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PubMed2000

Identification of Novel Regulatory Elements of Human Stem Cell Factor Gene in MCF Cell.

Human stem cell factor(hSCF)is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5'flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5x end deletions of hSCF gene. This study indicates that the region of -1190 -853 significantly enhanced the luc gene expression, while the region of -339 -162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both -1190 -853 and -339 -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 -853 and -339 -273 DNA fragments of the hSCF 5'flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.

Journal Article↗

PubMed1996

Evolution of promoter sequences: elements of a canonical promoter for prespore genes of Dictyostelium.

An attempt is made to define a minimal prespore promoter which contains all elements essential for correct regulation of expression of a prespore gene. The prespore genes of Dictyostelium are coregulated during development. Most begin transcription at the same early stage, and activity of all is restricted to prespore tissue during the later slug stage. Sequences 5' to the coding sequences of eight prespore genes were searched for all elements proposed to control transcription and for new elements. The meaningfulness of occurrences of elements and pairs of elements in prespore promoters was evaluated by comparison with frequencies of occurrences in promoters of other, nonprespore genes. These comparisons resulted in definition of a canonical prespore promoter, a stretch of about 200 nucleotides containing at least one of each of three elements. Certain limitations were found on the spacing of elements. Orientation of elements with respect to each other appeared unrestricted. All elements often occurred in multiple copies. This structure suggests that individual copies of each element are not conserved during evolution, but instead continually appear and disappear.

Animals↗

PubMed2002

Regulation of gene promoters of hypothalamic peptides.

In order to fulfill their roles in neuroendocrine regulation, specific hypothalamic neurons are devoted to produce and deliver biologically active peptides to the pituitary gland. The biosynthesis and release of peptides are strictly controlled by afferents to these hypothalamic neurons. Cell-specific expression and biosynthetic regulation largely relies on transcription from the gene promoter for which the 5(')-flanking regions of the peptidergic genes contain essential elements. Cell-specific transcription factors employ these regulatory elements to exert their control over the expression of the peptidergic gene. This article explores the properties of regulatory elements of the major hypothalamic peptides, somatostatin, growth hormone-releasing hormone, gonadotropin-releasing hormone, thyrotropin-releasing hormone, corticotropin-releasing hormone, vasopressin and oxytocin, and the transcription factors acting on them. These transcription factors are often endpoints of signal transduction pathways that can be activated by neurotransmitters or steroid hormones. Others are essential to provide cell-specific expression of the peptidergic gene during development and mature regulation.

Animals↗

PubMed1980

Elemental content of predigested liquid protein products.

Eight commercially available partially digested gelatin liquid products that have been used as a source of protein in dietary regimens for weight reduction and for protein supplementation were analyzed for their elemental composition. These products were prepared either by dry ashing or wet digestion techniques that were suitable for the analysis of individual elements by their respective analytical methods. Analytical methods used to assess the elemental content included inductively coupled argon plasma emission spectrometry for Ca, Mg, P, Fe, Zn, Cu, Mn, Ni, Mo, V, Be, Co, Cr, Tl, Al, Te, Sn and Sb; atomic absorption spectrophotometry for Na and K; hybride generation with atomic absorption spectrophotometry for Se and As; anodic stripping voltammetry for Cd and Pb; and ion selective electrode for F. The content of almost all of the elements in these products was extremely low compared with the amounts supplied daily for normal individuals by usual sources of dietary protein, the adult United States Recommended Daily Allowances, and typical intakes from the Total Diet Study. The following elements were not detectable in significant amounts in these products by inductively coupled argon plasma emission spectrometry: Mo, V, Be, Co, Cr, Tl, Al, Te, Sn, and Sb. The results suggest that use of these predigested liquid protein products as a sole source of nutriment will result in an inadequate intake of all of the essential elements. These products contained insignificant amounts of Ce and Pb, nonessential toxic elements.

Adult↗

PubMed1998

Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene.

