Biomedical subjects

R C Simmen

Publications and source records attributed to R C Simmen.

75 records · Page 5Linked to original sources

PubMed1985

The structural organization of the chicken calmodulin gene.

The structural organization of the entire chicken calmodulin (CaM) gene was determined by analysis of overlapping genomic clones obtained from Charon 4A and cosmid DNA libraries. These clones together span 39 kilobases of chicken genomic DNA. The CaM gene is 12 kilobases long and contains 8 exons interrupted by introns of highly variable size. The first intron (A) is only 19 base pairs (bp) long and divides the 5' untranslated region. Intron B separates the ATG from the first nucleotide of the triplet which encodes the NH2-terminal amino acid of CaM (Ala) whereas intron C separates the triplets encoding amino acids 10-11. Introns D, F, and G interrupt the Ca2+ binding subdomains II, III, and IV of CaM whereas intron E is localized in the linker region between the highly homologous NH2- and COOH-terminal halves of the protein. Primer extension studies using chicken brain poly(A+) mRNA and a fragment from the 5' untranslated region of a CaM cDNA identified the presumptive transcription initiation site (cap site) of the mRNA to be 103 bp 5' from the initiation condon ATG. A consensus sequence TATTTAA was localized 29 bp 5' from this cap site while a CCAAT sequence was located further 5' at position -58 bp. The structure of the CaM gene is strikingly similar to genes that encode other Ca2+ binding proteins from sea urchin, chicken, rat, and quail. These data suggest a conservation of genome organization related to the calcium binding and regulatory domains of Ca2+ binding proteins.

Animals↗

PubMed1984

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Animals↗

PubMed1984

Estrogen modulation of nuclear matrix-associated steroid hormone binding.

Estrogen regulation of the intranuclear distribution of estrogen-binding proteins has been examined in the chicken liver. Administration of estrogen [diethylstilbestrol (DES)] resulted in a rapid, dose-dependent increase in two types of estrogen-binding sites associated with the nuclear matrix. Type I appeared to be the estrogen receptor (ER; Kd for estradiol = 2.0 nM), whereas type II was equivalent to binding sites previously defined in rat uterine nuclei (Kd = 15 nM). The association of nuclear ER and type II sites with the nuclear matrix followed a time course corresponding to the maximal accumulation of nuclear ER in the liver. A 2-fold enrichment (per U protein) of nuclear ER in the matrix was obtained at a dose of 20 micrograms DES/0.2 kg compared to control values. Further enrichment was observed with higher doses of DES (200 and 2000 micrograms). The antiestrogens tamoxifen (tamoxifen-citrate) and zuclomiphine 2-[p-(chloro-1,2-diphenyl-vinyl)phenoxyl]citrate) were potent inhibitors (greater than or equal to 50%) of the estrogen-stimulated association of both ER and type II sites with the matrix. However, they did not increase total nuclear or matrix ER concentrations when administered alone. These data demonstrate that the association of ER with liver nuclear matrix is estrogen dependent and suggest that the biological effects of estrogen at the nuclear level may be mediated by this association.

Animals↗