Characterization of VSG gene expression site promoters and promoter-associated DNA rearrangement events

1991 ◽  
Vol 11 (5) ◽  
pp. 2467-2480 ◽  
Author(s):  
K Gottesdiener ◽  
H M Chung ◽  
S D Brown ◽  
M G Lee ◽  
L H Van der Ploeg
Keyword(s):  
Transcriptional Control ◽  
Transcription Unit ◽  
Surface Glycoprotein ◽  
Dna Rearrangement ◽  
Cell Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Variant Cell ◽  
Expression Site ◽  
Simple Sequence

The expressed variant cell surface glycoprotein (VSG) gene of Trypanosoma brucei is located at the 3' end of a large, telomeric, polycistronic transcription unit or expression site. We show that the region 45 kb upstream of the VSG gene, in the expression site on a 1.5-Mb chromosome, contains at least two promoters that are arranged in tandem, directing the transcription of the expression site. DNA rearrangement events occur specifically, at inactivation of the expression site, and these events delete the most upstream transcribed region and replace it with a large array of simple-sequence DNA, leaving the downstream promoter intact. Because of the placement of simple-sequence DNA, the remaining downstream promoter now becomes structurally identical to previously described VSG promoters. The downstream promoter is repetitive in the genome, since it is present at several different expression sites. Restriction fragment length polymorphism mapping allows grouping of the expression sites into two families, those with and those without an upstream transcription unit, and the DNA rearrangement events convert the expression sites from one type to the other. Deletion of the upstream transcription unit also leads to the loss of several steady-state RNAs. The findings may indicate a role for promoter-associated DNA rearrangement events, and/or interactions between tandemly arranged promoters, in expression site transcriptional control.

scholarly journals Characterization of VSG gene expression site promoters and promoter-associated DNA rearrangement events.

1991 ◽  
Vol 11 (5) ◽  
pp. 2467-2480 ◽  
Author(s):  
K Gottesdiener ◽  
H M Chung ◽  
S D Brown ◽  
M G Lee ◽  
L H Van der Ploeg
Keyword(s):  
Transcriptional Control ◽  
Transcription Unit ◽  
Surface Glycoprotein ◽  
Dna Rearrangement ◽  
Cell Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Variant Cell ◽  
Expression Site ◽  
Simple Sequence

The expressed variant cell surface glycoprotein (VSG) gene of Trypanosoma brucei is located at the 3' end of a large, telomeric, polycistronic transcription unit or expression site. We show that the region 45 kb upstream of the VSG gene, in the expression site on a 1.5-Mb chromosome, contains at least two promoters that are arranged in tandem, directing the transcription of the expression site. DNA rearrangement events occur specifically, at inactivation of the expression site, and these events delete the most upstream transcribed region and replace it with a large array of simple-sequence DNA, leaving the downstream promoter intact. Because of the placement of simple-sequence DNA, the remaining downstream promoter now becomes structurally identical to previously described VSG promoters. The downstream promoter is repetitive in the genome, since it is present at several different expression sites. Restriction fragment length polymorphism mapping allows grouping of the expression sites into two families, those with and those without an upstream transcription unit, and the DNA rearrangement events convert the expression sites from one type to the other. Deletion of the upstream transcription unit also leads to the loss of several steady-state RNAs. The findings may indicate a role for promoter-associated DNA rearrangement events, and/or interactions between tandemly arranged promoters, in expression site transcriptional control.

scholarly journals A proposed mechanism for promoter-associated DNA rearrangement events at a variant surface glycoprotein gene expression site.

1992 ◽  
Vol 12 (10) ◽  
pp. 4784-4795 ◽  
Author(s):  
K M Gottesdiener ◽  
L Goriparthi ◽  
J P Masucci ◽  
L H Van der Ploeg
Keyword(s):  
Gene Expression ◽  
Promoter Region ◽  
Protozoan Parasite ◽  
Surface Glycoprotein ◽  
Dna Rearrangement ◽  
Cell Surface Glycoprotein ◽  
Circular Dna ◽  
Glycoprotein Gene ◽  
Variant Cell ◽  
Expression Site

