Leaky Transcription of Variant Surface Glycoprotein Gene Expression Sites in Bloodstream African Trypanosomes
1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês
10.1074/jbc.274.24.16884
ISSN1083-351X
AutoresClara M. Alarcón, Mehrdad Pedram, John E. Donelson,
Tópico(s)Biochemical and Molecular Research
ResumoTrypanosoma brucei undergoes antigenic variation by periodically switching the expression of its variant surface glycoprotein (VSG) genes (vsg) among an estimated 20–40 telomere-linked expression sites (ES), only one of which is fully active at a given time. We found that in bloodstream trypanosomes one ES is transcribed at a high level and other ESs are expressed at low levels, resulting in organisms containing one abundant VSG mRNA and several rare VSG RNAs. Some of the rare VSG mRNAs come from monocistronic ESs in which the promoters are situated about 2 kilobases upstream of the vsg, in contrast to the polycistronic ESs in which the promoters are located 45–60 kilobases upstream of the vsg. The monocistronic ES containing the MVAT4 vsg does not include the ES-associated genes (esag) that occur between the promoter and thevsg in polycistronic ESs. However, bloodstream MVAT4 trypanosomes contain the mRNAs for many different ESAGs 6 and 7 (transferrin receptors), suggesting that polycistronic ESs are partially active in this clone. To explain these findings, we propose a model in which both mono- and polycistronic ESs are controlled by a similar mechanism throughout the parasite's life cycle. Certain VSGs are preferentially expressed in metacyclic versusbloodstream stages as a result of differences in ESAG expression and the proximity of the promoters to the vsg and telomere. Trypanosoma brucei undergoes antigenic variation by periodically switching the expression of its variant surface glycoprotein (VSG) genes (vsg) among an estimated 20–40 telomere-linked expression sites (ES), only one of which is fully active at a given time. We found that in bloodstream trypanosomes one ES is transcribed at a high level and other ESs are expressed at low levels, resulting in organisms containing one abundant VSG mRNA and several rare VSG RNAs. Some of the rare VSG mRNAs come from monocistronic ESs in which the promoters are situated about 2 kilobases upstream of the vsg, in contrast to the polycistronic ESs in which the promoters are located 45–60 kilobases upstream of the vsg. The monocistronic ES containing the MVAT4 vsg does not include the ES-associated genes (esag) that occur between the promoter and thevsg in polycistronic ESs. However, bloodstream MVAT4 trypanosomes contain the mRNAs for many different ESAGs 6 and 7 (transferrin receptors), suggesting that polycistronic ESs are partially active in this clone. To explain these findings, we propose a model in which both mono- and polycistronic ESs are controlled by a similar mechanism throughout the parasite's life cycle. Certain VSGs are preferentially expressed in metacyclic versusbloodstream stages as a result of differences in ESAG expression and the proximity of the promoters to the vsg and telomere. African trypanosomes are protozoan parasites that evade the immune response of their mammalian hosts by periodically switching the major protein on their surface, the variant surface glycoprotein (VSG) 1The abbreviations used are: VSG, variant surface glycoprotein; vsg , VSG gene; MVAT, metacyclic variant antigen type; ES, vsg expression site; esag , expression site-associated gene; ESAG, protein product of anesag ; PARP, procyclic acidic repetitive proteins; IFA, immunofluoresence assay; mAb, monoclonal antibody; SL, spliced leader; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction 1The abbreviations used are: VSG, variant surface glycoprotein; vsg , VSG gene; MVAT, metacyclic variant antigen type; ES, vsg expression site; esag , expression site-associated gene; ESAG, protein product of anesag ; PARP, procyclic acidic repetitive proteins; IFA, immunofluoresence assay; mAb, monoclonal antibody; SL, spliced leader; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction (for recent reviews, see Refs. 1Pays E. Nolan D.P. Mol. Biochem. Parasitol. 