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N L Eberhardt

Publications and source records attributed to N L Eberhardt.

At least 55 records · Page 3Linked to original sources
PubMed1992

Thyroid hormone responsiveness in human growth hormone-related genes. Possible correlation with receptor-induced DNA conformational changes.

Triiodothyronine (T3) induces the transcription of the human chorionic somatomammotropin (hCS) promoter transfected into rat pituitary (GC) cells, but does not stimulate the homologous human growth hormone (hGH) promoter. As demonstrated by forward and reverse mutagenesis, this differential T3 responsiveness is due to subtle structural differences in a T3 response element located between nucleotides -64 and -44 of the 5'-flanking DNA of the hGH and hCS promoters. Synthetic hCS(-70/-40) DNA binds thyroid hormone receptors with a 4-fold higher affinity than the corresponding hGH T3 response element, indicating that small differences in receptor binding properties are reflected by major differences in T3 responsiveness. Analysis of circular permutation fragments containing the native hGH and hCS or mutated hCS(-70/-40) sequences demonstrates that the thyroid hormone receptor induces DNA bending. The extent of bending shows a possible correlation with the function of these sequences, suggesting that the receptor-induced changes in DNA conformation may be required for thyroid hormone receptor action.

Animals↗
PubMed1992

A shell program for the design of PCR primers using genetics computer group (GCG) software (7.1) on VAX/VMS systems.

A number of software analysis packages for the design of PCR primers are available for PCs; however, software for users that depend on VAX/VMS operating systems is not available. By treating oligonucleotides as RNA molecules, I have designed an alternative means toward studying oligonucleotide interactions using software that is currently available from The Genetics Computer Group (GCG, Madison, WI). The oligonucleotide interactions with self and non-self are analyzed by the GCG FOLD program, a program which finds a secondary structure of minimum free energy for an RNA molecule. This approach allows the identification of self-priming primer pairs, and the interaction energies provide a guideline for the prediction of optimal PCR primers.

Base Sequence↗
PubMed1989

Human chorionic somatomammotropin and growth hormone gene expression in rat pituitary tumour cells is dependent on proximal promoter sequences.

Human placental chorionic somatomammotropin (hCS-A or hCS-B) and pituitary growth hormone (hGH-N) are related by structure and function. The hCS-A gene is expressed in rat pituitary tumour (GC) cells after gene transfer. Deletion of hCS-A 5'-flanking DNA reveals repressor activity upstream of nucleotide -132, and a region essential for expression in GC cells between nucleotides -94 and -61. The sequences in this region differ from the equivalent hGH-N gene DNA by one nucleotide, and include the binding site (-92 to -65) for a pituitary-specific factor (GHF-1), required for hGH-N expression in GC cells. Exchange of hGH-N with hCS-A gene DNA in this region maintains expression in GC cells. By contrast, modification of these sequences blocks expression. These data indicate that proximal promoter sequences, equivalent to those bound by GHF-1 on the hGH-N gene, are required for hCS-A expression in GC cells.

Animals↗
PubMed1988

The human growth hormone gene contains both positive and negative control elements.

A subset of DNA sequences in the 5'-flanking DNA of the human growth hormone (hGH) gene was examined by protein-DNA binding and gene transfer-expression experiments. Two adjacent cis-acting elements (I and II) were identified between nucleotides -308/-235 of the hGH gene that modulated the expression of a linked reporter gene in transfected HeLa cells. Elements I and II repressed gene expression whereas element II alone activated it. HeLa whole cell extracts contain two factors that bind hGH DNA carrying elements I and II. Factor I binds to single-stranded DNA, and its binding is correlated with repression of gene expression. Factor II binds between nucleotides -275/-257 of the hGH gene. This region is homologous to the binding site for the adenovirus major late transcription factor, and factor II binding to hGH DNA is competed by adenovirus major late promoter DNA, indicating that the hGH and major late adenovirus promoters share a transcription regulatory element.

Animals↗
PubMed1988

Effect of butyrate on thyroid hormone-mediated gene expression in rat pituitary tumour cells.