The expressed variant cell surface glycoprotein (VSG) gene of the protozoan parasite Trypanosoma brucei is invariably found at one of several telomeric VSG gene expression sites (ESs). The active ES in variant 118 clone 1 is found on a 1.5-Mb chromosome, and the promoter region is located more than 45 kb upstream of the VSG gene. We had previously shown that DNA rearrangement events occurred in the promoter region, specifically at inactivation of this ES (K. M. Gottesdiener, H.-M. Chung, S. L. Brown, M. G.-S. Lee, and L. H. T. Van der Ploeg, Mol. Cell. Biol. 11:2467-2477, 1991). In this report, we describe the cloning of the entire 17-kb promoter region, which revealed the presence of two identical 2.15-kb tandem promoter repeats separated by 13 kb of DNA. The two virtually identical promoter repeats both function efficiently in directing transcription in transient transfection assays in insect-form trypanosomes. We characterized the DNA rearrangement events that occur at ES inactivation, and by studying both of the reciprocal products of this recombination event, we infer that these result from direct (promoter) repeat recombination, formation of heteroduplex DNA, and a reciprocal exchange event that releases a circular DNA as a side product of the reaction. The finding of DNA recombinational events in a region of the VSG gene ES that encodes the promoter(s), and their relatively frequent occurrence at ES inactivation, suggests a possible role in ES control.

A proposed mechanism for promoter-associated DNA rearrangement events at a variant surface glycoprotein gene expression site

1992 ◽  
Vol 12 (10) ◽  
pp. 4784-4795
Author(s):  
K M Gottesdiener ◽  
L Goriparthi ◽  
J P Masucci ◽  
L H Van der Ploeg
Keyword(s):  
Gene Expression ◽  
Promoter Region ◽  
Protozoan Parasite ◽  
Surface Glycoprotein ◽  
Dna Rearrangement ◽  
Cell Surface Glycoprotein ◽  
Circular Dna ◽  
Glycoprotein Gene ◽  
Variant Cell ◽  
Expression Site

The expressed variant cell surface glycoprotein (VSG) gene of the protozoan parasite Trypanosoma brucei is invariably found at one of several telomeric VSG gene expression sites (ESs). The active ES in variant 118 clone 1 is found on a 1.5-Mb chromosome, and the promoter region is located more than 45 kb upstream of the VSG gene. We had previously shown that DNA rearrangement events occurred in the promoter region, specifically at inactivation of this ES (K. M. Gottesdiener, H.-M. Chung, S. L. Brown, M. G.-S. Lee, and L. H. T. Van der Ploeg, Mol. Cell. Biol. 11:2467-2477, 1991). In this report, we describe the cloning of the entire 17-kb promoter region, which revealed the presence of two identical 2.15-kb tandem promoter repeats separated by 13 kb of DNA. The two virtually identical promoter repeats both function efficiently in directing transcription in transient transfection assays in insect-form trypanosomes. We characterized the DNA rearrangement events that occur at ES inactivation, and by studying both of the reciprocal products of this recombination event, we infer that these result from direct (promoter) repeat recombination, formation of heteroduplex DNA, and a reciprocal exchange event that releases a circular DNA as a side product of the reaction. The finding of DNA recombinational events in a region of the VSG gene ES that encodes the promoter(s), and their relatively frequent occurrence at ES inactivation, suggests a possible role in ES control.

A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei

1992 ◽  
Vol 12 (3) ◽  
pp. 1218-1225
Author(s):  
P Paindavoine ◽  
S Rolin ◽  
S Van Assel ◽  
M Geuskens ◽  
J C Jauniaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trypanosoma Brucei ◽  
Membrane Fraction ◽  
Transcription Unit ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Yeast Cells ◽  
Variant Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Expression Site

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

scholarly journals Stable variant-specific transcripts of the variant cell surface glycoprotein gene 1.8 expression site in Trypanosoma brucei.

1988 ◽  
Vol 8 (2) ◽  
pp. 854-859 ◽  
Author(s):  
C Shea ◽  
L H Van der Ploeg
Keyword(s):  
Cell Surface ◽  
Surface Glycoprotein ◽  
Minimum Size ◽  
Cell Surface Glycoprotein ◽  
Glycoprotein Gene ◽  
Variant Cell ◽  
Expression Site ◽  
Specific Mrnas ◽  
Chromosome Specific Library ◽  
Nascent Transcripts

The structure and transcriptional regulation of the 1.8 variant cell surface glycoprotein (VSG) gene expression site located on a 430-kilobase (kb) chromosome was examined in a 430-kb-chromosome-specific library. Using 32P-labeled nascent transcripts generated by nuclear run-on, we selected recombinant clones derived from the 430-kb chromosome which were coordinately activated with the 1.8 VSG gene. The results show that a repetitive region with a minimum size of 27 kb is coordinately activated with the 1.8 VSG gene. As with the 1.8 VSG gene, transcription is by RNA polymerases that are insensitive to the drug alpha-amanitin at concentrations up to 1 mg/ml. Transcription results in the generation of several stable variant-specific mRNAs. These mRNAs most likely belong to a family of repetitive expression-site-associated genes.

scholarly journals A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei.

