Transcriptional Analysis of the Glutamate Dehydrogenase Gene in the Primitive Eukaryote, Giardia lamblia

We studied gene expression in the ancient eukaryote, Giardia lamblia, by taking advantage of assays developed recently in our laboratory, which allow new genetic analyses of this organism. We examined the transcription of a 2.2-kilobase segment of the Giardia genome that contains theglutamate dehydrogenase (GDH) gene and a portion of a second open reading frame encoding an uncharacterized gene. Nuclear run-on analyses showed that the genes are transcribed as two separate units spaced less than 200 base pairs apart, and transcription of the GDH gene initiates just 3–6 nucleotides upstream of its translation start codon. We characterized the GDH promoter by transfecting Giardia with DNA constructs that used the GDH upstream sequence to drive the expression of a luciferase reporter gene. By deletion and mutational analyses, we localized promoter function to three motifs within a 50-base pair region of the GDH upstream sequence. Using band shift assays and UV cross-linking, we demonstrated specific binding of a 68-kDa protein from Giardia nuclear extracts to short poly(T) tracts contained within two of the sequence motifs on single-stranded DNA from the promoter region. This report describes one of the first functional gene promoter and its cognate DNA-binding protein in this primitive eukaryote. We studied gene expression in the ancient eukaryote, Giardia lamblia, by taking advantage of assays developed recently in our laboratory, which allow new genetic analyses of this organism. We examined the transcription of a 2.2-kilobase segment of the Giardia genome that contains theglutamate dehydrogenase (GDH) gene and a portion of a second open reading frame encoding an uncharacterized gene. Nuclear run-on analyses showed that the genes are transcribed as two separate units spaced less than 200 base pairs apart, and transcription of the GDH gene initiates just 3–6 nucleotides upstream of its translation start codon. We characterized the GDH promoter by transfecting Giardia with DNA constructs that used the GDH upstream sequence to drive the expression of a luciferase reporter gene. By deletion and mutational analyses, we localized promoter function to three motifs within a 50-base pair region of the GDH upstream sequence. Using band shift assays and UV cross-linking, we demonstrated specific binding of a 68-kDa protein from Giardia nuclear extracts to short poly(T) tracts contained within two of the sequence motifs on single-stranded DNA from the promoter region. This report describes one of the first functional gene promoter and its cognate DNA-binding protein in this primitive eukaryote. variant-specific surface protein(s) glutamate dehydrogenase base pair(s) TATA box-binding protein kilobase(s) polymerase chain reaction dithiothreitol untranslated region polymerase The protozoan Giardia lamblia is a representative of the most primitive lineage within the eukaryotic domain (1.Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (590) Google Scholar, 2.Hashimoto T. Nakamura Y. Kamaishi T. Nakamura F. Adachi J. Okamoto K.-I. Hasegawa M. Mol. Biol. Evol. 1995; 12: 782-793PubMed Google Scholar, 3.Klenk H.-P. Zillig W. Lanzendorfer M. Grampp B. Palm P. Arch. Protistenk. 1995; 145: 221-230Crossref Scopus (31) Google Scholar). This protozoan is found in freshwater lakes and streams throughout the world, and is an intestinal parasite of humans and other mammals.Giardia has just two stages in its simple life cycle: a swimming trophozoite form, and an infective cyst form. The transformation of the trophozoite into the cyst is accompanied by the activation of gene transcription of cyst-specific genes (4.Lujan H.D. Mowatt M.R. Nash T.E. Microbiol. Mol. Biol. Rev. 1997; 61: 294-304Crossref PubMed Scopus (129) Google Scholar).Giardia trophozoites have the unusual ability to switch the make-up of the major protein on its cell surface by varying the expression of a repertoire of genes encoding these variant-specific surface proteins (VSPs).1Although antigenic variation in Giardia is commonly thought to be a mechanism to evade the immune response of the host, the biological significance of VSPs in Giardia has not been determined (5.Nash T. Parasitol. Today. 1992; 8: 229-234Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 6.Nash T.E. Phil. Trans. R. Soc. Lond. 1997; 352: 1369-1375Crossref PubMed Scopus (60) Google Scholar). The initiation of transcription is a key step in regulating gene expression and has been studied extensively in organisms ranging from yeast to man. Although results from these studies were presumed to be representative of all eukaryotes, little is known about this process in organisms lower than yeast on the evolutionary tree. In higher eukaryotes, the basal level of transcription by RNA polymerase II is dependent on the assembly of a set of general transcription factors on the core promoter which consists of either a TATA box, an initiator, or both (reviewed in Ref. 7.Roeder R.G. Trends Biochem. Sci. 1991; 16: 402-408Abstract Full Text PDF PubMed Scopus (347) Google Scholar). While the binding of the transcription factor TBP (TATA box-binding protein) to the TATA box has been well characterized, less is known about proteins which interact with the initiator. Since TBP is required for transcription from TATA-less promoters, it is possible that TBP can bind directly to the initiator, or alternatively, it is recruited indirectly to this region by its interaction with another protein which binds this motif (8.Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (564) Google Scholar). In some genes, additional factors and their specific recognition sites are used to modulate transcription regulation. For example, the mammalian β-globin and the histone H2B genes have an additional promoter element, called the CAAT box, located between 40 and 100 bp upstream of the transcription initiation site (reviewed in Ref. 9.La Thangue N.B. Rigby P.W.J. Hames B.D. Glover D.M. Transcription and Splicing, Frontiers in Molecular Biology. IRL Press, Oxford1988: 1-41Google Scholar). This element, with the consensus sequence GG(C/T)CAATCT, is bound by a family of transcription factors which up-regulate transcription of these genes. Since little information is available about promoter structure and function in lower eukaryotes, we were interested in determining the requirements of a “minimal” gene promoter in one of the lowest eukaryotes, G. lamblia. Furthermore, knowledge about the constitutive transcription of a housekeeping gene, such asGDH, would be a starting point for future studies on regulated gene expression, such as during the processes of encystation and antigenic variation in this medically important parasite. All transfection plasmids used were based on the constructs P11 and 3′Δ2 described previously (10.Yee J. Dennis P.P. J. Biol. Chem. 1992; 267: 7539-7544Abstract Full Text PDF PubMed Google Scholar, 11.Yee J. Nash T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5615-5619Crossref PubMed Scopus (73) Google Scholar). The constructs used in the nuclear run-on experiments are shown in Fig.1. Plasmid 1 represents the P11 construct, a pGEM-3Zf(−) vector (Promega) carrying a 2.29-kb genomic DNA fragment from G. lamblia (Portland-1 isolate) that contains the entire coding region of the GDH gene (1350 bp) and its accompanying 5′ (820 bp) and 3′ (120 bp) flanking sequences. Plasmids 2–7 contain subcloned DNA fragments generated from P11, by either restriction enzyme digests or PCR amplification. Plasmids CWP-1 and -2 are pCR II-based vectors (Invitrogen) that contain the first 400 bp of coding region subcloned from constructs containing the full-length cyst wall protein genes (12.Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 13.Mowatt M.R. Lujan H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (137) Google Scholar). Plasmid vectors, pGEM-3zf(−) and pCR II, which contain no inserts, were used as negative controls. The rRNA construct, carrying a 5.6-kb fragment of the Giardiaribosomal RNA operon, a from T. 15: PubMed Scopus Google Scholar). In the deletion the plasmids used are shown in the between the GDH and the luciferase genes within the 3′Δ2 by a Plasmid from the of the DNA fragment containing a portion of GDH 5′ region from of the GDH 5′ region from are contained within plasmids and In the DNA fragment containing the of the GDH gene in of the 5′ region in to bp in the In the sequence of the GDH 5′ region in is shown on the to were by this region in with containing within this sequence. were generated by of that and sites the 5′ and 3′ All constructs were by DNA The from one used for PubMed Scopus Google Scholar).Giardia were and to in 1 and 1 for on by with for 1 The were and into 100 of of transcription 1 1 and transcription to for of RNA to and RNA to The RNA used to containing fragments of the GDH gene and its and sequences. Giardia isolate) were and for as described previously J. Nash T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5615-5619Crossref PubMed Scopus (73) Google Scholar). Plasmid DNA and the were and on a The on for the were to of in a of the were as described previously J. Nash T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5615-5619Crossref PubMed Scopus (73) Google Scholar). For of the cell used with 100 of luciferase The luciferase of the in are of experiments and have within of the In of in and for luciferase RNA transfection with and RNA were as described of the RNA with units of for The by the of by for 5′ to of the luciferase to the RNA and with and A. J. PubMed Scopus Google Scholar). The were on a a DNA sequence generated from the 3′Δ2 the as a Nuclear extracts were from Giardia a of the of and 1991; PubMed Scopus Google Scholar).Giardia in and by the of on for a nuclear by of the cell for in an The nuclear in and on for with of the The for in an and the