1998; 91: 3-36Crossref PubMed Scopus (108) Google Scholar, 2Borst P. Bitter W. Blundell P.A. Chaves I. Cross M. Gerrits H. Van Leeuwen F. McCulloch R. Taylor M. Rudenko G. Mol. Biochem. Parasitol. 1998; 91: 67-76Crossref PubMed Scopus (69) Google Scholar, 3Cross G.A.M. BioEssays. 1996; 18: 283-293Crossref PubMed Scopus (203) Google Scholar, 4Donelson J.E. J. Biol. Chem. 1995; 270: 7783-7786Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 5Vanhamme L. Pays E. Microbiol. Rev. 1995; 59: 223-240Crossref PubMed Google Scholar). Individual VSG genes (vsg) are transcribed from 20–40 expression sites (ES), each of which is located near a chromosome telomere. The vsgs are maneuvered into the telomere-linked ESs by duplicative translocation or telomere exchange (6Donelson J.E. Rice-Ficht A.C. Microbiol. Rev. 1985; 49: 107-125Crossref PubMed Google Scholar, 7Rudenko G. McCulloch R. Dirks-Mulder A. Borst P. Mol. Biochem. Parasitol. 1996; 80: 65-75Crossref PubMed Scopus (58) Google Scholar). The switch involves either the arrival of a newly duplicatedvsg in an active ES (gene conversion) or the activation of another ES already containing a vsg (in situactivation). During their transmission between the tsetse fly vector and a mammalian host, African trypanosomes undergo a series of transformations and pre-adaptive changes (8Vickerman K. Br. Med. Bull. 1985; 41: 105-114Crossref PubMed Scopus (476) Google Scholar, 9Vickerman K. Tetley L. Hendry K.A. Turner C.M. Biol. Cell. 1988; 64: 109-119Crossref PubMed Scopus (156) Google Scholar). The VSG is first expressed at the metacyclic stage in the salivary glands of the tsetse fly as a pre-adaptive measure for entering an unknown host and initiating an infection. The metacyclic trypanosomes are a heterogeneous population expressing a small subset (15 to 20) of the vsg repertoire called the metacyclic variant antigen type (MVAT) vsgs (10Tetley L. Turner C.M. Barry J.D. Crowe J.S. Vickerman K. J. Cell Sci. 1987; 87: 363-372PubMed Google Scholar,11Turner C.M. Barry J.D. Maudlin I. Vickerman K. Parasitology. 1988; 97: 269-276Crossref PubMed Google Scholar). After trypanosomes enter the bloodstream of their host, they continue to express the MVAT vsgs for up to 7 days and then switch to expression of a non-MVAT vsg (12Esser K.M. Schoenbechler M.J. Gingrich J.B. J. Immunol. 1982; 129: 1715-1718PubMed Google Scholar). Occasionally, the MVAT vsgs are re-expressed in the bloodstream stage late in infection (reviewed in Ref. 13Donelson J.E. Hill K.L. El-Sayed N.M. Mol. Biochem. Parasitol. 1998; 91: 51-66Crossref PubMed Scopus (84) Google Scholar). A typical bloodstream ES is composed of a 45–60 kb polycistronic transcription unit that, in addition to the telomere-linkedvsg, contains a minimum of eight ES-associated genes (esag). In contrast, the MVAT vsg ESs are composed of monocistronic transcription units that are devoid ofesags. A common observation about trypanosome antigenic variation is that one and only one of the ESs is usually activated at a given time in a given bloodstream trypanosome (14Clayton C. Moldave K. Cohn W. Progress in Nucleic Acid Research and Molecular Biology. Academic Press, San Diego1992: 37-66Google Scholar). This conclusion is based primarily on Northern blots of RNA from pure trypanosome clones in which only one VSG mRNA species is detected and nuclear run-on assays showing that only one vsg is transcribed in isolated nuclei of a bloodstream trypanosome clone. Yet, if a secondvsg is placed in an activated ES using recombinant DNA techniques, equal amounts of both VSG molecules appear on the surface of bloodstream form trypanosomes (15Munoz-Jordan J.L. Davies K.P. Cross G.A. Science. 