Association of transcriptionally active vitellogenin II gene with the nuclear matrix of chicken liver

Supercoiled DNA loops linked to the nuclear matrix can be progressively cleaved with deoxyribonuclease I. The DNA which remains associated with the nuclear matrix can be purified and analysed for vitellogenin II sequence content by dot blot hybridization. Using this technique we show that vitellogenin II gene sequences are selectively associated with the nuclear matrix of liver but not with oviduct of laying hens. Following primary stimulation in immature chicks of vitellogenin synthesis with estradiol, the association of the gene with the nuclear matrix precedes vitellogenin mRNA synthesis. After 15 days when the level of vitellogenin mRNA has returned to zero, the gene is no longer preferentially associated with the nuclear matrix. At this time a second stimulation with estradiol results in a reassociation of the vitellogenin II gene with the nuclear matrix. In addition to the structural gene, both the 3' and 5' end flanking regions (1.5-2 kb) also bind to the nuclear matrix. However, beyond the limit of 1.5-2 kb upstream from the 5' end of the gene, there is no preferential binding of DNA to the nuclear matrix.     

Primary induction of vitellogenin synthesis in monolayer cultures of amphibian hepatocytes

Direct induction of vitellogenin production in cultured male amphibian hepatocytes by estradiol-17 beta has been accomplished. Liver cells were isolated from adult male bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium containing insulin and estradiol. Vitellogenin production was measured by direct immunoprecipitation from radioactively labeled secreted protein with a specific antiserum against vitellogenin. Significant quantities of vitellogenin were detected in the exported protein on the second day of hormone treatment. Vitellogenin production increased with duration of culture in the presence of estradiol until by the eighth day approximately 90% of secreted protein was vitellogenin. This response is largely comparable to that obtainable in vivo. Indirect immunofluorescence microscopy was used to identify cells synthesizing vitellogenin in response to estradiol. An increase in cytoplasmic fluorescence could be seen in cells throughout the cultures, with increasing time in the presence of estradiol. By the sixth day of treatment, the majority of cells showed significant fluorescence labeling. The results suggest that studies on the mechanisms underlying the primary activation of the vitellogenin gene may now be conducted under well defined conditions in a monolayer liver cell culture system.     

The major and minor chicken vitellogenin genes are each adjacent to partially deleted pseudogene copies of the other.

The major chicken vitellogenin gene (VTGII) has previously been cloned and sequenced. We now report the isolation of genomic clones that encompass a minor chicken vitellogenin gene (VTGIII) which is also expressed in the liver in response to estradiol. Our analysis reveals that a pseudogene for VTGII (psi VTGII) lies 1,426 base pairs upstream of this VTGIII gene. A reevaluation of published sequence data reveals that the converse is also true, namely, that a pseudogene for VTGIII (psi VTGIII) lies 1,345 base pairs downstream of the VTGII gene. Our results show that a 335-base-pair deletion has removed the psi VTGIII promoter and cap site but left residual estrogen response element in a region where nuclease-hypersensitive sites have been reported to be induced in response to estradiol.     

Improved method for purification of Xenopus laevis vitellogenin and demonstration of cross-reactivity among wide-range species by radioimmunoassay

This study was performed to improve the purification of Xenopus vitellogenin and establish the radioimmunoassay. The procedure of purification consisted of ammonium precipitation, DEAE-Sephadex chromatography and Sephadex G-200 gel chromatography. Using this procedure, 934 mg vitellogenin was purified from 49 ml of estradiol treated female Xenopus plasma (about 19 mg/ml). Vitellogenins purified from male and female plasma after a single injection of estradiol showed good correspondence in electrophoretic patterns and amino acid compositions, indicating that vitellogenin synthesis in the male occurs in four different genes as in the female. The radioimmunoassay for vitellogenin was established using an antibody in the plasma obtained from rabbits injected with purified Xenopus female vitellogenin. The titer was 20,000 times dilution of the plasma, and the minimum detectable amount of vitellogenin was 0.1 microgram. The cross-reactivity of this antibody with newt vitellogenin was about 65% and that of chick 6%. The cross-reaction was also observed in female bullfrog plasma. Vitellogenin content was increased gradually during the first 6 days after injection of estradiol in female and the elevated level of vitellogenin dropped afterward.     

A 21 KDa protein resides in the nucleosomes of micrococcal nuclease sensitive liver chromatin of Atlantic salmon treated with estradiol

Atlantic salmon was treated with 17-beta-estradiol to induce the process of vitellogenesis in liver. When the isolated liver nuclei were incubated with micrococcal nuclease at increasing enzyme/DNA ratios a 21 KDa protein appeared in the nuclease sensitive chromatin, the S-fraction. After HPLC gel filtration the 21 KDa protein resided with the oligo- and mononucleosomes. The S-fraction contained vitellogenin gene sequences at low nuclease/DNA ratios. The sequences were detectable also in the mononucleosomes derived from the S-fraction. After hormone treatment the vitellogenin gene exposed a higher sensitivity to micrococcal nuclease than the hormone-untreated controls. The results indicate that the 21 KDa protein took part in the hormone-mediated changes in gene expression by modulating the structure of estradiol responsive chromatin domains including the vitellogenin gene.     

Partial purification of estradiol receptor from Xenopus laevis liver and levels of receptor in relation to estradiol concentration

We have used ammonium sulphate precipitation followed by affinity chromatography to partially purify the estrogen receptor from Xenopus laevis liver which may control the genes for vitellogenin, the precursor of the egg yolk proteins. The rate at which receptor binds estradiol explains the kinetics of the induction of vitellogenin synthesis by estradiol, and the dissociation constant (0.5 X 10(-9) M) explains the concentration dependence of the response, which has a threshold of 10(-9) M estradiol, when 67% of the receptor is bound to estradiol. The estradiol concentration in male liver, which does not make vitellogenin, is 0.18 X 10(-9) M, sufficient to saturate 26% of the receptor, while in female liver, which makes vitellogenin continuously, the estradiol concentration is 3.5 X 10(-9) M, giving 88% saturation of receptor, suggesting that the proportion of occupied receptor decides whether or not the vitellogenin genes are active. In the physiological concentration range, estradiol modulates the level of receptor, which varies between 100 binding sites per nucleus in males and 440 in females, but artificially high concentrations of estradiol raise the level to approximately 1000 sites per nucleus. This suggests that the small increase in vitellogenin mRNA induced by physiological concentrations of estradiol is due to pre-existing receptor and that the much larger increases induced by very high concentrations depends on newly-synthesized receptor.