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Results for “regulatory element transcripts”

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PubMed1998

Proximal sequences of the aldolase A fast muscle-specific promoter direct nerve- and activity-dependent expression in transgenic mice.

Muscle activity is known to modulate the muscle fiber phenotype. Changes in muscle activity (normal or experimentally induced) lead to modifications of the expression status of several muscle-specific genes. However, the transcription regulatory elements involved in the adaptative response are mainly unknown. The aldolase A muscle-specific promoter, pM, is expressed in adult fast twitch muscle with a preferential expression in fast glycolytic-2B fibers. Its activity is induced during postnatal muscle maturation, suggesting a role of nerve and/or muscle activity. Indeed, denervation of gastrocnemius in newborn mice prevented the activation of the promoter in this muscle, despite the nerve-independent formation of 2B fibers. Although the nerve was necessary for pM onset during development, denervating the gastrocnemius in adults had only mild effects on pM activity. By contrast, a transgene including the pM proximal regulatory sequences that are sufficient to reproduce the 2B fiber-specific expression of the endogenous promoter was shown to be highly sensitive to both neonatal and adult denervation. Transgenes containing muscle-specific pM proximal promoter elements were used to delineate the regulatory elements involved in this response to innervation and changes in the contractile activity pattern. Nerve- and activity-dependent elements could be localized in the 130-base pair-long proximal promoter region of the human aldolase A gene.

Animals↗

PubMed1999

Inducible gene knockouts in the small intestinal and colonic epithelium.

We have developed two systems for performing Cre-mediated recombination of target genes in the rapidly self-renewing mouse small intestinal and colonic epithelium. When expression of Cre recombinase is placed directly under the control of transcriptional regulatory elements from a fatty acid-binding protein gene (Fabp), deletion of loxP flanked (floxed) DNA sequences is initiated as early as embryonic day 13.5, well before completion of intestinal morphogenesis. By embryonic day 16.5, Fabp-Cre also directs recombination in all cell layers of the transitional epithelium that lines the renal calyces and pelvis, ureters, and bladder. Fabp-Cre expression and recombination are maintained in both epithelia throughout adulthood. The second system allows recombination to be induced only in the gut and at any period during adulthood. This system uses Fabp regulatory elements to direct expression of a reverse tetracycline-regulated transactivator (rtTA). Another transgene encodes Cre under the control of tet operator sequences and a minimal promoter from human cytomegalovirus (tetO-P(hCMV)-Cre). In the absence of a doxycycline inducer, no basal recombination is detectable in the gut of adult tri-transgenic mice containing Fabp-rtTA, tetO-P(hCMV)-Cre, plus a floxed reporter gene. After 4 days of oral administration of doxycycline, recombination of the reporter is apparent in the small intestinal, cecal, and colonic epithelium. After doxycycline is withdrawn, the recombined locus persists for at least 60 days, indicating that recombination has occurred in epithelial cell progenitors that have long residency times in the proliferative units of the intestine (crypts of Lieberkühn). This inducible system should have a number of applications for examining gene function at selected times in postnatal life, under selected physiologic or pathophysiologic conditions.

Animals↗

PubMed2004

Temperature-dependent modification and activation of B-MYB: implications for cell survival.

B-MYB is a ubiquitous transcription factor with an essential role in mouse development. Because cells with a disrupted B-MYB gene cannot be obtained, it is still unknown what is the critical function(s) exerted by B-MYB in mammalian cells. In this study we have observed that reducing B-MYB expression in primary human fibroblasts by using RNA interference results in a partial block of the cells in the G(2) phase of the cell cycle and cell death. Surprisingly, suppressing B-MYB transcriptional activity with a dominant-negative molecule is without effect, suggesting that its transactivating function is not essential. Only human or murine fibroblasts exposed to high temperature are sensitized to cell death in the presence of dominant-negative B-MYB. This correlates with temperature-dependent binding of endogenous B-MYB to transcriptional regulatory elements of the stress-related gene ApoJ/clusterin. We find that regulation of ApoJ/clusterin by B-MYB is a pro-survival response to thermal stress. Thus, B-MYB is regulated by temperature to activate genes required for cell survival.