1996; 272: 1795-1797Crossref PubMed Scopus (49) Google Scholar). Furthermore, trypanosomes expressing two VSGs simultaneously occur naturally during the switch from one VSG to another (16Esser K.M. Schoenbechler M.J. Science. 1985; 229: 190-193Crossref PubMed Scopus (51) Google Scholar, 17Baltz T. Giroud C. Baltz D. Roth C. Raibaud A. Eisen H. Nature. 1986; 319: 602-604Crossref PubMed Scopus (44) Google Scholar). In earlier experiments, we found that about 4% of the cDNAs in a cDNA library of the MVAT4 bloodstream clone of Trypanosoma brucei rhodesiense encode the MVAT4 VSG (2000 out of 50,000 cDNAs screened), a result consistent with earlier estimates that the VSG and its mRNA represent 5–10% of the total protein and mRNA in bloodstream trypanosomes (6Donelson J.E. Rice-Ficht A.C. Microbiol. Rev. 1985; 49: 107-125Crossref PubMed Google Scholar). However, partial sequence determinations of about 500 random cDNAs in this same library revealed three cDNAs that encode non-MVAT4 VSGs based on known amino acid similarities shared among VSGs (18El-Sayed N.M. Alarcon C.M. Beck J.C. Sheffield V.C. Donelson J.E. Mol. Biochem. Parasitol. 1995; 73: 75-90Crossref PubMed Scopus (97) Google Scholar). This result led us to re-examine the assumption that in bloodstream trypanosomes all of the telomere-linked vsg ESs are silent except for one. We initially treated either bloodstream or cultured procyclic trypanosomes with ultraviolet (UV) irradiation which enhances the amounts of some pre-mRNAs and mRNAs in trypanosomes. The molecular mechanisms underlying this enhancement are not completely understood but UV irradiation has been shown to inhibit pre-mRNA processing and mRNA decay, as well as arrest RNA elongation (19Coquelet H. Tebabi P. Pays A. Steinert M. Pays E. Mol. Cell. Biol. 1989; 9: 4022-4025Crossref PubMed Scopus (32) Google Scholar, 20Coquelet H. Steinert M. Pays E. Mol. Biochem. Parasitol. 1991; 44: 33-42Crossref PubMed Scopus (28) Google Scholar). We found that telomere-linked ESs previously thought to be silent undergo low level transcription and that at least some of the resultant precursor transcripts are processed into mature VSG mRNAs containing the 5′ spliced leader (SL) and a 3′ poly(A). Two of the eight or more esags in the polycistronic ESs,esags 6 and 7, encode subunits of a heterodimeric transferrin receptor and are typically located immediately downstream of the ES promoters. Different ESs code for slightly different transferrin receptors possessing markedly different binding affinities for transferrin of different mammals (21Borst P. Bitter W. Blundell P. Cross M. McCulloch R. Rudenko G. Taylor M.C. Van Leeuwen F. Hide G. Mottram J.C. Coombs G.H. Holmes P.H. Trypanosomiasis and Leishmaniasis: Biology and Contol. British Society for Parasitology/CAB Int., Oxford1997: 109-131Google Scholar). Since only one ES is normally active in a trypanosome, the specific transferrin receptor encoded by that ES could have a significant effect on the ability of a given trypanosome clone to grow in the bloodstream of a given host (22Bitter W. Gerrits H. Kleft R. Borst P. Nature. 