Gretchen Ehrenkaufer

All Publications

• SPARKing academic technologies across the valley of death. Nature biotechnology Kim, J. S., Kargotich, S., Lee, S. H., Yajima, R., Garcia, A. A., Ehrenkaufer, G., Romeo, M., Maria, P. S., Grimes, K. V., Mochly-Rosen, D. 2024; 42 (2): 339-342

  View details for DOI 10.1038/s41587-024-02130-5

  View details for PubMedID 38361072

  View details for PubMedCentralID 6226120

• High-throughput phenotypic screen identifies a new family of potent anti-amoebic compounds. PloS one Sauvey, C., Meewan, I., Ehrenkaufer, G., Blevitt, J., Jackson, P., Abagyan, R. 2023; 18 (5): e0280232

  Abstract

  Entamoeba histolytica is a disease-causing parasitic amoeba which affects an estimated 50 million people worldwide, particularly in socioeconomically vulnerable populations experiencing water sanitation issues. Infection with E. histolytica is referred to as amoebiasis, and can cause symptoms such as colitis, dysentery, and even death in extreme cases. Drugs exist that are capable of killing this parasite, but they are hampered by downsides such as significant adverse effects at therapeutic concentrations, issues with patient compliance, the need for additional drugs to kill the transmissible cyst stage, and potential development of resistance. Past screens of small and medium sized chemical libraries have yielded anti-amoebic candidates, thus rendering high-throughput screening a promising direction for new drug discovery in this area. In this study, we screened a curated 81,664 compound library from Janssen pharmaceuticals against E. histolytica trophozoites in vitro, and from it identified a highly potent new inhibitor compound. The best compound in this series, JNJ001, showed excellent inhibition activity against E. histolytica trophozoites with EC50 values at 0.29 μM, which is better than the current approved treatment, metronidazole. Further experimentation confirmed the activity of this compound, as well as that of several structurally related compounds, originating from both the Janssen Jump-stARter library, and from chemical vendors, thus highlighting a new structure-activity relationship (SAR). In addition, we confirmed that the compound inhibited E. histolytica survival as rapidly as the current standard of care and inhibited transmissible cysts of the related model organism Entamoeba invadens. Together these results constitute the discovery of a novel class of chemicals with favorable in vitro pharmacological properties. The discovery may lead to an improved therapy against this parasite and in all of its life stages.

  View details for DOI 10.1371/journal.pone.0280232

  View details for PubMedID 37159460

• Stress Response in Entamoeba histolytica Is Associated with Robust Processing of tRNA to tRNA Halves. mBio Sharma, M., Zhang, H., Ehrenkaufer, G., Singh, U. 2023: e0345022

  Abstract

  tRNA-derived fragments have been reported in many different organisms and have diverse cellular roles, such as regulating gene expression, inhibiting protein translation, silencing transposable elements, and modulating cell proliferation. In particular, tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here, we report the presence of tRNA-derived fragments in Entamoeba, with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite-to-cyst conversion, with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves, as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments associated with Entamoeba Argonaute proteins, EhAgo2-2 and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoebas. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses, including encystation, suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba. IMPORTANCE In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small-RNA sequencing data sets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during the developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway, which mediates robust gene silencing in Entamoeba. We noticed that in response to heat shock, the protein translation levels were elevated in the parasites. This effect was reversed in the presence of an analog of leucine, which also reduced the levels of the tRNA halves in the stressed cells. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.

  View details for DOI 10.1128/mbio.03450-22

  View details for PubMedID 36809068

• Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds PLOS NEGLECTED TROPICAL DISEASES Sauvey, C., Ehrenkaufer, G., Shi, D., Debnath, A., Abagyan, R. 2021; 15 (2): e0008425

  Abstract

  Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300-400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.

