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Results for “regulatory element transcripts”

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PubMed2001

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

Animals↗

PubMed1997

Regulation of murine myelin proteolipid protein gene expression.

To identify putative sequences that direct cell type-specific expression and/or enhance proteolipid protein (PLP) gene expression, glial or nonglial cells were transfected with various PLP-luciferase constructs that collectively span the entire mouse PLP-specific DNA present in a transgene known to direct cell type specificity in transgenic mice. These constructs were transfected into murine oligodendrocyte cell lines that transcribe the PLP gene and, hence, should contain the requisite trans-acting factors necessary for PLP gene expression. Mouse NIH/3T3 fibroblasts were used as a nonglial model. We have finely mapped the PLP promoter region for transcriptional regulatory elements and demonstrate both positive and negative elements, none of which appear to extinguish expression in nonglial cells. The 5'-flanking PLP DNA tested did not enhance the basal herpes simplex-1 virus thymidine kinase (TK) promoter, nor did PLP sequences present in the distal half of intron 1. The 5' portion of intron 1 did enhance TK promoter activity, suggesting that this region of the gene may contain enhancer elements that modulate PLP gene expression; however, the enhancement did not appear to be cell type-specific. Intriguingly, a 541 bp region of the intron that significantly enhanced TK promoter activity contains multiple JC virus repeated elements and other elements known to be important in restricting the virus to oligodendrocytes. These results suggest that intron 1 sequences may modulate expression of the PLP gene.

3T3 Cells↗

PubMed1990

Dissecting the complexity of the nervous system by enhancer detection.

Enhancer detectors are DNA constructs which, when introduced into a eukaryotic genome, respond to nearby genomic transcriptional regulatory elements by means of a reporter gene, revealing the expression pattern of genes in their vicinity. Recent experiments in Drosophila suggest that enhancer detection is a powerful method to identify genes that are expressed in the nervous system. Since enhancer detectors allow a rapid molecular and genetic characterization of genes in their vicinity, the method will greatly facilitate the study of neural development and behavior.

Animals↗

PubMed2005

Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.

BACKGROUND: Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. METHODS: We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. RESULTS: For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels. CONCLUSIONS: Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.

Antigens, CD34↗

PubMed1998

Molecular cloning of the cDNA and the promoter of the hamster uteroglobin/Clara cell 10-kDa gene (ug/cc10): tissue-specific and hormonal regulation.

Using the polymerase chain reaction on first-strand cDNAs from lung as well as on genomic DNA, we have cloned and sequenced both the cDNA and 1.5 kb of the promoter of the gene coding for hamster uteroglobin/Clara cell 10-kDa protein (UG/CC10), a secretory protein mainly synthesized in the pulmonary Clara cells. The amino acid sequence deduced from the cDNA indicated a preprotein of 96 residues, 19 of which corresponded to the signal peptide. The promoter region contained transcriptional regulatory elements conserved between species. Northern blot and S1 resistance assays detected high expression of ug/cc10 in lung but not at all in other tissues. Expression in lung was positively controlled by glucocorticoids and was also subjected to a biphasic developmental regulation after birth. In agreement with the tissue-specific expression observed in vivo, the hamster promoter preferentially directed the expression of a reporter gene in cells derived from Clara cells. The results indicated that the tissue-specific expression and the regulation of ug/cc10 varied considerably between species but all of them had a high expression in lung.

Amino Acid Sequence↗

PubMed1994

lacZ expression in germline transgenic zebrafish can be detected in living embryos.

Use of transgenic technology in zebrafish has been limited by the inability to efficiently express transgenes in early embryos of F1 and subsequent generations and to rapidly detect transgenic fish. We generated transgenic fish by injecting fertilized eggs with the Escherichia coli lacZ gene under the control of the Xenopus elongation factor 1 alpha transcriptional regulatory element. Four of five lines of transgenic fish we obtained express the lacZ gene in early embryos. The pattern of expression was distinct for each line, with two lines showing extensive expression beginning at approximately the midblastula transition, one showing patchy expression and one showing expression almost exclusively in motor neurons. Expression patterns were stable through the F2 generation in the three lines studied to date. The availability of these lines facilitated the development of a reliable and rapid method for live-staining lacZ-expressing embryos using the substrate fluorescein-di-beta-D-galactopyranoside (FDG). Positive embryos of the two most highly lacZ-expressing lines could be identified after 2-3 min of staining in FDG and then picked out and raised. These observations should prove useful for a variety of studies in zebrafish.