Cell Cycle Proteins↗

PubMed2000

Promoter sequences of the putative Anopheles gambiae apyrase confer salivary gland expression in Drosophila melanogaster.

The saliva of blood-feeding arthropods contains an apyrase that facilitates hematophagy by inhibiting the ADP-induced aggregation of the host platelets. We report here the isolation of a salivary gland-specific cDNA encoding a secreted protein that likely represents the Anopheles gambiae apyrase. We describe also two additional members of the apyrase/5'-nucleotidase family. The cDNA corresponding to the AgApyL1 gene encodes a secreted protein that is closely related in sequence to the apyrase of the yellow fever mosquito, Aedes aegypti, and whose expression appears enriched in, but not restricted to, female salivary glands. The AgApyL2 gene was found searching an A. gambiae data base, and its expression is restricted to larval stages. We isolated the gene encoding the presumed A. gambiae apyrase (AgApy) and we tested its putative promoter for the tissue-specific expression of the LacZ gene from Escherichia coli in transgenic Drosophila melanogaster. All the transgenic lines analyzed showed a weak but unambiguous staining of the adult glands, indicating that some of the salivary gland-specific transcriptional regulatory elements are conserved between the malaria mosquito and the fruit fly. The availability of salivary gland-specific promoters may be useful both for studies on vector-parasite interactions and, potentially, for the targeted tissue-specific expression of anti-parasite genes in the mosquito.

5'-Nucleotidase↗

PubMed1993

Analysis of the Drosophila gene for the laminin B1 chain.

We have isolated and sequenced a Drosophila genomic DNA that encodes the entire coding region of the laminin B1 chain. The genomic DNA sequenced spans 11,787 bp, including a 1.1-kb 5'-flanking region, 5 exons, 4 introns, and a 1.4-kb 3'-flanking region. The open reading frame is within the two largest exons, the exons 3 and 4, while the first two and the last exons are much smaller and are untranslated. The structure of the Drosophila laminin B1 gene is similar to the Drosophila laminin B2 gene. Their exon-intron lengths and Eco RI, Pst I restriction maps are quite conserved. Both of their open reading frames are very compact, and their first introns are much larger than all of the rest of the introns. These results are consistent with the suggestion that the B1 and B2 genes could be derived from an ancestral gene. The similarity of the proximal 5'-flanking regions of the Drosophila B1 and B2 genes is 46.6%. Also, similar sequences of transcriptional regulatory elements, even though not site conserved, are found in both proximal 5'-flanking regions of the B1 and B2 genes. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 1,048 bp of 5'-flanking region shows a strong expression of chloramphenicol acetyltransferase (CAT) activity. The deletion clones that contain sequences between nucleotides -462 to +150, and -282 to +150 all show strong CAT activity. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can promote the expression of the laminin B1 gene.

Amino Acid Sequence↗

PubMed1993

Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors.

We have recently shown that direct injection of DNA can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. Vectors were designed specifically for vaccination by direct DNA injection and refined to improve plasmid production in Escherichia coli. The vectors consist of a pUC-19 backbone with the cytomegalovirus (CMV) IE1 enhancer, promoter, and intron A transcription regulatory elements and the BGH polyadenylation sequences driving the expression of the reporter gene CAT or influenza A nucleoprotein (NP) or hemagglutinin (HA). The respective vectors expressed high levels of chloramphenicol acetyltransferase (CAT) and NP in tissue culture, and yielded 14-15 mg of purified plasmid per liter of Escherichia coli culture. Immunization of mice with the NP and HA expression vectors resulted in protection from subsequent lethal challenges of influenza using either heterologous or homologous strains, respectively.

Animals↗

PubMed1994

Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit.

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.