These studies correlate the effects of (sodium) butyrate on intranuclear thyroid hormone receptor levels, with influences on both endogenous and transfected rat growth hormone (rGH) gene expression and regulation by L-triiodothyronine (T3). In rat anterior pituitary tumour (GH3) cells, 5.0 mM butyrate elicits a biphasic reduction in the number of nuclear T3 receptors. About 75% are depleted rapidly (t1/2 = 7 h), and the remaining receptors are depleted more slowly (t1/2 = 59 h). GH3 cells were treated with increasing concentrations of butyrate (0-5.0 mM), plus or minus 10 nM T3 for 48 h. Total cytoplasmic RNA, cellular protein and medium were analysed for rGH levels with radiolabelled rGH cDNA or antibodies. A greater than 50-fold increase in rGH mRNA level was seen after T3 treatment in the absence or presence of 0.1 mM butyrate. However, 1.0 and 5.0 mM butyrate decreased the stimulation of rGH mRNA levels by T3 to 10- and less than 2-fold, respectively. Control mRNA levels were decreased slightly by increasing butyrate concentrations; rGH mRNA level was 2- to 3-fold higher in the absence of 5 mM butyrate. The pattern of butyrate/T3 response displayed by both cellular and secreted rGH was similar to that seen with mRNA levels. Thus, the predominant effect of butyrate on T3-mediated regulation of growth hormone gene expression is at the level of transcription or mRNA accumulation. A hybrid gene containing 5'-flanking DNA from the rGH gene fused to the bacterial gene coding for chloramphenicol acetyl transferase (CAT), was used to transfect rat pituitary tumour cells with or without butyrate and T3 treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Regulated expression of chimaeric genes containing the 5'-flanking regions of human growth hormone-related genes in transiently transfected rat anterior pituitary tumor cells.

The expression and hormonal regulation of chimaeric genes containing the 5'-flanking regions of the normal human growth hormone (hGH-1), the variant hGH (hGH-2) and chorionic somatomammotropin (hCS-1) genes fused to the chloramphenicol acetyl transferase (CAT) gene has been examined after transient transfection into cultured rat pituitary (GC), and non-pituitary (HeLa and Rat 2) tumor cells. As assessed by levels of CAT activity, the hGH-1 and hCS-1 gene hybrids were expressed at 5- to 25-fold higher levels in GC cells than in HeLa or Rat 2 cells. The hGH-2 gene hybrid was expressed at very low levels in all 3 cell types. Triiodothyronine treatment of transiently transfected GC cells had little effect on CAT activity from the hGH-1 gene hybrid but increased CAT activity from the hCS-1 gene hybrid. A slight but significant increase in CAT expression was detected with both genes after dexamethasone treatment. The data indicate that elements present on the hGH-1 and hCS-1 genes' 5'-flanking DNA are required for the efficient expression of these genes in GC cells.

Animals↗
PubMed1987

The primary structure of coho salmon growth hormone and its cDNA.

Total RNA was extracted from coho salmon growth hormone (sGH) cell regions and used to synthesize double-stranded cDNA, which was inserted into a plasmid vector and used to transform Escherichia coli HB101. The total RNA was also separated according to size by electrophoresis on agarose gels and the fraction that directed the cell-free synthesis of protein in the size range of GHs of other species was isolated and used to screen the transformed colonies of E. coli. A clone containing the putative sGH cDNA was identified and its nucleotide sequence was determined. To verify that the cDNA was that of sGH, the GH cell region of coho pituitary glands was incubated in organ culture. The secreted GH was purified by HPLC and the sequence of its 42 amino-terminal amino acids was determined. Comparison of this sequence with the amino acid sequence derived from the cDNA showed that it encoded sGH. Medium containing the presumptive sGH as the only prominent protein was active in a GH radioreceptor assay that involved labeled bovine GH and pregnant mouse liver membranes: the sGH was approximately 10% as active as the bGH standard. RNA blotting analysis showed that sGH was the major species of RNA produced by the GH cell region of the salmon pituitary. The mRNA of sGH differed from those of human, rat, and bovine GH in that its 3'-untranslated region was unusually large (about 500 nucleotides) but the coding region showed significant homology with mammalian GHs and resembled them in having a strong (78%) preference for G and C in the third positions of the codons. The amino acid sequence of sGH showed 32-34% and 19-22% identical homology with mammalian GHs and prolactins, respectively. Several conserved regions between sGH and mammalian GH and PRL molecules were also revealed that could indicate conservation of structurally and/or functionally important domains. Hydropathy analysis disclosed that although sGH and the GH of a representative mammal (pig) had similar profiles in some regions, the sGH was overall more hydrophobic than the pig (p) GH. Similarities and differences, were also noted in the predicted secondary structure of sGH and pGH.

