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G Weisinger

Publications and source records attributed to G Weisinger.

29 records · Page 2Linked to original sources
PubMed1993

Cholinergic regulation of rat preproenkephalin RNA in the adrenal medulla.

Expression of the rat preproenkephalin (ppENK) gene involves transsynaptic cholinergic mechanisms. We evaluated the effects of cholinergic agonist treatments in vivo on the expression of adrenomedullary ppENK RNA. Cholinergic treatment with nicotinic + muscarinic receptor agonists resulted in a synergistic 100-fold rise in steady-state ppENK messenger RNA levels, but only a 30- to 35-fold rise in initiation of steady-state ppENK RNA transcripts. The levels of initiated ppENK steady-state RNA peaked at two days, whereas mature (1.45 kb) ppENK mRNA levels continued to rise, peaking at four days. This suggested that other transcriptional (attenuation or alternative splicing) or post-transcriptional (RNA stabilization) regulatory mechanisms must be operative. As multiple ppENK RNA start sites exist, we examined how usage of multiple sites was altered by cholinergic treatments. The predominant start site changed from E2 in the basal state, to E4 after primary cholinergic stimulation, to E3 after re-treatment. This represents novel example of differential usage of multiple RNA initiation start sites in vivo. Differences in initiated and mature transcripts are consistent with at least four mechanisms involved in control of cholinergic-induced ppENK RNA expression: (i) simply new initiation of RNA transcripts, (ii) differential usage of the multiple RNA start sites, (iii) stabilization of mRNA transcripts, and (iv) attenuation and/or alternative RNA splicing of RNA transcripts.

Adrenal Medulla↗
PubMed1992

Primary sequence of -1436 to +53 bp of the rat preproenkephalin gene putative Z-DNA and regulatory motifs.

We report novel sequence data extending -1436 bases 5' of the rat proenkephalin gene start site known as E4. We noted an interesting stretch of 58 bases of alternating pyrimidines that lies immediately adjacent to 71 bases of an alternating purine-pyrimidine Z-DNA-like sequence that lies between -694 bp and -566 bp. Multiple sequence homologies to putative cis-acting regulatory factor binding sites were identified by a computer aided sequence search.

Animals↗
PubMed1992

Tissue- and treatment-specific usage of multiple preproenkephalin transcriptional start sites.

The significance of the 5' heterogeneity of the transmitter gene ppEnk was evaluated by comparing start site usage (E1-E4) between 12 tissues from untreated rats, using primer extension analysis. In the basal state, we found that E3- and E4-initiated transcripts accounted for 80% of the total striatal RNA present compared with a preferential usage of the E2 start site in all other tissues. To determine whether this selective expression could be modified by biologically relevant pathways, rats were made hypoglycemic. After insulin shock, only E3 + E4-initiated transcripts increased (16-fold at 1 day) in the adrenal medulla but were unaffected in the striatum. As the effects of insulin shock on the adrenal medulla are mediated by cholinergic pathways and the striatum also receives cholinergic inputs, we also compared the effects of cholinergic drug treatments on start site usage in these two tissues. Rats were treated with cholinergic agonists (nicotine + oxotremorine) which induced adrenomedullary E2 and E3 + E4 transcripts (5- and 80-fold, respectively). This effect peaked at 2 days. In contrast, in the same animals, striatal ppEnk RNA (E3 + E4) increased only 10-15-fold after drug treatment. Hence it appears that biologically relevant whole animal stimuli (insulin shock or cholinergic agents) activate biochemical pathways, which affect start site usage in a tissue-specific fashion. Selective RNA start site usage suggests a biological significance, which may be important in the widespread tissue expression of this gene.

Adrenal Medulla↗
PubMed1990

Multiple preproenkephalin transcriptional start sites are induced by stress and cholinergic pathways.

A major control of gene expression occurs at the level of initiation of RNA transcription. In the nervous system this is reflected in part by 5' end RNA heterogeneity of neural transcripts. We now report the characterization of four preproenkephalin (ppEnk) RNA initiation sites in the rat brain striatum. In the adrenal medulla two ppEnk transcriptional start sites were detected. Moreover, cholinergic induction of ppEnk RNA initiation was observed in the adrenal medulla, but not in the striatum. The converse was true following handling stress. Our observations suggest that selective start site usage and stimulus evoked induction of specific RNA initiation is tissue-specific. We speculate that start site usage and induction may provide separate mechanisms through which neuronal gene expression is controlled in anatomically, as well as functionally distinct neurohumoral structures.

Adrenal Medulla↗
PubMed1989

Preproenkephalin DNA-binding proteins in the rat: 5' flanking region.

Various extracellular signals (i.e. transmitters, hormones, growth factors, etc.), together with their respective second-messenger pathways, regulate transmitter biosynthesis and neuronal function by altering gene expression. In this study we validated a protocol for isolating rat striatum and adrenal medullary nuclei for the purpose of extracting, identifying, and characterizing, nuclear regulatory factors which may serve a functional role in signal-transduction processes. Through gel retardation studies using a 299 base pair (bp) XmnI-SacI 32P-labeled probe (derived from the 5' untranslated region of the rat preproenkephalin gene), we show that different patterns of retained bands result from nuclear extracts derived from rat adrenal medulla and striatum (as well as from other tissue). These tissue differences may have biological significance since rat adrenal medullae have low basal enkephalin levels while the striatum has high levels of this peptide and its respective mRNA. Additionally, certain retained bands were common to both cytosolic and nuclear compartments, suggesting binding factors may be located in either cell space. An initial biochemical characterization of these factors was also undertaken. Generally, salt levels of 100 mM or more reduced factor binding while 10-50 mM sodium ion levels showed preferentially enhanced bands. Binding activity appeared optimal at pH 6.8. As all retained bands were abrogated by proteinase K treatment, these factors appear to have a significant protein component. Finally, of particular interest is that this 299 bp region contains many sequences showing over 80% sequence identity with several previously characterized transcriptional control elements (i.e. cAMP and phorbol ester inducible enhancers, GCN4, AP1, Sp1, CCAAT binding factor, ATF, and AP2). If binding is confirmed (footprint analysis) and function validated (transfection studies), the evolutionary significance of the apparent presence of gene regulatory sequences and functional element divergence of the DNA region between different species can be evaluated.

