAT activity as a marker for protein expression, cannot distinguish between alterations in transcription, mRNA processing or transcript stability. These findings suggested that pgs28 3 gene regulatory domains were either not recognized or mediate different signals in human cells. To determine if transcript processing was different in the two species, the sites of transcript polyadenylation in human cells was determined using 3 RACE (Rapid Amplification of cDNA Ends, Invitrogen) (Fig. 2). Polyadenylation occurred predominantly downstream of the first two eukaryotic polyadenylation consensus motifs (ATTAAA/AATAAA) approximately 69 and 95 nucleotides, respectively, from the pgs28 stop codon (Fig. 2). The second AATAAA motif was favored, perhaps due to
proximal downstream T-rich sequences, which are a component of endogenous metazoan cis-acting signals [8,21] (Fig. 2). The site of polyadenylation did not correlate with the plasmid from which the transcript was derived (Fig. 2). No transcripts with polyadenylation at the site reported for the pgs28 endogenous gene transcript were detected. The failure to detect transcripts polyadenylated at the endogenous pgs28 transcript polyadenylation site, despite the presence of a proximal ATTAAA eukaryotic polyadenylation consensus motif, further suggests that the cis-acting signals recognized by P. gallinaceum and human cells are inherently different. It is unclear why the CAT expression derived from plasmids containing the pgs28 3 gene flanking region is low in comparison to that derived from the pMLSISCAT control plasmid (Fig. 1). In both cases the human cells appear to be using classic hexamer consensus motifs to signal polyadenylation. It is possible that there are parasite-specific signals in the pgs28 3 gene flanking region which suppress or inhibit 3 gene processing in human cells. The cis-acting 3 -end-processing signals of yeast, animals and plants are different, but share an overall pattern [8,22]. There are also similarities in the trans-acting proteins required for 3 -end-processing. Although studies initially suggested that a more complex set of trans-acting proteins were responsible for cleavage and polyadenylation in yeast, it now seems likely that most components of the yeast apparatus will have clear mammalian counterparts [23]. This suggests that overall similarities between the 3 gene processing mechanisms of Plasmodium and those of other eukaryotes will emerge. A recent functional analysis of the cis-acting 3 UTR elements necessary for Pgs28 expression suggests that they share similarities with those of yeast or plants [24]. There are few studies of the trans-acting proteins involved in Plasmodium 3 -end-processing. The RNA polymerase II (RNA Pol II) of Plasmodium falciparum and Plasmodium berghei were cloned and sequenced in the pregenomic era [25,26]. Analysis of their carboxy-terminal domains (CTD), which couple transcription to 3 -end formation, revealed that they contain the basic structure of most eukaryotic CTD with tandem heptatpeptide repeats [27]. Several 3 -end-processing trans-acting protein homologues have been annotated in the P. falciparum genome database [28] (http://plasmodb.org/). Studies to verify their function, however, have not been reported. The accumulating data suggests that additional attempts to discover systems in which the differences between the 3 -end-processing mechanisms of Plasmodium and those of other eukaryotes can be elucidated are merited. This, combined with functional studies of putative trans-acting factors now available in the Plasmodium genome database will permit a better understanding of the parasite’s 3 gene regulatory apparatus. As the current studies demonstrate, the gene regulatory apparatus of Plasmodium may be sufficiently different from that of its human host to permit the development of chemotherapeutics which target it [29].