protein in the The protein of the determined by the as the The into in a and The nuclear protein by from a 1 of the Giardia stages during its and The used are shown in are for the and for the For the two were in to a of an in a for and to 1 single-stranded and were with and The containing the yeast site has the The containing the TATA box has the these were from fragment containing sequence from to into a vector fragment containing the promoter region of the GDH gene from the by and as in a PCR The were and for in a The PCR and its sequence by the DNA The fragment by the of the of either or with or in the PCR amplification. For for in a The GDH fragment to Transcription of RNA in from the fragment into the vector described The the site in the and transcription the promoter by the of and RNA The RNA by the of The DNA transcription by with (Promega) and the RNA to and were from contained of of nuclear and of in a of binding 100 1 In all and were the of the protein In the studies on single-stranded DNA the first 100 and on to its to the binding The were on for for 200 in with a of to were the and to for the for and to The binding and as were and in on were to UV by a set of reaction to a band shift the to and to the on the were The were in the of a with the set of binding and a the and to as described transcription of the Giardia contained within a genomic DNA we developed a nuclear run-on in this by a used for PubMed Scopus Google Scholar). In this Giardia were and RNA by the of The RNA and used to containing of the genomic fragment all of which the GDH in genomic were to DNA fragments containing the GDH coding region and and a portion of its sequence In no were for bp upstream of the start and for to a DNA fragment located 200 bp upstream which to a portion of an open reading frame encoding an uncharacterized gene. The negative of genes for two Giardia cyst wall and two vectors, and showed no the the In to Giardia to we to that transcription initiation from plasmids into Giardia is the as that from its genes. We examined the 5′ of RNA transcribed from the 3′Δ2 J. Nash T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5615-5619Crossref PubMed Scopus (73) Google which sequence to drive the expression of a luciferase RNA from the transfection and an specific to the luciferase portion of the and not The of that transcription from the initiates the major site as that determined previously for the gene (10.Yee J. Dennis P.P. J. Biol. Chem. 1992; 267: 7539-7544Abstract Full Text PDF PubMed Google Scholar). promoter within the GDH on luciferase from of this region contained on expression plasmids were in on luciferase were the sequence to within bp of the translation start luciferase this point and and to the sequence of the of GDH coding region in the transfection constructs and not luciferase The upstream sequence of the GDH gene, containing the region in the deletion from other Giardia genes in the base not varying from to nucleotides in found in all in the with the of the specific genes. The contains the transcription initiation site in genes which have been and with the translation start in all genes second in or on either DNA is located upstream and the We to this motif as the element, for the Giardia box, and its consensus sequence is were on the to the and the have promoter function in and to other promoter within the sequence. from Giardia with these constructs showed that within a TATA located between the and the have no on luciferase to the have a on luciferase In within the in a of luciferase This to this were to nucleotides that are not sites for transcription initiation luciferase the of nucleotides within this the the as the sequence. to an located between the and the element, a in luciferase to another to the and the upstream were in the the in luciferase than that with to either motifs and the promoter region contains binding sites for transcription a PCR fragment this region from a fragment of upstream sequence to as a in band shift assays the PCR fragment with proteins from a Giardia nuclear two called and were The is protein and as the protein The sites on the fragment were localized by short that this region in band shift assays The represents the region of the GDH sequence between and and contains the The represents the region of the GDH sequence between and and contains the the represents the region to between the two motifs and contains the first The and containing the and the were to for protein binding to the fragment the containing the region between the two motifs not Furthermore, of containing either the binding site for the yeast site or the not for protein binding to the fragment not the sequence of protein binding to the were to contain to the and the which were found to a in luciferase in an to contain to the in of this not for protein binding the in of the not for protein binding directly the protein binding ability of the and the the and containing of these motifs were and with