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Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum.

Young R, Taylor JE, Kurioka A, Becker M, Louis EJ, Rudenko G - BMC Genomics (2008)

Bottom Line: BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG.Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries.However, our data does not show a clear correlation between size of trypanosome host range and either number of BESs or extent of ESAG genetic diversity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford, OX1 3SY, UK. rosanna.young@green.ox.ac

ABSTRACT

Background: African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast.

Results: Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires.

Conclusion: With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the relatively narrow host range of this subspecies and its apparent long-term clonality. However, our data does not show a clear correlation between size of trypanosome host range and either number of BESs or extent of ESAG genetic diversity.

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Isolation of trypanosome BESs in yeast using Transformation Associated Recombination (TAR) cloning as described in[ 34]. A schematic of a typical BES is shown above with the promoter indicated with a flag, and different expression site associated genes (ESAGs) indicated with coloured boxes. Characteristic repeat arrays either upstream of the BES promoter (50 bp repeats) or adjacent to the telomeric VSG gene (70 bp repeats) are indicated with vertically hatched boxes. Trypanosome telomere repeats are indicated with white arrows. The linearised pEB4 TAR vector is shown below with yeast telomere repeats (black triangles) as well as a positive (+) and a negative (-) selectable marker flanking a BES promoter containing fragment. Recombination between the vector and genomic DNA on the BES promoter fragment results in a yeast artificial chromosome (YAC) which can be stably maintained.
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Figure 1: Isolation of trypanosome BESs in yeast using Transformation Associated Recombination (TAR) cloning as described in[ 34]. A schematic of a typical BES is shown above with the promoter indicated with a flag, and different expression site associated genes (ESAGs) indicated with coloured boxes. Characteristic repeat arrays either upstream of the BES promoter (50 bp repeats) or adjacent to the telomeric VSG gene (70 bp repeats) are indicated with vertically hatched boxes. Trypanosome telomere repeats are indicated with white arrows. The linearised pEB4 TAR vector is shown below with yeast telomere repeats (black triangles) as well as a positive (+) and a negative (-) selectable marker flanking a BES promoter containing fragment. Recombination between the vector and genomic DNA on the BES promoter fragment results in a yeast artificial chromosome (YAC) which can be stably maintained.

Mentions: We cloned repertoires of BES containing telomeres from three trypanosome subspecies: T. b. gambiense DAL 972 (genome strain currently being sequenced by the Sanger Research Institute), T. b. brucei EATRO 2340 and T. equiperdum STIB 818. T. b. brucei EATRO 2340 was originally thought to be a T. b. rhodesiense subspecies [35]. However, as we did not find evidence for the presence of SRA which is considered diagnostic for T. b. rhodesiense [12,13], this strain was tentatively redesignated to be a T. b. brucei (see Additional file 1 and Materials). The BES containing telomeres were isolated in yeast using a method relying on Transformation Associated Recombination (TAR) cloning [34,36]. Linearised yeast TAR vector pEB4, containing a BES promoter fragment and a yeast telomere, was cotransformed into yeast spheroplasts together with total trypanosome genomic DNA (Fig. 1). Recombination between the BES promoter within the vector and similar sequences within the trypanosome genomic DNA provides the yeast vector with a second (trypanosome derived) telomere, thereby stabilising the episome as a YAC (yeast artificial chromosome).


Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum.

Young R, Taylor JE, Kurioka A, Becker M, Louis EJ, Rudenko G - BMC Genomics (2008)

Isolation of trypanosome BESs in yeast using Transformation Associated Recombination (TAR) cloning as described in[ 34]. A schematic of a typical BES is shown above with the promoter indicated with a flag, and different expression site associated genes (ESAGs) indicated with coloured boxes. Characteristic repeat arrays either upstream of the BES promoter (50 bp repeats) or adjacent to the telomeric VSG gene (70 bp repeats) are indicated with vertically hatched boxes. Trypanosome telomere repeats are indicated with white arrows. The linearised pEB4 TAR vector is shown below with yeast telomere repeats (black triangles) as well as a positive (+) and a negative (-) selectable marker flanking a BES promoter containing fragment. Recombination between the vector and genomic DNA on the BES promoter fragment results in a yeast artificial chromosome (YAC) which can be stably maintained.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533676&req=5

Figure 1: Isolation of trypanosome BESs in yeast using Transformation Associated Recombination (TAR) cloning as described in[ 34]. A schematic of a typical BES is shown above with the promoter indicated with a flag, and different expression site associated genes (ESAGs) indicated with coloured boxes. Characteristic repeat arrays either upstream of the BES promoter (50 bp repeats) or adjacent to the telomeric VSG gene (70 bp repeats) are indicated with vertically hatched boxes. Trypanosome telomere repeats are indicated with white arrows. The linearised pEB4 TAR vector is shown below with yeast telomere repeats (black triangles) as well as a positive (+) and a negative (-) selectable marker flanking a BES promoter containing fragment. Recombination between the vector and genomic DNA on the BES promoter fragment results in a yeast artificial chromosome (YAC) which can be stably maintained.
Mentions: We cloned repertoires of BES containing telomeres from three trypanosome subspecies: T. b. gambiense DAL 972 (genome strain currently being sequenced by the Sanger Research Institute), T. b. brucei EATRO 2340 and T. equiperdum STIB 818. T. b. brucei EATRO 2340 was originally thought to be a T. b. rhodesiense subspecies [35]. However, as we did not find evidence for the presence of SRA which is considered diagnostic for T. b. rhodesiense [12,13], this strain was tentatively redesignated to be a T. b. brucei (see Additional file 1 and Materials). The BES containing telomeres were isolated in yeast using a method relying on Transformation Associated Recombination (TAR) cloning [34,36]. Linearised yeast TAR vector pEB4, containing a BES promoter fragment and a yeast telomere, was cotransformed into yeast spheroplasts together with total trypanosome genomic DNA (Fig. 1). Recombination between the BES promoter within the vector and similar sequences within the trypanosome genomic DNA provides the yeast vector with a second (trypanosome derived) telomere, thereby stabilising the episome as a YAC (yeast artificial chromosome).

Bottom Line: BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG.Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries.However, our data does not show a clear correlation between size of trypanosome host range and either number of BESs or extent of ESAG genetic diversity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford, OX1 3SY, UK. rosanna.young@green.ox.ac

ABSTRACT

Background: African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast.

Results: Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires.

Conclusion: With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the relatively narrow host range of this subspecies and its apparent long-term clonality. However, our data does not show a clear correlation between size of trypanosome host range and either number of BESs or extent of ESAG genetic diversity.

Show MeSH
Related in: MedlinePlus