1998; 391: 499-502Crossref PubMed Scopus (128) Google Scholar). The MVAT4 vsg ES does not contain either of transferrin receptor subunits. However, the bloodstream MVAT4 clone does express functional transferrin receptors and its ESAG 6 mRNA level is comparable to that in MITat 1.2 trypanosomes (23Steverding D. Overath P. Mol. Biochem. Parasitol. 1996; 78: 285-288Crossref PubMed Scopus (19) Google Scholar), which express the polycistronic 221 vsg ES (22Bitter W. Gerrits H. Kleft R. Borst P. Nature. 1998; 391: 499-502Crossref PubMed Scopus (128) Google Scholar, 24Cully D.F. Ip H.S. Cross G.A.M. Cell. 1985; 42: 173-182Abstract Full Text PDF PubMed Scopus (126) Google Scholar). We found that sequences coding for multiple members of the esag 6 and 7 families are readily detected in an MVAT4 cDNA library and that these sequences are derived from elsewhere in the genome. The implications of these findings for antigenic variation and control of the vsg ESs are incorporated in a model for the “leaky” transcription of the telomere-linked ESs. Bloodstream trypanosome clones MVAT4, MVAT5-Rx2, MVAT7, and WRATat 1.1 from the WRATat serodeme of T. brucei rhodesiense (25Campbell G.H. Esser K.M. Wellde B.T. Diggs C.L. Am. J. Trop. Med. Hyg. 1979; 28: 974-983Crossref PubMed Scopus (23) Google Scholar) were grown and isolated from rats as described previously (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar, 27Lu Y. Alarcon C.M. Hall T. Reddy L.V. Donelson J.E. Mol. Cell. Biol. 1994; 14: 3971-3980Crossref PubMed Google Scholar, 28Lu Y. Hall T. Gay L.S. Donelson J.E. Cell. 1993; 72: 397-406Abstract Full Text PDF PubMed Scopus (52) Google Scholar). The WRATat 1.1 trypanosome clone is the progenitor of the MVAT4, 5, and 7 clones. As described below, the purity of each of these four trypanosome clones was determined to be more than 99% with respect to the VSG being expressed. Procyclic trypanosomes were derived from the bloodstream clone MVAT5-Rx2 or MVAT7 and were maintained in culture as described (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar). MVAT4 bloodstream parasites were collected from infected rats and incubated with a 1:20 dilution of monoclonal antibodies (mAb) specific for the VSG expressed by MVAT4, MVAT5, and MVAT7 bloodstream trypanosomes. In a total of 10 fields of the microscope each of about 500 parasites present was individually scored for binding to the mAbs. Of these organisms more than 99% bound to the MVAT4 mAb and none bound to the MVAT5 or MVAT7 mAbs. Then a total of 100 fields (containing about 5,000 parasites) were systematically inspected by eye and no parasites were found that bound to the MVAT5 or MVAT7 mAbs. When about 5,000 MVAT5-Rx2 bloodstream parasites were examined by the same procedure, ≥99% of those parasites were recognized by the MVAT5 mAb and none were observed to bind the MVAT4 or MVAT7 mAbs. Likewise, when about 5,000 WRATat 1.1 bloodstream parasites and a similar number of MVAT7 bloodstream parasites were incubated with the MVAT4- and MVAT5-specific mAbs, none of these bloodstream organisms were found to be recognized by these mAbs. Parasites were collected, treated or untreated with UV irradiation, and their nuclei isolated using a protocol provided by Dr. Etienne Pays and described previously (19Coquelet H. Tebabi P. Pays A. Steinert M. Pays E. Mol. Cell. Biol. 1989; 9: 4022-4025Crossref PubMed Scopus (32) Google Scholar, 20Coquelet H. Steinert M. Pays E. Mol. Biochem. Parasitol. 