Establishment and characterization of Xenopus oviduct cells in primary culture

Based on a previously established procedure for Xenopus hepatocytes, we describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response [A. P. Wolffe, J. F. Glover, and J. R. Tata (1984) Exp. Cell Res. 154, 581], from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the same characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes [A. J. Perlman, A. P. Wolffe, J. Champion, and J. R. Tata (1984) Mol. Cell. Endocrinol. 38, 51]. Unlike the latter in primary culture [M. P. R. Tenniswood, P. F. Searle, A. P. Wolffe, and J. R. Tata (1983) Mol. Cell. Endocrinol. 30, 329], oviduct cell cultures did not actively metabolize either estradiol or progesterone (t1/2 approximately equal to 55 and 7 h, respectively). The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfil the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.     

Protein synthesis by membrane-bound and free ribosomes of secretory and non-secretory tissues

1. Methods for the separation of membrane-bound and free ribosomes from rat brain (cortex) and skeletal muscle were described and the preparations characterized by chemical analysis and electron microscopy. The attachment of ribosomes to membranes is not an artifact of the separation procedure. 2. The rate of incorporation of l-[(14)C]leucine into protein in vitro by the membrane-bound and free ribosomes from these two predominantly non-protein-secreting tissues is compared with that by similar preparations from rat liver. With all three tissues the initial rate was higher for the membrane-bound preparations. 3. By using the technique of discharging nascent polypeptide chains by incubation with puromycin followed by treatment with sodium deoxycholate (Redman & Sabatini, 1966), a major difference was observed for the vectorial discharge of nascent protein synthesized both in vivo and in vitro on membrane-bound ribosomes from liver, on the one hand, and brain and muscle, on the other. Whereas a large part of nascent protein synthesized on membrane-bound liver ribosomes was discharged into the membranous vesicles (presumably destined for export from the cell), almost all nascent protein from membrane-bound ribosomes from brain and muscle was released directly into the supernatant. Incorporation of [(3)H]puromycin into peptidyl-[(3)H]puromycin confirmed these findings. There was thus no difference between membrane-bound and free ribosomes from brain on the one hand, and from free polyribosomes from liver on the other, as far as the vectorial release of newly synthesized protein was concerned. 4. Incubation with puromycin also showed that the nascent chains, pre-formed in vivo and in vitro, are not involved in the attachment of ribosomes to membranes of the endoplasmic reticulum. 5. The differences in vectorial discharge from membrane-bound ribosomes from liver as compared with brain and muscle are not due to the different types of messenger RNA in the different tissues. Polyphenylalanine synthesized on incubation with polyuridylic acid was handled in the same way as polypeptides synthesized with endogenous messenger. 6. It is concluded that there is a major difference in the attachment of ribosomes to the membranes of the endoplasmic reticulum of secretory and non-secretory tissues, which results in a tissue-specific difference in the vectorial discharge of nascent proteins.     

Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.     

The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri).

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 X 10(-2) and 1 X 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.     

Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin.     

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

Sub-nuclear fractionation. I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of ${}^3\text{H-actinomycin D}$, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].