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D R Morris

Publications and source records attributed to D R Morris.

At least 19 recordsLinked to original sources
PubMed1992

Cell-specific translation of S-adenosylmethionine decarboxylase mRNA. Regulation by the 5' transcript leader.

The mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) has an unusual distribution in polysomes from cells of T lymphocyte origin. It associates predominantly with monosomes and small polysomes with none located in the preribosomal or ribonucleoprotein pool. In sharp contrast, it associates broadly with larger polysomes in several nonlymphoid cell lines, including fibroblasts and the adrenal carcinoma line, Y1. The AdoMetDC 5'-transcript leader (5'-TL) is highly conserved between human and bovine mRNAs. It has a length of about 330 nucleotides and contains a 21-nucleotide upstream open reading frame (uORF) approximately 14 nucleotides downstream of the cap site. The AdoMetDC 5'-TL, when used to replace the 5'-TL of the human growth hormone gene in a chimeric expression construct, causes a suppressed polysomal distribution of the chimeric mRNA identical to that of the endogenous AdoMetDC mRNA in the T cell line, Jurkat. In contrast, mRNA encoded by the same chimeric construct, when expressed in Y1 cells, mimics the broad polysomal distribution of the endogenous AdoMetDC mRNA. Mutations that remove the uORF, or prevent its initiation, abolish the translational suppression in T cells, establishing that the uORF is a negative element that modulates the cell-specific polysomal distribution of AdoMetDC mRNA.

Adenosylmethionine Decarboxylase↗
PubMed1992

Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A.

Transcription of the ornithine decarboxylase (ODC) gene is rapidly elevated by activation of protein kinase A (PKA). The additive influence of three cis-acting elements is responsible for this regulation in an adrenal carcinoma cell line. Two sites, CRE2 at -48 base pairs (bp) relative to the start of transcription and CRE3 at +95 bp, are identical to the core motif of the cAMP-responsive element (CRE) of the somatostatin gene and are conserved in the mouse, rat, and human ODC genes. Mutation of CRE2 resulted in a substantial decrease in basal promoter activity, as well as a 5-fold decrease in inducibility of the ODC promoter by PKA. CRE3 did not contribute to the basal activity of the ODC promoter, but mutation of this site resulted in a 2-fold decrease in inducibility by PKA. Deletion of a 45-bp sequence (GC-box) located 5' of CRE2, also resulted in a 2-fold decrease in inducibility of the ODC promoter. DNase I protection revealed the presence of protein binding at CRE2, the TATA box, and the GC-box of the ODC promoter. Mutation of CRE2 resulted in loss of protection of this sequence, as well as the 3' extension of the footprint over the TATA box, without affecting interactions at the GC box. Antibodies to the well characterized CRE-binding protein CREB recognized proteins binding to CRE2, suggesting that binding of CREB, or an antigenically related protein, is important for the activity of CRE2. Additionally, recombinant CREB bound to a DNA probe containing the CRE2 sequence.

Adrenal Gland Neoplasms↗
PubMed1992

A novel DNA binding activity is elevated in thymocytes expressing high levels of H-2Dd after radiation leukemia virus infection.

Resistance to radiation leukemia virus-induced leukemia is mediated by gene(s) in the H-2D region of the MHC; a clear correlation exists between disease resistance and increased H-2Dd expression on the thymocyte surface. We have investigated the molecular basis for this stimulation of H-2Dd class I expression. Elevated H-2 mRNA and H-2 transcription are demonstrated in the infected thymocytes as compared to normal thymocytes indicating that the elevation of H-2 surface expression is the result of transcriptional activation. Gel mobility assays performed with nuclear extracts of normal and infected thymocytes and sequences 5' of the H-2Dd gene show that specific binding occurs with both extracts; the binding differs both quantitatively and qualitatively, however. DNase I protection analysis detects a protein binding site that is protected only by extracts from infected cells. The protected region contains a sequence similar to the AP-1 consensus sequence. Gel shift competition assays and UV photo-cross-linking to an oligonucleotide containing this sequence demonstrate that specific binding of an H-2 binding factor 1 occurs and that this factor is not the AP-1 binding complex. This novel binding factor, activated in vivo, might also be involved in the normal regulation of H-2 gene expression by recognizing the highly conserved binding sequence (TGACGCG) found in the 5' flanking region of many MHC class I genes. This is the first demonstration of the parallel stimulation of a DNA binding activity and increased transcription occurring in thymocytes after infection with a leukemogenic retrovirus.