Animals↗

PubMed2002

Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain.

Previous studies demonstrated that the rat neuron-specific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken beta-actin hybrid (CBA) promoter-based systems. NSE promoter-driven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10(7) particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted detection of cells distal to the injection site that had retrogradely transported the vector from their terminal areas. Incorporating the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) into a CBA promoter vector induced greater expression levels in the hippocampus, as measured by stereological estimates of cell numbers and by Western blots, which demonstrated an 11-fold increase. Incorporation of the WPRE also improved transgene expression in primary neuronal cultures. The increased efficiency obtained with vector elements such as the CBA promoter and the WPRE may enhance the ability to genetically modify larger portions of the brain while requiring smaller doses and volumes.

Actins↗

PubMed1993

Structures and chromosomal localizations of two rat genes encoding S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a ubiquitous enzyme in eukaryotic cells and responds to a wide variety of stimuli affecting growth. To provide a framework for understanding the molecular basis of the mechanisms responsible for regulating the expression of this enzyme activity, we recently cloned and sequenced the rat gene for AdoMetDC. Now we have isolated another, slightly different AdoMetDC gene from a rat genomic library. Comparison of the two genes shows a high degree of conservation of sequence and structural organization. The two genes share the following characteristics: (1) both are approximately 16 kb in size, have identical exon/intron boundaries, and exhibit similar intron/exon structural organization; and (2) the nucleotide sequences are highly conserved in the coding regions and in many introns. Analysis of mouse-rat somatic cell hybrids has localized both rat genes to chromosome 20. The most interesting feature of these genes is that their 5' flanking regions are totally different. The promoter activities of the 5' regulatory regions were assessed by transient gene expression assays in Rat-2 cells after fusion to the chloramphenicol acetyltransferase gene. Transient transfections with the chimeric DNAs demonstrated that these fragments were able to function as efficient promoters, indicating that the diverging 5' regions of two AdoMetDC genes contain functional, but different, regulatory transcription elements.

Adenosylmethionine Decarboxylase↗

PubMed1994

Structure and regulation of the murine Clara cell secretory protein gene.

Clara cell secretory protein (CC10 or CCSP) is an abundant component of airway secretions and has the ability to bind small hydrophobic molecules. Genomic clones were isolated for the murine gene coding for Clara cell secretory protein. Nucleotide sequence analysis revealed that the gene was composed of three exons that span 4316 bp. Organization of the murine CCSP gene was very similar to that of the rabbit gene that codes for the biochemically related uteroglobin protein. Messenger RNAs for both genes were coded for by three exons, counterparts of which encode similar structural and functional protein domains. Transcriptional regulatory elements in the 5' flanking DNA were conserved between species, as were the functional properties of these elements when characterized in assays of promoter function. These data support the notion that Clara cell secretory protein genes from the rat and mouse, and the uteroglobin gene in rabbit, represent interspecies homologues.

Amino Acid Sequence↗

PubMed1998

Chromosomal localization and genomic characterization of the mouse melastatin gene (Mlsn1).

We recently described a novel gene, melastatin, whose expression is inversely correlated with melanoma aggressiveness. Chromosomal localization of this gene places it on mouse chromosome 7 and in the 15q13-q14 region of the human genome. Although expression patterns and chromosomal localization in the mouse are consistent with involvement of melastatin mutations in the mouse ruby-eye-2 defect, congenic analysis showed genetic segregation of the two loci. Cloning of the full-length human cDNA revealed a much larger transcript than we had previously identified, corresponding to a 1533-amino-acid protein product with homology to members of the transient receptor potential (Trp) family of calcium channels. The mouse melastatin gene contains 27 exons and spans at least 58 kb of genomic DNA. The promoter region of Mlsn1 contains four potential microphthalmia binding sites including an M box, a transcriptional regulatory element unique to genes with a restricted melanocytic expression pattern. A 1-kb PvuII fragment from this region was capable of driving high levels of luciferase expression in B16 melanoma cells.

Amino Acid Sequence↗

PubMed1992

Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes.

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into plasma cells. CD19 is a member of the Ig gene superfamily with two extracellular Ig-like domains separated by a non-Ig-like domain, and with an extensive approximately 240 amino acid cytoplasmic domain. In this study, Southern blot analysis revealed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned approximately 8 kilobases (kb) of DNA in human and approximately 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5' of the putative translation initiation AUG codon was well conserved in sequence between human and mouse and contained potential transcription regulatory elements. In addition, the 3' untranslated regions (UT) of the CD19 genes following the termination codon were conserved in sequence. The high level of conservation of nucleotide sequences between species in all exons and 5' and 3' UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.

