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PubMed1997

Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression.

Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is a key enzyme in the synthesis of glucose in the liver and kidney and of glyceride-glycerol in white adipose tissue and the small intestine. The gene for the cytosolic form of PEPCK (PEPCK-C) is acutely regulated by a variety of dietary and hormonal signals, which result in alteration of synthesis of the enzyme. Major factors that increase PEPCK-C gene expression include cyclic AMP, glucocorticoids, and thyroid hormone, whereas insulin inhibits this process. PEPCK-C is absent in fetal liver but appears at birth, concomitant with the capacity for gluconeogenesis. Regulatory elements that control transcription of the PEPCK-C gene in liver, kidney, and adipose tissue have been delineated, and many of the transcription factors that bind to these elements have been identified. Transgenic mice have been especially useful in elucidating the physiological roles of specific sequence elements in the PEPCK-C gene promoter and in demonstrating the key role played at these sites by the isoforms of CAAT/enhancer binding protein in patterning of PEPCK-C gene expression during the perinatal period. The PEPCK-C gene provides a model for the metabolic control of gene transcription.

Animals↗

PubMed1993

Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene.

The structure of the mouse natriuretic peptide type-B (BNP) gene was determined by isolating and sequencing genomic clones. The mouse BNP gene was structurally similar to other natriuretic peptide genes and comprised three exons and two introns. Expression of the mouse BNP gene was found only in cardiac tissue as determined by ribonuclease protection analyses. Initiation of transcription was 31 bp downstream from a consensus TATA box as determined by primer extension analysis of cardiac RNA. Comparative DNA sequence analysis identified several DNA elements with potential transcriptional regulatory function. Comparative amino acid sequence analysis showed that the N-terminal portion of the mouse and rat BNP precursors was more conserved than the C-terminal 45-amino-acid sequence that constitute the bioactive BNP-45 peptide. The proteolytic processing site (RXXR-S) generating bioactive BNPs was highly conserved among all BNP precursors and was identical to the consensus site of furin, a calcium-dependent serine endoprotease. Finally, the BNP gene was mapped using recombinant inbred DNA and a polymerase chain reaction-based restriction fragment-length polymorphism assay to mouse chromosome 4 near the atrial natriuretic factor (Anf) locus. No recombination event between Bnp and Anf was evident in the 39 recombinant inbred and inbred strains examined. This physical linkage between the two natriuretic peptide genes expressed in cardiac tissue may be important for their transcriptional regulation.

Amino Acid Sequence↗

PubMed1997

Human insulin-like growth factor I (IGF-I) produced in the mammary glands of transgenic rabbits: yield, receptor binding, mitogenic activity, and effects on IGF-binding proteins.

Insulin-like growth factor I (IGF-I) has acute insulin-like metabolic effects and long-term anabolic actions offering a range of important therapeutic applications. To evaluate a system for large-scale production of this peptide in the mammary glands of transgenic livestock, we generated transgenic rabbits carrying fusion genes in which a synthetic DNA coding for human IGF-I (hIGF-I) was placed under the transcriptional control of regulatory elements isolated from the bovine alpha S1-casein (alpha S1-cas) gene. Western blot analysis of milk from alpha S1-cas-hIGF-I transgenic rabbits demonstrated production of high amounts of mature hIGF-I peptide (7.6 kDa). Quantitative analysis by RIA revealed hIGF-I levels between 50 and 300 micrograms/ml milk. Recombinant hIGF-I purified from the milk of alpha S1-cas-hIGF-I transgenic rabbits bound to IGF-I receptors on human IM-9 lymphoblasts and stimulated DNA synthesis by growth-arrested MG-63 human osteosarcoma cells as efficiently as hIGF-I produced in Escherichia coli. Ligand blot analysis of milk serum revealed the presence of 45-kDa, 30-kDa, and 23-kDa IGF-binding proteins (IGFBPs). The 30-kDa IGFBP was shown to be IGFBP-2 by immunoprecipitation using an antiserum raised against human IGFBP-2. Secretion of IGFBP-2 was markedly stimulated by hIGF-I overproduction in alpha S1-cas-hIGF-I transgenic rabbits. The latter displayed slightly increased milk yield, but no significant changes in total protein content or overall milk protein composition, and reared their offspring without any problems or clinical signs of impaired welfare, even after multiple lactations. Our results indicate that high amounts of biologically active hIGF-I can be produced in the mammary glands of alpha S1-cas-hIGF-I transgenic rabbits. Local production of hIGF-I in mammary tissue is associated with increased secretion of IGFBP-2, which may prevent major biological effects by high levels of hIGF-I on the mammary gland.