1991; 44: 33-42Crossref PubMed Scopus (28) Google Scholar, 26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar). Previous titration experiments indicated that 50 mJ/cm2 was an appropriate dose of UV irradiation to use for RNA run-on analyses. The nuclei were stored at −80 °C until used. They were thawed, incubated with [α-32P]UTP, and their RNAs isolated for use as probes in Southern blots as described (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar). In some experiments α-amanitin (200–500 μg/ml) was added to the nuclei prior to incubation. All other recombinant DNA techniques, such as Northern blots, subcloning of restriction fragments, DNA sequencing,etc. were conducted using standard procedures (29Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Sequence alignments were conducted using the HIBIO MacIntosh DNASIS program (Hitachi) and Lazergene software (DNA*Star). Interpretation of the data shown below depends heavily upon the extent to which the cloned bloodstream MVAT4, MVAT5-Rx2 and WRATat 1.1 parasites were free from contamination with parasites expressing other VSGs. To examine this question, IFA of the bloodstream trypanosomes were conducted using mAb directed against various VSGs as described under “Experimental Procedures.” In the case of the MVAT4 bloodstream parasites, whose RNA was used for the Northern blots shown in Fig. 3, more than 99% of the organisms were recognized by the MVAT4 mAb, and of the 5,000 organisms examined none were recognized by the MVAT5 or MVAT7 mAbs despite the fact that low levels of MVAT5 and 7 VSG RNAs was present. Similarly, the WRATat 1.1 parasites, whose nuclei were used for the run-on experiments shown in Fig. 2, were not contaminated with parasites expressing MVAT4, 5, or 7 VSGs at a level detectable by IFA.Figure 2Southern blots probed with run-on RNA prepared from nuclei of WRATat 1.1 bloodstream (bloodform) trypanosomes or procyclic trypanosomes. Cells were UV irradiated and/or treated with α-amanitin as indicated below the autoradiograms. Plasmids shown in the photograph of the ethidium bromide (EtBr)-stained agarose gel were cleaved with various restriction enzymes to excise their cloned inserts. In some cases, the inserts or the vector were cleaved into multiple fragments, i.e. the M5 insert was excised as fragments a-d. The lanes contain DNA fragments corresponding to the brackets shown in Fig. 1,i.e. lane 1.1 contains fragment 1.1 shown in Fig.1; lane M4 contains fragment M4, etc. Lane T contains a fragment encoding tubulin; lane P contains a fragment encoding PARP; lane M contains standard marker DNA fragments. The 3-kb fragment in each lane is the linearized vector.View Large Image Figure ViewerDownload (PPT) Fig. 1depicts the expression sites and transcripts for four different basic copy vsgs expressed in the WRATat serodeme of T. brucei rhodesiense. Each of the four telomere-linkedvsgs is shown as it exists in the genome of WRATat 1.1 trypanosomes. The WRATat 1.1 vsg is preceded by an upstream “barren” region of 25 or more kilobases that is composed predominately of 70-bp repeats, similar to that of several other characterized bloodstream vsg ESs. Its transcription unit appears to be very large and initiated upstream of the 70-bp repeats, consistent with the expression of two other documented bloodstreamvsgs whose primary transcripts are 60 and 45 kb, respectively (30Kooter J.M. van der Spek H.J. Wagter R. d'Oliveira C.E. van der Hoeven F. Johnson P.J. Borst P. Cell. 1987; 51: 261-272Abstract Full Text PDF PubMed Scopus (164) Google Scholar, 31Johnson P.J. Kooter J.M. Borst P. Cell. 1987; 51: 273-281Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 32Pays E. Tebabi P. Pays A. Coquelet H. Revelard P. Salmon D. Steinert M. Cell. 1989; 57: 835-845Abstract Full Text PDF PubMed Scopus (200) Google Scholar). The genes for MVAT VSGs 4, 5, and 7 are typically expressed during the metacyclic stage, the final developmental stage of the parasite in its insect vector (33Lenardo M.J. Rice-Ficht A.C. Kelly G. Esser K.M. Donelson J.