Animals↗

PubMed1994

Chromosomal localization, genomic structure, and allelic polymorphism of the human CD79 alpha (Ig-alpha/mb-1) gene.

The germline DNA sequence of the human CD79 alpha (Ig-alpha/mb-1) gene was determined by polymerase chain reaction sequencing of a cosmid clone derived from an arrayed human chromosome 19 library. The CD79 alpha gene was localized to chromosome 19q13.2; this localization places the gene within the CEA-like gene cluster with the following gene order: -CEA-CGM1-CD79 alpha-RPS11-ATP1A3-BGP-CGM9-. The genomic organization of the human CD79 alpha gene resembles the mouse counterpart with five exons interrupted by four introns. Computer analyses suggest the presence of transcription regulatory elements known to be important in the regulation of mouse CD79 alpha (AP-1, EBF, AP-2, MUF2, and SP-1 sites), as well as elements not found in the mouse gene (an NK-kappa B binding site and a series of E-box motifs). Similar to the mouse gene, the 5' flanking region of human CD79 alpha lacks a TATA box; however, unlike mouse CD79 alpha, a classical octamer motif could not be identified in the human gene. Finally, a new Rsa I restriction fragment length polymorphism was defined in the non-coding regions of the human gene.

Amino Acid Sequence↗

PubMed1996

Characterization of human papillomavirus type 57b: transforming activity and comparative sequence analysis as probes for biological determinants associated with high-risk oncogenic viruses.

The association of human papillomavirus type 57 (HPV-57) with premalignant and malignant tumors of the nasal cavity was previously reported (Wu et al., Lancet 341, 522, 1993). We determined the complete nucleotide sequence of HPV-57b (GenBank 37537), which was molecularly cloned from a benign fungiform papilloma, and compared it with other HPV types and HPV-57a, which was cloned from an inverted papilloma of the maxillary sinus by de Villiers et al. (Virology 171, 248. 1989). Comparative and phylogenetic analysis of amino acid sequences of the HPV-57b oncogenes E5, E6, and E7 were performed with HPV-6, 11, 16, and 18. Phylogenetic trees using the Jotun-Hein algorithm indicated a closer relationship of HPV-57b E5 and E7 with corresponding genes of HPV-18. Signature pattern analysis of these two oncogenes was also in agreement with a closer relatedness to HPV-16 and 18 oncogenes, which are associated with a high risk for malignant progression. Compared with 7861 bp of HPV-57a, HPV-57b had 7868 bp as well as differences in the restriction enzyme sites and the open reading frames, including at least five additional ones. To investigate the oncogenic potential of HPV-57b, NIH 3T3 and REF52 cells were cotransfected with two plasmids: pKP54. HPV-57b, which contains the HPV-57b genome, and pMT.neo.1, which confers resistance to G418. After selection in culture medium containing G418, 58% of the G418r NIH 3T3 colonies and 47% of the G418r REF52 colonies exhibited morphological transformation. These results indicate that the transcriptional regulatory elements and the oncoproteins of HPV-57b are active in vitro to induce cellular transformation, as are other high-risk HPV types.

3T3 Cells↗

PubMed2004

All roads lead to arginine: the squid protamine gene.

The protamine of squid is one of the most arginine-rich protamines (77%, mol/mol). It possesses a leading sequence that is posttranslationally removed during spermatogenesis in a manner that is analogous to that observed in some of its vertebrate protamine counterparts. In this paper we describe the gene sequence of the protamine of the squid Loligo opalescens. This represents the first complete gene sequence ever reported for an invertebrate protamine. Like those of vertebrate protamines, the messenger RNA is polyadenylated but the gene does not contain an intron. The promoter region contains the major transcriptional regulatory elements (CRE, TATA box, and CAP) that are also characteristic of the vertebrate protamine genes. It is unclear whether the similarities of protamines in species from both the deuterostome and the protostome branches represent the result of phylogenetic conservation or evolutionary convergence.

Amino Acid Sequence↗

PubMed1997

Genetic and functional complementation of the HSV1 UL27 gene and gB glycoprotein by simian alpha-herpesvirus homologs.