  View details for DOI 10.1371/journal.pntd.0008425

  View details for Web of Science ID 000617380500002

  View details for PubMedID 33556060

  View details for PubMedCentralID PMC7895358

• Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control. BMC genomics Zhang, H., Ehrenkaufer, G. M., Hall, N., Singh, U. 2020; 21 (1): 879

  Abstract

  BACKGROUND: The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27nt antisense sRNA populations which associate with EhAgo2-2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward.RESULTS: In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3'-end of the 27nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27nt and 31nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27nt sRNAs with 5'-polyphosphate (5'-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31nt sRNAs associate with EhAgo2-2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite.CONCLUSION: We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

  View details for DOI 10.1186/s12864-020-07275-6

  View details for PubMedID 33297948

• Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity PLOS NEGLECTED TROPICAL DISEASES Ehrenkaufer, G., Li, P., Stebbins, E. E., Kangussu-Marcolino, M. M., Debnath, A., White, C., Moser, M. S., DeRisi, J., Gisselberg, J., Yeh, E., Wang, S. C., Company, A., Monti, L., Caffrey, C. R., Huston, C. D., Wang, B., Singh, U. 2020; 14 (3)

  View details for DOI 10.1371/journal.pntd.0008150.r004

  View details for Web of Science ID 000528655400050

• Characterization of extracellular vesicles from Entamoeba histolytica identifies roles in intercellular communication that regulates parasite growth and development. Infection and immunity Sharma, M. n., Morgado, P. n., Zhang, H. n., Ehrenkaufer, G. n., Manna, D. n., Singh, U. n. 2020

  Abstract

  Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins including proteins associated with vesicle formation, cell-signaling, metabolism, and cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27nt in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNAi pathway proteins, including Argonaute were also present in amebic EVs. Interestingly, we found that the amebic EVs impact inter-cellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveals that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication amongst parasites and influence growth kinetics.

  View details for DOI 10.1128/IAI.00349-20

  View details for PubMedID 32719158

• Entamoeba stage conversion: progress and new insights. Current opinion in microbiology Manna, D. n., Ehrenkaufer, G. M., Lozano-Amado, D. n., Singh, U. n. 2020; 58: 62–68

  Abstract

  Entamoeba histolytica, an anaerobic protozoan, is an important global health problem. This parasite has a biphasic life cycle consisting of a dormant cyst stage which is environmentally resistant and transmits the infection, and the proliferative trophozoite stage which is motile and causes invasive disease. The stage conversion process remains poorly understood despite being central to amoebic biology. In this review, we will highlight recent progress in our understanding of Entamoeba stage conversion including dissecting transcriptome analysis in development, characterization of transcriptional networks, demonstration of epigenetic regulation, and role of small molecules that regulate Entamoeba development.

  View details for DOI 10.1016/j.mib.2020.09.005

  View details for PubMedID 33032142

• The NAD+ Responsive Transcription Factor ERM-BP Functions Downstream of Cellular Aggregation and Is an Early Regulator of Development and Heat Shock Response in Entamoeba. Frontiers in cellular and infection microbiology Manna, D. n., Lozano-Amado, D. n., Ehrenkaufer, G. n., Singh, U. n. 2020; 10: 363