Amino Acid Sequence↗
PubMed1987

The human growth hormone gene locus: structure, evolution, and allelic variations.

Genomic clones containing the closely related genes for human growth hormone (hGH) and chorionic somatomammotropin (hCS) were obtained from genomic bacteriophage lambda and cosmid libraries. The entire GH/CS chromosomal locus was reconstructed utilizing overlapping restriction fragments characterized from the isolated clones. The hGH/hCS locus contains two GH genes and three CS genes spanning 48 kb of DNA in the order: 5'-(hGH-1/hCS-5/hCS-1/hGH-2/hCS-2)-3', confirming analysis of cosmid clones obtained from a different human library (Barsh et al., 1983). To complete the characterization of the hCS genes, the nucleotide sequence of the hCS-5 gene was determined. Sequence analysis revealed a mutation of the 5' splice site at the exon II-intron B boundary, suggesting that the hCS-5 gene is a pseudogene. The nucleotide sequence of an allelic variant of the hCS-2 gene was determined and found to contain a single amino acid substitution and the deletion of a single codon. The hGH/hCS gene locus was further characterized by the localization of at least 27 Alu-type repetitive sequences and identification of three unique sequences in the vicinity of several hGH and hCS genes which define the probable breakpoints of the evolutionary duplication units. These data, combined with the nucleotide sequences of all five GH and CS genes, indicate that the hGH/hCS gene locus has evolved by duplication mechanisms. Evidence for the occurrence of at least one gene conversion event involving the hCS-1 gene precursor and the hCS-2 gene was found, indicating that the hGH/hCS gene locus has evolved by concerted mechanisms. The structure of the hCS genes is discussed in light of recent studies of CS genes from other mammalian species.

Alleles↗
PubMed1986

The human growth hormone gene is negatively regulated by triiodothyronine when transfected into rat pituitary tumor cells.

Human growth hormone (hGH) gene expression and regulation by thyroid and glucocorticoid hormones were examined after its stable introduction into rat anterior pituitary tumor cells. Transcripts from the hGH gene were correctly initiated and could be positively regulated by glucocorticoids. In contrast to the endogenous rat GH gene and a transfected, modified rat GH gene, the hGH gene was negatively regulated by triiodothyronine. Analysis of hGH gene 5'-flanking deletions indicated that a distal regulatory element is required for efficient transcription. Thus promoter, glucocorticoid, and thyroid hormone control elements are present on the transfected hGH gene, whose expression may be negatively controlled by thyroid hormone.

Animals↗
PubMed1986

Mechanisms of glucocorticoid hormone action.

This report summarizes our studies, in context with the results of other laboratories, of the molecular mechanisms of glucocorticoid hormone action. The receptors for these steroids are comprised of single polypeptide chains of about 90,000 molecular weight. Binding of agonist steroids to the receptor induces a conformational change to an active receptor form that is followed by a second change in the glucocorticoid-receptor complex, termed activation, that alters the charge of the complex and results in its binding to specific sites on the DNA termed glucocorticoid regulatory elements (GREs). The GRE on the human metallothionein-IIA gene is located in the 5'-flanking DNA. It can function independently of the gene's promoter, and when ligated upstream from the herpes simplex virus (HSV) thymidine kinase (TK) gene promoter, can activate it. The binding of the glucocorticoid-receptor complex to the GRE probably alters chromatin structure over a limited span to facilitate RNA polymerase action. The regulation by glucocorticoids of growth hormone gene expression is more complex. The steroid appears to elicit both transcriptional and posttranscriptional influences that are also affected by thyroid hormone. Also the glucocorticoid influences appear to be exerted in part through DNA structures located downstream from the transcriptional initiation site. A GRE has been defined in intron A of the hGH gene through gene transfer and DNA binding experiments. Finally, gene transfer experiments suggest that pituitary-specific factors influence the ability of glucocorticoids to affect GH gene expression.

