Archives of Medical Research, Volume 31, Issue 4, Pages S269-S270, July 2000, Authors:Suchismita ... more Archives of Medical Research, Volume 31, Issue 4, Pages S269-S270, July 2000, Authors:Suchismita Das; Anuradha Lohia.
Fidelity in transmission of genetic characters is ensured by the faithful duplication of the geno... more Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite,Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered inE. histolytica.
1. Mol Biochem Parasitol. 1996 Nov 25;82(2):257-60. Overexpression of P-glycoprotein gene 1 by tr... more 1. Mol Biochem Parasitol. 1996 Nov 25;82(2):257-60. Overexpression of P-glycoprotein gene 1 by transfected Entamoeba histolytica confers emetine-resistance. Ghosh SK, Lohia A, Kumar A, Samuelson J. Department of Tropical ...
Earlier studies have established two unusual features in the cell division cycle of Entamoeba his... more Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1× to 6× or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8–12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20–25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1× and 2× unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors – monastrol and HR22C16 analogues.
L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synth... more L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants. Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba. However, there is no report of I-1-P synthase or its gene from these organisms. The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants. Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica. Simultaneously, we have purified and characterised the native enzyme from E. histolytica trophozoites and the cloned gene product from Escherichia coli. The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis. Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.
Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not... more Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 °C or 37 °C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length.
An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP... more An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP-binding protein, most similar to the ras homologue rho. The Eh rho1 open reading frame was 208 amino acids long and encoded a 23-kDa protein similar to Saccharomyces cerevisiae RHO1-RHO4 and CDC42 and human rhoA, rac1, and G25K gene products. This similarity was greatest at the NH2 terminus of Eh rho1 where two GTP-binding sites and a possible effector site were conserved. A cysteine residue at the COOH terminus of Eh rho1 was followed by eight hydrophobic amino acids rather than the three hydrophobic amino acids present in other ras family proteins.
The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark... more The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark in the cell cycle of most well-studied eukaryotes and ensures that the progeny do not have more than the normal complement of genes and chromosomes. An exception to this rule has been described in cancer cells that occasionally become polyploid as a result of failure to restrain S phase despite the failure to undergo complete mitosis. Here, we describe the cell division cycle of the human pathogen, Entamoeba histolytica, which routinely accumulates polyploid cells. We have studied DNA synthesis in freshly subcultured cells and show that, unlike most eukaryotes, Entamoeba cells reduplicate their genome several times before cell division occurs. Furthermore, polyploidy may occur without nuclear division so that single nuclei may contain 1-10 times or more genome contents. Multinucleated cells may also accumulate several genome contents in each nuclei of one cell. Thus, checkpoints that normally prevent DNA reduplication until after cytokinesis in most eukaryotes are not observed in E. histolytica.
The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometr... more The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometry in asynchronous and partially synchronised cells. Dynamic changes in the DNA synthesis and DNA content of axenically growing trophozoites were observed by using 5-bromo-2&#39;-deoxyuridine (BrdU) uptake and DNA specific fluorochromes. It was observed that DNA synthesis in these cells continues beyond the typical S-phase stop point when DNA duplication is complete. Asynchronously growing E. histolytica cells could be synchronised by serum starvation followed by serum re-addition. BrdU incorporation in synchronised cells showed that cell synchrony is maintained for at least one generation time, in which the G1 phase lasts for 2-3 h and the S-phase lasts for 5-6 h. Analysis of our results revealed that E. histolytica trophozoites, growing in axenic medium, are made up of a heterogenous population of euploid and polyploid cells. The number of polyploid cells increases with age of the cells in culture. Expression of putative cell cycle and signal transduction markers was studied using specific antibodies and changes in their expression levels have been correlated with changes in the DNA content. Based upon our results we could identify G1, S and G2 phases of the cell cycle of E. histolytica and also predict the mechanism underlying the generation of polyploidy in these cells, which may have significant effects on its biology and pathogenesis.
Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica.... more Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.
We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic... more We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic protozoan Entamoeba histolytica. The full length genomic and cDNA clones for the Eh MCM3 gene have been characterised. The Eh MCM3 gene is much smaller than the Saccharomyces cerevisiae MCM3 gene and other eukaryotic homologs of the MCM3/P1 family. The predicted Eh Mcm3 protein was 597 amino acids long and showed 37 and 46% positional identity with the Sc Mcm3 and the mouse P1 homologs respectively. While proceeding along the chromosome from the Eh MCM3 gene, we have identified a homolog (Eh PAK) of the murine p21 activated kinase (Rn KPAK), or S. cerevisiae STE20. Eh PAK lies 126 bp upstream of the Eh MCM3 gene. The predicted Eh p21 activated kinase protein was 459 amino-acids long and showed 33% positional identity with the murine p21 activated kinase and its yeast homolog Ste20. Analysis of cDNA and genomic sequences shows that the 3&#39; untranslated region (UTR) of the Eh PAK mRNA and the 5&#39; UTR of the Eh MCM3 mRNA are transcribed from a common 40 bp genomic segment. This is the first report of an amoeba gene being physically linked to a second gene such that their transcripts are overlapping and there is no non-transcribed intergenic region between the two genes. Primer extension studies have confirmed that unlike most E. histolytica genes, which have short 5&#39; UTRs, the Eh MCM3 mRNA has a 126 bp long 5&#39; UTR and the Eh PAK mRNA has a 265 bp long 5&#39; UTR.