  Abstract

  Entamoeba histolytica is a protozoan parasite and a major cause of dysentery and diarrheal disease in developing countries. Disease transmission from one host to another occurs via cysts which can survive in environmental extremes and are transmitted through contaminated food and water. Recent studies in our lab identified a novel transcription factor, Encystation Regulatory Motif- Binding Protein (ERM-BP), which is responsive to NAD+ and has an important role in encystation. The key residues important for ERM-BP function were demonstrated in vitro using recombinant protein. In this study we demonstrate the in vivo functional consequences of mutations in key domains and their impact on Entamoeba encystation. Our results show that mutations in the DNA binding domain (ERM-BP-DBM) and in the nicotinamidase domain (ERM-BP-C198A) lead to protein mis-localization in both trophozoites and cysts and significantly reduce encystation efficiency. Additionally, we showed that silencing of ERM-BP significantly decreased the size and number of multi-nucleated giant cells (MGC) that form during encystation, indicating that ERM-BP functions upstream of the cellular aggregation that precedes stage conversion. Dissection of epistatic interactions between ERM-BP and a second encystation-related transcription factor, NF-Y revealed that ERM-BP is upstream of NF-Y in controlling the developmental cascade and appears to be one of the earliest regulators of development identified to date in Entamoeba. We also demonstrated that ERM-BP is upregulated during heat stress in Entamoeba, another condition which increases intracellular NAD+ levels and that overexpression of ERM-BP makes E. histolytica and E. invadens parasites more resistant to heat stress. Overexpression of ERM-BP in E. histolytica also induced the formation of cyst-like quadrinucleated cells and formation of MGCs. Overall, our work has identified an important role of ERM-BP in Entamoeba stress response and links an NAD+-responsive transcription factor to both development and heat shock response. Characterization of stress and developmental cascades are important avenues to investigate for Entamoeba, an important human parasitic pathogen.

  View details for DOI 10.3389/fcimb.2020.00363

  View details for PubMedID 32766170

  View details for PubMedCentralID PMC7379229

• Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia. International journal for parasitology. Drugs and drug resistance Kangussu-Marcolino, M. M., Ehrenkaufer, G. M., Chen, E., Debnath, A., Singh, U. 2019; 11: 80–94

  Abstract

  The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.

  View details for DOI 10.1016/j.ijpddr.2019.10.003

  View details for PubMedID 31707263

• Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization. mSphere Zhang, H., Tran, V., Manna, D., Ehrenkaufer, G., Singh, U. 2019; 4 (5)

  Abstract

  The RNA interference (RNAi) pathway regulates gene expression in many eukaryotic organisms. Argonaute (Ago) proteins, together with bound small RNAs (sRNAs), are key effectors that mediate gene silencing function. However, there is limited knowledge of Ago proteins and their functions in nonmodel systems. In the protozoan parasite Entamoeba histolytica, RNAi is a robust means for stable gene silencing mediated via large populations of antisense sRNAs. Here, we report functional characterization of three Ago proteins in E. histolytica (EhAgo2-1, EhAgo2-2, and EhAgo2-3). Our data show that each EhAgo protein has a distinct subcellular localization and binds 27-nucleotide (nt) sRNAs and that the localization of EhAgo proteins is altered in response to stress conditions. Via mutagenesis analyses, we demonstrated that the Ago PAZ (Piwi/Argonaute/Zwille) domain in all three EhAgos is essential for sRNA binding. With mutation of the PAZ domain in EhAgo2-2, there was no effect on the nuclear localization of the protein but a strong phenotype and a growth defect. We further show that EhAgo2-2 contains an unusual repetitive DR-rich (aspartic acid, arginine-rich) motif region which functions as a nuclear localization signal (NLS) and is both necessary and sufficient to mediate nuclear localization. Overall, our data delineate the localization and sRNA binding features of the three E. histolytica Ago proteins and demonstrate that the PAZ domain is necessary for sRNA binding. The repetitive DR-rich motif region in EhAgo2-2 has not previously been defined in other systems, which adds to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.IMPORTANCE The protozoan parasite Entamoeba histolytica, which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovered that three E. histolytica Ago proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) all bind 27-nt small RNAs and have distinct subcellular localizations, which change in response to stress conditions. The EhAgos bind small RNA populations via their PAZ domains. An unusual repetitive DR-rich motif region is identified in EhAgo2-2 that functions as a nuclear localization signal. Our results show for the first time an active nuclear transport process of the EhAgo2-2 RNA-induced silencing complex (RISC) in this parasite. These data add to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.