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6642-6646Crossref PubMed Scopus (49) Google Scholar, 34Lenardo M.J. Esser K.M. Moon A.M. Van der P.L.H. Donelson J.E. Mol. Cell. Biol. 1986; 6: 1991-1997Crossref PubMed Scopus (42) Google Scholar). Previously, however, we cloned rarely occurring bloodstream parasites expressing each of these VSGs (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar, 28Lu Y. Hall T. Gay L.S. Donelson J.E. Cell. 1993; 72: 397-406Abstract Full Text PDF PubMed Scopus (52) Google Scholar). In the MVAT4 bloodstream clone, the MVAT4 vsg is expressed from the same site as that depicted in Fig. 1 (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar). In contrast, in three independently isolated MVAT5 bloodstream clones (called MVAT5-Rx1, -Rx2, and -Rx3), the MVAT5 vsg shown in Fig. 1 is duplicated and expressed from another telomere-linked ES containing upstream 70-bp repeats similar to those in front of the WRATat 1.1 vsg (27Lu Y. Alarcon C.M. Hall T. Reddy L.V. Donelson J.E. Mol. Cell. Biol. 1994; 14: 3971-3980Crossref PubMed Google Scholar,28Lu Y. Hall T. Gay L.S. Donelson J.E. Cell. 1993; 72: 397-406Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Likewise, in an MVAT7 bloodstream clone, the MVAT7 vsgis expressed from a duplicated gene copy (35Kim K.S. Donelson J.E. J. Biol. Chem. 1997; 272: 24637-24645Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) but in this case, the duplicated segment includes the promoter shown in Fig. 1. About 2 kb upstream of each of the three telomere-linked MVATvsgs shown in Fig. 1 is a 70–80 bp sequence, indicated by a flag, that possesses promoter activity when placed in front of a luciferase reporter gene on a plasmid that is transfected transiently into trypanosomes (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar, 35Kim K.S. Donelson J.E. J. Biol. Chem. 1997; 272: 24637-24645Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 36Nagoshi Lu Y. Alarcon C.M. Donelson J.E. Mol. Biochem. Parasitol. 1995; 72: 33-45Crossref PubMed Scopus (26) Google Scholar). The MVAT5 and 7 vsgpromoters share more than 90% identity, and the MVAT4 vsgpromoter has about 50% identity with the other two (13Donelson J.E. Hill K.L. El-Sayed N.M. Mol. Biochem. Parasitol. 1998; 91: 51-66Crossref PubMed Scopus (84) Google Scholar, 36Nagoshi Lu Y. Alarcon C.M. Donelson J.E. Mol. Biochem. Parasitol. 1995; 72: 33-45Crossref PubMed Scopus (26) Google Scholar). The MVAT4 and 7 vsg promoters are used by the bloodstream trypanosomes expressing these two vsgs (26Alarcon C.M. Son H.J. Hall T. Donelson J.E. Mol. Cell. Biol. 1994; 14: 5579-5591Crossref PubMed Scopus (67) Google Scholar, 35Kim K.S. Donelson J.E. J. Biol. Chem. 1997; 272: 24637-24645Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The MVAT5 promoter depicted in Fig. 1 is not used by the duplicated MVAT5 vsgin bloodstream MVAT5 trypanosomes because the upstream boundary of the duplicated segment occurs between the indicated promoter and the start codon (27Lu Y. Alarcon C.M. Hall T. Reddy L.V. Donelson J.E. Mol. Cell. Biol. 1994; 14: 3971-3980Crossref PubMed Google Scholar). However, this MVAT5 promoter and the other two MVAT promoters indicated in Fig. 1 are thought to be active in metacyclic trypanosomes expressing that particular vsg (13Donelson J.E. Hill K.L. El-Sayed N.M. Mol. Biochem. Parasitol. 1998; 91: 51-66Crossref PubMed Scopus (84) Google Scholar, 34Lenardo M.J. Esser K.M. Moon A.M. Van der P.L.H. Donelson J.E. Mol. Cell. Biol. 1986; 6: 1991-1997Crossref PubMed Scopus (42) Google Scholar, 36Nagoshi Lu Y. Alarcon C.M. Donelson J.E. Mol. Biochem. Parasitol. 