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T J Ley

Publications and source records attributed to T J Ley.

At least 73 records · Page 4Linked to original sources
PubMed1992

Characterization of the 5' untranslated region of the human c-fgr gene and identification of the major myelomonocytic c-fgr promoter.

In this study, we have characterized the 5' region of the human c-fgr proto-oncogene and identified the major myelomonocytic c-fgr promoter. Seven distinct 5' untranslated exons were identified and localized to a region extending 13 kb upstream from the first coding exon. Two major promoters were identified, one utilized exclusively in Epstein-Barr virus (EBV)-infected B-lymphocyte cell lines, and the other functional only in myelomonocytic cells. Differential promoter utilization and alternative splicing of the 5' untranslated exons give rise to at least six distinct c-fgr mRNA species that differ only in their 5' untranslated regions. Two major mRNAs were identified, c-fgr A and c-fgr 4; these two mRNAs were detected exclusively in EBV-infected B-lymphocyte cell lines and myelomonocytic cells respectively. We have previously demonstrated that c-fgr is transcriptionally activated in U937 cells treated with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or cycloheximide (CHX). We now show that a DNA fragment extending from -772 to +97 (with respect to the transcription initiation site upstream from exon M4) is responsive to TPA but not CHX treatment in U937 cells. These results suggest that TPA and CHX induce c-fgr mRNA accumulation by different mechanisms.

Base Sequence↗
PubMed1992

Neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation.

Human mononuclear phagocytes have the capacity to participate directly in extracellular matrix turnover via the secretion of neutral proteinases. These neutral proteinases include the serine proteinases, elastase and cathepsin G and the metalloproteinases, interstitial collagenase, 92 kD type IV collagenase, 72 kD type IV collagenase and stromelysin. Mononuclear phagocytes also produce the counter-regulatory metalloproteinase inhibitor, TIMP (tissue inhibitor of metalloproteinases). We have studied the capacity of normal human mononuclear phagocytes and of the human monocytic tumor line U937 to elaborate proteinases and inhibitors. The serine proteinases, elastase and cathepsin G, are present only at the earliest stages of mononuclear phagocyte differentiation (U937 cells in the basal state, freshly isolated peripheral blood monocytes) and are stored within intracellular granules. As human mononuclear phagocytes differentiate (U937 cells exposed to phorbol esters, human monocytes cultured in vitro), the cellular content of these serine proteinases declines rapidly. Accompanying the acquisition of a more differentiated state, the ability for regulated secretion of the neutral metalloproteinases is attained. This capacity is acquired in a sequential manner, with secretion of the 92 kD type IV collagenase observed at earlier states of differentiation while release of stromelysin requires a fully differentiated and LPS (lipopolysaccharide)-stimulated alveolar macrophage. Interstitial collagenase and 72 kD type IV collagenase are synthesized at intermediate stages of differentiation. In comparison to human fibroblasts, human mononuclear phagocytes produce approximately 10-30% of the interstitial collagenase, 10% of the stromelysin and 1-2% of the 72 kD type IV collagenase on a per cell basis. Synthesis of the 92 kD type IV collagenase is restricted to the inflammatory cell (but also occurs in neutrophils and keratinocytes).

Adult↗
PubMed1991

The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice.

The human CSP-B/CGL-1 gene is the homologue of the mouse granzyme B/CCPI gene and encodes a cytotoxic T-lymphocyte-specific serine protease. We have used regulatory sequences upstream from the CSP-B gene to drive human growth hormone gene expression in transgenic mice. Eleven founder mice were screened for transgene expression in activated T-cells. Expression was detected in 10 mice; levels of expression were integration site-dependent. The transgene was not expressed in resting lymphocytes but could be activated by treatment with concanavalin A or interleukin-2, indicating that CSP-B regulatory sequences are responsive to signals originating at either the T-cell receptor or the interleukin-2 receptor. Transgene expression was detected at the whole organ level only in lymph nodes and small intestine, where endogenous mouse CCPI mRNA was also present. The time course of transgene activation in T-lymphocytes was similar to that of the mouse CCPI gene. No differences in levels of expression of the transgene were observed in activated lymphocyte populations that had been depleted of either CD4+ or CD8+ cells; in contrast, the mouse CCPI gene was expressed primarily in CD8+ cells. Six CD4+ T-cell clones with Th0, Th1, or Th2 phenotypes were generated from a transgenic animal. All clones expressed moderate to high levels of the transgene, but only three clones expressed mouse CCPI, indicating that the transgene is disregulated in CD4+ T-cell subsets. The CSP-B regulatory unit represents a novel reagent for targeting gene expression to activated T-lymphocytes.

