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Genetic exchange in Trypanosoma brucei: evidence for mating prior to metacyclic stage development.

Tait A, Macleod A, Tweedie A, Masiga D, Turner CM - Mol. Biochem. Parasitol. (2006)

View Article: PubMed Central - PubMed

Affiliation: Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow Biomedical Research Centre, 120, University Place, Glasgow G12 8TA, UK. gvwa01@udcf.gla.ac.uk

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It is well established that genetic exchange occurs between Trypanosoma brucei parasites when two stocks are used to infect tsetse flies under laboratory conditions and a number of such crosses have been undertaken... Both cross and self-fertilisation can take place, with the products of mating being the equivalent of F1 progeny in a Mendelian system... One of the remaining unknowns about this system of genetic exchange is the life cycle stage at which mating takes place... The development from procyclic to metacyclic is via a succession of morphologically distinct stages, the most prominent of which are the mesocyclic and epimastogote stages in the proventiculus and salivary glands, respectively... Gibson and Bailey, using parental stocks tagged with selectable markers, provided evidence that mating does not occur between procyclic stage trypanosomes in the mid-gut of the tsetse fly, in contrast to a previous report... The genotypes of single metacyclic trypanosomes (amplified vegetatively in mice) obtained from crosses, clearly show that they are the products of mating... DNA was prepared for AFLP analysis from the parental and progeny clones after growth in immuno-suppressed mice (independent preparations from those used for the microsatellite analysis)... A section of one gel is shown in Fig. 1 to illustrate the differences observed between the parental stocks and the inheritance of these differences in the resulting progeny... When these data are combined with the multilocus genotypes generated from the micro- and minisatellite data, there is clear evidence that one pair of clones from the 927 × 247 cross and two pairs from the 386 × 247 cross are identical in terms of genotype... While a proportion of these markers will be genetically linked, there is a sufficiently large number of independent markers to make the conclusion of identity a robust one... However, in both crosses most metacyclic stage clones are unique therefore suggesting there is very limited vegetative growth between mating and the development of the metacyclic otherwise many identical clones would be observed... In order to explain the results from the two crosses, taken together, mating cannot take place between the metacyclic stages but is most likely to occur between the epimastigotes with limited subsequent mitotic division between mating and the development of the metacyclic stage... Firstly, pre-metacyclics are relatively rare and this is thought to be a very transient phase in development... Secondly, in other flagellates, the process of gamete fusion that leads to syngamy is first mediated via flagella attachment and it is only in the attached epimastigote phase of the life cycle that T. brucei has the required flagellum–substratum and flagellum–flagellum interactions.

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Autoradiograph of labelled AFLP gel with DNA samples digested with HindIII and Taq1. Each primer had a two-nucleotide extension (H-CA and T-AA) for selective amplification and fragments were separated on a 6% acrylamide gel. Tracks: 1-STIB 247; 2-STIB 386; 3-F9/45 mcl 2; 4-F9/45 mcl 11; 5-F492/50 mcl 12; 6-F492/50 mcl 13; 7-F9/45 mcl 9; 8-F9/45 mcl 10. Arrows indicate fragments that are heterozygous for polymorphisms in one of the two parental stocks and segregate in the progeny.
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fig1: Autoradiograph of labelled AFLP gel with DNA samples digested with HindIII and Taq1. Each primer had a two-nucleotide extension (H-CA and T-AA) for selective amplification and fragments were separated on a 6% acrylamide gel. Tracks: 1-STIB 247; 2-STIB 386; 3-F9/45 mcl 2; 4-F9/45 mcl 11; 5-F492/50 mcl 12; 6-F492/50 mcl 13; 7-F9/45 mcl 9; 8-F9/45 mcl 10. Arrows indicate fragments that are heterozygous for polymorphisms in one of the two parental stocks and segregate in the progeny.