  View details for DOI 10.1128/mSphere.00580-19

  View details for PubMedID 31619501

• An NAD(+)-dependent novel transcription factor controls stage conversion in Entamoeba ELIFE Manna, D., Lentz, C., Ehrenkaufer, G., Suresh, S., Bhat, A., Singh, U. 2018; 7

  View details for DOI 10.7554/eLife.37912

  View details for Web of Science ID 000448684300001

• High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY Ehrenkaufer, G. M., Suresh, S., Solow-Cordero, D., Singh, U. 2018; 8

  View details for DOI 10.3389/fcimb.2018.00276

  View details for Web of Science ID 000441933600001

• Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens. Infection and immunity Suresh, S., Ehrenkaufer, G., Zhang, H., Singh, U. 2016; 84 (4): 964-975

  Abstract

  Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission.Entamoeba invadensis the model system to studyEntamoebadevelopmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools inE. invadenshas limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway inEntamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence twoE. invadensgenes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis ofEntamoebastage conversion is now possible, representing an important technical advance for the system.

  View details for DOI 10.1128/IAI.01161-15

  View details for PubMedID 26787723

  View details for PubMedCentralID PMC4807475

• High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress PLOS ONE Zhang, H., Ehrenkaufer, G. M., Manna, D., Hall, N., Singh, U. 2015; 10 (8)

  Abstract

  The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  View details for DOI 10.1371/journal.pone.0134481

  View details for Web of Science ID 000359062300039

  View details for PubMedID 26248204

• Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns INTERNATIONAL JOURNAL FOR PARASITOLOGY Manna, D., Ehrenkaufer, G. M., Singh, U. 2014; 44 (11): 837-845

  Abstract

  Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

  View details for DOI 10.1016/j.ijpara.2014.06.008

  View details for Web of Science ID 000343361100009

• Small RNA pyrosequencing in the protozoan parasite Entamoeba histolytica reveals strain-specific small RNAs that target virulence genes BMC GENOMICS Zhang, H., Ehrenkaufer, G. M., Hall, N., Singh, U. 2013; 14

  Abstract

  Small RNA mediated gene silencing is a well-conserved regulatory pathway. In the parasite Entamoeba histolytica an endogenous RNAi pathway exists, however, the depth and diversity of the small RNA population remains unknown.To characterize the small RNA population that associates with E. histolytica Argonaute-2 (EhAGO2-2), we immunoprecipitated small RNAs that associate with it and performed one full pyrosequencing run. Data analysis revealed new features of the 27nt small RNAs including the 5'-G predominance, distinct small RNA distribution patterns on protein coding genes, small RNAs mapping to both introns and exon-exon junctions, and small RNA targeted genes that are clustered particularly in sections of genome duplication. Characterization of genomic loci to which both sense and antisense small RNAs mapped showed that both sets of small RNAs have 5'-polyphosphate termini; strand-specific RT-PCR detected transcripts in both directions at these loci suggesting that both transcripts may serve as template for small RNA generation. In order to determine whether small RNA abundance patterns account for strain-specific gene expression profiles of E. histolytica virulent and non-virulent strains, we sequenced small RNAs from a non-virulent strain and found that small RNAs mapped to genes in a manner consistent with their regulation of strain-specific virulence genes.We provided a full spectrum analysis for E. histolytica AGO2-2 associated 27nt small RNAs. Additionally, comparative analysis of small RNA populations from virulent and non-virulent amebic strains indicates that small RNA populations may regulate virulence genes.

  View details for DOI 10.1186/1471-2164-14-53

  View details for Web of Science ID 000316679600001

  View details for PubMedID 23347563

  View details for PubMedCentralID PMC3610107

• The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation GENOME BIOLOGY Ehrenkaufer, G. M., Weedall, G. D., Williams, D., Lorenzi, H. A., Caler, E., Hall, N., Singh, U. 2013; 14 (7)

  Abstract

  Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion.We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains.Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.

  View details for DOI 10.1186/gb-2013-14-7-r77

  View details for Web of Science ID 000328194900009

• Transient and stable transfection in the protozoan parasite Entamoeba invadens MOLECULAR AND BIOCHEMICAL PARASITOLOGY Ehrenkaufer, G. M., Singh, U. 2012; 184 (1): 59-62

  Abstract

  Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.