Animals↗
PubMed1991

Functional properties of the beta-globin locus control region in K562 erythroleukemia cells.

In this report, we compare the function of the human beta-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for "classical" enhancer activity, (2) a colony assay that detects "productive integration events," and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had approximately 10% as much colony assay activity as the entire mu LAR. However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma-neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested.

Base Sequence↗
PubMed1991

Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2.

We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.

Blotting, Southern↗
PubMed1991

1,25-Dihydroxyvitamin D3 induces monocytic differentiation of the PLB-985 leukemic line and promotes c-fgr mRNA expression.

1,25-dihydroxyvitamin D3 [1,25(OH)2D] is known to stimulate maturation of the human promyelocytic line HL-60 and murine bone marrow precursor cells along a monocyte/macrophage pathway. In this study, the steroid's effects on the PLB-985 leukemic line were examined. We found that 1,25(OH)2D induces monocytic differentiation of PLB-985 as manifested by morphological appearance, histochemical staining, and changes in cell surface antigen expression. Additionally, there was acquisition of functional monocyte characteristics including phagocytic activity and superoxide anion production via the respiratory burst pathway. Steady state levels of mRNA derived from the leukocyte-specific proto-oncogene c-fgr were also increased by the steroid. Thus, 1,25(OH2)D effectively differentiates PLB-985 cells along a monocytic pathway, providing a useful model of macrophage differentiation.

Calcitriol↗
PubMed1991

A novel c-fgr exon utilized in Epstein-Barr virus-infected B lymphocytes but not in normal monocytes.

The fgr proto-oncogene encodes a nonreceptor protein-tyrosine kinase, designated p55c-fgr. In this study, we have isolated human fgr cDNA molecules from normal monocyte mRNA templates. Nucleotide sequence analysis of the longest fgr cDNA revealed a 5' untranslated region of 927 bp which included two Alu-like repeats as well as three translation stop codons immediately upstream of the initiator for p55c-fgr synthesis. Within genomic DNA, these sequences were distributed over 13 kbp as three distinct 5' untranslated exons. Previous studies have shown that Epstein-Barr virus (EBV) increases c-fgr mRNA levels in B lymphocytes. By comparing the nucleotide sequence reported for transcripts isolated from EBV-infected B lymphocytes with those of our monocyte cDNA as well as genomic DNA, we identified a novel untranslated exon utilized only in EBV-infected cells. The transcriptional initiation sites of fgr mRNA expressed in EBV-converted cells were mapped and shown to reside within a region identified as an intron for fgr mRNA that is expressed in normal myelomonocytic cells. Furthermore, the region of the fgr locus upstream of the novel exon displayed properties of a transcriptional promoter when transfected into heterologous cells. We conclude from all of these findings that activation of the fgr gene by EBV is achieved by mechanisms distinct from those normally regulating its programmed expression in myelomonocytic cells.

B-Lymphocytes↗
PubMed1990

Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells.

We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated "A gamma-neo" transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

Beta-Globulins↗
PubMed1990

Developmental regulation of the human cathepsin G gene in myelomonocytic cells.

Cathepsin G is a neutral serine protease that is found in the azurophil granules of neutrophils and monocytes. Previous experiments had demonstrated that cathepsin G is actively produced by the promonocytic U937 cell line, and that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of these cells toward macrophages resulted in a reduction of cathepsin G activity. In this study, we have analyzed the mechanism of this TPA-induced down-regulatory event. Using in situ hybridization techniques, we demonstrated that cathepsin G mRNA is detectable only at the promyelocytic stage of myeloid development. Using U937 promonocytic cells as a model, we demonstrated; 1) cathepsin G protein levels decline in TPA-treated cells; 2) this decline was due to a nearly complete loss of cathepsin G mRNA in cells treated with TPA for 24 h; and 3) the rate of cathepsin G mRNA loss with TPA treatment was similar to that with actinomycin D. These results suggested that cathepsin G transcription was down-regulated within several hours of TPA addition. This was directly tested by performing nuclear run-off assays of TPA-treated U937 cells; cathepsin G transcription was shown to be strand-specific, and declined within 4 h of TPA addition. Cathepsin G transcription was essentially undetectable 8 or more hours after TPA treatment, suggesting that down-regulation is predominantly transcriptional. Cycloheximide treatment of U937 cells resulted in a partial block of TPA-mediated cathepsin G down-regulation, indicating that continuous protein synthesis is required for down-regulation to occur. A newly synthesized protein or proteins may therefore be required for the transcriptional down-regulation of cathepsin G during the normal development of promyelocytes or promonocytes.