Amino Acid Sequence↗

PubMed1998

Structural characterization of the rat cysteine-rich intestinal protein gene and overexpression of this LIM-only protein in transgenic mice.

Cysteine-rich intestinal protein (CRIP) has a double zinc-finger motif called the LIM domain. The most elementary member of the Group 2 LIM-only protein family, CRIP was initially identified as a developmentally regulated intestinal gene. Subsequently, it was found to be highly expressed in immune cells. The structural portion of the rat CRIP gene is comprised of five exons extending over i.8 kb, with the two zinc-finger motifs of the LIM domain being divided among the first three exons. In addition to transcriptional regulatory elements previously identified in the promoter, consensus sequences for AP-1, AP2, Sp-1, and a glucocorticoid response element are located within the first intron. We have developed a line of transgenic mice that overexpress the rat CRIP gene with an expression profile that mirrors that of the endogenous gene. Driven by the homologous rat CRIP promoter, expression increased threefold to sevenfold in intestine, thymus, spleen, and lung over endogenous levels. The transgenic mice had only about 50% of the white blood cell count found in nontransgenic animals. Differential leukocyte counts showed transgenic animals had proportionately fewer lymphocytes and more monocytes, eosinophils, neutrophils. Flow cytometry data suggested that mice overexpressing CRIP have more CD4+/CD8+ thymic lymphocytes. These data suggest that CRIP plays a significant role in differentiation or maturation of cells with rapid turnover such as those found in the intestine and immune system.

Amino Acid Sequence↗

PubMed1996

Long-term secretion of therapeutic proteins from genetically modified skeletal muscles.

Protein delivery from genetically modified skeletal muscle has been reported previously. However, a stable and prolonged secretion was obtained in immunocompromised or newborn animals only. To evaluate the clinical relevance of this approach, we have transduced myoblasts from an adult beta-glucuronidase-deficient (MPS VII) mouse with retroviral vectors carrying either the human beta-glucuronidase cDNA or the murine erythropoietin (Epo) cDNA. The cells were then grafted into the tibialis anterior muscle of adult immunocompetent MPS VII recipients. Protein expression was controlled either by ubiquitous or muscle-specific transcriptional regulatory elements. Animals were analyzed over an 8-month period. The in situ detection of beta-glucuronidase activity revealed up to 60% of genetically modified myofibers in the recipient muscles. The human desmin promoter and enhancer showed the highest in vivo expression. Secretion of beta-glucuronidase induced a disappearance of lysosomal storage lesions in the liver and spleen of recipient animals. Delivery of Epo led to a permanent increase of hematocrit values over 3 months. These results showed that the transplantation of genetically modified myoblasts allowed a sustained secretion of recombinant proteins at therapeutic levels in immunocompetent adult mice. They suggest that the approach may be considered for human applications.

Animals↗

PubMed2005

Prediction of regulatory modules comprising microRNAs and target genes.

MOTIVATION: MicroRNAs (miRNAs) are small endogenous RNAs that can play important regulatory roles via the RNA-interference pathway by targeting mRNAs for cleavage or translational repression. We propose a computational method to predict miRNA regulatory modules (MRMs) or groups of miRNAs and target genes that are believed to participate cooperatively in post-transcriptional gene regulation. RESULTS: We tested our method with the human genes and miRNAs, predicting 431 MRMs. We analyze a module with genes: BTG2, WT1, PPM1D, PAK7 and RAB9B, and miRNAs: miR-15a and miR-16. Review of the literature and annotation with Gene Ontology terms reveal that the roles of these genes can indeed be closely related in specific biological processes, such as gene regulation involved in breast, renal and prostate cancers. Furthermore, it has been reported that miR-15a and miR-16 are deleted together in certain types of cancer, suggesting a possible connection between these miRNAs and cancers. Given that most known functionalities of miRNAs are related to negative gene regulation, extending our approach and exploiting the insight thus obtained may provide clues to achieving practical accuracy in the reverse-engineering of gene regulatory networks. AVAILABILITY: A list of predicted modules is available from the authors upon request.