  View details for DOI 10.1016/j.molbiopara.2012.04.007

  View details for Web of Science ID 000305112200011

  View details for PubMedID 22561071

  View details for PubMedCentralID PMC3358517

• Entamoeba histolytica Genomic Analyses ANAEROBIC PARASITIC PROTOZOA: GENOMICS AND MOLECULAR BIOLOGY Singh, U., Ehrenkaufer, G. M., Clark, C. G., Johnson, P. J., Adam, R. D. 2010: 157–73

  View details for Web of Science ID 000277629500006

• A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica CELLULAR MICROBIOLOGY Ehrenkaufer, G. M., Hackney, J. A., Singh, U. 2009; 11 (6): 898-910

  Abstract

  Conversion between a cyst and trophozoite stage is essential to disease transmission and pathogenesis in the parasitic protist Entamoeba histolytica. A transcriptomic analysis of E. histolytica cysts and trophozoites has recently been accomplished, but the molecular basis of the regulation of encystation is not known. We have now identified a developmentally regulated Myb protein (belonging to the SHAQKY family of Myb proteins), which controls expression of a subset of amoebic stage-specific genes. Overexpression of the nuclear localized Myb protein resulted in a transcriptome that overlapped significantly with the expression profile of amoebic cysts. Analysis of promoters from genes regulated by the Myb protein identified a CCCCCC promoter motif to which amoebic nuclear protein(s) bind in a sequence-specific manner. Chromatin immunoprecipitation demonstrated that the E. histolytica Myb protein binds to promoters of genes which contain the CCCCCC motif and which are regulated by the Myb protein. This work is the first identification of a transcription factor, which regulates expression of a subset of stage-specific genes in E. histolytica. Identification of transcriptional regulatory networks that control developmental pathways will provide novel insights into the biology of this important human pathogen.

  View details for DOI 10.1111/j.1462-5822.2009.01300.x

  View details for Web of Science ID 000265883500005

  View details for PubMedID 19239479

  View details for PubMedCentralID PMC3424735

• Recent insights into Entamoeba development: Identification of transcriptional networks associated with stage conversion INTERNATIONAL JOURNAL FOR PARASITOLOGY Singh, U., Ehrenkaufer, G. M. 2009; 39 (1): 41-47

  Abstract

  Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death globally. The parasite life cycle alternates between the trophozoite form, which is motile and causes invasive disease and the cyst stage, which is environmentally resistant and transmits infection. Understanding the triggers that initiate stage conversion is an important yet understudied area of investigation. Recent progress in dissecting the transcriptional networks that regulate E. histolytica development is outlined in this paper.

  View details for DOI 10.1016/j.ijpara.2008.09.004

  View details for Web of Science ID 000262735000004

  View details for PubMedID 18938171

  View details for PubMedCentralID PMC2648836

• Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis CELLULAR MICROBIOLOGY Vicente, J. B., Ehrenkaufer, G. M., Saraiva, L. M., Teixeira, M., Singh, U. 2009; 11 (1): 51-69

  Abstract

  Upon host infection, the protozoan parasite Entamoeba histolytica is confronted with reactive oxygen and nitrogen species and must survive these stresses in order to cause invasive disease. We analysed the parasite's response to oxidative and nitrosative stresses, probing the transcriptional changes of trophozoites of a pathogenic strain after a 60 min exposure to H2O2 (1 mM) or a NO donor (dipropylenetriamine-NONOate, 200 microM), using whole-genome DNA microarrays. Genes encoding reactive oxygen and nitrogen species detoxification enzymes had high transcriptional levels under basal conditions and upon exposure to both stresses. On a whole-genome level, there was significant modulation of gene expression by H2O2 (286 genes regulated) and dipropylenetriamine-NONOate (1036 genes regulated) with a significant overlap of genes modulated under both conditions (164 genes). A number of transcriptionally regulated genes were in signalling/regulatory and repair/metabolic pathways. However, the majority of genes with altered transcription encode unknown proteins, suggesting as yet unraveled response pathways in E. histolytica. Trophozoites of a non-pathogenic E. histolytica strain had a significantly muted transcriptional response to H2O2 compared with the pathogenic strain, hinting that differential response to oxidative stress may be one factor that contributes to the pathogenic potential of E. histolytica.