The MIP gene, the founder of the MIP family of channel proteins, is specifically expressed in fiber cells of the ocular lens and expression is regulated temporally and spatially during development. We previously found that a DNA fragment containing 253 bp of 5'-flanking sequence and 42 bp of exon 1 of the human MIP gene contains regulatory elements responsible for lens-specific expression of the MIP gene. In this report we have analyzed the function of overlapping Sp1 and AP2 binding sites present in the MIP promoter. Using DNase I footprinting analysis we found that purified Sp1 and AP2 transcription factors interact with several domains of the human MIP promoter sequence -253/+42. Furthermore, addition of purified Sp1 to Drosophila nuclear extracts activates in vitro transcription from the MIP promoter -253/+42. This promoter activity is competed by oligonucleotides containing domains footprinted with Sp1. Using promoter-reporter gene ( CAT ) constructs we found that the sequence -39/-70 contains a cis regulatory element essential for promoter activity in transient assays in lens cells. EMSA analysis showed that lens nuclear extracts contain factors that bind to the MIP 5'-flanking sequence containing overlapping Sp1 and AP2 binding domains at positions -37/-65. Supershift experiments with lens nuclear extracts indicated that Sp3 is also able to interact with this regulatory element, suggesting that Sp1 and Sp3 may be involved in regulation of transcription of the MIP gene in the lens.

Amino Acid Sequence↗

PubMed2005

Indicators for chemicals: sources, impacts and policy performance.

BACKGROUND: Different types of indicators have been developed to describe the impact of chemicals on society and environment. Due to the high number of substances and their different types of use, most of these indicators are directed to specific areas of interest--regarding workplace safety, environmental health or consumer health. They address a specific subset of chemicals and can be used for monitoring enterprise-specific, national or international management measures. MAIN FEATURES: A survey of existing indicators for chemicals has shown that indicators already exist for a remarkable number of problem fields. As soon as the release and the environmental fate of chemicals are taken into account, the complexity of the approaches increases considerably. The distinction between indicators for drivers, pressures, state, impacts and responses, as proposed by the European Environmental Agency, supports the identification of proper indicators for a specific type of problem. DISCUSSION AND CONCLUSIONS: No single indicator exists which is able to cover the whole range of chemicals and their applications. Several indicator approaches cover at least a subset of the most relevant substances. If they are intended to be used for European monitoring, robust data must be provided by EU Member States. Chemicals in enterprises (ancillary inputs as well as process chemicals) are an important element of in-plant material flow management--in terms of occupational safety and health as well as environmental protection. Existing indicators for hazardous chemicals can be a valuable tool for process and product refinement regarding hazardous chemicals, especially for enterprises. OUTLOOK: Indicators for production and impact of chemicals, as well as policy performance indicators, are essential elements in order to monitor the management of chemicals. They have to be established for the national and for the EU level.

Data Collection↗

PubMed2003

Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control.

Neural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. We have investigated the basis of the specific neural crest expression of Krox20 and identified a cis-acting enhancer element (NCE) located 26 kb upstream of the gene that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins and which responded to factors expressed throughout the neural crest. Consistent with this the NCE was strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevented the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organisation also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning.

Animals↗

PubMed2003

Transcriptional regulation of Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa: dissection of key promoter elements.

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) is an adaptor molecule expressed in all hemopoietic cell lineages except mature B cells and is known to play critical roles in the function of T cells, mast cells, and platelets and in vascular differentiation. Although great progress has been achieved in our understanding of SLP-76 function, little is known about the mechanisms regulating its expression. In this study we report the initial characterization of essential elements that control SLP-76 transcription. We identify several DNase I-hypersensitive sites in the SLP-76 locus, with a prominent site located in its promoter region. This site exists in T cells and monocytic cells, but not in B cells or fibroblasts. Using transient transfection assays, we identify a 507-bp fragment containing the 5'-untranslated region of the first exon and the immediate upstream sequence that confers transcriptional activation in T cells and monocytic cells, but not in B cells. Analysis of the 5' ends of SLP-76 transcripts reveals differential regulation of SLP-76 transcription initiation between T cells and monocytic cells. Mutational and gel-shift analyses further indicate a critical role within this region for a binding site for Ets family transcription factors. The present study provides the first data to address the mechanisms controlling SLP-76 transcription by providing evidence for several key cis-regulatory elements in the promoter region.