Algorithms↗

PubMed1990

Disruption of thymocyte development and lymphomagenesis induced by SV40 T-antigen.

The lck gene encodes a membrane-associated protein tyrosine kinase that is expressed specifically in lymphoid cells, especially thymocytes. Structural analysis of the murine and human lck genes previously identified conserved 5' flanking sequences that were proposed to represent transcriptional regulatory elements. Here we demonstrate that a murine lck promoter construct containing these sequences directs the expression of the SV40 T-antigen gene in lymphoid cells. Remarkably, expression of SV40 T-antigen in transgenic animals dramatically disturbs thymic development, resulting in preferential loss of CD4+CD8+ thymocytes. In contrast, immature cells lacking both CD4 and CD8 markers are present in near-normal numbers. Thus SV40 T-antigen expression appears partially to arrest thymopoiesis. Mice bearing the lck-SV40 transgene develop readily explantable thymic tumors at 12-18 weeks of age. Fluorocytometric analyses of lck-SV40 tumor cells reveal that immature thymocytes are frequently immortalized. The lck-SV40 mouse may therefore provide materials for the in vitro investigation of thymocyte differentiation.

Animals↗

PubMed1992

Triple helix formation by purine-rich oligonucleotides targeted to the human dihydrofolate reductase promoter.

The ability of oligodeoxynucleotides to form specific triple helical structures with critical regulatory sequences in the human dihydrofolate reductase (DHFR) promoter was investigated. A battery of purine-rich oligonucleotides targeted to the two purine.pyrimidine strand biased regions near the DHFR transcription initiation site was developed. The stable triple helical structures formed by binding of the oligonucleotides to the native promoter double helix were dominated by G*G.C triplets, with interspersed C*C.G and A*A.T alignments. Mismatches between the oligonucleotide and the purine-rich strand of the target significantly destabilized third strand binding, and a G*A.T alignment was particularly unfavorable. Formation of a pur.pur.pyr triple helical structure results in a localized limitation of access to the native double helical DNA and produces sequence dependent conformational alterations extending several nucleotides beyond the triplex-duplex boundary. Although they differ only by the insertion of two A.T base pairs, the distal and proximal purine.pyrimidine regions can be targeted individually due to the high degree of sequence specificity of triple helical alignment. Triplex formation overlapping any of three consensus transcriptional regulatory elements and collectively covering 50% of the DHFR core promoter is now possible with this set of oligonucleotides.

Base Sequence↗

PubMed1997

Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: I. Structural analyses of the gene and multiple sizes of mRNA.

The structure of rat CYP51 gene encoding sterol 14-demethylase was examined. The CYP51 gene spanned about 18 kb and contained 10 exons. The copy number of CYP51 in the rat genome was determined to be one. In addition, one CYP51 processed (intron-less) pseudogene covering the coding and ca. 600-bp 3'-noncoding sequences of CYP51 cDNA was found in the rat genome. Multiple transcription initiation sites were predicted by primer extension and 5'-RACE methods using poly(A)+ RNA from liver, ovary, and testis, and the major ones were located at 126 and 123 nucleotides upstream from the initiation ATG codon. The primer extension also showed several minor sites around the major ones. In addition to these sites, other minor initiation sites were also predicted at around 330 and 460 nucleotides upstream from the initiation ATG codon. No TATA box was found in the putative promoter region, but multiple GC boxes were found around the cap sites, supporting the previously inferred housekeeping nature of CYP51 gene and the existence of the multiple transcription initiation sites. A few consensus transcription regulatory elements such as CRE were found in the 5'-flanking region. Four polyadenylation signals were found in the 3'-noncoding region by the 3'-RACE method. Three of them were used to generate 3.1-, 2.7-, and 2.3-kb mRNAs in liver and ovary. The remaining one was used only in testis to generate 1.9-kb mRNA having an unusually short trailer sequence, suggesting a specific regulatory mechanism for generating CYP51 mRNA in testis different from that in liver and ovary.