Cathepsin G↗
PubMed1990

Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions.

DNA sequences located in a region 6-18 kilobases (kb) upstream from the human epsilon-globin gene are known as the locus-activating region (LAR) or dominant control region. This region is thought to play a key role in chromatin organization of the beta-like globin gene cluster during erythroid development. The beta-globin LAR activates linked globin genes in transiently or stably transfected erythroleukemia cells and in erythroid cells of transgenic mice. Since the human beta-globin LAR is functional in mice, we reasoned that critical LAR sequence elements might be conserved between mice and humans. We therefore cloned murine genomic sequences homologous to one portion of the human LAR (site II, positions -11,054 to -10,322 with respect to the human epsilon gene). We found that this murine DNA fragment (mouse LAR site II) and sequences homologous to human LAR sites I and III are located upstream from the mouse beta-like globin gene cluster and determined that their locations relative to the cluster are similar to that of their human counterparts. The homologous site II sequences are 70% identical between mice and humans over a stretch of approximately 800 base pairs. Multiple core sequences with greater than 80% identity were present within this region. Transient and stable transfection assays of K562 erythroleukemia cells demonstrated that both human and mouse LAR elements contain enhancer activity and confer hemin inducibility on a linked human gamma-globin promoter. These results suggest that primary structural elements--and the spatial organization of these elements--are important for function of the beta-globin LAR.

Animals↗
PubMed1990

A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human alpha/delta T-cell receptor locus.

The chymotrypsin-like family of serine protease genes includes several members that are expressed exclusively in subsets of hematopoietic cells. For example, human neutrophil elastase and cathepsin G are expressed only in myelomonocytic precursors, and cytotoxic-T-cell serine proteases are found only in cytotoxic lymphocytes. We have used a cathepsin G cDNA probe to clone two cathepsin G-like genes (designated CGL-1 and CGL-2) from a human genomic library. We have determined that CGL-1 is identical to a previously identified gene (known as CCPI, CTLA I, or cytotoxic serine protease B) that is expressed only in activated cytotoxic T lymphocytes. We show here that cathepsin G, CGL-1, and CGL-2 are linked on an approximately 50-kilobase locus found on human chromosome 14 at band q11.2. This gene cluster maps to the same chromosomal band as the alpha and delta T-cell receptor genes; this region is involved in most chromosomal translocations and inversions that are specifically associated with T-cell malignancies.

Blotting, Southern↗
PubMed1990

Function of transfected globin promoters and the globin locus activator in K562 erythroleukemia cells.

We have examined the importance of cis-acting regulatory elements within the human gamma-globin gene promoter and the globin locus activating region in K562 cells. A gamma-globin or beta-globin promoter fragments were fused with the neomycin phosphotransferase gene in a plasmid-based vector (gamma-neo or beta-neo) and transiently transfected by electroporation into K562 cells. Correctly initiated gamma-neo or beta-neo transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation and then determined that a gamma-globin promoter fragment extending from -299 and +36 was active in the assay but that a beta-globin promoter extending from -375 to +46 was inactive. Deletion of the gamma-globin promoter to -199 did not affect promoter function, but deletion to -160 reduced promoter strength to 70% of that of control. Additional deletion to position -130 reduced promoter strength to 19% of the control value, and to position -61, 8.7% of the control value. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (HPFH), -202 C----G, -196 C----T and -117 G----A, were not overexpressed in K562 cells, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only in adult red cells. When the beta-globin locus activating region (LAR) was added to a wild-type or an HPFH gamma-neo plasmid, the abundance of correctly initiated gamma-neo transcripts increased dramatically. However, beta-neo expression could not be activated by the LAR in K562 cells. These studies should allow us to further dissect the interactive roles of globin promoters and enhancers in K562 cells.

Cell Line↗
PubMed1990

Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.