Animals↗

PubMed1992

Gonadotrope-specific expression of the human follicle-stimulating hormone beta-subunit gene in pituitaries of transgenic mice.

The molecular basis for the cell-specific and hormonal regulation of the pituitary gonadotropin beta-subunit genes is unknown. To identify the essential DNA regulatory elements of the human FSH beta (hFSH beta) gene, we introduced a 10-kilobase cloned fragment encompassing the gene into the germline of transgenic mice. We showed by a combination of Northern blot hybridization, RIA, and immunofluorescence histochemistry that the hFSH beta gene was expressed exclusively in mouse pituitary gonadotropes. Human FSH beta was actively secreted, presumably as an interspecies heterodimer, with mouse alpha-subunit, and no free hFSH beta was detected in serum. Male transgenic mice consistently had higher basal pituitary expression and secretion of hFSH beta than the female transgenic mice. Castration combined with testosterone replacement caused a marked reduction in hFSH beta gene expression in transgenic males, but had no effect on the pituitary levels of FSH in normal male mice. These experiments demonstrate that the essential DNA regulatory elements and gonadotrope transcription factors required for cell-specific expression of the FSH beta gene must be conserved between human and mouse. There appear to be important differences in the hormonal regulation of the gene between species, however.

Animals↗

PubMed1989

Genetic structure of the abd-A gene of Drosophila.

We report the embryonic and adult phenotypes of a number of mutations of the abd-A gene of the bithorax complex. Some of them result in loss of abd-A function in the whole abd-A domain and are usually lethal. These probably eliminate or inactivate abd-A protein products. Other mutations affect only part of the abd-A domain. These are viable, appear to map outside the abd-A transcription unit, and presumably alter the normal spatial regulation of abd-A products. We propose a model of abd-A structure based on a protein-coding region and two cis-regulatory regions. Regulatory region 1, 3' to the transcription unit, contains positive and negative regulatory elements. Regulatory region 2, 5' to the transcription unit, establishes the correct level of abd-A activity in the abdominal metameres.

Animals↗

PubMed2006

Use of chromatin immunoprecipitation (ChIP) to detect transcription factor binding to highly homologous promoters in chromatin isolated from unstimulated and activated primary human B cells.

The Chromatin Immunoprecipiation (ChIP) provides a powerful technique for identifying the in vivo association of transcription factors with regulatory elements. However, obtaining meaningful information for promoter interactions is extremely challenging when the promoter is a member of a class of highly homologous elements. Use of PCR primers with small numbers of mutations can limit cross-hybridization with non-targeted sequences and distinguish a pattern of binding for factors with the regulatory element of interest. In this report, we demonstrate the selective in vivo association of NF-kappaB, p300 and CREB with the human Igamma1 promoter located in the intronic region upstream of the Cgamma1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology.

Journal Article↗

PubMed2001

Identification of Schizosaccharomyces pombe genes that encode putative homologues of Saccharomyces cerevisiae mediator complex subunits.

The mediator complexes transduce regulatory information from upstream regulatory elements to the transcription machinery in organisms ranging from yeasts to humans. By a genome-wide search we identified 14 ORFs and genes in the genome of the fission yeast Schizosaccharomyces pombe that encode putative homologues of Saccharomyces cerevisiae mediator subunits. The Sch. pombe proteins are smaller and appear to form a mediator of lower complexity, which is consistent with the hypothesized ancient origin of fission yeasts.

Amino Acid Sequence↗

PubMed1992

Immunolocalization of mineralocorticoid receptor in human kidney, pancreas, salivary, mammary and sweat glands: a light and electron microscopic immunohistochemical study.

