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J T Schanke

Publications and source records attributed to J T Schanke.

7 recordsLinked to original sources
PubMed1998

SP1/SP3-binding sites and adjacent elements contribute to basal and cyclic adenosine 3',5'-monophosphate-stimulated transcriptional activation of the rhesus growth hormone-variant gene in trophoblasts.

Transcriptional activation of the rhesus monkey GH-variant gene in syncytiotrophoblasts is developmentally regulated by trophoblast-specific and cAMP-responsive mechanisms. Progressive deletions of 5'-flanking DNA defined the most proximal 140 bp as the minimal region retaining full cAMP-stimulated mGH-V transcription. To identify the regions of this promoter critical for transcription, transient transfections of reporter plasmids containing systematic 10 base mutations throughout this proximal region were performed. Mutation of the region from -140/-131 decreased transcription in syncytiotrophoblasts by 50%, and gel mobility-shift analyses demonstrated that Sp1 and Sp3 bound to a region containing a GGGAGG motif at -136/-131. Mutation of the -62/-53 region decreased transcriptional activation by 66-99%, and Sp1 and Sp3 bound to a GGTGGG motif overlapping this region (at -65/-60). Selective mutation of this Sp1/Sp3 site decreased basal transcription by approximately 80%, and cAMP-stimulated transcription by up to 75% (with the greatest effect in primary syncytiotrophoblast cultures), indicating that the Sp1/Sp3 site is critical for transcriptional activation. Mutations in the regions adjacent to the Sp1/Sp3 sites (-130/-111 and -52/-43) also dramatically reduced (by 75%) transcriptional activation in trophoblasts. We conclude that two Sp1/Sp3 sites as well as additional elements directly adjacent to these sites contribute to trophoblast-specific cAMP-responsiveness of the mGH-V proximal promoter.

Animals↗
PubMed1997

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Amino Acid Sequence↗
PubMed1994

An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells.

Animals↗
PubMed1994

Organization of the transcription factor binding sites in the kappa Ig intron enhancer. Effects of position, orientation, and spacing.

The kappa Ig intron enhancer is comprised of multiple sequence motifs known to bind trans-acting factors that activate gene expression. A species comparison reveals a high level of conservation of the organization of the transcription factor binding sites within the enhancer. The importance of the conserved organization of the kappa intron enhancer was examined by using topologic mutations that disrupt the position, orientation, and spacing of individual binding sites within the enhancer. The effects of these changes were monitored by their effects on reporter gene activity at two distinct stages of B cell development. Previously, mutational analysis indicated the kappa B and kappa E2 sequence motifs to be the most crucial sites for intron enhancer function. We have demonstrated that intron enhancer activity is dependent on the position of the kappa B and kappa E2 sequence motifs within the enhancer. Intron enhancer function is, however, independent of kappa B and kappa E2 binding site orientation and is flexible in spacing requirements among binding sites.

Animals↗