  View details for DOI 10.1111/j.1462-5822.2008.01236.x

  View details for Web of Science ID 000262150100005

  View details for PubMedID 18778413

  View details for PubMedCentralID PMC3418052

• Small RNAs with 5 '- Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica PLOS PATHOGENS Zhang, H., Ehrenkaufer, G. M., Pompey, J. M., Hackney, J. A., Singh, U. 2008; 4 (11)

  Abstract

  Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5'-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5'-polyphosphate siRNAs extends to single-celled eukaryotic organisms.

  View details for DOI 10.1371/journal.ppat.1000219

  View details for Web of Science ID 000261481200023

  View details for PubMedID 19043551

  View details for PubMedCentralID PMC2582682

• Transcriptional Regulatory Networks in Entamoeba histolytica CURRENT DRUG TARGETS Ehrenkaufer, G. M., Singh, U. 2008; 9 (11): 931-937

  Abstract

  Expression profiling with microarray technology has revolutionized exploration of transcriptional regulatory networks on a genome-wide scale. This approach has been successfully applied to the study of Entamoeba histolytica, which causes dysentery and liver abscesses and is a leading parasitic cause of death globally. A variety of microarray platforms have been developed for this system including those generated from genomic DNA, long oligonucleotides, and short oligonucleotides. Using these tools researchers have identified parasite genes whose transcript abundance is differentially regulated during stress, host invasion, and stage conversion. Additionally, novel virulence factors have been identified by identifying genes that are highly expressed in virulent but with low expression in non-virulent Entamoeba strains. All combined, these studies have provided new data on molecular aspects of amebic biology, pathogenic potential and stage conversion and provide investigators with the first insights into potential novel drug targets against amebic disease.

  View details for Web of Science ID 000261495900003

  View details for PubMedID 18991605

• Identification of developmentally regulated genes in Entamoeba histolytica Ehrenkaufer, G. M., Singh, U. AMER SOC TROP MED & HYGIENE. 2007: 300-301

  View details for Web of Science ID 000250758201506

• Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica BMC GENOMICS Ehrenkaufer, G. M., Eichinger, D. J., Singh, U. 2007; 8

  Abstract

  Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known.In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)).This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

  View details for DOI 10.1186/1471-2164-8-216

  View details for Web of Science ID 000248701500001

  View details for PubMedID 17612405

  View details for PubMedCentralID PMC1940012

• Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite CELLULAR MICROBIOLOGY Ehrenkaufer, G. M., Haque, R., Hackney, J. A., Eichinger, D. J., Singh, U. 2007; 9 (6): 1426-1444

  Abstract

  Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite strains derived from recent human infections and whole-genome transcriptional profiling, we determined that 1439 genes (approximately 15% of annotated genes) were potentially developmentally regulated. Genes enriched in cysts (672 in total) included cysteine proteinases and transmembrane protein kinases, which may be involved in signal transduction. Genes enriched in trophozoites (767 in total) included genes typically thought of as important in tissue invasion by trophozoites, including the Gal/GalNAc lectin light subunit and cysteine protease 1. Putative regulators of differentiation including possible G-protein coupled receptors, signal transduction proteins and transcription factors were identified. A number of E. histolytica stage-specific genes were also developmentally regulated in the reptilian parasite E. invadens, indicating that they likely have conserved functions in Entamoeba development. These advances lay the groundwork for dissection of the molecular signals that initiate stage conversion and development of novel diagnostic and therapeutic measures targeting E. histolytica cysts.