The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined.

Base Sequence↗
PubMed1989

Nucleotide sequence and regulation of a human 90-kDa heat shock protein gene.

In order to define the mechanisms responsible for the regulated expression of a human 90-kDa heat shock protein (HSP90) gene, we have determined the complete genomic sequence of the gene encoding HSP90 beta, including 1102 base pairs upstream of the transcription initiation site. This gene consists of 12 exons and 11 introns. The exons range in size from 99 to 396 base pairs and the introns range from 91 to 1433 base pairs. Analysis of the 5' promoter region revealed TATA and CAAT consensus sequences. Additionally, Sp1 factor binding sites and presumptive heat shock element consensus sequences were found surrounding the transcription initiation site. Three out of the six identified potential heat shock elements reside within the first intron. The ability of the promoter region to respond to heat shock was examined by constructing a plasmid containing the 5' end of the HSP90 beta gene driving a neomycin phosphotransferase reporter gene. This plasmid was used to stably transform human K562 erythroleukemia cells. With heat shock, the abundance of HSP-neo transcripts increased, demonstrating that the promoter region of this gene contains the cis-acting DNA elements required for the heat shock response.

Base Sequence↗
PubMed1989

Genomic organization and chromosomal localization of the human cathepsin G gene.

Cathepsin G is a 26,000-Da serine protease that is found in the azurophil granules of neutrophils and monocytes. The cathepsin G gene is expressed at high levels in U937 promonocytic cells, but is down-regulated with phorbol-induced differentiation. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic fibroblast library using cathepsin G cDNA, and obtained two lambda clones that contained the cathepsin G locus. The cathepsin G gene spans 2.7 kilobase pairs of genomic DNA and consists of 5 exons and 4 introns. The genomic organization of cathepsin G is similar to that of human neutrophil elastase, rat mast cell protease II, murine adipsin, and murine cytotoxic T-cell serine proteases, with protease catalytic residues located near the borders of exons 2, 3, and 5. Using in situ hybridization techniques, we localized cathepsin G to chromosome 14q11.2, a site that is near the alpha/delta T-cell receptor complex. Cathepsin G transcription is abolished in U937 nuclei with 2 micrograms/ml alpha-amanitin, indicating that this gene is probably transcribed by RNA polymerase II. The 5' end of the cathepsin G gene was defined by primer extension and S1 nuclease protection assays. A TATA box is found at position -29, and a CAAT box is found at -69 with respect to the transcription initiation site. Having defined the genomic structure and chromosomal location of cathepsin G, we are now attempting to identify the DNA elements in or near this gene that mediate its tissue and development-specific pattern of expression.

Amino Acid Sequence↗
PubMed1989

Analysis of epitope expression and the functional repertoire of recombinant complement receptor 2 (CR2/CD21) in mouse and human cells.

We transfected human complement receptor 2 (CR2/CD21) cDNA containing eukaryotic expression constructs into CR2-negative mouse L cells and human K562 erythroleukemia cells. We subsequently selected stably transformed cells that expressed human CR2, as assessed by flow microfluorimetry analysis and immunoprecipitation of 125I-labeled surface membranes using the monoclonal anti-CR2 antibody, HB5. Utilizing flow microfluorimetry analysis, epitopes recognized by anti-CR2 mAb HB5, OKB7, B2, and four other anti-CR2 antibodies were detected on CR2 expressing transfectants but not parental cells. In addition, CR2 expressing transfected cells efficiently formed rosettes with sheep erythrocyte intermediates bearing human C3bi and C3d, but not C4b or C3b, consistent with the known ligand specificity of CR2. CR2 containing transfectants were also demonstrated to specifically bind EBV. Infection with EBV of CR2 expressing L cells and K562 cells resulted in mean expression of Epstein-Barr nuclear Ag (EBNA) at 48 h in 0.35% of CR2 expressing L cells and 3.7% of CR2 expressing K562 cells. Parental L cells and K562 cells did not express EBNA after EBV infection. These results indicate that CR2 alone is sufficient to transfer both C and EBV receptor functions to heterologous cells. In addition, expression of EBNA was found to be significantly higher in human K562 than mouse L cells, both expressing the same recombinant receptor. These results suggest that mechanisms other than CR2 binding lead to inefficient EBV infection and/or EBNA synthesis in mouse fibroblasts.

Animals↗