Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5' upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a beta-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.
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PMID:Novel DNA binding proteins participate in the regulation of human neurofilament H gene expression. 127 52

Using a synthetic peptide that encompasses the zinc finger domain of the eukaryotic transcription factor Sp1, we produced a number of monoclonal antibodies that specifically reacted with the target antigen. Analysis by competitive inhibition assay of five of the monoclonal antibodies revealed that they all recognized a dominant epitope in the synthetic peptide and reacted strongly to recombinantly synthesized beta-galactosidase-Sp1 fusion polypeptide. To determine cellular distribution of Sp1-like molecules, cytoplasmic and nuclear proteins from human lung fibroblasts (HFL-1) and a human rhabdomyosarcoma cell line (A204) were immunoblotted and reacted with our antibodies. In addition to the well characterized 95 Kd and 105 Kd proteins, considered to be the authentic Sp1 polypeptide, a number of other cellular proteins reacted with these antibodies. Immunofluorescence staining of the cells with mAb to the zinc finger of Sp1 also revealed cell-specific differences in intracellular distribution of Sp1-like molecules. Both cytoplasmic and nuclear staining was readily observed in the rhabdomyosarcoma cells. In contrast, while some HFL-1 cells exhibited staining of only cytoplasm, both cytoplasmic and nuclear immunofluorescence was seen in others.
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PMID:Generation of monoclonal antibodies to the zinc finger domain of the eukaryotic transcription factor Sp1. 201 Nov 20

As the major proteins of adult keratinocytes, keratins provide biochemical markers for exploring mouse epidermal embryogenesis. Here, we used a modified method of whole-mount in situ hybridization to track skin-specific expression of endogenous keratin mRNAs throughout embryogenesis. To monitor transcriptional regulation, we coupled this with beta-galactosidase expression of a human epidermal keratin promoter-driven transgene. These studies have radically changed our perception of how the program of gene expression becomes established during epidermal development. Specifically, we have discovered that (1) basal keratin (K5 and K14) genes are first detected at E9.5 in a highly regional fashion, and surprisingly as early as the single layered ectodermal stage; (2) the early patterns do not correlate with morphogenesis per se, but rather with regional variations in the embryonic origin of underlying mesenchyme, supporting morphogenetic criteria that early inductive cues are mesenchymal; (3) epidermal keratin genes are expressed in periderm, supporting the notion that this layer arises from ectodermal stratification, even though it is simple epithelial-like in morphology and is subsequently sloughed during development; (4) later embryonic patterns of K5 and K14 gene expression parallel proliferative capacity and not stratification; and (5) K1 and K10 mRNAs are first detected as early as E13.5, and their patterns correlate with differentiation and not stratification. These patterns of epidermal gene expression led us to explore whether potential transcriptional regulators of these genes are expressed similarly. We show that AP2 (but not Sp1) cRNAs hybridize in a pattern similar to, but preceding that of basal keratin cRNAs. Finally, using gene expression in cultured cells, we demonstrate that AP2 has a strong inductive effect on basal keratin expression in a cellular environment that does not normally possess AP2 activity.
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PMID:Programming gene expression in developing epidermis. 752 78

The herpes simplex virus type 1 (HSV-1) major capsid protein VP5 gene (UL19) is expressed with beta gamma (gamma 1 [leaky late]) kinetics. We have previously described the construction of recombinant HSV-1 in which the VP5 promoter was engineered to control the expression of the bacterial beta-galactosidase gene as a reporter (C.-J. Huang, S. A. Goodart, M. K. Rice, J. F. Guzowski, and E. K. Wagner, J. Virol. 67:5109-5116, 1993). Here we describe further mutational analysis in recombinant viruses. We have precisely defined the boundaries of the VP5 promoter and identified two regions important for both the level and the kinetics of expression. The 5' boundary was located at -48 relative to the initiation site of transcription by analyzing a series of nested deletions in the upstream sequence, and although a number of cis-acting sites influencing transient expression have been identified upstream of this point, these sites have no role in promoter activity during productive infection. Deletion of an Sp1-binding site located between -48 and the TATA box at -30 greatly reduced VP5 promoter activity late but not early after infection. A cis-acting element whose sequence resembles the human immunodeficiency virus type 1 initiator was located between -2 and +10 in the VP5 sequence by characterizing a series of deletions and site-directed block mutations downstream the TATA box. This element defines the 3' limit of the VP5 promoter, and like the upstream element, disruption of this element also inhibited promoter activity late in the productive cycle.
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PMID:The herpes simplex virus type 1 major capsid protein (VP5-UL19) promoter contains two cis-acting elements influencing late expression. 805 55