Adaptor Proteins, Signal Transducing↗

PubMed2002

[The development of conjugal dissatisfaction in the context of a restructured family].

What factors influence the quality of marital relationships in stepfamilies? How does marital dissatisfaction develop in these families, and what elements can help prevent it? These are the questions which this article tries to answer. We first identify three areas where difficulties occur: individual, relational, and social. By using Social Learning Theory, we then explain how the interaction of factors specific to these areas contributes to the development of marital dissatisfaction in remarried couples. Finally, 3 essential elements for preventing difficulties emerge from the analysis: (a) information, (b) communicative abilities and (c) support.

Communication↗

PubMed2000

Stoichiometry in producer-grazer systems: linking energy flow with element cycling.

All organisms are composed of multiple chemical elements such as carbon, nitrogen and phosphorus. While energy flow and element cycling are two fundamental and unifying principles in ecosystem theory, population models usually ignore the latter. Such models implicitly assume chemical homogeneity of all trophic levels by concentrating on a single constituent, generally an equivalent of energy. In this paper, we examine ramifications of an explicit assumption that both producer and grazer are composed of two essential elements: carbon and phosphorous. Using stoichiometric principles, we construct a two-dimensional Lotka-Volterra type model that incorporates chemical heterogeneity of the first two trophic levels of a food chain. The analysis shows that indirect competition between two populations for phosphorus can shift predator-prey interactions from a (+, -) type to an unusual (-, -) class. This leads to complex dynamics with multiple positive equilibria, where bistability and deterministic extinction of the grazer are possible. We derive simple graphical tests for the local stability of all equilibria and show that system dynamics are confined to a bounded region. Numerical simulations supported by qualitative analysis reveal that Rosenzweig's paradox of enrichment holds only in the part of the phase plane where the grazer is energy limited; a new phenomenon, the paradox of energy enrichment, arises in the other part, where the grazer is phosphorus limited. A bifurcation diagram shows that energy enrichment of producer-grazer systems differs radically from nutrient enrichment. Hence, expressing producer-grazer interactions in stoichiometrically realistic terms reveals qualitatively new dynamical behavior.

Animals↗

PubMed1998

Regulation of the expression of the sn-glycerol-3-phosphate dehydrogenase gene in Drosophila melanogaster.

P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.

Age Factors↗

PubMed1997

Control of cell-type specific gene expression in Dictyostelium by the general transcription factor GBF.

To understand how positional information within an organism specifies patterning during development, we are analyzing spatially regulated gene expression in Dictyostelium. CAR3 is a member of the cAMP, 7-span receptor family which directs the transition from unicellular to multicellular organism and regulates cellular differentiation and pattern formation. CAR3 mRNA is expressed maximally at 8-10 hours of development, as individual cells aggregate and differentiate, and is accumulated to equivalent levels in all cells. CAR3 is also induced in shaking cultures by response to extracellular cAMP. We now show, by extensive mutagenesis, that the maximum length of contiguous sequences required for accurate spatiotemporal regulation of CAR3 is approx. 350 bp. These sequences include three significant elements located in upstream and transcribed regions. Arrays of G-boxes (GBF regulatory sites) are centered near positions -165 and +50 and, although either is sufficient for induction by cAMP and expression in prespore cells, both are required for expression in prestalk cells. Another GC-rich element near position -80 is required for maximal expression of prespore-specific constructs, although full-length promoters carrying clustered mutations through the -80 region are still expressed in all cells, but with slightly reduced expression. Spatiotemporal expression of CAR3 during development, thus, requires cell-specific combinatorial interactions of multiple but redundant regulatory components. These essential elements are located in upstream and transcribed regions. However, most surprisingly, a primary control for spatial patterning of CAR3 expression appears to be mediated by GBF, a general transcription factor expressed ubiquitously during Dictyostelium development following early aggregation.

Animals↗

PubMed2006

The antibody repertoire in evolution: chance, selection, and continuity.

