Isolation and characterization of the mouse metallothionein-I gene.

Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli chi 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 380-base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The metallothionein-I insert was nick-translated and used to screen both a mouse myeloma and a mouse embryo DNA library in bacteriophage lambda.

Regulation of gene transcription by estrogen and progesterone. Lack of hormonal effects on transcription by Escherichia coli RNA polymerase.

Estrogen and progesterone markedly stimulate transcription of ovalbumin and conalbumin (transferrin) genes in chick oviduct as measured by hybridization of labeled RNA synthesized in isolated nuclei to immobilized plasmid DNA containing these gene sequences. Using this direct assay for specific gene transcription, we explored the basis of previous reports indicating that steroid hormones also cause changes in oviduct chromatin structure that can be detected by Escherichia coli RNA polymerase. We observed no effect of these hormones on the ability of E.

Commitment of chick oviduct tubular gland cells to produce ovalbumin mRNA during hormonal withdrawal and restimulation.

Acute withdrawal of estrogen from chicks leads to a precipitous decline in egg white protein synthesis and egg white mRNAs in the oviduct. In this paper we explore the biochemical basis of this phenomenon as well as the capacity of the "withdrawn" tubular gland cells to be restimulated with steroid hormones. During withdrawal, the decline in ovalbumin mRNA was closely correlated with the decline in nuclear estrogen receptors.

Glucocorticoid induction of egg white mRNAs in chick oviduct.

Glucocorticoids induce ovalbumin and conalbumin mRNA in oviducts from chicks withdrawn from prior estrogen treatment. The magnitude and the kinetics of the responses obtained, either in vivo or in vitro, are comparable to those obtained with estrogen or progesterone. With cultured oviducts, 1 nM dexamethasone is sufficient for half-maximal accumulation of nuclear receptors and partial induction of both mRNAs, while maximal levels of receptors and both mRNAs are achieved with 30 to 100 nM dexamethasone.

The chicken transferrin gene. Restriction endonuclease analysis of gene sequences in liver and oviduct DNA.

The organization of the chicken transferrin gene and its surrounding sequences was determined by a comparison of the restriction map of the cloned structural gene (double-stranded cDNA) with the map of the transferrin gene in genomic DNA. This analysis reveals a complex arrangement of the transferrin gene in which structural sequences are interrupted by a minimum of 6 intervening sequences, and span at least 10 kilobases in genomic DNA. Furthermore, comparison of the DNA from individual chickens of the same breed indicates considerable allelic diversity in the restriction sites for Eco RI, HindIII, and Bam HI.

Transferrin gene expression. Effects of nutritional iron deficiency.

Nutritional iron deficiency was produced experimentally by raising newly hatched chicks on an iron-deficient diet for several weeks. During this time, hematocrit and hemoglobin values declined, iron stores were depleted, and the circulating level of transferrin increased 2- to 4-fold. The increase in serum transferrin was related to a similar increase in the rate of transferrin synthesis in liver.

Cloning of an almost full-length chicken conalbumin double-stranded cDNA.

Chicken conalbumin double-stranded cDNA (con-dscDNA) was synthesized from a laying hen oviduct mRNA preparation enriched for conalbumin mRNA (con-mRNA). The dscDNA was inserted by blunt-end ligation into the Sal I site of plasmid pBR322 which had been repaired with DNA polymerase I to create Taq I sites on each side of the inserted fragment. After bacterial transformation, one hybrid recombinant, pBR322-con1, which contains the largest inserted dscDNA (about 2350 bp) was shown to hybridize specifically to the RNA which is translated into conalbumin.

Regulation of procollagen synthesis during the development of chick embryo calvaria. Correlation with procollagen mRNA content.

During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins.

NH2-terminal sequence of the chick proalpha1(I) chain synthesized in the reticulocyte lysate system. Evidence for a transient hydrophobic leader sequence.

Translation of chick procollagen mRNA in a reticulocyte lysate system yields a larger proalpha1(I) chain than is observed in vivo. The NH2-terminal sequence of this putative precursor, determined by automated radiosequencing, is Met-Phe-Ser-Phe-Val-X-Ser-Arg-Leu-Leu-Leu-Leu-Ile-Ala-Ala-X-X-Leu-Leu. This sequence closely resembles the transient hydrophobic leader (signal) sequences observed on most secreted proteins.

Precursor of egg white ovomucoid. Amino acid sequence of an NH2-terminal extension.

The translation of ovomucoid mRNA in a reticulocyte lysate protein-synthesizing system yields a precursor form which contains an NH2-terminal extension of 23 amino acid residues. Edman degradation of radioactive translation products (pre-ovomucoid) identified the following sequence: formula : (see text), where the initiator methionine (in parentheses) is the only residue cleaved from the NH2 terminus during cell-free synthesis and the vertical line indicates the site at which pre-ovomucoid is cleaved in vivo to yield ovomucoid. The precursor sequence differs from those of two other proteins (pre-lysozyme and pre-conalbumin) secreted by the same cell, but resembles these and other secretory protein "signal peptides" in both length and hydrophobicity.

Comparison of the NH2-terminal sequence of ovalbumin as synthesized in vitro and in vivo.

The sequence of the NH2-terminal 35 residues of chicken ovalbumin was found to be identical with that of the product translated in vitro from the corresponding mRNA. Together with our previous results (Palmiter, R.D.

Estrogenic activity of the insecticide kepone on the chicken oviduct.

Kepone induces ovalbumin and conalbumin synthesis in explants of chick oviduct in vitro by acting as a weak estrogen. It binds to the nuclear estrogen receptor and is antagonized by the antiestrogen tamoxifen. Kepone also induces egg white protein synthesis in vivo by direct interaction with estrogen receptors and by indirectly increasing the concentration of progesterone in the serum.

Identical precursors for serum transferrin and egg white conalbumin.

The NH2-terminal sequences of egg white conalbumin and chicken serum transferrin were examined and found to be identical. Conalbumin, when synthesized in a rabbit reticulocyte cell-free translation system, was found to contain an NH2-terminal extension of 19 amino acid residues. Sequential Edman degradation of this precursor (pre-conalbumin) labeled with radioactive amino acids revealed the following sequence: formula see text: The vertical line indicates the site at which pre-conalbumin is cleaved to yield authentic conalbumin.

Ovalbumin: a secreted protein without a transient hydrophobic leader sequence.

Ovalbumin mRNA was translated in a reticulocyte lysate. The primary translation product starts with methionine derived from Met-tRNAf. When the nascent polypeptide is about 20 residues long, this methionine is removed.

Prevention of NH2-terminal acetylation of proteins synthesized in cell-free systems.

The NH2 terminus of ovalbumin is acetylated in cell-free protein-synthesizing systems as it is in vivo. The acetyl group is derived from acetyl-CoA and it is incorporated during translation. Acetylation can be prevented by metabolizing the available acetyl-CoA to citrate with the addition of citrate synthase and oxalacetate to the translation system.

Precursor of egg white lysozyme. Amino acid sequence of an NH2-terminal extension.

Lysozyme mRNA was translated in a reticulocyte lysate with mixtures of radioactive amino acids. The in vitro product isolated by immunoprecipitation was shown by gel electrophoresis, peptide mapping, and sequence analysis to be larger than lysozyme synthesized in vivo. An NH2-terminal extension was completely sequenced by automated Edman degradation; the phenylthiohydantoins from each cycle were separated by high pressure liquid chromatography and quantitated by scintillation spectroscopy.