M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of beta-galactosidase alpha-complementation.

A DNA fragment encoding the transposon Tn9 chloramphenicol acetyltransferase gene (cat) was inserted into M13 phage and pUC plasmid cloning vehicles. When the cat gene was inserted in the same orientation as the lacZ gene, two new polypeptides were produced. One polypeptide possessed chloramphenicol acetyltransferase activity, while the other expressed beta-galactosidase alpha-donor activity. Both new polypeptides were translated from a hybrid messenger RNA initiating from the lac promoter. These observations may help explain why not all inserts produce white plaques.

Estrogen regulates the number of RNA polymerase II molecules in rooster liver.

Administration of estrogen to roosters causes an increase in the activities of DNA-dependent RNA polymerases I and II in nuclei isolated from liver. In an effort to determine the cause of this increased transcriptional activity, we have examined the activity of RNA polymerase II that we have solubilized from nuclei of normal, estrogen-stimulated, and estrogen-withdrawn roosters. In addition, we have measured the actual numbers of RNA polymerase II molecules per nuclear equivalent of DNA in livers of roosters in each estrogenic state by the technique of [3H]amanitin-binding. The administration of estrogen is attended by a 2-fold increase in enzymatic activity of solubilized RNA polymerase II per liver nucleus within 24 h. In addition, there is a 2-fold increase in the number of RNA polymerase II molecules per nucleus in the livers of these animals after the administration of estrogen. During withdrawal from estrogen for 14 days, the activities of RNA polymerases I and II in isolated nuclei and the activity of solubilized RNA polymerase II return to the unstimulated levels. Moreover, the [3H]amanitin-binding capacity of nuclear extracts from the livers of roosters in various stages of hormonal stimulation closely mimics the RNA polymerase II activity of the same extracts. These observations indicate that estrogen exerts a rigid control over the population of RNA polymerase II molecules in avian liver.

Phosphorylation of rat ascites tumor non-histone chromatin proteins. Differential phosphorylation by two cyclic nucleotide-independent protein kinases and comparison to in vivo phosphorylation.

The substrate specificity of two cyclic nucleotide-independent protein kinases purified from both rat ascites tumor cells and calf thymus has been examined. The protein kinases, designated casein kinase I and II, were purified to near homogeneity and are free of contaminating protein substrates. Non-histone chromatin proteins, purified from Novikoff ascites tumor cells and free of protein kinase and activity, were phosphorylated with casein kinases I and II and the spectrum of labeled polypeptides determined. Phosphorylation of non-histone chromatin proteins with casein kinase I from either source, resulted in the same pattern of 32P-labeled polypeptides when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Similarly, casein kinase II from either source phosphorylated an identical set of polypeptides. The distribution of 32P-labeled non-histone chromatin proteins phosphorylated by casein kinase I differed from that labeled by casein kinase II suggesting that each protein kinase phosphorylates a specific subset of non-histone chromatin proteins. The differences in protein substrate specificity of casein kinase I and casein kinase II was confirmed by two-dimensional polyacrylamide gel electrophoresis. Non-histone chromatin proteins, labeled with 32P in vivo, were analyzed by the same methods and the distribution of labeled polypeptides compared to in vitro phosphorylation patterns. A number of prominent in vitro phosphorylated polypeptides appear identical with non-histone chromatin proteins labeled with 32P in vivo. These results suggest that casein kinases I and II may function in vivo in the phosphorylation of non-histone chromatin proteins.

Specific transcription in chicken liver chromatin by endogenous RNA polymerase II. Comparison of an estrogen-inducible gene with a constitutively expressed gene.

We have developed a system for the in vitro transcription of specific genes in rooster liver chromatin by endogenous RNA polymerase II that maintains the specificity of transcription in vivo. Radioactive transcripts synthesized in vitro were identified and quantitated by hybridization to a vast excess of cloned cDNA. The cDNA preparations employed corresponded to vitellogenin mRNA, the synthesis of which is responsive to estrogen stimulation in vivo, and chicken serum albumin mRNA, the synthesis of which is not significantly affected by estrogen stimulation in vivo. Comparing the pattern of transcription of the albumin and vitellogenin genes in chromatin from the liver of the normal rooster with the pattern in chromatin from the liver of the estrogen-stimulated rooster, we found that prior estrogen treatment of the rooster is attended by a slight decrease in the differential rate of transcription of the albumin gene and approximately a 10-fold increase in the differential rate of transcription of the vitellogenin gene. Because this pattern of transcription reflects the estrogen-induced changes in transcription observed in vivo, chromatin preparations from the livers of normal and estrogen-stimulated roosters can be used to investigate regulation of specific gene transcription at the molecular level in vitro.

Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.

In vitro translation of avian vitellogenin messenger RNA.

Administration of 17beta-estradiol to roosters induced the synthesis of vitellogenin in the liver. The mRNA that specifies this protein has been purified from the livers of estrogen-treated roosters and has been shown to have a molecular weight of 2.3 X 10(6) (Deeley, R.G., Gordon, J.I., Burns, A.T.H., Mullinix, K.P., Bina-Stein, M., and Goldberger R.F. (1977) J. Biol. Chem. 252, 8310-8319). In order to rigorously establish the identity of the polypeptide specified by this mRNA, we used a staphylococcal nuclease-treated, mRNA-dependent wheat germ cell-free translation system capable of synthesizing polypeptides as large as vitellogenin (monomer Mr = 240,000). Vitellogenin mRNA directs the in vitro synthesis of a polypeptide with the following features: (a) it co-migrates with authentic vitellogenin in SDS-polyacrylamide gels; (b) it is highly enriched for serine but is not phosphorylated; (c) it is immunoprecipitated by purified, monospecific, anti-vitellogenin antibody; and (d) it has an unusual cyanogen bromide cleavage pattern characteristic of vitellogenin. The most striking characteristic of the cyanogen bromide cleavage products is an extremely large polypeptide (Mr = 90,000) that contains two phosvitins. The kinetics of incorporation of serine and methionine into vitellogenin synthesized in the wheat germ cell-free translation system indicates that the phosvitins are located near the COOH-terminal portion of the molecule.

Comparative study of hen yolk phosvitin and plasma vitellogenin.

Vitellogenin, the only phosphoprotein detectable in the plasma of laying hens, is present at an approximate concentration of 1 mg/mL and can be isolated by chromatography on diethylaminoethylcellulose. Vitellogenin has a molecular weight of 235 000--240 000 and contains approximately 3% phosphorus by weight. Evidence that this protein is the precursor of phosvitins includes its ability to act as an acceptor for phosphate with a phosvitin specific kinase, the generation of a peptide similar to phosvitin by trypsinization, and the presence of distinctive peptides of multiple clustered phosphoserine upon partial acid hydrolysis. This partial sequence similarity between phosvitins and vitellogenin has not been previously reported. The phosphorus content and amino acid composition of vitellogenin are consistent with a model which contains two phosvitins and one lipovitellin. The total molecular weights of these proteins (28 000 + 34 000 + 170 000 = 232 000) are close to that of vitellogenin.