Mentions: To test the identity of these pairs of clones further, genome wide markers were used. Previous work, using the technique of amplified fragment length polymorphism (AFLP [13,14]), has been undertaken to construct linkage maps of TREU 927 and STIB 386 using some of the metacyclic derived progeny and provides genome wide analyses of polymorphic segregating markers [2]. DNA was prepared for AFLP analysis from the parental and progeny clones after growth in immuno-suppressed mice (independent preparations from those used for the microsatellite analysis). A section of one gel is shown in Fig. 1 to illustrate the differences observed between the parental stocks and the inheritance of these differences in the resulting progeny. The parental clones (247 and 386) show a series of identical bands as well as differences in which a band is present in one parent but not the other and some of these bands segregate in the F1 progeny clones as a presence/absence (Fig. 1 arrows). This can be interpreted as the homozygous absence of a polymorphic site in one parent (therefore no amplification product) with the second parent being heterozygous for that polymorphism. These polymorphisms arise from the presence/absence of a restriction site, insertion/deletion or single base change in the sequence matching the 3′ extension of one primer. This polymorphism will then segregate into the progeny, which will inherit one ‘absent’ allele from one parent and either the amplified band or ‘absent’ allele from the second parent [2,13]. Applying this technique to the detection of differences between the three parental clones (247, 386 and 927), using the restriction enzyme pairs EcoR1/Mse1 and HindIII/Taq1, and screening the F1 progeny clones to detect markers that segregate, 157 segregating polymorphic fragments were detected in the 247 × 386 cross and 185 in the 247 × 927 cross. Fig. 1 illustrates the different patterns of segregation of the parental bands in progeny that differ in genotype (lanes 3–7), as well as the identity of clones F9/45 mcl 9 and 10 (lanes 7 and 8). These data allow a detailed genetic fingerprint of each progeny clone to be generated and a comparison between the clones in terms of their similarity. The AFLP data confirmed the findings with the min- and microsatellite markers. In the cross 927 × 247, seven of the metacyclic derived clones were confirmed to be of distinct, independent genotypes and two clones were identical to each other (AFLP genotype A, Table 1). These data indicate that most of the metacyclic stage clones are genotypically distinct and that the metacyclic stage is of hybrid genotype and therefore a product of mating (Table 1). AFLP analysis of six of the metacyclic derived clones from the 247 × 386 cross, showed that five were of unique genotype and two were identical for all AFLP markers (AFLP genotype J, Table 1). When these data are combined with the multilocus genotypes generated from the micro- and minisatellite data, there is clear evidence that one pair of clones from the 927 × 247 cross and two pairs from the 386 × 247 cross are identical in terms of genotype. While a proportion of these markers will be genetically linked, there is a sufficiently large number of independent markers to make the conclusion of identity a robust one.


Genetic exchange in Trypanosoma brucei: evidence for mating prior to metacyclic stage development.

Tait A, Macleod A, Tweedie A, Masiga D, Turner CM - Mol. Biochem. Parasitol. (2006)

Autoradiograph of labelled AFLP gel with DNA samples digested with HindIII and Taq1. Each primer had a two-nucleotide extension (H-CA and T-AA) for selective amplification and fragments were separated on a 6% acrylamide gel. Tracks: 1-STIB 247; 2-STIB 386; 3-F9/45 mcl 2; 4-F9/45 mcl 11; 5-F492/50 mcl 12; 6-F492/50 mcl 13; 7-F9/45 mcl 9; 8-F9/45 mcl 10. Arrows indicate fragments that are heterozygous for polymorphisms in one of the two parental stocks and segregate in the progeny.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2311417&req=5

