Estrogen-responsive genes encoding egg yolk proteins vitellogenin and apolipoprotein II in chicken are differentially regulated by selective estrogen receptor modulators.

In a hen, large quantities of the egg yolk proteins, apolipoprotein II (apo-II) and vitellogenin (VG), are expressed in the liver and transported to the oviduct during egg production. Estrogenic stimulation of the hepatic expression of apo-II and VG is due to both transcriptional increase and mRNA stabilization. The nucleolytic degradation of apo-II messenger RNA (mRNA) is prevented by estrogen-regulated mRNA-stabilizing factor (E-RmRNASF). Gene-specific effects of a select panel of selective estrogen receptor modulators (SERMs) on the hepatic expression of the estrogen-responsive genes encoding apo-II, VG, and E-RmRNASF in the chicken liver were investigated. In the present study, 6-week-old roosters were treated with the vehicle, estrogen, the SERMs genistein, resveratrol, tamoxifen, pterostilbene, raloxifene, catechin, and clomiphene or a combination of estrogen and a 200-fold excess of each of the SERMs. Results from mRNA stabilization studies conducted to investigate the stimulation of expression of E-RmRNASF in the liver by these agents showed that the expression of E-RmRNASF in the liver was stimulated by estrogen and the SERMs genistein, resveratrol, tamoxifen, pterostilbene, and catechin but not by the vehicle, clomiphene or raloxifene. The expression of apo-II and VG from the aforementioned treatments was determined by Northern blot analysis, RNase protection assays, and Western blot analysis. The transcription and protein expression of both apo-II and VG genes were seen in response to treatment with estrogen but not with the SERMs or combinations of estrogen and each of the SERMs. The SERMs that stimulated the expression of E-RmRNASF antagonized the stimulation of the expression of both apo-II and VG by estrogen, demonstrating a gene-specific, selective regulation of the aforementioned genes in the chicken liver by the SERMs. The above panel of SERMs may likely have adverse effects on egg production.

Bam HI cleaves the self complementary dodecamer d-CGCGGAGCCGCG, before the two G's and possibly binds in the DNA major groove.

Oligodeoxynucleotides with GT or GA mispairs within the Bam HI recognition sequence (GGATCC), have been prepared. Binding and cleavage of the native vis a vis the mismatch substrates by Bam HI are analysed. UV melting curves and CD spectra of the oligomers suggest a double stranded B-DNA conformation. The enzyme Bam HI binds with varying affinities to the oligomers except the one with the GT wobble base pair. Bam HI cleaves the cognate sequence, GGATCC, between the two Guanines but cleaves GGAGCC before the guanines. The unusual cleavage is due to a local distortion in the DNA structure. Kinetic analysis of the cleavage reactions using the 35S labeled hexadecamers, d-ATGGCGGATCCGCCAT and d-ATGGCGGAGCCGCCAT, as substrates gives Km values 11.08 nM and 1.16 nM with corresponding Kcat of 11.04 and 0.62 min-1 respectively. The results are consistent with the binding of Bam HI in the major groove.

Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants Km and Kcat for BamHI restriction endonuclease: an empirical and iterative approach.

An assay using a very small amount of 35S-labeled deoxyoligonucleotide as a substrate for the determination of Km and Kcat for the restriction enzyme BamHI is described. Two synthetic deoxyoligonucleotides, ATGGCGGATCCGC and ATGGCGGAGCCGC, containing the cognate and a mismatch BamHI sequence, respectively, were labeled by an end-filling reaction using the Klenow fragment of DNA polymerase and [35S]dATP to generate the labeled self-complementary substrates. The dependence of BamHI hydrolysis on substrate concentration was investigated using mixtures of a fixed amount of radiolabeled substrate and varying amounts of cold-labeled substrate over a wide range. The apparent competitive inhibition observed due to the phenomenon of carrier dilution was analytically corrected by an empirical as well as an iterative approach to give Km values comparable to those reported in the literature. We have found that the values obtained using the empirical formula are very close to the precise values obtained through iteration. Our procedure has used isotopic dilution to advantage to make the assay less expensive and can be applied effectively to any enzyme-substrate reaction in which the substrate and the product have radioactive labels. The method would be especially useful for a rapid analysis and comparison of kinetic constants of various mutant enzymes or substrates.