All jawed vertebrates contain the genetic elements essential for the function of the adaptive/combinatorial immune response, have diverse sets of natural antibodies resulting from segmental gene recombination, express comparable functional repertoires and can produce specific antibodies following appropriate immunization. Profound variability occurs in the third hypervariable (CDR3) segments of light and heavy chains even within antibodies of the same ostensible specificity. Germline VH and VL elements, as well as the joining (J) segments are highly conserved among the distinct vertebrate species. Conservation is particularly noted among the VH3-like sequences of all jawed vertebrates in the FR2 and FR3 segments, as well as in the FGXGT(R or K)L J-segment characteristic of light chains and TCRs and the WGXGT(uncharged)VT JH segments. Human VH3-53 and Vlambda6 family orthologs may be present over the entire range of vertebrates. Models of the three-dimensional structures of shark VH/VL combining sites indicate similarity in framework structure and comparable CDR usage to those of man. Although carcharhine shark VH regions show greater than 50% identity to the human VH germline prototype, searches of lower deuterostome and invertebrate databases fail to detect molecules with significant relatedness. Overall, antibodies of jawed vertebrates show tremendous individual diversity, but are constructed incorporating design features that arose with the evolutionary emergence of the jawed vertebrates and have been conserved through at least 450 million years of evolutionary time.

Amino Acid Sequence↗

PubMed1988

Identification of regions essential for extrachromosomal replication and maintenance of an endogenous plasmid in Dictyostelium.

Initial experiments with the endogenous 12.3 kb Dictyostelium discoideum plasmid Ddp1 led to the generation of a large shuttle vector, Ddp1-20. In addition to Ddp1, this vector contains pBR322 and a gene fusion that confers G418 resistance in Dictyostelium cells. We have shown that Ddp1-20 replicates extrachromosomally in Dictyostelium cells and can be grown in Escherichia coli cells (1). We have now examined deletions within this vector to identify the elements essential for extrachromosomal replication and stable maintenance of the plasmid. We find that a 2.2 kb fragment is sufficient to confer stable, extrachromosomal replication with a reduction in copy number from about 40 to approximately 10-15 copies per cell. Vectors containing additional Ddp1 sequences have a higher copy number. The 2.2 kb region contains none of the complete, previously identified transcription units on Ddp1 expressed during vegetative growth or development. These results suggest that gene products expressed by Ddp1 are not essential for replication, stability, or partitioning of the plasmid between daughter cells. Vectors carrying only the 2.2 kb fragment plus the gene fusion conferring G418 resistance transform Dictyostelium cells with high efficiency using either calcium phosphate mediated transformation or electroporation. Finally, we have examined the relative levels of expression of actin promoters driving neoR genes when in extrachromosomal or integrating vectors.

Chromosome Deletion↗

PubMed2000

Spatial and temporal expression of a Polysphondylium spore-specific gene.

In the cellular slime mold Polysphondylium spherical masses of cells are periodically released from the base of the culminating sorogen. These whorls undergo a morphogenetic transformation from spherical to radial symmetry, marked by the early emergence of a radially symmetric prepattern on the whorl surface. In previous experiments, morphogenesis was followed by observing prestalk cell markers. Here we describe the isolation and characterization of a spore coat gene whose expression pattern is the negative image of the prestalk pattern. To study the molecular mechanism of sp-45 gene regulation, we have cloned and analyzed the sp-45 promoter. Deletion analysis localized a single positive regulatory element (PRE) to a 106-bp fragment between positions -246 and -352 of the upstream coding sequence. This fragment can be further divided into a promoter-proximal and promoter-distal PRE and a 29-bp sequence between them. The distal PRE can regulate prespore expression when fused to a nonfunctioning basal promoter. The distal PRE contains two adjacent essential elements, a Gr box (GTGATATAGTGG) and a TA box (TAATATATT). Each element can drive prespore cell-specific reporter gene expression independently when incorporated into a nonfunctional promoter. Our results also show that prespore cell-specific gene expression is solely under positive regulation, with no evidence for spore-specific enhancers or cis-acting negative regulatory elements. By fusing GFP to the C-terminus of sp-45, we have demonstrated that the graded gene expression of SP45 in the sorogen is regulated by a sequence lying within the sp-45 coding sequence. The temporal and spatial expression pattern of this protein, taken together with the prestalk expression pattern, demonstrates unambiguously that the radial symmetries that emerge in the whorl are established by a system of positional coordinates and that cell sorting plays little if any role in this process.