nuclear protein extracts a both in the of nuclear a with to the of the both can with other with which contains the region between the two motifs not for protein This that the two containing the and the element, bind to the protein or proteins in the nuclear The of the protein bound to by UV of the binding with and were to UV and one set of reaction to a band shift The major band that with were from the and a with the set of binding The of this showed that the major band from both the of band of this in the binding In a of the were from band shift of binding that were not The DNA from these to on a the were in the of this not the 68-kDa protein can bind single-stranded a of experiments single-stranded as either or in band shift assays the single-stranded were used as in band shift both and containing the and the of the bind proteins Although we that the previously with the binding of the 68-kDa protein to the it is possible that the protein bound to single-stranded DNA from the this which not bind protein and to the which bind protein this to a a of of the from this that of the with in the not this used in the band shift no In the with the containing and a the 68-kDa protein to bind single-stranded and from the not with the single-stranded were used as to protein binding to the PCR the that were shown to bind protein and were to for that not bind protein not for binding Since the of both the and the are of and we the of an of and poly(T) DNA as in the binding While poly(T) an and all protein binding to the not for binding the 68-kDa protein can bind to RNA were transcribed in an promoter from a fragment of the GDH upstream sequence which contains the region used in the band shift of this RNA not protein binding to the fragment the RNA and with proteins from the nuclear were these not be by an of from promoter region not the initiation of transcription in we a nuclear run-on PubMed Scopus Google to transcribed from a genomic DNA fragment in Giardia studies that Giardia have short that are nucleotides in Microbiol. Rev. 1991; PubMed Google Scholar). these analyses are of and assays on it is the 5′ of these to sites of transcription initiation or to from the of In the such as analyses of promoter have been by the of in which a sequence is to the 5′ of RNA as it is transcribed J. Mol. Biol. 1991; PubMed Scopus Google Scholar). Using nuclear run-on assays on a genomic DNA fragment containing the Giardia GDH gene, we localized transcription initiation of gene to a region that with the sites that were determined previously by and This that the GDH and from other Giardia genes, are not and have the all eukaryotic Although experiments with have been with RNA from the results are as to the have 5′ T. N. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). it is not known translation initiates in these other eukaryotic required to allow and a sequence to binding translation can a of organisms that is to be the of all eukaryotic have short that are and B. Dennis P. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, Dennis P.P. J. 1989; 8: PubMed Scopus Google Scholar, P.P. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Since analyses as the most to (1.Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (590) Google it is that these organisms have to translation of their In our nuclear run-on assays we a of RNA from an uncharacterized open reading located less than 200 bp upstream from the major GDH transcription start site This upstream is a of a separate transcription no were in the region between this and the one encoding GDH and Although this is the first that two such spaced are transcribed in it that of open reading within genomic DNA fragments M. Palm P. Grampp B. Peattie D.A. Zillig W. 1992; PubMed Scopus Google A. N. M. M. and M. in Molecular A. J. and S. be other of units within The that the is with genes is by which an of of DNA within this genome A. D.A. 15: PubMed Scopus Google and by results from genome A. N. M. M. and M. in Molecular Scholar). We that this within Giardia genes no have been found in of its genes Furthermore, the of Giardia are the the in both and 15: PubMed Scopus Google Scholar, A. D.A. 15: PubMed Scopus Google Scholar). In a we developed a to with DNA constructs containing a for gene We found that the of luciferase in the of upstream sequence in of the on the constructs J. Nash T.