1995; 72: 33-45Crossref PubMed Scopus (26) Google Scholar). The promoter lying far upstream of the WRATat 1.1 vsg has not been identified. While analyzing the VSG transcripts in nuclei of bloodstream trypanosomes expressing these VSGs, we noticed that nascent RNA from supposedly silent, telomere-linked vsgs could be detected if the bloodstream parasites were first treated with UV irradiation. The use of UV irradiation for mapping transcription units is based on the inability of RNA polymerase to traverse pyrimidine dimers created in the DNA by the irradiation. Thus, synthesis of a long RNA is more sensitive to UV inactivation than is synthesis of a short RNA (19Coquelet H. Tebabi P. Pays A. Steinert M. Pays E. Mol. Cell. Biol. 1989; 9: 4022-4025Crossref PubMed Scopus (32) Google Scholar, 31Johnson P.J. Kooter J.M. Borst P. Cell. 1987; 51: 273-281Abstract Full Text PDF PubMed Scopus (220) Google Scholar). In addition, it has been shown that UV irradiation also modifies pre-mRNA processing and inhibits mRNA decay in trypanosomes (19Coquelet H. Tebabi P. Pays A. Steinert M. Pays E. Mol. Cell. Biol. 1989; 9: 4022-4025Crossref PubMed Scopus (32) Google Scholar, 20Coquelet H. Steinert M. Pays E. Mol. Biochem. Parasitol. 1991; 44: 33-42Crossref PubMed Scopus (28) Google Scholar). An example of the use of nascent RNA from the nuclei of UV-treated parasites to probe Southern blots of different vsgs is shown in Fig. 2. The agarose gel shown in theleft panel contained restriction fragments derived from (i) the four vsgs or their upstream regions (see Fig. 1), and (ii) the genes for tubulin (lane T) and PARP (lane P). Nitrocellulose filters of the gel were probed with run-on [32P]RNA from nuclei prepared from bloodstream WRATat 1.1 trypanosomes (top row of autoradiograms) or from procyclic trypanosomes (bottom row) derived from bloodstream trypanosome clone MVAT5-Rx2. Before nuclei isolation, the parasites were subjected to either no UV irradiation or to 50 mJ/cm2UV irradiation. In addition, the nuclei incubations with [α-32P]UTP were conducted in the absence or presence of α-amanitin (200 μg/ml). The WRATat 1.1 bloodstream RNA synthesized in the absence of both UV irradiation and α-amanitin (Fig. 2, top panel, labeled 0 mJ/cm2) hybridized the genes for the WRATat 1.1 VSG and tubulin, as expected. This bloodstream run-on RNA also hybridized to the PARP gene, as has been shown previously (37Clayton C.E. Fueri J.P. Itzhaki J.E. Bellofatto V. Sherman D.R. Wisdom G.S. Vijayasarathy S. Mowatt M.R. Mol. Cell. Biol. 1990; 10: 3036-3047Crossref PubMed Scopus (107) Google Scholar, 38Graham S.V. Barry J.D. Mol. Biochem. Parasitol. 1991; 47: 31-42Crossref PubMed Scopus (23) Google Scholar), even though mature PARP mRNA is not present in bloodstream trypanosomes. In addition, some hybridization to the MVAT7 vsg can be detected, and very weak signals to the MVAT4 and 5 vsgs also are present upon long exposure (not shown). When α-amanitin was present during the nuclei incubation, transcription of the tubulin genes was greatly reduced, but transcription of the PARP genes and the WRATat 1.1 vsg was relatively unaffected, indicating that their transcription is mediated by an α-amanitin-resistant RNA polymerase complex, as demonstrated previously (39Kooter J.M. Borst P. Nucleic Acids Res. 1984; 12: 9457-9472Crossref PubMed Scopus (157) Google Scholar, 40Rudenko G. Chung H.-M.M. Pham V.P. Van der Ploeg L.H.T. EMBO J. 1991; 10: 3387-3397Crossref PubMed Scopus (88) Google Scholar, 41Zomerdijk J.C.B.M. Kieft R. Borst P. Nature. 