Amino Acid Sequence↗

PubMed2002

Comparison of the post-transcriptional regulation of the mRNAs for the surface proteins PSA (GP46) and MSP (GP63) of Leishmania chagasi.

MSP (GP63) and PSA (GP46) are abundant 63- and 46-kDa glycolipid-anchored proteins on the surface of the promastigote form of most Leishmania species. MSP is a zinc metalloprotease that confers resistance to host complement-mediated lysis. PSA contains internal repeats of 24 amino acids, and its function is unknown. The steady state levels of mRNAs for both glycoproteins are regulated post-transcriptionally, resulting in about a 30-fold increase as Leishmania chagasi promastigotes grow in vitro from logarithmic phase to stationary phase. Previous studies showed the 3'-untranslated regions (3'-UTRs) of these mRNAs are essential for this post-transcriptional regulation. These two 3'-UTRs of 1.0 and 1.3 kilobases were cloned immediately downstream of a beta-galactosidase reporter gene in a plasmid, and segments were systematically deleted to examine which portions of the 3'-UTRs contribute to the post-transcriptional regulation. The 92-nucleotide segment of greatest similarity between the two 3'-UTRs was deleted without loss of regulation, but the segments flanking this similarity region have positive regulatory elements essential for the regulation. We propose that similar, but non-identical, molecular mechanisms regulate the parallel expression of these two L. chagasi mRNAs despite their lack of sequence identity. These post-transcriptional mechanisms resemble the mechanism recently suggested for the regulation of mRNAs encoding the dipeptide (EP) and pentapeptide (GPEET) repeat proteins in Trypanosoma brucei that involves interactions between positive and negative regulatory elements in the 3'-UTR.

3' Untranslated Regions↗

PubMed1995

Regulatory elements in the promoter region of the renal kallikrein gene in normotensive vs hypertensive rats.

The renal kallikrein-kinin system has been implicated in the pathogenesis of hypertension. The expression level of the renal kallikrein gene in the kidney is significantly lower in spontaneously hypertensive rats (SHR) as compared with that of normotensive (SD and WKY) rats. Deletion analysis showed that the fragment -356/-188 of the promoter contains a transcriptional silencer(s) and the GC rich region located between -77 and -187 is the minimal essential element for directing the expression of the CAT reporter gene in mouse L cells. In the kidney of normotensive vs hypertensive rats, the nuclear protein factors NF1/CTF and SP1 bind differently to the renal kallikrein promoter, but similarly in the salivary gland. The differential transcriptional regulation of the rat renal kallikrein gene in the kidney may be responsible for the genetic difference between normotensive and hypertensive rats.

Animals↗

PubMed2006

E-learning for healthcare students: developing the communities of practice framework.

AIM: This paper presents research considering whether healthcare students were able to develop characteristics of communities of practice when engaged in an online module. BACKGROUND: Little is known about whether the communities of practice framework can be applied to online learning, with no previous consideration of its potential use within healthcare education. METHODS: Using a case study approach the research, completed in 2004, had two phases. A questionnaire was administered to a group of 109 healthcare students to gain information on which to base sampling for the subsequent phase. Phase 2 employed three strands of data collection: five students completed an online diary, the online interactions of seven students were captured on a discussion board and three students were interviewed. Data were analysed using a form of pattern matching. FINDINGS: Students were able to develop essential elements of communities of practice: mutual engagement, joint enterprise and shared repertoire, though this was not uniformly seen. Particular issues emerged for the online community, including enabling access to the online environment to support mutual engagement. The development of trust was also threatened by difficulties of presenting identities online. Joint enterprise was hampered by the online situation, although the virtual classroom proved essential for supporting endeavour. Not all students were committed to their groups. There was some evidence of group members developing shared repertoire, as routines of group working emerged. Professional understanding and computer skills were also enhanced. CONCLUSION: The framework can be applied to supporting online learning internationally amongst students and has applicability to professional groups. Those intending to employ the framework should ensure that students can gain access to the community and have the computer skills to engage. Course design should be considered to ensure support for developing the essential components of communities of practice.

Adult↗