Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

Virulence factors of Entamoeba histolytica.

Recent studies have increased our knowledge of Entamoeba histolytica cell biology and gene regulation. In the ameba, dominant-negative mutations in the Gal/GalNAc lectin affect adhesion and cytolysis, whereas mutations in meromyosin affect cytoskeletal function. Studying these mutant proteins has improved our understanding of the role of these proteins in E. histolytica virulence. The characterization of the CP5 cysteine protease and the induction of apoptosis in host target cells has led to a better comprehension of the mechanisms by which trophozoites can lyse target cells.

Association of UNP, a ubiquitin-specific protease, with the pocket proteins pRb, p107 and p130.

The murine Unp gene encodes a widely expressed ubiquitin-specific protease. The predicted sequence of the UNP protein features motifs common to viral oncoproteins through which these proteins interact with the retinoblastoma gene product pRb, as well as the related 'pocket proteins' p107 and p130. We have explored the possibility that UNP interacts with pocket proteins, and report here that such associations can be detected in vitro and in cells. Associations of UNP and pocket proteins are sensitive to site-directed mutations in a manner directly analogous to those documented in viral oncoproteins. We conclude that within cells UNP does physically associate with pRb, and can also associate with p107 and p130.

Characterization of the ubiquitin-specific protease activity of the mouse/human Unp/Unph oncoprotein.

The ubiquitin-specific proteases (Ubps) are a family of largely dissimilar enzymes with two major conserved sequence regions, containing either a conserved cysteine residue or two conserved histidine residues, respectively. The murine Unp oncoprotein and its human homologue, Unph, both contain regions similar to the conserved Cys and His boxes common to all the Ubps. In this study we show that Unp and Unph are active deubiquitinating enzymes, being able to cleave ubiquitin from both natural and engineered linear ubiquitin-protein fusions, including the polyubiquitin precursor. Mutation of the conserved Unp Cys and His residues abolishes this activity, and identifies the likely His residue in the catalytic triad. Unp is tumorigenic when overexpressed in mice, leading to the suggestion that Unp may play a role in the regulation of ubiquitin-dependent protein degradation. We have demonstrated here that the high-level expression of Unp in yeast does not disrupt the degradation of the N-end rule substrate Tyr-beta-galactosidase (betagal), the non-N-end rule substrate ubiquitin-Pro-betagal, or the degradation of abnormal, canavanine-containing proteins. These data suggest that Unp is not a general modulator of ubiquitin-dependent proteolysis. However, Unp may have a role in the regulation of the degradation of a specific, as yet undescribed, substrate(s).

Genomic structure of Unp, a murine gene encoding a ubiquitin-specific protease.

The murine Unp gene encodes a ubiquitin-specific protease, a member of a family of enzymes that includes the product of the human tre-2 oncogene. The Unp gene has previously been mapped to chromosome 9. We have cloned in bacteriophage a 50 kilobase region of chromosome 9 containing the Unp gene, and have determined the nucleotide sequence of the gene. The gene has 22 exons, distributed over 47.4 kb. A processed ribosomal S2 pseudogene was identified in the third intron of the Unp gene. Expression of Unp is driven by a GC-rich, 'housekeeping' type promoter.

The estrogenic and antiestrogenic properties of tamoxifen in GH4C1 pituitary tumor cells are gene specific.