  View details for DOI 10.1111/j.1462-5822.2006.00882.x

  View details for Web of Science ID 000246577400006

  View details for PubMedID 17250591

• Identification of putative transcriptional regulatory networks in Entamoeba histolytica using Bayesian inference NUCLEIC ACIDS RESEARCH Hackney, J. A., Ehrenkaufer, G. M., Singh, U. 2007; 35 (7): 2141-2152

  Abstract

  Few transcriptional regulatory networks have been described in non-model organisms. In Entamoeba histolytica seminal aspects of pathogenesis are transcriptionally controlled, however, little is known about transcriptional regulatory networks that effect gene expression in this parasite. We used expression data from two microarray experiments, cis-regulatory motif elucidation, and a naïve Bayesian classifier to identify genome-wide transcriptional regulatory patterns in E. histolytica. Our algorithm identified promoter motifs that accurately predicted the gene expression level of 68% of genes under trophozoite conditions. We identified a promoter motif ((A)/(T)AAACCCT) associated with high gene expression, which is highly enriched in promoters of ribosomal protein genes and tRNA synthetases. Additionally, we identified three promoter motifs (GAATGATG, AACTATTTAAACAT(C)/(T)C and TGAACTTATAAACATC) associated with low gene expression. The promoters of a large gene family were highly enriched for these motifs, and in these genes the presence of >/=2 motifs predicted low baseline gene expression and transcriptional activation by heat shock. We demonstrate that amebic nuclear protein(s) bind specifically to four of the motifs identified herein. Our analysis suggests that transcriptional regulatory networks can be identified using limited expression data. Thus, this approach is applicable to the multitude of systems for which microarray and genome sequence data are emerging.

  View details for DOI 10.1093/nar/gkm028

  View details for Web of Science ID 000246294700013

  View details for PubMedID 17355990

  View details for PubMedCentralID PMC1874630

• Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression BMC GENOMICS Ali, I. K., Ehrenkaufer, G. M., Hackney, J. A., Singh, U. 2007; 8

  Abstract

  In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown.In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (approximately 95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 microM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities.This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.

  View details for DOI 10.1186/1471-2164-8-7

  View details for PubMedID 17207281

• DNA methylation has limited effect on gene expression in Entamoeba histolytica Ali, I. K., Ehrenkaufer, G. M., Hackney, J. A., Singh, U. AMER SOC TROP MED & HYGIENE. 2006: 179

  View details for Web of Science ID 000242343901056

• Microarray mediated transcriptome comparison of Entamoeba histolytica trophozoites in vivo and in vitro Gilchrist, C. A., Houpt, E., Trapaidze, N., Fei, Z., Crasta, O., Sobral, B., Asgharpour, A., Evans, C., Martino-Catt, S., Baba, D. J., Stroup, S., Hamano, S., Ehrenkaufer, G., Okada, M., Singh, U., Nozaki, T., Mann, B. J., Petri, W. A. AMER SOC TROP MED & HYGIENE. 2006: 112-113

  View details for Web of Science ID 000242343900385

• Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome MOLECULAR AND BIOCHEMICAL PARASITOLOGY Gilchrist, C. A., Houpt, E., Trapaidze, N., Fei, Z., Crasta, O., Asgharpour, A., Evans, C., Martino-Catt, S., Baba, D. J., Stroup, S., Hamano, S., Ehrenkaufer, G., Okada, M., Singh, U., Nozaki, T., Mann, B. J., Petri, W. A. 2006; 147 (2): 163-176

