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Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping.

Kumari V, Iyer LR, Roy R, Bhargava V, Panda S, Paul J, Verweij JJ, Clark CG, Bhattacharya A, Bhattacharya S - BMC Genomics (2013)

Bottom Line: Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes.The results obtained by our method correlated with the data from other typing methods.Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACT

Background: The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains.

Results: We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies.

Conclusions: Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.

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Detection and validation of EhSINE1 polymorphic loci 13, 17, 19 and 42. (A) Schematic representation of primers designed from different loci. The hollow box represents the EhSINE1 element; flanking genes have not been shown for simplicity. (B) PCR was performed using genomic DNA of HM-1:IMSS (H) and Rahman (R) strains as template, using primers from sequences flanking the EhSINE1 copy as shown in the schematic representation. The size of amplicons was determined by electrophoresis in 1% agarose gels (Top panel). Of 4 SINE1 unoccupied sites found computationally two were tested (13 and 42). Two more (17 and 19) were evaluated by PCR and Southern Blotting in Rahman. The sizes of amplicons obtained are indicated on the right, with arrows. The amplicon from strain Rahman was shorter by ~550 bp (the size of EhSINE1) at loci 13, 17 and 19, but was longer at locus 42 (explained in the text). The absence of EhSINE1 was further confirmed by Southern blotting with EhSINE1 probe, which failed to hybridize with the amplicons from strain Rahman (Bottom panel).
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Figure 3: Detection and validation of EhSINE1 polymorphic loci 13, 17, 19 and 42. (A) Schematic representation of primers designed from different loci. The hollow box represents the EhSINE1 element; flanking genes have not been shown for simplicity. (B) PCR was performed using genomic DNA of HM-1:IMSS (H) and Rahman (R) strains as template, using primers from sequences flanking the EhSINE1 copy as shown in the schematic representation. The size of amplicons was determined by electrophoresis in 1% agarose gels (Top panel). Of 4 SINE1 unoccupied sites found computationally two were tested (13 and 42). Two more (17 and 19) were evaluated by PCR and Southern Blotting in Rahman. The sizes of amplicons obtained are indicated on the right, with arrows. The amplicon from strain Rahman was shorter by ~550 bp (the size of EhSINE1) at loci 13, 17 and 19, but was longer at locus 42 (explained in the text). The absence of EhSINE1 was further confirmed by Southern blotting with EhSINE1 probe, which failed to hybridize with the amplicons from strain Rahman (Bottom panel).

Mentions: Of the eight predicted polymorphic loci in strain Rahman we validated experimentally six using PCR (Figures 3 and 4) with primers designed from the flanking sequences of EhSINE1/EhSINE2 in HM-1:IMSS (Additional file 1: Figure S1 and Additional file 2: Table S1). The absence of SINE sequences was inferred from the size of the amplicon (smaller by the size of SINE) and by Southern hybridization using a SINE sequence as a probe. The amplicon sizes in Rahman from three EhSINE1 polymorphic loci (13, 17 and 19) were smaller by about 550 bp suggesting that indeed these sites lacked EhSINE1. This was also confirmed by Southern hybridization (Figure 3B, bottom panel). In contrast, the amplicon size of another polymorphic EhSINE1 locus (42) was actually larger by 1.5 kb in Rahman. Probing a Southern blot of the amplicon using EhSINE1-flanking sequences from locus 42 confirmed that the amplified region in Rahman indeed belonged to the same locus (Additional file 3: Figure S2). However, two different sets of primers designed using the HM-1:IMSS sequence at this locus failed to produce an amplicon in Rahman. Therefore it appears that this locus may have undergone multiple changes and is not a simple case of SINE absence. We did not analyse this locus further. The two predicted EhSINE2 polymorphic loci (18 and 50) were also validated using PCR and Southern hybridization (Figure 4 ii and iii). At both loci the amplicons from Rahman were 700 bp shorter (the size of EhSINE2).


Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping.