We have examined the effects of the antiestrogen tamoxifen (TAM) and the estrogen 17 beta-estradiol (E2) on several estrogen-regulated responses in GH4C1 pituitary tumor cells. After 5 days of treatment with either TAM (1.0 microM) or E2 (1.0 nM), the level of PRL mRNA was markedly increased when measured by the cytosolic dot blot procedure. In contrast, only E2 was able to increase the levels of beta-actin mRNA and cytosolic protein, suggesting that this estrogen may stimulate cell proliferation over the course of treatment. This apparent difference in the abilities of TAM and E2 to stimulate GH4C1 cell proliferation was examined directly. TAM had no effect on cell proliferation as evidenced by its inability to increase cellular DNA or deoxythymidine triphosphate incorporation by nuclei isolated from treated cells. In contrast, E2 stimulated cell proliferation as evidenced by increases in cellular DNA and deoxythymidine triphosphate incorporation by isolated nuclei. The abilities of TAM and E2 to induce progesterone receptor (PR) and PR mRNA were also examined. TAM was unable to increase the levels of PR or PR mRNA, whereas E2 was effective in both of these regards. When added in combination with E2, TAM acted as a classical antiestrogen, partially blocking the induction of PR by E2. To determine whether the inabilities of TAM to stimulate cell proliferation and induce PR were a function of TAM concentration, dose-response experiments were performed. TAM at concentrations ranging from 10(-8)-10(-6) M was effective in inducing PRL mRNA, but at none of the tested concentrations was TAM effective in stimulating cell proliferation or inducing PR.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient expression of luciferase in Entamoeba histolytica driven by the ferredoxin gene 5' and 3' regions.

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 x 10(3) relative light units per second (RLU s-1) when assayed representing a stimulation of 18,000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5' and 3' ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.

Epidermal growth factor induces prolactin mRNA in GH4C1 cells via a protein synthesis-dependent pathway.

Prolactin (PRL) gene expression is regulated through a complex network of signal transduction pathways activated by various hormones and growth factors. Estrogens regulate PRL gene transcription in vivo through both direct and indirect, protein synthesis-dependent, mechanisms. Therefore, we hypothesized that other stimulators of PRL gene transcription might also act via protein synthesis-dependent mechanisms. To test this hypothesis, we examined, in GH4C1 rat pituitary tumor cells, the effects of protein synthesis inhibitors on the induction of PRL mRNA by known stimulators of PRL gene transcription. Whereas induction by epidermal growth factor (EGF) was abolished by cycloheximide and puromycin, increases in PRL mRNA caused by thyrotropin releasing hormone, 12-O-tetradecanoylphorbol 13-acetate, forskolin, or dibutyryl cyclic AMP were unaffected. These data suggest that the induction of PRL mRNA by EGF may require the induced synthesis of an intermediary regulatory protein.

Estradiol and triiodothyronine increase production of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) by GH4C1 rat pituitary tumor cells.

The main purpose of this study was to examine the effect of 17 beta-estradiol (E2) on the production of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBP) by GH4C1 cells, a pituitary tumor cell line that displays many phenotypic properties of the anterior pituitary lactotroph. At a low population density (10,500 cells/cm2), E2 stimulated production of IGF-I by 4.2-fold. At this density, the antiestrogen tamoxifen (TAM) had no significant effect, whereas triiodothyronine (T3), which has been demonstrated to increase the level of IGF-I mRNA in the parental GH3 cell line, stimulated IGF-I production by 3.3-fold. Both E2 and T3 also stimulated GH4C1 cell proliferation at this population density. At a four-fold higher population density (42,000 cells/cm2), E2, TAM and T3 had little effect on IGF-I production. E2 failed to stimulate proliferation of GH4C1 cells at high density, and T3 stimulated proliferation to a lesser extent than observed at the low density. At the low population density, E2 and T3 stimulated production of IGFBP-3 by 6- and 11-fold, respectively. At high density, the abilities of E2 and T3 to stimulate IGFBP-3 production were somewhat reduced. TAM had no effect on IGFBP-3 production at either population density. These data indicate that E2 and T3 stimulate production by GH4C1 cells of IGF-I through a mechanism that is sensitive to changes in population density.

Identification of nerve growth factor-responsive sequences within the 5' region of the bovine preprotachykinin gene.

The production of substance P and the mRNA encoding its precursor (preprotachykinin, PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion (drg) neurons. To explore the mechanism by which NGF regulates the production of PPT mRNA, we have transfected PC12 cells and F11 cells with plasmids containing the bovine PPT promoter linked to the reporter gene chloramphenicol acetyltransferase (CAT). We have identified (i) functional elements within the PPT promoter which are necessary for expression in the absence of NGF and (ii) two separate regions, each of approximately 250 bp, which confer NGF responsiveness. Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes.

Evidence for the Role of Cyclic AMP-Responsive Elements in Human Virus Replication and Disease.