Base Sequence↗
PubMed1992

Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells.

Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.

Adrenal Cortex↗
PubMed1992

A regulatory cis element and a specific binding factor involved in the mitogenic control of murine ribosomal protein L32 translation.

The mRNA encoding ribosomal protein L32 redistributes from untranslated subribosomal particles into polysomes after mitogenic activation of quiescent T-lymphocytes and fibroblasts. To identify the regions of the L32 mRNA which are important in regulating its cytoplasmic location we constructed a plasmid containing the murine L32 cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat promoter and introduced this construct into murine 3T3 fibroblasts. The mRNA transcribed from the RSV-L32 construct redistributed from subribosomal particles into polysomes in response to mitogenic activation in a manner similar to endogenous L32 mRNA. A conserved polypyrimidine region present at the 5' terminus of all ribosomal protein mRNAs is required for translational regulation of L32 mRNA since deletion of this sequence resulted in a mRNA that was not sequestered in subribosomal particles in quiescent cells. A radioactive RNA probe containing the first 34 nucleotides of the L32 5'-untranslated region, including the polypyrimidine region, specifically interacted with a protein of about 56 kDa. This protein did not bind detectably to RNA probes lacking the polypyrimidine sequence. Binding activity was similar in protein extracts made from resting and activated cells, suggesting that binding of the 56-kDa protein as measured in this assay is not regulated. This protein is a member of what may be an emerging family of polyribopyrimidine-binding proteins with diverse biochemical functions.

3T3 Cells↗
PubMed1992

Sequestered end products and enzyme regulation: the case of ornithine decarboxylase.

The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools.

Animals↗
PubMed1991

Activation of dense human tonsilar B cells. Induction of c-myc gene expression via two distinct signal transduction pathways.

Antibodies to surface Ig or to the B cell marker CD20 trigger resting human B cells in similar yet distinct ways. Either antibody induces five-fold increases in the expression of the protooncogene, c-myc, as detected with semi-quantitative Northern blot assays. The induction of c-myc mRNA by anti-IgM or anti-CD20 is blocked by inhibitors of protein kinase C (PKC) such as staurosporine and by pretreatment of B cells with phorbol esters to reduce cellular PKC levels. This suggests that PKC is involved in the pathways stimulated by both anti-IgM and anti-CD20. However, anti-CD20, unlike anti-IgM, does not activate significant increases in inositol triphosphate or intracellular-free calcium. Further, anti-CD20-triggered elevation of c-myc mRNA is inhibited by pertussis and cholera toxins, whereas the pathway initiated by anti-IgM if anything is stimulated by pertussis toxin and unchanged by cholera toxin. Further differences in the nature of these two signals were seen when the expression of adhesion/recognition molecules were examined. Anti-IgM consistently induces increased expression of the adhesion molecules CD54 (I-CAM-1) and B7/BB-1 on B cells, but anti-CD20 does not. Yet both anti-CD20 and anti-IgM increase class II MHC, CD18 (LFA-1 beta-chain) and LFA-3 levels. These data suggest that the way in which B cells are activated may influence their surface phenotype and possibly subsequent migration or cell-cell interactions.

Antibodies, Anti-Idiotypic↗
PubMed1991

A new perspective on ornithine decarboxylase regulation: prevention of polyamine toxicity is the overriding theme.