1992 ◽  
Vol 12 (3) ◽  
pp. 1218-1225 ◽  
Author(s):  
P Paindavoine ◽  
S Rolin ◽  
S Van Assel ◽  
M Geuskens ◽  
J C Jauniaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trypanosoma Brucei ◽  
Membrane Fraction ◽  
Transcription Unit ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Yeast Cells ◽  
Variant Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Expression Site

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

scholarly journals A similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of Trypanosoma brucei

1991 ◽  
Vol 11 (3) ◽  
pp. 1473-1479
Author(s):  
M Berberof ◽  
A Pays ◽  
E Pays
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Gene Transcription ◽  
Transcription Unit ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Expression Site ◽  
Similar Gene

The genes for the variant surface glycoprotein (VSG) and procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.

scholarly journals A similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of Trypanosoma brucei.

1991 ◽  
Vol 11 (3) ◽  
pp. 1473-1479 ◽  
Author(s):  
M Berberof ◽  
A Pays ◽  
E Pays
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Gene Transcription ◽  
Transcription Unit ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Expression Site ◽  
Similar Gene

The genes for the variant surface glycoprotein (VSG) and procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.

Stable variant-specific transcripts of the variant cell surface glycoprotein gene 1.8 expression site in Trypanosoma brucei

1988 ◽  
Vol 8 (2) ◽  
pp. 854-859
Author(s):  
C Shea ◽  
L H Van der Ploeg
Keyword(s):  
Cell Surface ◽  
Surface Glycoprotein ◽  
Minimum Size ◽  
Cell Surface Glycoprotein ◽  
Glycoprotein Gene ◽  
Variant Cell ◽  
Expression Site ◽  
Specific Mrnas ◽  
Chromosome Specific Library ◽  
Nascent Transcripts

The structure and transcriptional regulation of the 1.8 variant cell surface glycoprotein (VSG) gene expression site located on a 430-kilobase (kb) chromosome was examined in a 430-kb-chromosome-specific library. Using 32P-labeled nascent transcripts generated by nuclear run-on, we selected recombinant clones derived from the 430-kb chromosome which were coordinately activated with the 1.8 VSG gene. The results show that a repetitive region with a minimum size of 27 kb is coordinately activated with the 1.8 VSG gene. As with the 1.8 VSG gene, transcription is by RNA polymerases that are insensitive to the drug alpha-amanitin at concentrations up to 1 mg/ml. Transcription results in the generation of several stable variant-specific mRNAs. These mRNAs most likely belong to a family of repetitive expression-site-associated genes.

scholarly journals DNA rearrangements associated with multiple consecutive directed antigenic switches in Trypanosoma brucei.

1996 ◽  
Vol 16 (7) ◽  
pp. 3615-3625 ◽  
Author(s):  
M Navarro ◽  
G A Cross
Keyword(s):  
Trypanosoma Brucei ◽  
Short Range ◽  
Direct Analysis ◽  
Transcription Unit ◽  
The Other ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Polycistronic Transcription ◽  
Tangible Evidence ◽  
Expression Site

Changes in variant surface glycoprotein (Vsg) expression allow Trypanosoma brucei to elude the immune response. The expressed vsg is always located at the telomeric end of a polycistronic transcription unit known as an expression site (ES). Although there are many ESs, only one is active at any particular time. The mechanisms regulating ES transcription and switching are unknown. Chromosome rearrangements within or upstream of the ES have been described to occur in occasional switch events, but no changes have been consistently associated with switching. We inserted the drug resistance genes neo and ble, conferring resistance to G418 and phleomycin, respectively, 1 kb downstream of "silent" ES promoters. This demonstrated that short-range transcription could be achieved from a silent ES promoter. From one initial transformant clone, panels of independent consecutive on-off-on switch clones were generated and analyzed. The first activation of the neo-targeted ES was always associated with deletion of the upstream tandem promoter in this ES, but no further rearrangements were detected in consecutive off-on switches of this ES. On the other hand, direct analysis of ES promoters showed that deletions and duplications occurred elsewhere. Activation of a ble-tagged 300-kb chromosome could not be achieved, but phleomycin-resistant clones could be obtained. One such clone arose from recombination between three ESs. Taken together, our experiments suggest that ES switching may occur after a period of chromosomal interactivity that may or may not leave tangible evidence in the form of detectable sequence changes.

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