  Abstract

  A genome-wide transcriptional analysis of Entamoeba histolytica was performed on trophozoites isolated from the colon of six infected mice and from in vitro culture. An Affymetrix platform gene expression array was designed for this analysis that included probe sets for 9435 open reading frames (ORFs) and 9066 5' and 3' flanking regions. Transcripts were detected for > 80% of all ORFs. A total of 523 transcripts (5.2% of all E. histolytica genes) were significantly changed in amebae isolated from the intestine on Days 1 and 29 after infection: 326 and 109 solely on Days 1 and 29, and 88 on both days. Quantitative real-time reverse transcriptase PCR confirmed these changes in 11/12 genes tested using mRNA isolated from an additional six mice. Adaptation to the intestinal environment was accompanied by increases in a subset of cell signaling genes including transmembrane kinases, ras and rho family GTPases, and calcium binding proteins. Significant decreases in mRNA abundance for genes involved in glycolysis and concomitant increases in lipases were consistent with a change in energy metabolism. Defense against bacteria present in the intestine (but lacking from in vitro culture) was suggested by alterations in mRNA levels of genes similar to the AIG1 plant antibacterial proteins. Decreases in oxygen detoxification pathways were observed as expected in the anaerobic colonic lumen. Of the known virulence factors the most remarkable changes were a 20-35-fold increase in a cysteine proteinase four-like gene, and a 2-3-fold decrease in two members of the Gal/GalNAc lectin light subunit family. Control of the observed changes in mRNA abundance in the intestine might potentially rest with four related proteins with DNA binding domains that were down-regulated 6-16-fold in the intestinal environment. In conclusion, the first genome-wide analysis of the transcriptome of E. histolytica demonstrated that the vast majority of genes are transcribed in trophozoites, and that in the host intestine trophozoites altered the expression of mRNAs for genes implicated in metabolism, oxygen defense, cell signaling, virulence, antibacterial activity, and DNA binding.

  View details for DOI 10.1016/j.molbiopara.2006.02.007

  View details for Web of Science ID 000237525600003

  View details for PubMedID 16569449

• Transcription of the Dictyostelium glycogen phosphorylase-2 gene is induced by three large promoter domains DEVELOPMENTAL GENETICS Favis, R., McCaffery, Ehrenkaufer, G., Rutherford, C. L. 1998; 23 (3): 230-246

  Abstract

  The promoter of the Dictyostelium glycogen phosphorylase-2 (gp2) gene possesses a profound AT-bias, typical of promoters in this organism. To understand how Dictyostelium achieves specificity during transcriptional regulation under the constraint of this highly biased nucleotide composition, we have documented the changes in chromatin structure associated with developmental induction of gp2 gene expression. DNase I hypersensitive analyses indicated the presence of several developmentally regulated nuclease-sensitive sites located upstream of the start codon: two strong sites at approximately -250 bp and -350 bp and three substantially weaker sites at -290 bp, -445 bp, and -505 bp. In vitro footprint analyses using nuclear extracts derived from several stages of development (corresponding to varying levels of gp2 expression) revealed three large regions of occupation that were developmentally regulated and corresponded to these nuclease-sensitive sites: -227 to -294 bp (domain 1), -327 to -383 bp (domain 2), and -416 to -534 bp (domain 3). The presence and the extent of the three regulatory domains was confirmed by in vivo footprint analyses spanning the same developmental time points. Southwestern analyses using probes encompassing these footprints demonstrated that probes corresponding to domains 1 and 3 both interacted with 83 and 77 kDa peptides. The domain 3 probe also interacted with a 92 kDa peptide, while only a 62 kDa peptide is recognized by the domain 2 probe. In all cases, peptides capable of binding these probes were found in nuclear extracts derived from differentiated cells and not in undifferentiated cell nuclear extract. Using nuclear extract from differentiated cells and probes corresponding to the three domains, gel mobility shift analyses detected ladders of retarded bands for both domains 1 and 3 and three major retarded bands for domain 2. These results suggest that specificity in transcriptional activation in the AT-rich promoters of Dictyostelium may be achieved by requiring multiple protein-DNA and/or protein-protein interactions to occur before induction can proceed.

  View details for Web of Science ID 000077186000008

  View details for PubMedID 9842717