Plastins are a family of human actin-binding proteins (isoforms) which are abundantly expressed in all normal replicating mammalian cells. One isoform, L-plastin, is constitutively expressed at high levels in hemopoietic cell types while T-plastin is constitutively expressed in all non-hemopoietic cells of solid tissues that have replicative potential (fibroblasts, endothelial cells, epithelial cells, melanocytes, etc.). L-plastin is, however, constitutively synthesized in many types of malignant human cells of solid tissues suggesting that its expression is induced during tumorigenesis. The frequency of L-plastin induction in some cancers of the steroid-regulated female reproductive tract (breast, ovary, uterus, and placenta) appears to be especially high (79% in a limited survey). To learn the mechanism of L-plastin gene activation accompanying tumorigenesis, we have begun to characterize the promoter and regulatory elements of the L-plastin gene. Transcription initiation from this promoter was found to occur at multiple sites and as near as 10 base pairs from the 3'-side of the TATAAA box. The promoter and its flanking DNA were cloned and sequenced to identify potential regulatory elements that participate in the induction of the L-plastin gene in neoplastic cells. Examination of upstream sequences revealed the existence of two potential progesterone, one potential estrogen, and four potential Ets-1 responsive elements flanking the promoter. A 315-base pair fragment spanning the TATAAA box and a potential Sp1-binding site exhibited maximum promoter activity using CAT as a reporter while longer promoter fragments extending into upstream flanking sequences spanning the hormone receptor-response elements exhibited reduced promoter activity. An expression vector, pHLPPr-1-neo, was constructed using a 5.1-kilobase pair EcoRI-HindIII fragment of the L-plastin gene that contained the potential upstream regulatory elements, the TATAAA box, and part of the first exon. This promoter could direct the constitutive expression of the reporter beta-galactosidase at high frequency in transfected colonies of transformed cells that express L-plastin constitutively; by contrast, this promoter was virtually inactive in transfected colonies of normal fibroblasts and it exhibited a low frequency of constitutive activation in transfected colonies of in vitro SV40-transformed fibroblasts which did not exhibit L-plastin expression. The utility of this recombinant promoter in determining the mechanism(s) that leads to activation of the L-plastin gene in tumor cells is discussed. The potential significance of regulation of the L-plastin gene by reproductive hormones in cancers arising in hormone-responsive tissues is also discussed.
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PMID:Characterization of the human L-plastin gene promoter in normal and neoplastic cells. 842 53

Protective protein/cathepsin A (PPCA) is a lysosomal serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. Its deficiency in humans leads to the lysosomal storage disorder galactosialidosis (GS). The pathologic manifestations in patients relate primarily to the severe deficiency of neuraminidase, and the physiological significance of cathepsin A activity remains unclear. The mouse model of GS, which closely resembles the human phenotype, shows that cells from numerous tissues, especially the central nervous system (CNS), are affected by this disease. To study the site and level of expression of PPCA mRNA in murine and human tissues, we analyzed the promoter regions of the corresponding genes. Their 5' genomic regions were strikingly similar in both organization and sequence. A single 1.8-kb PPCA transcript is present in humans, whereas mouse tissues have a major 1.8-kb and a minor 2.0-kb transcript, both of which are differentially expressed. These two mouse mRNA species differ only in their 5' untranslated region (UTR). The larger mRNA, unique to mouse, is transcribed from an upstream TATA-box-containing promoter, which is absent in the human gene. The downstream promoter, which transcribes the 1.8-kb mRNA common to human and mouse, has characteristics of housekeeping gene promoters and contains putative Sp1 binding sites and three USF/MLTF sequences. In vitro studies demonstrated that expression from the downstream promoter is higher than that from the upstream murine-specific promoter. In situ hybridization of mouse tissue sections identified regions of the brain that preferentially express the 2.0-kb transcript. Our results imply that PPCA mRNA distribution and regulation in murine tissues differs from that in human tissues.
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PMID:Identification of the promoters for the human and murine protective protein/cathepsin A genes. 917 65

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.
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PMID:A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice. 927 25

Functional analysis of two promoters controlling early herpes simplex virus type 1 (HSV-1) transcripts encoding the UL37 and UL50 (dUTPase) proteins are described in this report. Transcripts expressed under the control of these promoters were found to be expressed early regardless of the position of the transcription unit within the viral genome. Despite this, wt dUTPase mRNA was 6-10 times more abundant than the UL37 transcript both in wt and recombinant viruses. This same difference in transcript abundance was seen when a reporter gene (beta-galactosidase) was controlled by the two promoters in recombinant viruses in the heterologous glycoprotein C (gC) locus. Thus, both the kinetics and relative abundance of UL50 and UL37 transcripts are a direct function of their respective promoter regulatory elements. Characterization of mutated UL37 and UL50 promoters in recombinant viruses showed that the functional modules important for expression from these promoters are concentrated upstream of the transcription start site; however the extent and composition of these modules in terms of the cis-acting elements they contain was different for each. For the UL37 promoter, both a HiNF-P factor binding site (-53 to -58 bp) and the TATA homology (-22 to -27) were required for any detectable expression, while an Sp1 binding site at -123 augmented this but was not absolutely required. In contrast, the only functional elements crucial for expression from the UL50 promoter were the TATA box (-25 to -31) and an Sp1 binding site at -117 bp relative to the cap site. Despite differences in detail, when the functional architecture of these two early promoters were compared to the extensively characterized HSV-1 thymidine kinase (UL23) promoter, class-specific similarities are clearly apparent.
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PMID:Functional modules important for activated expression of early genes of herpes simplex virus type 1 are clustered upstream of the TATA box. 965 2

The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (-230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the -230/+126 base pair promoter express beta-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.
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PMID:Combinatorial cis-acting elements control tissue-specific activation of the cardiac troponin I gene in vitro and in vivo. 973 4

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.
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PMID:Transcriptional regulation of the Bmp2 gene. Retinoic acid induction in F9 embryonal carcinoma cells and Saccharomyces cerevisiae. 988 May 12


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