RNA metabolism in chick oviduct during oestrogen stimulation

The amount and the labelling of RNA were studied in chick oviduct after secondary stimulation of pre-treated chicks with oestrogen. The weight of the oviduct increases over 2 fold in 20 h of oestrogen action using a dose of 1 mg. During the hormonal treatment the amount of cytoplasmic and nuclear RNA increases about 3 and 1.5 fold, respectively. The amount of poly(A) containing RNA increases somewhat less than that of RNA lacking poly(A). The proportion of poly(A) containing species of the total cytoplasmic and nuclear RNA decreases from 5.8% to 4.4% and from 7.8% to 6.3%, respectively. Hydroxyurea largely prevents the effect of oestrogen in increasing the amount of RNA. Incorporation of [3H]uridine in RNA in vitro is stimulated about 2.5 fold in cytoplasmic RNA lacking poly(A) and only 1.2 fold in RNA containing poly(A) in 20 h of oestrogen action. In nuclear RNA the labelling of the two species is stimulated about 1.7 fold. Hydroxyurea fails to interfere with the oestrogen-induced stimulation in the labelling of RNA. Cytoplasmic and nuclear RNA containing poly(A) is distributed in a peak of about 20 S in agarose-acrylamide gels.     

Effect of a skeleton photoperiod on the daylength-dependent response to oestrogen in canaries (Serinus canarius)

We have analyzed the sequence complexity and diversity of poly(A)-containing mRNA derived from two highly differentiated chicken tissues. Two independent approaches were used in our analyses. The first involves the annealing of cDNA copies of mRNA to a vast excess of the template RNA; the second procedure uses hybridization between highly radioactive single-copy genomic DNA and mRNA. The results obtained using these two experimental approaches are in good accord and reveal the presence of 12,000-15,000 diverse mRNA species in both chicken liver and oviduct. In both cell types, the kinetics of annealing of cDNA to its template mRNA demonstrate discrete frequency classes with most of the different mRNA species present in fewer than 10 copies per cell. 70% of oviduct mRNA, however, consists of about 10 abundant RNA species, which probably are responsible for the synthesis of the egg white proteins. The diversity of mRNA species in chicken liver and oviduct was further studied by heterologous annealing reactions between cDNA or singlecopy genomic DNA and a vast excess of mRNA. These studies demonstrate that 85% of the different mRNA sequences detected are present in both liver and oviduct, and suggest that the vast majority of the information expressed as mRNA is required for the maintenance of cellular functions common to all tissues.     

Evidence that vitamin A is not required for the biosynthesis of ovalbumin in chicks

Four-day-old pullets fed a vitamin A-deficient diet were stimulated daily with 1 mg 17beta-estradiol-3-benzoate/day for 6 to 19 days. The onset of vitamin A deficiency had no effect on oviduct growth in these chicks; even though vitamin A-deficient chicks showed a severe decline in growth rate while controls (fed the same diet supplemented with retinyl palmitate) continued to grow, estrogen stimulated resulted in similar oviduct size. Ovalbumin concentrations of estrogen-stimulated chicks were determined by immunoprecipitation of the soluble protein supernatant fraction of oviduct. The concentration of ovalbumin in oviducts of chicks fed a vitamin A-supplemented diet was similar in the concentration in oviducts of chicks fed a vitamin A-deficient diet. The incorporation of [3H]glucosamine and 14C-amino acids into immunoprecipitable ovalbumin, following the in vitro incubation of minced oviduct, indicated that ovalbumin synthesis was not affected by vitamin A deficiency. The specific activity of incorporated [3H]glucosamine, the 14C-amino acid incorporation into ovalbumin, the relative rate of ovalbumin synthesis, and the relative effiency of [3H]glucosamine incorporation into ovalbumin were each similar between the two diet groups. The relative efficiency of [3H]glucosamine incorporation into sodium dodecyl sulfate and dithiothreitol extractable membranous proteins of oviduct was not affect by vitamin A deficiency.     

Translation of ovalbumin mRNA in Xenopus laevis oocytes. Characterization of the system and effects of estrogen on injected mRNA populations

Ovalbumin messenger RNA (mRNAov) purified from hen oviduct was injected into Xenopus laevis oocytes. The oocytes were incubated in culture medium containing [3H]leucine. Analysis of the oocyte cytosol on Sephadex G-15O columns demonstrated a peak of radioactivity which cochromatographed with authentic ovalbumin. Radioactive protein contained in this peak was precipitated by ovalbumin antiserum, coelectrophoresed with ovalbumin on sodium dodecyl sulfate (SDS) and urea gels at pH 8.7, and eluted with the protein at the same pH (4.8) on CM-cellulose chromatography. Injection of increasing amounts of mRNAov was found to elicit a linear response in terms of ovalbumin synthesis. Moreover, there was linear incorporation of radioactivity into microinjected oocytes over a minimum period of 91 h. Less than 1 ng mRNAov was detected in this system. Ovalbumin mRNA activity was present in RNA preparations from chicks treated with estrogen but was undetectable in animals withdrawn from the hormone. This study constitutes an initial demonstration of a steroid hormone-induced alteration in mRNA population as assayed in intact viable heterologous cells.     