Amino Acid Sequence↗

PubMed1997

Phylogenetic analysis of cystic fibrosis transmembrane conductance regulator gene in mammalian species argues for the development of a rabbit model for cystic fibrosis.

The species-specific pattern of cystic fibrosis transmembrane conductance regulator (CFTR) expression was investigated in order to identify species closely related to man which can be used as potential cystic fibrosis (CF) animal models. To this purpose, the nucleotide sequences of the CFTR promoter region of eight mammalian species representing four different orders (Primates, Artiodactyla, Lagomorpha and Rodentia) were analyzed. Distance matrices and unrooted trees of the CFTR promoter region sequences yielded two deeply separated groups, one including man (Homo sapiens), nonhuman primates (Hylobates lar, Macaca fascicularis, Saimiri sciureus), cow (Bos taurus), and rabbit (Oryctolagus cuniculus) and the other including the rodents (Rattus norvegicus, Mus musculus). Divergences between rodent and nonrodent groups have been observed in putative cis transcriptional regulatory elements and can be involved in the differences of pattern of expression between these two groups. Comparison of the available CFTR cDNA sequences enabled us to root the tree with a noneutherian outgroup and to perform a phylogenetic analysis. This analysis did not detect any base composition bias and supported polyphyletic Glires. Although a long-branch attraction artifact cannot be completely excluded, these findings converge toward the recent statement (Graur, Duret, and Gouy 1996) that Lagomorpha is more closely related to Primates than to Rodentia. In addition, the phenylalanine residue in exon 10 involved in the most common CF mutation in man is conserved in rabbit. These phylogenetic analyses as well as anatomical and developmental data suggest that, once rabbit embryonic stem cells become available, the rabbit will provide a suitable tool for both gene transfer and pharmacological investigations and could lead to a better CF model than the current murine models.

Animals↗

PubMed1998

In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation.

An in vivo gene transfer technique for living mouse testes was used to develop a novel transient expression assay system for transcriptional regulatory elements of spermatogenic specific genes. The combination of DNA injection into seminiferous tubules and subsequent in vivo electroporation resulted in an efficient and convenient assay system for gene expression during spermatogenesis. The transfer of the firefly luciferase reporting gene driven by the Protamine-1 (Prm-1) enhancer region revealed a significant increase in the activity of the reporter enzyme. Histochemical studies of the transfer of the lacZ gene driven by the Prm-1 enhancer showed specific lacZ expression only in haploid spermatid cells in adult testes, corresponding with the expression pattern of endogenous Prm-1. We were able to detect long-lasting transgene expression in the transfected spermatogenic cells. A group of spermatogenic differentiating cells maintained the transfected lacZ expression after more than 2 mo of transfection, suggesting that spermatogenic stem cells and/or spermatogonia could also incorporate foreign DNA and that the transgene could be transmitted to the progenitor cells derived from a transfected proliferating germ cell.

Animals↗

PubMed2002

Regulation of herpes simplex virus 1 replication using tumor-associated promoters.