Using a polyclonal antibody against a synthetic fusion protein corresponding to 167 amino acids of the N-terminal region of the human renal mineralocorticoid receptor (MR), an immunohistochemical study was performed to investigate the intraglandular and intracellular localization of the receptor in human kidney, salivary gland, pancreas, mammary gland and sweat gland both at the light and electron microscopic levels. In the kidney, immunoreactivity was observed in distal convoluted tubules, branches of Henle's loop, and collecting tubules in the renal cortex, and papillary and Henle's loops' ducts in the renal medulla. No significant differences in the distribution of immunoreactivity were observed using different fixatives (10% neutral formalin, 100% methanol, 4% paraformaldehyde, PLP (periodate-lysine-2% paraformaldehyde) solution and Zamboni solution) and processing methods (paraffin embedding and frozen sectioning). Immunoreactivity in the kidney was observed in both the cytoplasm and nucleus, with cytoplasmic staining predominant, regardless of the methods of tissue preparation. Immunoelectron microscopy, employing a pre-embedding method, demonstrated the presence of immunoreaction precipitates in nuclei, endoplasmic reticulum, perinuclear cisternae, free cytoplasm and cell membranes. In nuclei, immunoreactivity was observed in euchromatin but not in heterochromatin, which is consistent with an association of MR with specific DNA regulatory elements located in transcriptionally active euchromatin. In other organs, MR was expressed in cells of the excretory ductal system where mineralocorticoids are known to play a role in electrolyte homeostasis.

Animals↗

PubMed2026

Developmental patterning of adipose tissue by abd-A and Abd-B homeotic genes in Drosophila melanogaster.

The Bithorax Complex (BX-C) homeobox proteins specify segmental identities along the anterior-posterior axis during Drosophila embryogenesis. Differential expression of the BX-C genes abd-A and Abd-B distinguishes abdominal from thoracic adipocytes, yet the mechanism regulating this heterogeneity remains poorly understood. Here, we identify cis-regulatory elements (CREs) and transcription factors that direct abdominal-specific expression of abd-A and Abd-B in the larval fat body. Fine-mapping analyses identified a 627-bp CRE within the Abd-B locus and a ~6-kb CRE within the abd-A locus sufficient to drive heterogeneous expression. Yeast one-hybrid screening combined with functional analyses identified Lola, Lolal, and Combgap as key repressors of Abd-B, whereas Piragua (Prg) and Seven up (Svp) function as transcriptional activators, indicating that adipocyte heterogeneity in postembryonic adipose tissue is actively regulated. In turn, lola and prg are repressed by Abd-B, whereas lolal and svp are activated, forming a feedback circuit further modulated by Wnt signaling, which promotes lola and lolal expression while repressing svp. CUT&RUN analyses suggest that these interactions are direct, with dTCF/Pan and Abd-B occupancy detected at target loci. Together, our findings define a transcriptional circuit that regulates Abd-B gene transcription to pattern adipose tissue and may establish the developmental basis of fat depot specialization.

Abd-B↗

PubMed2001

Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene.

Previously we identified and cloned the cDNA for a new protein, apolipoprotein L (apoL), present in plasma and mainly associated with large high density lipoprotein particles. Using 5' rapid amplification of cDNA ends, RT-PCR and comparison with three Human Genome Project and three expressed sequence tag sequences, we have characterized the gene for apoL and for three additional, highly homologous proteins that constitute a new family of proteins that display no homology with previously described apolipoproteins. The genes for all four proteins, apoL-I, apoL-II, apoL-III, and apoL-IV, are located at chromosome 22q12.1-13.1 within a 127,000-bp region. The apoL-I gene is in the opposite orientation to the other three. All four genes have TATA-less promoters, which contain putative sterol regulatory elements, suggesting that transcription of these genes may be coordinated with that of the low density lipoprotein receptor and genes in pathways involving the synthesis of triglycerides and cholesterol. The gene family has a consensus eight-exon structure with alternative splice sites that could produce as many as eight distinct gene products. The apoL-II and apoL-III genes have alternative transcriptional start sites as a result of additional 5' exons. apoL-I, apoL-II, and apoL-III are expressed to the highest degree in the lung. Other tissues with high expression are the pancreas, prostate, spleen, liver, and placenta. Four clustered common polymorphisms, three of which altered the protein sequence, were found in apoL-I, all in linkage disequilibrium, and describing two haplotypes: the more common Lys166/Ile244/Lys271 and the rarer Glu166/Met244/Arg271.

Alternative Splicing↗

PubMed2001

Mediators of inflammation and acute phase response in the liver.