Kumari V, Iyer LR, Roy R, Bhargava V, Panda S, Paul J, Verweij JJ, Clark CG, Bhattacharya A, Bhattacharya S - BMC Genomics (2013)

Detection and validation of EhSINE1 polymorphic loci 13, 17, 19 and 42. (A) Schematic representation of primers designed from different loci. The hollow box represents the EhSINE1 element; flanking genes have not been shown for simplicity. (B) PCR was performed using genomic DNA of HM-1:IMSS (H) and Rahman (R) strains as template, using primers from sequences flanking the EhSINE1 copy as shown in the schematic representation. The size of amplicons was determined by electrophoresis in 1% agarose gels (Top panel). Of 4 SINE1 unoccupied sites found computationally two were tested (13 and 42). Two more (17 and 19) were evaluated by PCR and Southern Blotting in Rahman. The sizes of amplicons obtained are indicated on the right, with arrows. The amplicon from strain Rahman was shorter by ~550 bp (the size of EhSINE1) at loci 13, 17 and 19, but was longer at locus 42 (explained in the text). The absence of EhSINE1 was further confirmed by Southern blotting with EhSINE1 probe, which failed to hybridize with the amplicons from strain Rahman (Bottom panel).
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Related In: Results  -  Collection

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Figure 3: Detection and validation of EhSINE1 polymorphic loci 13, 17, 19 and 42. (A) Schematic representation of primers designed from different loci. The hollow box represents the EhSINE1 element; flanking genes have not been shown for simplicity. (B) PCR was performed using genomic DNA of HM-1:IMSS (H) and Rahman (R) strains as template, using primers from sequences flanking the EhSINE1 copy as shown in the schematic representation. The size of amplicons was determined by electrophoresis in 1% agarose gels (Top panel). Of 4 SINE1 unoccupied sites found computationally two were tested (13 and 42). Two more (17 and 19) were evaluated by PCR and Southern Blotting in Rahman. The sizes of amplicons obtained are indicated on the right, with arrows. The amplicon from strain Rahman was shorter by ~550 bp (the size of EhSINE1) at loci 13, 17 and 19, but was longer at locus 42 (explained in the text). The absence of EhSINE1 was further confirmed by Southern blotting with EhSINE1 probe, which failed to hybridize with the amplicons from strain Rahman (Bottom panel).
Mentions: Of the eight predicted polymorphic loci in strain Rahman we validated experimentally six using PCR (Figures 3 and 4) with primers designed from the flanking sequences of EhSINE1/EhSINE2 in HM-1:IMSS (Additional file 1: Figure S1 and Additional file 2: Table S1). The absence of SINE sequences was inferred from the size of the amplicon (smaller by the size of SINE) and by Southern hybridization using a SINE sequence as a probe. The amplicon sizes in Rahman from three EhSINE1 polymorphic loci (13, 17 and 19) were smaller by about 550 bp suggesting that indeed these sites lacked EhSINE1. This was also confirmed by Southern hybridization (Figure 3B, bottom panel). In contrast, the amplicon size of another polymorphic EhSINE1 locus (42) was actually larger by 1.5 kb in Rahman. Probing a Southern blot of the amplicon using EhSINE1-flanking sequences from locus 42 confirmed that the amplified region in Rahman indeed belonged to the same locus (Additional file 3: Figure S2). However, two different sets of primers designed using the HM-1:IMSS sequence at this locus failed to produce an amplicon in Rahman. Therefore it appears that this locus may have undergone multiple changes and is not a simple case of SINE absence. We did not analyse this locus further. The two predicted EhSINE2 polymorphic loci (18 and 50) were also validated using PCR and Southern hybridization (Figure 4 ii and iii). At both loci the amplicons from Rahman were 700 bp shorter (the size of EhSINE2).

Bottom Line: Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes.The results obtained by our method correlated with the data from other typing methods.Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACT

Background: The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains.

Results: We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies.

Conclusions: Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.

Show MeSH
Related in: MedlinePlus