We review the involvement of the cyclic AMP responsive DNA element (CRE) and the ATF/CREB (activating transcription factor/CRE binding protein) family of transcription factors in the regulation and pathology of clinically important viruses that infect humans, including the herpesviridae, adenoviridae, parvoviridae, hepadnaviridae, and retroviridae families. CRE sequences found in specific regulatory elements of human viruses are listed, and the functional evidence for CRE activity, in the form of DNA binding assays, mutational studies, transfection and transcriptional activation experiments, or in vitro transcription assays, is summarized. Manipulation of cellular processes is required for virus replication in human cells following infection. A primary target of many viruses is the cellular transcription machinery, and several human viruses contain transcriptional activator and repressor proteins that affect cellular transcription. Through their effect on cellular transcription, viral genes alter the pattern of cellular gene expression, and thereby affect the differentiation state and cell cycle progression of the infected cell. We summarize evidence demonstrating that the CRE and its binding proteins are involved in the activity of the viruses, implicating their function in the pathogenesis of human diseases. The targeting of specific transcription factor pathways as a potential therapeutic approach is discussed. Copyright 1996 S. Karger AG, Basel

Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis.

The ubiquitin-proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G9 3A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild-type or dominant-negative mutant ubiquitin (Ub(K48R)). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9-12% of total ubiquitin. Mice expressing the G9 3A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G9 3A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild-type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.

Escherichia coli rep gene: sequence of the gene, the encoded helicase, and its homology with uvrD.

The sequence of a 2.67-kilobase section of the Escherichia coli chromosome that contains the rep gene has been determined. This gene codes for a protein of predicted Mr 72,800, a DNA helicase, which is also a single-stranded DNA-dependent ATPase. The sequenced region contains an open reading frame of the correct length and orientation to encode the Rep protein. A secondary structure for the protein can be formulated from the amino acid sequence. We have compared both the primary and the secondary structures of Rep with other proteins and find the greatest homology between Rep and E. coli helicase II, the product of the uvrD gene.

A single-stranded DNA binding protein which interacts with sequences within the bovine preprotachykinin promoter: regulation by nerve growth factor.

The production of substance P (SP) and the mRNA encoding its precursor preprotachykinin (PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion neurons. We have shown previously that two regions of the bovine PPT promoter are capable of mediating the induction by NGF of the downstream structural gene in transfected PC12 cells. Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes. We show here that PC12 cells contain a single-stranded DNA binding protein (SSBP-PC12) which can interact specifically with this site. Binding activity was increased by NGF treatment of PC12 cells.

A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.

Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.

Control of ferredoxin and Gal/GalNAc lectin gene expression in Entamoeba histolytica by a cis-acting DNA sequence.

The ferredoxin (fdx) and lectin (hgl5) promoters of Entamoeba histolytica contain the DNA sequence motif TATTCTATT (URE3). Previously we showed that mutation of the URE3 motif in the hgl5 lectin promoter results in an increase in promoter reporter activity. Mutation of this motif in the fdx promoter led to a 40-to-50% decrease in fdx promoter activity as measured by reporter gene activity and abundance of mRNA. E. histolytica nuclear proteins exhibited sequence-specific binding to the URE3 motif in electrophoretic mobility shift assays. These results support the regulation of both ferredoxin and lectin promoters by a nuclear protein(s) which recognizes the URE3 motif.

Escherichia coli rep gene: identification of the promoter and N terminus of the rep protein.

The functional Escherichia coli rep gene, which encodes the Mr 67,000 Rep helicase, has been localized within a 2.55-kilobase sequence. Its regulatory region has been characterized by the use of rep-lacZ fusions. The direction of transcription of the rep gene is clockwise on the E. coli chromosome, as are the nearby ilvC and rho genes. The sequence of the rep control region was determined, and putative regulatory sequences were identified; no evidence for autoregulation of expression was obtained. Transcription of the gene was not enhanced during the SOS response. The location of the promoter and the beginning of the protein were confirmed by S1 nuclease mapping of the 5' end of rep mRNA and determination of the NH2-terminal sequence of the rep protein.