Archives of Medical Research, Volume 31, Issue 4, Pages S269-S270, July 2000, Authors:Suchismita ... more Archives of Medical Research, Volume 31, Issue 4, Pages S269-S270, July 2000, Authors:Suchismita Das; Anuradha Lohia.
Fidelity in transmission of genetic characters is ensured by the faithful duplication of the geno... more Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite,Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered inE. histolytica.
1. Mol Biochem Parasitol. 1996 Nov 25;82(2):257-60. Overexpression of P-glycoprotein gene 1 by tr... more 1. Mol Biochem Parasitol. 1996 Nov 25;82(2):257-60. Overexpression of P-glycoprotein gene 1 by transfected Entamoeba histolytica confers emetine-resistance. Ghosh SK, Lohia A, Kumar A, Samuelson J. Department of Tropical ...
Earlier studies have established two unusual features in the cell division cycle of Entamoeba his... more Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1× to 6× or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8–12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20–25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1× and 2× unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors – monastrol and HR22C16 analogues.
L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synth... more L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants. Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba. However, there is no report of I-1-P synthase or its gene from these organisms. The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants. Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica. Simultaneously, we have purified and characterised the native enzyme from E. histolytica trophozoites and the cloned gene product from Escherichia coli. The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis. Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.
Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not... more Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 °C or 37 °C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length.
An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP... more An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP-binding protein, most similar to the ras homologue rho. The Eh rho1 open reading frame was 208 amino acids long and encoded a 23-kDa protein similar to Saccharomyces cerevisiae RHO1-RHO4 and CDC42 and human rhoA, rac1, and G25K gene products. This similarity was greatest at the NH2 terminus of Eh rho1 where two GTP-binding sites and a possible effector site were conserved. A cysteine residue at the COOH terminus of Eh rho1 was followed by eight hydrophobic amino acids rather than the three hydrophobic amino acids present in other ras family proteins.
The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark... more The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark in the cell cycle of most well-studied eukaryotes and ensures that the progeny do not have more than the normal complement of genes and chromosomes. An exception to this rule has been described in cancer cells that occasionally become polyploid as a result of failure to restrain S phase despite the failure to undergo complete mitosis. Here, we describe the cell division cycle of the human pathogen, Entamoeba histolytica, which routinely accumulates polyploid cells. We have studied DNA synthesis in freshly subcultured cells and show that, unlike most eukaryotes, Entamoeba cells reduplicate their genome several times before cell division occurs. Furthermore, polyploidy may occur without nuclear division so that single nuclei may contain 1-10 times or more genome contents. Multinucleated cells may also accumulate several genome contents in each nuclei of one cell. Thus, checkpoints that normally prevent DNA reduplication until after cytokinesis in most eukaryotes are not observed in E. histolytica.
The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometr... more The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometry in asynchronous and partially synchronised cells. Dynamic changes in the DNA synthesis and DNA content of axenically growing trophozoites were observed by using 5-bromo-2&#39;-deoxyuridine (BrdU) uptake and DNA specific fluorochromes. It was observed that DNA synthesis in these cells continues beyond the typical S-phase stop point when DNA duplication is complete. Asynchronously growing E. histolytica cells could be synchronised by serum starvation followed by serum re-addition. BrdU incorporation in synchronised cells showed that cell synchrony is maintained for at least one generation time, in which the G1 phase lasts for 2-3 h and the S-phase lasts for 5-6 h. Analysis of our results revealed that E. histolytica trophozoites, growing in axenic medium, are made up of a heterogenous population of euploid and polyploid cells. The number of polyploid cells increases with age of the cells in culture. Expression of putative cell cycle and signal transduction markers was studied using specific antibodies and changes in their expression levels have been correlated with changes in the DNA content. Based upon our results we could identify G1, S and G2 phases of the cell cycle of E. histolytica and also predict the mechanism underlying the generation of polyploidy in these cells, which may have significant effects on its biology and pathogenesis.
Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica.... more Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.
We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic... more We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic protozoan Entamoeba histolytica. The full length genomic and cDNA clones for the Eh MCM3 gene have been characterised. The Eh MCM3 gene is much smaller than the Saccharomyces cerevisiae MCM3 gene and other eukaryotic homologs of the MCM3/P1 family. The predicted Eh Mcm3 protein was 597 amino acids long and showed 37 and 46% positional identity with the Sc Mcm3 and the mouse P1 homologs respectively. While proceeding along the chromosome from the Eh MCM3 gene, we have identified a homolog (Eh PAK) of the murine p21 activated kinase (Rn KPAK), or S. cerevisiae STE20. Eh PAK lies 126 bp upstream of the Eh MCM3 gene. The predicted Eh p21 activated kinase protein was 459 amino-acids long and showed 33% positional identity with the murine p21 activated kinase and its yeast homolog Ste20. Analysis of cDNA and genomic sequences shows that the 3&#39; untranslated region (UTR) of the Eh PAK mRNA and the 5&#39; UTR of the Eh MCM3 mRNA are transcribed from a common 40 bp genomic segment. This is the first report of an amoeba gene being physically linked to a second gene such that their transcripts are overlapping and there is no non-transcribed intergenic region between the two genes. Primer extension studies have confirmed that unlike most E. histolytica genes, which have short 5&#39; UTRs, the Eh MCM3 mRNA has a 126 bp long 5&#39; UTR and the Eh PAK mRNA has a 265 bp long 5&#39; UTR.
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