fig1: Autoradiograph of labelled AFLP gel with DNA samples digested with HindIII and Taq1. Each primer had a two-nucleotide extension (H-CA and T-AA) for selective amplification and fragments were separated on a 6% acrylamide gel. Tracks: 1-STIB 247; 2-STIB 386; 3-F9/45 mcl 2; 4-F9/45 mcl 11; 5-F492/50 mcl 12; 6-F492/50 mcl 13; 7-F9/45 mcl 9; 8-F9/45 mcl 10. Arrows indicate fragments that are heterozygous for polymorphisms in one of the two parental stocks and segregate in the progeny.
Mentions: To test the identity of these pairs of clones further, genome wide markers were used. Previous work, using the technique of amplified fragment length polymorphism (AFLP [13,14]), has been undertaken to construct linkage maps of TREU 927 and STIB 386 using some of the metacyclic derived progeny and provides genome wide analyses of polymorphic segregating markers [2]. DNA was prepared for AFLP analysis from the parental and progeny clones after growth in immuno-suppressed mice (independent preparations from those used for the microsatellite analysis). A section of one gel is shown in Fig. 1 to illustrate the differences observed between the parental stocks and the inheritance of these differences in the resulting progeny. The parental clones (247 and 386) show a series of identical bands as well as differences in which a band is present in one parent but not the other and some of these bands segregate in the F1 progeny clones as a presence/absence (Fig. 1 arrows). This can be interpreted as the homozygous absence of a polymorphic site in one parent (therefore no amplification product) with the second parent being heterozygous for that polymorphism. These polymorphisms arise from the presence/absence of a restriction site, insertion/deletion or single base change in the sequence matching the 3′ extension of one primer. This polymorphism will then segregate into the progeny, which will inherit one ‘absent’ allele from one parent and either the amplified band or ‘absent’ allele from the second parent [2,13]. Applying this technique to the detection of differences between the three parental clones (247, 386 and 927), using the restriction enzyme pairs EcoR1/Mse1 and HindIII/Taq1, and screening the F1 progeny clones to detect markers that segregate, 157 segregating polymorphic fragments were detected in the 247 × 386 cross and 185 in the 247 × 927 cross. Fig. 1 illustrates the different patterns of segregation of the parental bands in progeny that differ in genotype (lanes 3–7), as well as the identity of clones F9/45 mcl 9 and 10 (lanes 7 and 8). These data allow a detailed genetic fingerprint of each progeny clone to be generated and a comparison between the clones in terms of their similarity. The AFLP data confirmed the findings with the min- and microsatellite markers. In the cross 927 × 247, seven of the metacyclic derived clones were confirmed to be of distinct, independent genotypes and two clones were identical to each other (AFLP genotype A, Table 1). These data indicate that most of the metacyclic stage clones are genotypically distinct and that the metacyclic stage is of hybrid genotype and therefore a product of mating (Table 1). AFLP analysis of six of the metacyclic derived clones from the 247 × 386 cross, showed that five were of unique genotype and two were identical for all AFLP markers (AFLP genotype J, Table 1). When these data are combined with the multilocus genotypes generated from the micro- and minisatellite data, there is clear evidence that one pair of clones from the 927 × 247 cross and two pairs from the 386 × 247 cross are identical in terms of genotype. While a proportion of these markers will be genetically linked, there is a sufficiently large number of independent markers to make the conclusion of identity a robust one.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow Biomedical Research Centre, 120, University Place, Glasgow G12 8TA, UK. gvwa01@udcf.gla.ac.uk

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

It is well established that genetic exchange occurs between Trypanosoma brucei parasites when two stocks are used to infect tsetse flies under laboratory conditions and a number of such crosses have been undertaken... Both cross and self-fertilisation can take place, with the products of mating being the equivalent of F1 progeny in a Mendelian system... One of the remaining unknowns about this system of genetic exchange is the life cycle stage at which mating takes place... The development from procyclic to metacyclic is via a succession of morphologically distinct stages, the most prominent of which are the mesocyclic and epimastogote stages in the proventiculus and salivary glands, respectively... Gibson and Bailey, using parental stocks tagged with selectable markers, provided evidence that mating does not occur between procyclic stage trypanosomes in the mid-gut of the tsetse fly, in contrast to a previous report... The genotypes of single metacyclic trypanosomes (amplified vegetatively in mice) obtained from crosses, clearly show that they are the products of mating... DNA was prepared for AFLP analysis from the parental and progeny clones after growth in immuno-suppressed mice (independent preparations from those used for the microsatellite analysis)... A section of one gel is shown in Fig. 1 to illustrate the differences observed between the parental stocks and the inheritance of these differences in the resulting progeny... When these data are combined with the multilocus genotypes generated from the micro- and minisatellite data, there is clear evidence that one pair of clones from the 927 × 247 cross and two pairs from the 386 × 247 cross are identical in terms of genotype... While a proportion of these markers will be genetically linked, there is a sufficiently large number of independent markers to make the conclusion of identity a robust one... However, in both crosses most metacyclic stage clones are unique therefore suggesting there is very limited vegetative growth between mating and the development of the metacyclic otherwise many identical clones would be observed... In order to explain the results from the two crosses, taken together, mating cannot take place between the metacyclic stages but is most likely to occur between the epimastigotes with limited subsequent mitotic division between mating and the development of the metacyclic stage... Firstly, pre-metacyclics are relatively rare and this is thought to be a very transient phase in development... Secondly, in other flagellates, the process of gamete fusion that leads to syngamy is first mediated via flagella attachment and it is only in the attached epimastigote phase of the life cycle that T. brucei has the required flagellum–substratum and flagellum–flagellum interactions.

Show MeSH
Related in: MedlinePlus