The acute phase response is a generalized response of the organism to multiple disturbances of its physiological homeostasis. It consists of local and systemic reactions. Inflammatory processes are the main causes for the initiation of these defence mechanisms. Responsible mediators for the acute phase response are predominantly cytokines, whereby the liver is the predominant target organ. Changes in hepatocyte gene expression profiles result in dramatic changes in serum concentrations of specific plasma proteins, called acute phase proteins. IL-6 was identified as the principal mediator of this reaction. Via its cellular signal transducer gp130 IL-6 induces DNA-binding of STAT transcription factors on regulatory elements of target genes. While IL-6 dependent processes are mainly conferred to be protective other inflammatory cytokines are attributed to be cytotoxic for the liver. TNF-alpha was shown to be involved in several models of liver failure as a mediator for both cytotoxicity and cell proliferation. TNF-alpha leads via caspases to the onset of apoptosis, the so-called programmed cell death. On the other hand it activates NF-kappaB thereby triggering inflammatory processes. In this review we display the relevance for intracellular actions of both cytokines in several models of liver injury. Especially we refer to the T-cell mediated Concanavalin A induced liver failure and to liver regeneration induced by CCL4 and partial hepatectomy. Both cytokines contribute in concert to a cellular balance during these pathophysiological conditions.

Acute-Phase Reaction↗

PubMed2002

[Androgens and increased lipogenesis in prostate cancer. Cell biologic and clinical perspectives].

A search for novel and biologically relevant androgen-regulated genes and processes in the human prostate led to the intriguing observation that androgens provoke a remarkable and coordinated increase in the expression of several genes involved in triglyceride and cholesterol synthesis in various prostatic adenocarcinoma cell lines. This coordinated activation was shown to be the result of a novel and indirect pathway in which androgens cause activation of a secondary transcription regulator, Sterol Regulatory Element Binding Protein (SREBP), a pivotal factor in the control of intracellular lipid homeostasis. The biological relevance of increased lipogenesis in the biology of prostate cancer is underlined by recent immunocytochemical investigations on needle biopsies showing an increase in the expression of Fatty Acid Synthase (FAS) in 94% of the tumor-lesions examined. This increase is already evident in the earliest recognizable lesions (Prostatic Intra-epithelial Neoplasia; PIN) and is more pronounced in tumors with a higher Gleason score, suggesting that increased FAS expression may serve both as an early tumor marker and as a marker of tumor progression. As in tumor cell-lines, increased FAS expression in prostate tumors seems to be only part of a more general and coordinated activation of lipogenic pathways. Further studies revealed that lipogenesis in prostate tumor cells can be enhanced not only by androgens but also by growth factors and by tumor-associated disturbances in signal transduction pathways. EGF, for instance, is also able to activate lipogenesis via the SREBP pathway and activation of the P13 kinase system by inactivation of PTEN (a phenomenon observed in some 50% of the prostate cancers) also causes increased lipogenesis. The early and nearly universal activation of lipogenesis in prostate cancers (and also in various other tumors) suggests that this may be a fundamental event in the development of the tumoral phenotype, an element that certainly merits further investigation. In addition, there are serious indications that interference with enhanced lipogenic activity in tumor cells may cause tumor cell death and delayed tumor development, suggesting that increased lipogenic activity in tumor cells may open a novel avenue for therapeutic intervention.

Adenocarcinoma↗

PubMed1992

Regulation of opioid gene expression: a model to understand neural plasticity.

The recent finding that neurotransmitters and drugs that affect neurotransmission have important influences on gene expression suggests that drug-induced alterations in gene expression may underlie many long-term effects of addictive drugs, for example, dependence and drug-seeking behaviors. These long-term adaptive responses to opiate drugs have been particularly difficult to understand at a mechanistic level. Data presented here indicate that the gene encoding the opioid precursor proenkephalin is highly regulated by neural activity, second-messenger pathways, and PKA. These observations raise the possibility that drugs of abuse (e.g., opiates acting through opiate receptors) may act at the genetic level to modulate the expression of endogenous opiates and that these effects may underlie one component of the brain's long-term adaptive response to exogenous opiates. The transgenic animals described above can be used to investigate opiate drug-induced changes in proenkephalin gene expression, allowing rapid analysis of changes in proenkephalin gene expression in highly restricted populations of neurons in a fashion previously impossible. In addition, by analyzing the effects of specific enhancer mutations on tissue-specific and transsynaptic regulation of proenkephalin expression, transgenic models will permit mechanistic investigations within the intact nervous system that cannot otherwise be undertaken. Investigation of mechanisms underlying this process requires the analysis of intracellular signaling pathways, responsive DNA regulatory elements, and the transcription factors transducing synaptic signals into gene regulation. In the studies described herein, we demonstrate that AP-1 complexes consisting of different Jun proteins differentially regulate proenkephalin transcription at the CRE-2 element. c-Jun constitutively activates proenkephalin transcription, whereas JunD activates in a fashion completely dependent on the activation of second-messenger pathways and the cAMP-dependent PKA. JunB alone has no effect on proenkephalin gene expression, yet this molecule effectively blocks activation mediated by JunD and, hence, may act as a repressor. These data are consistent with a model (figure 4) in which preexisting JunD mediates the rapid cAMP-dependent activation of the proenkephalin enhancer, whereas IEGs such as JunB or c-Fos mediate the protein synthesis-dependent inactivation. Because c-Jun activates proenkephalin transcription constitutively, induction of c-Jun may lead to a further and prolonged activation of proenkephalin gene expression. Hence, the ratio of c-Jun to JunB induction may determine whether proenkephalin is repressed or further activated.