OBJECTIVE: To investigate use of transcriptional regulatory elements (promoters) for tumor-associated antigens to achieve HSV-1 replication preferentially in cells that overexpress the tumor-associated antigens. SUMMARY BACKGROUND DATA: An important advantage of replicating viruses for cancer therapy is their ability to simultaneously destroy tumor cells by replication and release progeny virion to infect and destroy adjacent cancer cells. This strategy requires regulation of the viral life cycle to obtain robust replication in neoplastic cells and minimize replication in nonneoplastic cells. METHODS: Promoters for the human carcinoembryonic antigen (CEA) and MUC1/DF3 tumor-associated antigens were characterized and cloned into HSV-1 mutants as heterologous promoters regulating expression of two different HSV-1 genes. Viral replication in tumor cells and cytotoxicity was quantified with in vitro assays. Antineoplastic efficacy was characterized in a flank tumor xenograft model. RESULTS: Several CEA promoters were cloned and characterized using luciferase reporter assays. The most specific promoter was used to construct and isolate two different HSV-1 mutants in which critical genes are regulated by this promoter (ICP4 and gamma(1) 34.5). Similarly, the promoter for the DF3/MUC1 tumor-associated antigen was cloned into a third HSV-1 mutant such that it regulates expression of gamma(1) 34.5. Regulation of ICP4 expression by the CEA promoter during HSV-1 infection overly attenuates viral replication. Regulation of gamma(1) 34.5 expression by either the CEA promoter or the MUC1/DF3 promoter during HSV-1 infection modulates viral replication, with preferential replication in cells that overexpress the corresponding tumor-associated antigen. A single intratumoral inoculation of an HSV-1 mutant with the MUC1/DF3 promoter regulating gamma(1) 34.5 expression results in significant antineoplastic activity in MUC1-positive pancreatic carcinoma xenografts as compared to mock inoculation. CONCLUSIONS: Promoters for tumor-associated antigens may be incorporated into the HSV-1 genome to regulate HSV-1 replication. The choices of HSV-1 gene and tumor-associated promoter are important determinants of success of this strategy. Because of its preferential replication in MUC1-positive tumors, an HSV-1 mutant with the MUC1/DF3 promoter regulating gamma(1) 34.5 expression will undergo further examination as a novel cancer therapy agent.

Animals↗

PubMed1991

The nucleotide sequence of the infectious cloned DNA components of potato yellow mosaic virus.

The complete nucleotide sequence of a Venezuelan isolate of potato yellow mosaic virus (PYMV) has been determined, showing it to be typical of subgroup I geminiviruses in that it is whitefly-transmitted, has a circular, bipartite ssDNA genome and possesses bidirectionally orientated open reading frames (ORFs). The two genomic components have little sequence similarity apart from a common region of 268 nucleotides (nt) which is almost identical. Analysis of ORFs revealed six potential coding regions encoding proteins of Mr greater than 10K, four in PYMV A (2593 nt) and two in PYMV B (2547 nt), which are preceded by regulatory transcription elements and have polyadenylation signals present at the ends. Amino acid sequence alignments of PYMV DNA ORF-encoded proteins with those encoded by other previously sequenced geminivirus ORFs show that PYMV is closely related to those geminiviruses isolated from the New World, especially in the putative coat protein gene regions.

Amino Acid Sequence↗

PubMed2004

Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa.

Phylogenetic analysis of hepatitis B virus (HBV) has led to its classification into eight genotypes, A to H. The dominant genotype in South Africa is genotype A, which consists of two subgenotypes, A1 and A2. Subgenotype A1 (previously subgroup A') predominates over subgenotype A2 (previously subgroup A minus A'). The complete genome of HBV isolated from 18 asymptomatic carriers of the virus and five acute hepatitis B patients was amplified; the resulting amplicons were cloned and sequenced. All acute hepatitis isolates belonged to subgenotype A1 and had no distinguishing mutations relative to the isolates from asymptomatic carriers, which had a distribution of ten subgenotype A1, two subgenotype A2 and six genotype D. The presence of the previously described amino acid residues that distinguish subgenotype A1 (subgroup A') from the remainder of genotype A in the S and polymerase genes was confirmed. Moreover, the large number of subgenotype A1 isolates sequenced allowed identification in the other open reading frames of additional nucleotide and amino acid changes that are characteristic of subgenotype A1. In particular, nucleotide mutations at positions 1809-1812 that alter the Kozak sequence of the precore/core open reading frame, and A(1888) in the precore region, were found exclusively in subgenotype A1 isolates. Unique sequence alterations of the transcriptional regulatory elements were also found in subgenotype A1 isolates. The mean nucleotide divergence of subgenotype A1 was greater than that of subgenotype A2, suggesting that this subgenotype has been endemic for a longer time in the South African black population than had subgenotype A2.

Carrier State↗