Utilizing co-transfection of DNA from glycoprotein gB- strain of HSV1 and cloned fragments of several simian alpha-herpesviruses containing the UL26, UL27 (gB glycoprotein), and UL28 gene homologs, replication-competent recombinant viruses were produced. Genetic analysis of one HSV1/SA8 recombinant (HSV1/SgB) demonstrated the presence of SA8 DNA comprising the entire UL27 (gB) gene and parts of the UL28 and UL26 ORFs in an otherwise HSV1 genome. The recombinant was shown to express the SA8 gB and p40 proteins (UL27 & UL26.5 gene products, respectively); all other proteins were indistinguishable from those of HSV1. The recombinant behaved like SA8 in gB-specific virus neutralization and cell surface antibody binding assays, while plaque morphology and replication kinetics were very similar to HSV1. Despite its overwhelming HSV1 genetic constitution, the recombinant displayed a pathogenic phenotype in mice very different from the parental HSV1. While HSV1 produced corneal disease in ocularly infected mice and readily spread to the nervous system. HSV1/SgB was markedly impaired in both respects. These results demonstrate the functional equivalency of the cercopithecine monkey virus gB glycoproteins and genes (including transcriptional regulatory elements) in HSV1, the functional nature of HSV1/SA8 chimeric UL28 and UL26 genes/proteins, and that UL28, gB and/or p40 proteins may effect the pathogenicity of HSV1.

3T3 Cells↗

PubMed1995

Molecular cloning and sequence analysis of a seed-expressed acyl carrier protein (ACP) gene from Brassica campestris (Agrani).

We describe here the nucleotide sequence of the Brassica campestris ACPSF1 gene which encodes a seed-expressed acyl carrier protein (ACP). The 3600 bp sequence consists of 1740 bp upstream of the translation start codon, 828 bp spanning the coding region which is interrupted by three introns and 1032 bp downstream of the stop codon. Using a ACPSF1 gene-specific probe, transcripts could be detected in developing seeds, but not in leaves. The gene is now the only known member that represents group I seed-expressed ACP multigene family of Brassica species. The 5' flanking sequence of the ACPSF1 gene was examined for putative transcriptional regulatory elements. A sequence alignment of the 5' flanking regions of the available seed-expressed ACP genes of Brassica species showed some conserved regions which might have some common regulatory significance.

Acyl Carrier Protein↗

PubMed1990

The human pregnancy-specific glycoprotein genes are tightly linked on the long arm of chromosome 19 and are coordinately expressed.

The pregnancy-specific glycoprotein (PSG) genes encode a group of proteins which are found in large amounts in placenta and maternal serum. In situ hybridization analyses of metaphase chromosomes reveal that all the human pregnancy-specific glycoprotein (PSG) genes are located on the long arm of chromosome 19 (19q13.2-13.3), overlapping the region containing the closely-related carcinoembryonic antigen (CEA) gene subgroup. Higher resolution analyses indicate that the PSG genes are closely linked within an 800kb SacII restriction endonuclease fragment. This has been confirmed through restriction endonuclease mapping and DNA sequence analyses of isolated genomic clones, which show that at least some of these genes are located in very close proximity. Further, these studies have helped to identify a new member of the PSG gene subfamily (PSG7). DNA/RNA hybridization analyses, using gene-specific oligonucleotide probes based on published sequences, showed that five from six PSG genes tested are coordinately transcribed in the placenta. Due to the close proximity of these genes and their coordinated expression pattern, common transcriptional regulatory elements may exist.

Base Sequence↗

PubMed1992

DNA binding factors which interact with the Sp1 site of the chicken delta 1-crystallin promoter are developmentally regulated.

Transcription of the delta 1-crystallin gene is developmentally regulated in the embryonic chicken lens. Previous work defined a positive transcription regulatory element between positions -120 and -43 of the delta 1-crystallin promoter. This region contains a putative Sp1 binding site (-78 to -71), adjacent to a CAAT box (-70 to -67). Gel retardation assays using lens nuclear extracts revealed two protein-DNA complexes which involved the Sp1 site. The formation of the complexes increased from day 6 to day 11 of embryogenesis (period of lens organogenesis) peaked between days 11 and 15, then decreased in a non-parallel manner until hatching (day 21). A point mutation in the Sp1 binding site of the delta 1-crystallin promoter abolished formation of one of the complexes (complex 1, slower in mobility), while point mutations in the CAAT box had no effect on the formation of either complex. Studies using purified Sp1 protein and increasing amounts of embryonic chicken lens nuclear extracts showed cooperativity in the formation of both complexes, more remarkable with complex 1.

Aging↗