Animals↗

PubMed1993

Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms' tumor locus.

Colony-stimulating factor-1 (CSF-1) is a member of the immediate early gene family, which is expressed in mitogen-stimulated quiescent fibroblasts. The biological effects of CSF-1 are multifaceted and include stimulation of the proliferation and differentiation of myeloid progenitors and activity of circulating monocytes and tissue-specific macrophages. Ablation of circulating levels of biologically active CSF-1 in mice leads to osteopetrosis and sterility, thus implicating a role for CSF-1 in bone remodeling and implantation. Identification of regulatory elements and cognate transcription factors that bind the csf-1 promoter and mediate such diverse expression patterns is of great interest. We identified a sequence element at -273 to -265 (relative to the transcription initiation site) in the murine csf-1 promoter, which contains overlapping consensus sequences for the Wilms' tumor protein (WT1), EGR-1, SP1, and SP3 proteins. WT1 and EGR-1 proteins produced in vitro bound to this sequence, and co-transfection of wt1 with a csf-1-cat reporter plasmid resulted in repression of promoter activity. Interestingly, nuclear extracts prepared from serum-stimulated C3H10T1/2 cells contained predominantly SP1 and SP3 binding activities, which recognized the -273 to -265 site. Thus repression of the csf-1 promoter by WT1 at this site may involve competition between SP1 family transcriptional activators and the WT1 repressor. Colony-stimulating factor-1 may be a physiologically relevant target gene for regulation by the WT1 transcription factor.

Animals↗

PubMed1997

beta43': An enhancer displaying neural-restricted activity is located in the 3'-untranslated exon of the rat nicotinic acetylcholine receptor beta4 gene.

Members of a neuronal nicotinic acetylcholine receptor subunit gene cluster ordered beta4, alpha3, alpha5 in the vertebrate genome are expressed in highly restricted patterns in the PNS and CNS. Nothing is known, however, about the regulatory elements that control transcription of these genes in selected neuronal cell populations. We report here a novel enhancer, designated beta43', that is positioned in the beta4 3'-untranslated exon. It is composed of two nearly identical 37 bp direct repeats that are separated by 6 bp. Multimerization of the enhancer upstream of the alpha3 minimal promoter results in synergistic activation. Analysis in different cell types, including three neural lines and primary keratinocytes, shows that beta43' is preferentially active in the neural line PC12, which expresses all members of the cluster. Mobility shift assays reveal a cell-type-specific complex, which forms with the first repeat of the enhancer and PC12 extracts. Complexes co-migrating with the PC12 cell complex are not detected with extracts from other lines, which suggests that PC12 cells contain a differentially expressed factor that may be important for the restricted activity of beta43'. The cell-type-specific activity of the beta43' enhancer suggests that it is important for regulating restricted expression patterns of one or more clustered neuronal acetylcholine receptor genes. Its location within the beta4 gene may be a selective pressure for maintaining tight linkage of clustered neuronal nAchR genes.

Animals↗

PubMed1997

[Usefulness of glutathione S-transferase as a tumor marker].