The polyamines are essential cellular components for growth. Control of a key regulated enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), as a function of growth, is an area of intense interest. A unique regulatory property of ODC is the short half-life of the protein, which has been suggested to be an important factor in rapid activation of polyamine biosynthesis after cells are mitogenically stimulated. In this paper, it is argued that the biological significance of the short half-life of ODC is unrelated to the rate of its induction to a new steady state by growth factors, which is in fact limited by the relatively long half-life of the ODC mRNA. Instead, I suggest that the rapid turnover of ODC protein becomes of significance when cells cease growth and expeditious downregulation of the enzyme is important in preventing polyamine overproduction, which would result in cytotoxicity in the arrested cells. Although mitogenic activation of ODC expression has been studied extensively, there is very little known about the mechanisms controlling downregulation of polyamine biosynthesis during the arrest of animal cell growth. These considerations suggest that this would be a fertile area of future inquiry.

Animals↗
PubMed1990

Specific regulation by endogenous polyamines of translational initiation of S-adenosylmethionine decarboxylase mRNA in Swiss 3T3 fibroblasts.

S-Adenosylmethionine decarboxylase (AdoMetDC) activity was elevated 18.8-fold in Swiss 3T3 fibroblasts which were depleted of cellular polyamines by using the inhibitor difluoromethylornithine (DFMO). Although the cellular level of AdoMetDC mRNA and the half-life of active AdoMetDC protein were also increased (4.3- and 1.5-fold respectively), together they could not account for the magnitude of the increase in AdoMetDC activity. These data suggested that the translation of AdoMetDC mRNA must be increased in the polyamine-depleted cells to account fully for the elevation in activity. The cellular distribution of AdoMetDC mRNA was examined in the polyamine-depleted cells, and it was found almost exclusively associated with large polysomes. In contrast, AdoMetDC mRNA in untreated controls was very heterogeneous, with the proportion associated with monosomes equal to that associated with large polysomes. The shift of the AdoMetDC message into large polysomes occurred within 18 h after addition of DFMO to the cultures and could be reversed by adding exogenous putrescine. The effect of polyamine depletion on AdoMetDC translation was specific, since there was no change in the distribution in polysomes of either actin mRNA or the translationally controlled mRNA encoding ribosomal protein S16 in the DFMO-inhibited cells. Thus the translational efficiency of AdoMetDC mRNA in vivo is regulated either directly or indirectly by the concentration of intracellular polyamines through a mechanism involving translational initiation, which results in a change in the number of ribosomes associated with this mRNA.

Adenosylmethionine Decarboxylase↗
PubMed1990

Simultaneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of eukaryotic initiation factor 4E after mitogenic stimulation of Swiss 3T3 cells.

Ribosomal protein L32 mRNA moved from messenger ribonucleoprotein particles into polysomes following serum activation of quiescent Swiss 3T3 cells. This redistribution of the mRNA into a translationally active state began by 1 h and was complete by 3 h after activation. In contrast, actin mRNA showed no translational control, being found predominantly in polysomes in both quiescent and activated cultures. The phosphorylation state of eukaryotic initiation factor (eIF) 4E, which binds mRNA caps, was examined in parallel. eIF-4E phosphorylation was elevated by 1 h following serum activation and reached a peak by 3-5 h. Treatment of resting cells with phorbol ester also simultaneously stimulated eIF-4E phosphorylation and the movement of L32 mRNA into polysomes. These results are consistent with a model in which mitogen-induced phosphorylation increases the pool of active eIF-4E molecules, which in turn cause the recruitment of translationally controlled mRNAs to actively synthesizing ribosomes.

Actins↗
PubMed1990

Cell type-specific mechanisms of regulating expression of the ornithine decarboxylase gene after growth stimulation.

Ornithine decarboxylase (ODC) mRNA is strongly induced by mitogenic activation of resting Swiss 3T3 fibroblasts and T lymphocytes. Nuclear run-on analysis revealed a low level of nascent transcripts in resting fibroblasts that was elevated upon activation. In contrast, there was a high level of transcription across the entire ODC gene in resting T cells, which remained unchanged upon activation. The stability of the mature ODC message was found to be unaffected by mitogenic stimulation. These results indicate that ODC mRNA levels are regulated transcriptionally in Swiss 3T3 cells and posttranscriptionally within the nucleus of T lymphocytes in response to mitogenic stimuli. In this unique situation, the mitogenic induction of a single gene, ODC, is regulated by two very distinct, cell-specific mechanisms.

