Body color development and genetic analysis of hybrid transparent crucian carp (Carassius auratus).

The aim of this study was to investigate the genetic mechanism of the transparent trait in transparent crucian carp. We observed body color development in transparent crucian carp larvae and analyzed heredity of color in hybrids produced with red crucian carp, ornamental carp, and red purse carp. The results showed that the body color of the newly hatched larvae matured into the adult pattern at approximately 54 days post-hatching. Two inter-species reciprocal crosses between transparent crucian carp and red crucian carp, and self-cross F1 of transparent crucian carp and self-cross F1 of red marking transparent crucian carp were conducted, and results indicated that the transparent-scaled trait is dominant over the normal-scaled trait. Furthermore, the transparent trait is a quantitative trait. All offspring in the four inter-genera reciprocal crosses of transparent crucian carp with ornamental carp and red purse carp were hybrids of common carp and crucian carp, and had a relatively low survival rate of 10-20%. Moreover, the transparent-scaled trait was observed to be dominant over the normal-scaled trait in the hybrid fish. In conclusion, our results suggest that the genetic mechanism underlying the color of goldfish is complex and requires further investigation.

The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors.

Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH(2)-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms alpha, delta, zeta, and mu, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKC alpha by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.

Transcriptional silencing by unliganded thyroid hormone receptor beta requires a soluble corepressor that interacts with the ligand-binding domain of the receptor.

Unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of genes bearing thyroid hormone response elements in their promoters. Binding of hormonal ligand to the receptor releases the transcriptional silencing and leads to gene activation. Previous studies showed that the silencing activity of TR is located within the C-terminal ligand-binding domain (LBD) of the receptor. To dissect the role of the LBD in receptor-mediated silencing, we used a cell-free transcription system containing HeLa nuclear extracts in which exogenously added unliganded TRbeta repressed the basal level of RNA polymerase II-driven transcription from a thyroid hormone response element-linked template. We designed competition experiments with a peptide fragment containing the entire LBD (positions 145 to 456) of TRbeta. This peptide, which lacks the DNA-binding domain, did not affect basal RNA synthesis from the thyroid hormone response element-linked promoter when added to a cell-free transcription reaction mixture. However, the addition of the LBD peptide to a reaction mixture containing TRbeta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone. An LBD peptide harboring point mutations, which severely impair receptor dimerization, also inhibited efficiently the silencing activity of TR, indicating that the relief of repression by the LBD was not due to the sequestration of TR or its heterodimeric partner retinoid X receptor into inactive homo- or heterodimers. We postulate that the LBD peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extracts and is essential for efficient receptor-mediated gene repression. We have identified the region from positions 145 to 260 (the D domain) of the LBD as a potential binding site of the putative corepressor. We observed further that a peptide containing the LBD of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LBD may bind to the same corepressor activity as the TR LBD. Interestingly, the RAR LBD complexed with its cognate ligand, all-trans retinoic acid, failed to compete for transcriptional silencing by TRbeta, indicating that the association of the LBD with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supporting the existence of the corepressor activity in the HeLa nuclear extracts. Our studies demonstrated that the silencing activity of TR was greatly reduced in the nuclear extracts preincubated with immobilized, hormone-free glutathione S-transferase-LBD fusion proteins, indicating that the corepressor activity was depleted from these extracts through protein-protein interactions with the LBD. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the other hand, failed to deplete the corepressor activity from the nuclear extracts, indicating that ligand binding to the LBD disrupts its interaction with the corepressor. From these results, we propose that a corepressor binds to the LBD of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing. Ligand binding to TR results in the release of the corepressor from the LBD and triggers the reversal of silencing by allowing the events leading to gene activation to proceed.

[Effect of opioid peptides on progesterone production by rat luteal cells in vitro].

The effect of exogenous opioid peptides on progesterone production by incubated rat luteal cells was studied. beta-endorphin (beta-EP) stimulated progesterone production in a dose-dependant manner (10(-8)-10(-6) mol/L); dynorphin exhibited a stimulatory effect only at 10(-6) mol/L, while Met-enkephalin had no substantial effect at dose from 10(-10)-10(-6) mol/L. mu-opioid receptor agonists DAGO and morphine also stimulated progesterone production. The stimulatory actions of beta-EP, DAGO and morphine were reversed completely by naloxone. On account of the fact that the beta-EP level in rat plasma is lower than that in the ovary, it seemed that beta-EP may be an intra-ovarian luteotrophic factor and be involved in the regulation of progesterone production. This action of beta-EP may be mediated by mu-opioid subtype receptors.

Ligand modulates the interaction of thyroid hormone receptor beta with the basal transcription machinery.

We investigated the molecular mechanisms underlying the transcriptional silencing and the hormone-induced activation of target genes by thyroid hormone receptor beta (TR-beta). We developed a cell-free transcription system containing HeLa cell nuclear extracts in which unliganded human TR-beta represses basal transcription from a promoter bearing thyroid hormone response elements. Binding of hormonal ligand to the receptor reverse this transcriptional silencing. Specific binding of TR-beta to the thyroid hormone response element at the target promoter is crucial for silencing. Studies employing TR-beta mutants indicate that the silencing activity is located within the C-terminal rather than the N-terminal domain of the receptor. Our studies reveal further that unliganded TR-beta inhibits the assembly of a functional transcription preinitiation complex (PIC) at the target promoter. We postulate that interaction with TR-beta impairs the function(s) of one or more assembling transcriptional complexes during the multistep assembly of a PIC. Consistent with this hypothesis, we observe that, in the absence of thyroid hormone, TR-beta or a heterodimer of TR-beta and retinoid-X-receptor undergoes direct protein-protein interactions with the transcription factor IIB-TATA binding protein complex, an early intermediate during PIC assembly. Binding of hormone to TR-beta dramatically reduces the interaction between the receptor and the transcription factor IIB-TATA binding protein complex. We propose that the role of ligand is to facilitate the assembly of functional PICs at the target promoter by reducing nonproductive interactions between TR-beta and the initiation factors.