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5615-5619Crossref PubMed Scopus (73) Google that are contained in the upstream sequence of the GDH gene. In this we determined that the major site of transcription initiation within the GDH upstream region contained on the is the as that from the gene not which the of to gene Giardia with reporter plasmids containing of the GDH upstream sequence showed that bp of DNA sequence upstream of the for the GDH gene is required to luciferase short in Giardia would allow DNA between genes, and would in the of its region and a motif were in an of the upstream sequence of the GDH gene from other Giardia genes not The sequence and of these that be of a Giardia gene have been recently in the promoter of gene J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these are from the DNA upstream of the surface genes This with their regulated expression during the process of antigenic that genes have a promoter that is from that of other genes. In the parasite in which antigenic variation the genes are transcribed by a RNA polymerase that has and nuclear than the one used to other protein encoding genes Full Text PDF PubMed Scopus Google Scholar, G. J. 1989; 8: PubMed Scopus Google Scholar, R. P. 1991; PubMed Scopus Google Scholar, Rev. Microbiol. 1997; PubMed Scopus Google Scholar). By the in Giardia be transcribed by a polymerase and have a promoter that is than the one for the GDH gene and other genes. the specific which to promoter within the bp) upstream we examined of to this region contained on constructs into Giardia sequence motifs were in this of these to the and and the motifs that were previously by their sequence in the of upstream not The is by an in the on This is a of this to a second bp not luciferase the consensus sequence of this is not as well as the and in the upstream of other Giardia genes not the three in the GDH the sequence has the on gene This with its that it is of the found in higher 1989; Full Text PDF PubMed Scopus Google Scholar). Since most Giardia genes are to in the Microbiol. Rev. 1991; PubMed Google one advantage of an is that it the of the promoter region to transcription this contain the binding site for a protein in the of the transcription This is by the that the specific of and nucleotides within this is important for promoter in transfection assays which a binding site for a protein is contained on this In the analyses of another ancient eukaryote, the gene examined contain not TATA A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of TATA in the of both Giardia that the of this motif is by the motif during the of the eukaryotic shift assays were used to possible sites for protein binding on the Giardia GDH a fragment containing the GDH promoter region and with proteins from a Giardia nuclear two and were Since the protein and the higher our is that are two of protein binding to the Using of the promoter as in band shift assays showed that one of the sites is contained within the motif and a second binding site is located on the motif within the the with the fragment in the band shift assays protein binding one of these and the represents protein binding both the and the were used as in band shift we found that can with other for protein binding The that the contain binding sites for the protein is by the UV which showed that both the and the bind a 68-kDa protein from the nuclear Since this is than of the in and M. R.G. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar, T. B.D. R. 61: Full Text PDF PubMed Scopus Google Scholar, N. R. Science. PubMed Scopus Google the of this binding protein from Giardia to known transcription factors in other be it is and band shift experiments showed that the 68-kDa protein can bind single-stranded DNA from the GDH promoter in a Although the protein to be bind in experiments we demonstrated that the protein is binding single-stranded DNA which has within the the protein binding to the fragment be to single-stranded in this DNA the 68-kDa protein can bind both DNA containing the and the DNA containing the and The binding of the 68-kDa protein to the short poly(T) tracts contained within the and the on these single-stranded DNA as the poly(T) DNA is an in the binding the 68-kDa protein bind RNA to the region used in the DNA binding which can bind specific on single-stranded DNA not on RNA have been characterized and shown to have in the of transcription in other T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (164) Google Mol. Biol. 1991; PubMed Scopus Google Scholar, Mol. Biol. 1992; 12: PubMed Scopus Google Scholar). results that we have characterized one of the first in a eukaryote. Since GDH genes in other eukaryotes, and protein encoding genes in are transcribed by RNA II, it is to that we have a promoter in Giardia that is with the transcription by a In our nuclear run-on assays in this we that the transcription of genes, in to encoding is to of as as 1 R. Mowatt and T. in to the of RNA II transcription in higher the of RNA are not known as have been our results a genetic this to have a genome with spaced a short and simple gene as well as transcription initiation to the start of the region. This with in its and gene regulation. is to that these are of that are by this genomic to be in other primitive eukaryotes, such as this can be We for on the assays and the of the containing the yeast binding site and the We T. for the of the Giardia rRNA We are to M. for the UV and with the of the We S. for the reading of this

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