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Fig. 1. (A) Plasmids containing pgs28 3 gene regulatory elements. Plasmids containing pgs28 3 UTR elements for expression in a mammalian cell line were derived from the previously described pMLSISCAT plasmid, which contains the SV40 promoter, the coding region of the chloroamphenicol acetyl transferase (CAT) gene and the SV40 late polyadenylation signal [30]. The pMLSISCAT plasmid was deleted of its SV40 late polyadenylation signal by digestion with SalI and XhoI and religated to create MLSISCAT dtl. MLSISCAT dtl was used as the parent vector for insertion of pgs28 3 gene flanking sequences which were derived from the previously described pgs28.1LUC and pgs28.1LUC DTR plasmids [6]. MLSISCAT pgs28 contains 727 bp of the pgs28 3 gene flanking region of pgs28.1LUC, which includes seven eukaryotic polyadenylation consensus motifs and a T-rich element. The 3 gene flanking regions of the MLSISCAT dtl DTR and MLSISCAT dtl 110 plasmids were both derived from the Pgs28.1LUC DTR plasmid which lacks the T-rich element. MLSISCAT dtl DTR contains the 602 bp 3 gene flanking region of the pgs28.1LUC DTR plasmid, which includes all seven eukaryotic polyadenylation sites but lacks the T-rich element. MLSISCAT dtl 110 contains the proximal 210 bp of the pgs28 3 gene flanking region [6]. The expression cassette of the indicated plasmid is diagrammed. SV405 promoter elements and the CAT gene coding region are indicated by gray and hatched boxes, respectively. 3 gene flanking elements are indicated by white boxes. The letter P indicates eukaryotic polyadenylation consensus motifs (ATTAAA/AATAAA). The pgs28 T-rich element is indicated by a black labeled box. (B) CAT Expression in T293 cells. Human embryonic kidney cells (T293) were grown as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% calf serum. At 70% confluency, cultures were co-transfected with 2 g pRSVLUC and 5 g of the specific MLSISCAT derived test plasmid in triplicate by the previously described Calcium Phosphate method [31]. The pRSVLUC plasmid, which contains a luciferase expression cassette, was used as an internal control as previously described [32]. The cells were incubated at 37 ◦ C for 48 h, washed two times with phosphate buffered saline (PBS), then incubated at room temperature 5 min with 1 ml TEN solution (0.04 M Tris–HCl [pH 7.4], 1 mM EDTA, 0.15 M NaCl). Pelleted cells were resuspended in 100 l of 0.25 M Tris–HCl pH 7.8, lysed by freeze-thawing and the cellular extracts assayed for LUC activity using the the Luciferase assay system (Promega) and for CAT using standard methods [31]. Levels of 14 C chloroamphenicol conversion were quantitated using the Storm Phosphoimager (Molecular Dynamics). In each experiment, LUC and CAT assays were performed in triplicate and the two most closely correlated values were averaged. Luciferase activity was used to normalize CAT values to a maximal value of 1. Data from three independent experiments are presented. Error bars represent the standard deviation from the mean.
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Fig. 2. (A) Determination of polyadenylation sites. 3 RACE analysis was performed using total RNA purified from transfected T293 cells using the TRI reagent (Molecular Research Center Inc.). RNA samples were DNase treated (RQ1 Rnase-Free Dnase, Promega). First strand cDNA synthesis was performed on 2 g of total RNA using the AP primer (5 -GGC CACGCG TCG ACT AGT AC(T)17 -3 ) according to the manufacturer’s instructions (Invitrogen Life Technologies). PCR reactions were incubated at 94 ◦ C for 3 min, followed by 35 cycles at 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 60 ◦ C for 1 min. Reactions were then incubated at 60 ◦ C for 1 min. In the first round of PCR amplification 5 l of the cDNA reaction mixture was used with the CAT gene specific 5 291 primer (5 -GTT ATT GGT GCC CTT AAA CGC CTG-3 ) and the 3 UAP primer (5 -CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3 ) (Invitrogen Life Technologies). A second round of nested PCR was performed using a gel plug extract of a 300 bp agarose gel electrophoresis band or 2 l of the total PCR reaction with the CAT gene specific 5 291-int primer (5 -CGC CTG AAT AAG TGA TAA TAA GCG G-3 ) and the 3 primer AUAP (5 -GGC CAC GCG TCG ACT AGT AC-3 ) (Invitrogen Life Technologies). PCR products of approximately 250 bp and 300 bp derived from MLSISCAT pgs28 series plasmids and pMLCISCAT transcripts, respectively, were resolved in a 2% agarose gel. Purified PCR products were cloned then sequenced. The sequences are shown with the corresponding pgs28 DNA sequence given in italics beginning with the indicated eukaryotic polyadenylation consensus motif downstream of the stop codon. The eukaryotic polyadenylation consensus motif is underlined. Variations in the site of polyadenylation may be due to degradation of RNA prior to reverse transcription or artifacts introduced during PCR amplification. (B) Schematic of pgs28 polyadenylation sites. The pgs28 DNA sequence downstream of the stop codon is diagrammed. Eukaryotic polyadenylation consensus motifs (AATAAA/ATTAAA) are represented as P-labeled boxes numbered from the stop codon. AATAAA and ATTAAA motifs are represented as black and stippled boxes, respectively. The T-rich element is indicated by a hatched box and the stop codon by a labeled arrowhead. Major and minor polyadenylation sites of MLSISCAT plasmid-derived transcripts are indicated by arrows or arrowheads with asterisks, respectively. The site of pgs28 polyadenylation in P. gallinaceum cells in indicated by a Pg*-labeled arrow.
Acknowledgements This work is supported by grant #AI 46494 from the National Institute of Health awarded to Linnie M. Golightly which includes a Minority Supplement to Silvia V. Brown. Raphael Oguariri is supported by a grant from the Fogarty Foundation. The authors wish to thank Raphael Oguariri, Laura Pologe, and Erik Falck-Pedersen for critically reviewing this manuscript.
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