Geert Ab

Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein

The gene encoding the major vitellogenin from chicken has been completely sequenced and its exon-intron organization has been established. The gene is 20,342 base-pairs long and contains 35 exons with a combined length of 5787 base-pairs. They encode the 1850-amino acid pre-peptide of vitellogenin, which is the precursor of the mature yolk proteins, the serine-rich and heavily phosphorylated phosvitin and the lipovitellin. The 217-amino acid phosvitin polypeptide occupies an internal position (residue 1112 through 1328) within the vitellogenin molecule. The 125,000 and 30,000 Mr lipovitellin polypeptides are encoded by the sequences at the N-terminal and the C-terminal sides of the phosvitin section, respectively. The main features of the gene and protein sequences, and the evolutionary implications, are discussed.

Expression-linked demethylation of 5-methylcytosines in the chicken vitellogenin gene region.

We have studied the methylation status of the estradiol-controlled chicken vitellogenin (Vtg) gene, which is expressed in the liver. A 30-kb region was investigated, containing 17 HpaII and 18 HhaI sites, of which 21 are in the 22-kb gene. Of these 21 sites, 9 were found to be demethylated in laying-hen liver relative to immature chicken liver. Outside the transcribed region, only one site was found to be relatively undermethylated in laying-hen liver. This site, at 0.6 kb in front of the gene, is, as shown earlier, also demethylated in rooster or immature chicken liver upon primary hormone stimulation, as well as in the non-expressing estradiol target organ oviduct. In this respect, this site sharply contrasts with those in the transcribed region, which appear to become demethylated only upon prolonged transcription of the gene.

Specific Stimulation of Ribosomal RNA Synthesis in E. coli by a Protein Factor

SummaryRibosomal RNA synthesis in a purified system is stimulated by a crude protein fraction prepared from E. coli. The positive effector which is not associated with RNA polymerase, nor is the sigma factor, increases the initiation frequency on a rRNA operon. The additional rRNA synthesis is inhibited by ppGpp to the same extent as the basal one.The evidence presented points to the existence of a positive control element for rRNA synthesis, which activity depends upon the physiological state of the cell.

A MUTATION IN THE RNA-POLYMERASE BETA'-SUBUNIT CAUSING DEPRESSED RIBOSOMAL-RNA SYNTHESIS IN ESCHERICHIA-COLI

SummaryMacromolecular synthesis in an Escherichia coli mutant with a temperature-sensitive β′ subunit of RNA polymerase was analysed. At the non-permissive temperature ribosomal RNA synthesis is strongly reduced while messenger RNA synthesis is affected to only a slight extent. The overall protein synthesis is only slightly affected. We conclude that the β′ subunit is involved in promoter recognition and plays a role in transcriptional selectivity.

Splicing pathways of the chicken apo very low density lipoprotein II (pre)messenger RNA.

The precursor‐mRNA transcribed from the chicken apo very low density lipoprotein II gene was identified. This gene which is under full estrogen control and only expressed in the liver, possesses three introns. Splicing intermediates were characterized by hybridization with intron‐specific probes, and by electron microscopy of R‐loops. The introns appear to be excised in a non‐obligatory order, but at different rates.

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.