Animals↗
PubMed1989

The role of lymphocytes and monocytes in hematopoietic growth factor production by peripheral blood mononuclear cells.

Stimulated peripheral blood mononuclear cells (MNC) are one of the richest described physiologic sources of colony-stimulating activity. To understand the molecular basis for, and the cellular sources of, this MNC activity, we cultured purified human lymphocytes and monocytes for 2 hr to 6 days and examined colony-stimulating factor (CSF) gene activity by Northern blot analysis. We show that MNC are capable of expressing messenger RNA for macrophage (M)-CSF, granulocyte (G)-CSF, GM-CSF, and multi-CSF when stimulated with mitogens. The time courses of induction of these genes differ, with G-CSF induction preceding that of the other CSFs. In addition, the spectra of CSFs produced by cell populations enriched for lymphocytes, monocytes, or macrophages differ. The implications of these findings for the selective activation of hematopoiesis are discussed.

Cells, Cultured↗
PubMed1989

Transforming growth factor-beta blocks proliferation but not early mitogenic signaling events in T-lymphocytes.

Transforming growth factor-beta (TGF-beta) inhibits the proliferation of T-lymphocytes in response to activation with mitogenic lectin. The influence of TGF-beta on elevation of cytosolic Ca2+, induction of proliferation-associated mRNA species, and total cellular RNA content has been studied. The cells seem to exit G0 when activated in the presence of TGF-beta, but they arrest in mid-G1 phase.

Acridine Orange↗
PubMed1988

Mitogenic signaling pathways regulating expression of c-myc and ornithine decarboxylase genes in bovine T-lymphocytes.

Expression of the c-myc and ornithine decarboxylase (ODC) genes is elevated early after mitogenic activation of T-lymphocytes, and regulation of the two genes seems to be coupled to transmembrane signaling pathways that are in part different. The evidence is consistent with protein kinase C (PKC) being both necessary and sufficient to induce expression of the ODC gene in response to treatment of T-cells with either the mitogen concanavalin A (Con A) or biologically active phorbol esters. Furthermore, there seems to be no involvement of events dependent on calmodulin (CaM) in the regulation of ODC in these cells. The situation with c-myc is more complex. In contrast to ODC, transcription of this gene is not stimulated by treatment of resting T-cells with phorbol esters alone, but the cells respond to phorbol esters in combination with the calcium ionophore ionomycin. Induction of the c-myc gene by Con A is inhibited by CaM antagonists. These results are consistent with a model in which transcriptional activation of the c-myc gene in resting T-lymphocytes requires two signals, one from PKC and the other involving CaM.

Animals↗
PubMed1987

Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen-activated lymphocytes.

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.

Actins↗
PubMed1987

Aggregation of DNA by analogs of spermidine; enzymatic and structural studies.

A homologous series of spermidine analogs, with defined abilities to replace the natural polyamine in supporting cell growth, was examined for its influence on the structure of supercoiled, aggregated DNA and on the ability of the DNA aggregates to act as substrates for various enzymes. The concentration of amine necessary to aggregate negatively supercoiled Col E1 DNA was progressively increased as the diaminobutane moiety of spermidine was extended beyond 5 methylene groups. 1H- and 31P-NMR spectroscopy suggested that less rigid DNA aggregates were formed by spermidine analogs than by spermidine itself. Spermidine and its analogs differentially modulated the activities of bacterial and mammalian type I topoisomerases and EcoRI restriction endonuclease on aggregated DNA in a manner reminiscent of the abilities of the amines to stimulate cell growth. When DNA was not aggregated, the influence of the various amines on these reactions was almost identical. These results are discussed in relation to the structures of the DNA aggregates in the presence of the various triamines.

Animals↗