1991; 353: 772-775Crossref PubMed Scopus (81) Google Scholar). In this particular experiment, transcription of the MVAT7 vsg in WRATat 1.1 bloodstream nuclei also appears to be reduced by α-amanitin, but this result is probably because of a flaw in the blot with no α-amanitin since in other experiments it repeatedly was unaffected by α-amanitin (see top row, second panel). The run-on RNA isolated from WRATat 1.1 bloodstream nuclei exposed to UV irradiation displayed a different hybridization profile (Fig. 2,top panels labeled 50 mJ/cmx). Very little hybridization occurred to the WRATat 1.1 vsg, indicating that the initiation site for its transcript is located far upstream. Likewise, tubulin transcription was greatly reduced by this amount of irradiation, but PARP transcription was not, reflecting the smaller size of the primary PARP transcripts. In contrast, hybridization to the MVAT4, 5, and 7 vsgs increased relative to that of RNA synthesized without UV treatment. In addition, no hybridization occurred to fragments that lie upstream of the MVATvsg promoters, i.e. fragments U4 and U7, indicating that the transcription was initiated at these promoters. Supporting this conclusion is the finding that the UV-enhanced transcription is relatively unaffected by α-amanitin (Fig. 2,last panel, top row), indicating that it is mediated by the same α-amanitin-resistant RNA-polymerase complex that synthesizes VSG and PARP RNAs (37Clayton C.E. Fueri J.P. Itzhaki J.E. Bellofatto V. Sherman D.R. Wisdom G.S. Vijayasarathy S. Mowatt M.R. Mol. Cell. Biol. 1990; 10: 3036-3047Crossref PubMed Scopus (107) Google Scholar, 39Kooter J.M. Borst P. Nucleic Acids Res. 1984; 12: 9457-9472Crossref PubMed Scopus (157) Google Scholar, 42Brown S.D. Huang J. Van der Ploeg L.H.T. Mol. Cell. Biol. 1992; 12: 2644-2652Crossref PubMed Scopus (72) Google Scholar). The same experiments were conducted with nuclei from procyclic trypanosomes (Fig. 2, bottom row). These hybridizations showed that the α-amanitin-resistant PARP RNA was unaffected by UV treatment, similar to that seen in bloodstream nuclei. Likewise, tubulin transcription was diminished by α-amanitin, similar to that observed in bloodstream nuclei, although in this particular experiment it was reduced less by UV irradiation than in other experiments and in the bloodstream examples shown in the top row. In procyclic trypanosomes no transcription of any of the vsgs was observed under any of the conditions (all four panels in thebottom row). Thus, UV treatment does not enhance detection of RNA from the telomere-linked MVAT vsgs in procyclic organisms as it does in bloodstream trypanosomes. Similar experiments using nuclei from other bloodstream trypanosome clones yielded results similar to that shown in the top rowof Fig. 2. For example, run-on RNA prepared from UV-treated bloodstream MVAT4 nuclei hybridized to the MVAT5 and MVAT7 vsgs, and run-on RNA from UV-treated bloodstream MVAT7 nuclei hybridized to the MVAT4 and 5 VSG genes (not shown). However, these UV-treated run-on RNAs from the MVAT trypanosome clones did not hybridize to the WRATat 1.1 vsg, as expected since UV irradiation decreases the amount of its very long transcript (Fig. 2, top panel). Thus, UV irradiation of bloodstream trypanosomes increases the amount of run-on RNA from those unexpressed, telomere-linked MVATvsgs whose promoters are located about 2 kb from the start codon. The simplest explanation of this observation is that the promoters are close enough to these genes that UV treatment enhances their RNA abundance, rather than diminishes it as is the case for the WRATat 1.1 VSG RNA whose promoter is far upstream. Therefore, in the bloodstream stage, the monocistronic ESs appear to retain a low level act
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