Adenosine Triphosphate↗
PubMed1986

The 5'-flanking sequences of the human growth hormone gene contain a cell-specific control element.

The 5'-flanking sequences of the human growth hormone (hGH) gene contain a cell-specific control element. Hybrid genes containing truncated 5'-flanking DNA fragments from the hGH gene fused to the chloramphenicol acetyl transferase (cat) gene were examined using transient transfection of rat anterior pituitary (GC) and nonpituitary cell lines (HeLa, Rat 2, and KB); preferential expression of these gene hybrids was only observed in GC cells. Deletions through the 5'-flanking sequences of the hGH gene revealed that the region containing nucleotides -230 to -180 is required for efficient cat gene expression in GC cells. This region of DNA is highly homologous to a region of the rGH gene that contains a tissue-specific control element. A hybrid gene containing the tissue-specific control element, but lacking the proximal promoter elements, of the hGH gene upstream from the promoter of the thymidine kinase gene (TKp) from herpes simplex virus ligated to the cat gene produced more CAT activity than the TKp.cat gene in GC cells but not in HeLa cells. These data suggest that the tissue-specific control element located in the 5'-region of the hGH gene can act in the presence of a heterologous promoter and is specific for expression in pituitary cells.

Acetyltransferases↗
PubMed1985

Interaction of triiodothyronine-receptor complexes with simian virus 40 minichromosomes in monkey kidney CV-1 cells.

Thyroid hormone-responsive tissues contain chromatin-localized receptors that bind to DNA and may associate preferentially with actively transcribed chromatin. To study such receptor-chromatin localization, we have used cultured CV-1 cells permissive for simian virus 40 (SV40), in which viral minichromosomes can be separated from the cellular chromatin. CV-1 cells were found to contain intranuclear thyroid hormone-binding sites with an affinity for T3 and T4 and a site concentration similar to those in other thyroid hormone-responsive tissues. When these cells were infected with SV40 or an SV40-human GH gene recombinant, T3 did not affect SV40 replication, early or late gene transcription, or human GH gene expression. However, in both cases, these infections resulted in the association of about 7.5% of the total specific T3-binding activity with the SV40 minichromosome, representing about 1 receptor molecule/65 minichromosomes and a 10-fold enrichment over the cellular chromatin-associated activity (4.3 fmol/micrograms SV40 minichromosomal DNA vs. 0.43 fmol/micrograms chromosomal DNA); 30% of this could be covalently cross-linked to the minichromosome with dissuccinimidyl suberate. The minichromosomes were also found to be transcriptionally active. Thus, thyroid hormone receptors interact preferentially with the SV40 minichromosome, possibly owing to their tendency to associate with transcriptionally active chromatin. This system provides an alternate approach to study the association of thyroid hormone receptors with defined chromosomal segments.

Animals↗
PubMed1984

Analysis of a major human chorionic somatomammotropin gene. Evidence for two functional promoter elements.

The sequence of one of the two major expressed human chorionic somatomammotropin genes (hCS-1) was determined. The hCS-1 gene and the human growth hormone gene (hGH-1) share 92% nucleotide sequence homology in their 5'- and 3'-flanking regions, introns and exons. This finding, in addition to the existence of multiple closely linked hGH and hCS genes, suggests that these genes are evolving by concerted mechanisms. S1 nuclease, hybridization, and primer extension analysis of placental poly(A+) RNA demonstrated the presence of two functional initiation sites within the hCS-1 and/or the hCS-2 gene(s). The majority (about 95%) of the transcripts initiate 30 nucleotides downstream from the TATAAA sequence, and about 5% of the transcripts initiate 30 nucleotides downstream from a CATAAA sequence located 55 nucleotides 5' to the TATAAA sequence. The presence of two functional promoter elements as well as direct repeated sequences flanking the TATAAA sequence and exon I of the hCS genes are consistent with the hypothesis (Cooke, N. E., and Baxter, J. D. (1982) Nature (Lond.) 297, 603-606) that an important regulatory element may have been inserted into the gene in a separate evolutionary event. An analysis of other direct repeats, internal homology, and homology between other growth hormone and chorionic somatomammotropin genes offers a more extensive conceptualization of how this gene family evolved.

Amino Acid Sequence↗