Adrenal Medulla↗
PubMed1989

Ontogeny of the opiate phenotype: an approach to defining transsynaptic mechanisms at the molecular level in the rat adrenal medulla.

Transmitter phenotypic expressions is a dynamic cellular process governed by multiple interactions with the neuronal environment. During sympathoadrenal development the arrival of presynaptic nerve terminals at the adrenal chromaffin cell (in the immediate postnatal period), coincides with the acquisition and subsequent development of a variety of transmitter biosynthetic capacities. Data discussed herein supports the contention that synaptic connections serve a central role in triggering the ontological cascade. Disruption of the normal timing of innervation events is detrimental to subsequent function and results in permanent deficiencies in development. In addition, alteration of transmitter biosynthetic regulatory mechanisms appears to reside at the level of gene expression. In view of this, additional molecular approaches are necessary to further elucidate the fundamental basis of neuronal transmitter phenotypic plasticity. Our approach to this problem represents a logical extension of previous research in this area and ultimately, will involve characterizing transcription activator molecules important in transmitter gene expression at various ontological ages.

Adrenal Medulla↗
PubMed1988

Multiple negative elements upstream of the murine c-myc gene share nuclear factor binding sites with SV40 and polyoma enhancers.

We previously identified a 716 bp DNA segment, 424 to 1140 bp 5' of the murine c-myc gene, which exhibited the properties of a transcriptional 'dehancer' because it negated the effects of the SV40 enhancer (Remmers et al., 1986). Here, we show that this 716 bp 'dehancer region' is composed of multiple negative elements each of which functions at a distance of > 1 Kb to inhibit the SV40 enhancer. One of these negative elements displays lymphoid cell specificity. HeLa cell nuclear factor binding sites are found within these negative effector sequences. One of these binding sites is related to that of the Py EF-C transcription factor which recognizes a short dyad symmetry element within the polyoma enhancer. Interestingly, a dimer of the Py EF-C recognition sequence exhibited a strong 'dehancer effect' in the pSV2CAT expression vector. These negative elements may function by interfering with transcription factors required for SV40 enhancer activity.

Animals↗
PubMed1986

Protein that induces cell differentiation causes nicks in double-stranded DNA.

The growth and differentiation of myeloid hematopoietic cells are regulated by different macrophage and granulocyte inducing proteins, those that induce growth and others that induce differentiation. The proteins that induce differentiation but not those that induce growth bind to double-stranded DNA. We now report that purified myeloid cell differentiation-inducing protein causes single strand breaks (nicks) in double-stranded DNA. This DNA nicking may initiate the changes in gene expression that are required for differentiation.

Animals↗
PubMed1985

Multimeric complexes of differentiation-inducing protein bound to DNA.

Myeloid hematopoietic precursor cells are induced to differentiate by the macrophage and granulocyte differentiation-inducing protein MGI-2 (DF). This differentiation-inducing protein bound to double-stranded but not to single-stranded mammalian DNA. The bound MGI-2 was not eluted by high salt, but was eluted by sodium dodecyl sulfate (SDS). MGI-2 also bound to double-stranded E. coli DNA, but with this DNA the bound MGI-2 was eluted by high salt. This indicated a difference in the binding affinities of MGI-2 to mammalian and E. coli DNA. MGI-2 bound to DNA was examined by electron microscopy. The results indicate that MGI-2 formed a multimeric complex with double-stranded DNA and that the size of the complex was correlated with the strength of protein binding to the DNA. The multimeric complex bound to DNA was disrupted by deoxyribonuclease. The data indicated that binding of this differentiation-inducing protein to DNA involves the formation of a multimeric complex in which the monomers are held together by DNA. It is suggested that the formation of such multimeric complexes of MGI-2 and DNA may allow activation of the multiple pathways of gene expression that is required for differentiation.

Animals↗
PubMed1983

DNA-binding protein that induces cell differentiation.

Macrophage and granulocyte-inducing (MGI) proteins regulate the growth and differentiation of myeloid hematopoietic cells. One class of these proteins (MGI-1) induces cell growth and another class (MGI-2) induces cell differentiation. Results obtained with DNA-cellulose column chromatography have shown that the differentiation-inducing protein MGI-2 can bind to double-stranded cellular DNA, but that there was no such binding under the same conditions by the growth-inducing protein MGI-1. DNA binding may thus be used to separate MGI-2 from MGI-1. The MGI-2 from mouse bound to DNA from mouse and calf. There were different elution peaks of the MGI-2 bound to DNA suggesting a heterogeneity of MGI-2 molecules, and the last peak eluted from the DNA cellulose column was enriched for one of the molecular forms of MGI-2. After one further step of purification by polyacrylamide gel electrophoresis, this molecular form of MGI-2 was active at a concentration of 6.5 X 10(-11) M. In normal development MGI-1 induces MGI-2. This induction of a DNA-binding differentiation-inducing protein by a growth-inducing protein is an efficient mechanism for the normal coupling of growth and differentiation. It is suggested that this may also be a mechanism for the normal coupling of growth and differentiation in other types of cells.

Cell Differentiation↗