The glutathione S-transferases (GSTs), a family of multifunctional proteins, catalyze the glutathione conjugation reaction with electrophilic compounds biotransformed from xenobiotics, including carcinogens, and are grouped into four classes, Alpha, Mu, Pi and Theta. Some of these forms are suggested to act to prevent carcinogenesis by detoxifying proximate or ultimate carcinogens. In neoplastic cells, specific forms are known to be expressed and have been known to participate in their resistance to anticancer drugs. In this article, we review recent findings regarding the respective molecular forms involved in carcinogenesis and their usefulness as tumor markers. GST M1 and GST T1 genes are polymorphic in the population and losses of these genes have been suggested as possible markers for greater susceptibility to lung cancer among smokers and several other cancers. Since many GST inducers prevent rodent chemical carcinogenesis, potential chemopreventive agents have been screened by their induction capabilities. However, reliable markers useful to predict results of prospective chemopreventive trials in populations are not established. Immunohistochemical studies have revealed that many cancers, histologically classified as adenocarcinomas or squamous cell carcinomas, express GST P1-1. Its expression is regulated at transcriptional level and regulatory elements of the gene have been clarified. However, transacting factors responsible for expression in cancer tissues remain to be clarified. In addition, stability of GST P1 mRNA is suggested to be partly responsible in some cell lines. Plasma or serum GST P1-1 levels are increased in 30-50% of patients with cancers of the gastrointestinal tract. This form is also suggested to participate in resistance to anticancer drugs such as cisplatin and daunorubicin, and its expression in cancer tissues may be of prognostic value in cancer patients. Further studies on this enzyme family are clearly needed to obtain a better understanding of cancer prevention and therapy.

Animals↗

PubMed1996

Structure and chromosomal localization of the human prostasin (PRSS8) gene.

Prostasin, denoted as PRSS8, is a newly identified human serine proteinase that shares high sequence identity with acrosin, plasma kallikrein, and hepsin (Yu et al., 1994, 1995). In the present study, a full-length PRSS8 gene has been isolated and characterized. A 7-kb PRSS8 gene fragment has been sequenced, including a 1.4-kb 5'-flanking region, the 4.4-kb PRSS8 gene, and a 1.2-kb 3'-flanking region. The gene consists of six exons and five introns based on comparison with its cDNA sequence. The sizes of these exons are 417, 18, 163, 272, 167, and 899 bp, while those of the introns are 243, 1763, 271, 85, and 92 bp. A number of potential regulatory elements have been revealed in the 5'-flanking region, including an AP2 site, two erythroid-specific promoter elements, and a sterol regulatory element. In addition, there are a variant GC box and a variant AP1 site in the promoter region. The transcription initiation site of the PRSS8 gene has been defined at the G residue and its adjacent A residue in a sequence CTCATGACT, which is similar to an initiator element CTCANTCT. Between the transcription initiation site and these putative regulatory elements, there is an AC-rich repetitive sequence that spans over 300 bp. Human PRSS8 is a single-copy gene and has been localized on chromosome 16p11.2 by in situ hybridization.

Amino Acid Sequence↗

PubMed2000

Inhibition of gene expression in Entamoeba by the transcription of antisense RNA: effect of 5' and 3' regulatory elements.

Down regulation of gene expression by antisense RNA is one of the ways to investigate the specific contribution of certain components to the physiology and activities of a cell. A successful inhibition of gene expression in Entamoeba trophozoites was achieved in stable transfectants by using hybrid plasmid constructs containing promotors that produce transcripts which do not bind to polysomes. Different promotors were found to be required for Entamoeba histolytica or Entamoeba dispar. In E. histolytica one of the two copies (g34) of the gene coding for ribosomal protein L21 was previously found to be transcribed but not translated. Inhibition of gene expression was obtained by placing in a transfection vector, the amoebapore A gene, in its antisense orientation, under the control of the g34 promotor. Transfectants of E. histolytica were shown to accumulate antisense transcripts and inhibit amoebapore synthesis. In contrast, transfectants with plasmid constructs in which the amoebapore gene was placed under the control of the gLE3 promotor of RP-L21, which is known to be translated, did not accumulate antisense transcript or inhibit gene expression. Maximal inhibition of amoebapore expression was obtained when the antisense construct also included the 5' and 3' untranslated regions of the amoebapore gene. In E. dispar the opposite situation was found, plasmid constructs containing the promotor regions of the gLE3 copy, which were shown to be poorly translated, were more efficient in inhibiting the synthesis of a 30 kDa surface-specific antigen than a construct with the g34 promotor element.

3' Untranslated Regions↗