Can SINEs: a family of tRNA-derived retroposons specific to the superfamily Canoidea

A repetitive element of approximately 200 bp was cloned from harbour seal (Phoca vitulina concolour) genomic DNA. The sequence of the element revealed putative RNA polymerase III control boxes, a poly A tail and direct terminal repeats characteristic of SINEs. Sequence and secondary structural similarities suggest that the SINE is derived from a tRNA, possibly tRNA-alanine. Southern blot analysis indicated that the element is predominately dispersed in unique regions of the seal genome, but may also be present in other repetitive sequences, such as tandemly arrayed satellite DNA. Based on slot-blot hybridization analysis, we estimate that 1.3 x 10(6) copies of the SINE are present in the harbour seal genome; SINE copy number based on the number of clones isolated from a size-selected library, however, is an order of magnitude lower (1-3 x 10(5) copies), an estimate consistent with the abundance of SINEs in other mammalian genomes. Database searches found similar sequences have been isolated from dog (Canis familiaris) and mink (Mustela vison). These, and the seal SINE sequences are characterized by an internal CT dinucleotide microsatellite in the tRNA-unrelated region. Hybridization of genomic DNA from representative species of a wide range of mammalian orders to an oligonucleotide (30mer) probe complementary to a conserved region of the SINE confirmed that the element is unique to carnivores of the superfamily Canoidea.     

Induction of avidin in the chick oviduct by tissue damage. Effect of promethazine chloride, CaCl2 and hydrocortisone on local induction

The local effect of the mechanical induction of avidin by ligature was studied in diethylstilbestrol-primed chicks. The highest induction of avidin was always found in the immediate vicinity of the silk ligature of the oviduct. The locality of the induction was highly dependent on the position of the ligature. The nonligated parts of the ligated oviduct also showed a slight avidin induction. These results indicate a strictly local effect of avidin induction by ligature. An antihistamine, promethazine chloride, has a potentiating effect on the avidin induction by ligature when administered after the ligature. On the other hand, membrane stabilization by hydrocortisone or CaCl2 did not influence the ligature-induced avidin synthesis. On the basis of these results it is concluded that the avidin induction is not mediated by histamine activation or membrane damage.     

Size distribution of rat liver nuclear RNA containing mRNA sequences

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98% formamide. Complementary DNA (cDNA) was prepared from the 8--14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that: 1. All abundant 8--14-S poly(A)-containing mRNAs have larger nuclear precursor molecules; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length. 2. 70% of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S. 3. The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8--14-S mRNA.     

Poly (A)-rich ribonucleoprotein complexes from HeLa cell messenger RNA

Polyribosomal messenger RNA from HeLa cells contain 3'-OH-terminal polyadenylate sequences approximately 133 nucleotides in length (weight average). When analyzed at the ribonucleoprotein level of organization these poly(A)-rich sequences are found to contain tightly bound proteins. These proteins remain associated with the poly(A)-rich RNA during affinity chromatography of RNase A and T1-digested polyribosomes on poly(U)-Sepharose in 0.5 M NaCl, and co-elute from the column with the RNA at 50% formamide. Controls establish that the co-purification of the proteins with poly(A) on poly(U)-Sepharose requires the molecular integrity of the poly(A). Polyacrylamide gel electrophoresis resolves the poly(A)-specific proteins into two components of 74,000 and 62,000 molecular weight. The larger protein is the same size as that previously reported to be associated with poly(A)-rich sequences in HeLa heterogeneous nuclear RNA (Kish, V.M., and Pederson, T. (1975), J. Mol. Biol. 95, 227-238). It is concluded that both HeLa nuclear and polyribosomal poly(A) sequences have a protein (62,000 molecular weight) associated with poly(A) appears to be confined only to messenger RNA.     

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.     

Effects of estrogen on gene expression in the chick oviduct. Isolation and fractionation of chromatin non-histone proteins

Non-histones isolated from hen oviduct chromatin have been fractionated by a variety of methods. Chromatin was dissociated in 2 M NaC1, 5 M Urea, 0.1% beta-mercaptoethanol and 0.01 M Tris - HC1, pH 8.3, and the DNA removed by ultracentrifugation. After desalting by gel filtration the chromatin proteins were separated into three distinct fractions by stepwise elution with 0.10 M NaC1, 0.25 M Na C1 and 15% guanidine - HC1 from Bio-Rex 70 columns. Fractions I and II contain only non-histones and Fraction III contains histones plus a small amount of non-histones. Further fractionation of the non-histones was achieved by ammonium sulfate precipitation and DEAE-cellulose chromatography for Fraction I and phosphocellulose chromatography and gel filtration on Bio-Gel A-15 m for Fraction II. The histone and non-histones present in Fraction III were separated by gel filtration on Bio-Gel A-0.5 m. All fractionation methods have been used preparatively with reasonable recoveries of protein (greater than or equal to 60%). The fractions have been characterized by acrylamide gel electrophoresis. The integrity of the histones was maintained during the fractionation procedure indicating that proteolytic degradation was unlikely to have occurred. There was no selective loss of chromatin proteins during the ultracentrifugation and desalting steps and the non-histones were separated into distinct fractions with enrichment of some species not apparent prior to fractionation of the chromatin proteins.     

Partial purification of a progesterone-inducible messenger RNA (avidin) from hen oviduct

The messenger RNA (mRNA) for avidin, which represents less than 0.05% of the total cellular proteins, was partially purified from hen oviduct, and the presence of avidin mRNA was shown to depend upon prior stimulation by progesterone. A total nucleic acid extract was subjected to oligo (dT)-cellulose chromatography, followed by Sepharose 4B chromatography, preparative agarose gel electrophoresis, and sucrose gradient centrifugation. The relative purity of each preparation was assessed by translation in a wheat-germ system; avidin messenger RNA activity was measured by specific immunoprecipitation of synthesized proteins. Avidin mRNA was separated from the bulk of the total messenger RNA activity of the oviduct and from all ribosomal RNAs to produce greater than a 1000-fold enrichment of avidin mRNA activity compared with total cellular RNA. Based on the translation assay, the most highly purified fraction contained about 2.5% avidin messenger RNA. Avidin mRNA activity was absent in partially purified mRNA obtained from estrogen-stimulated chick oviducts, but was detected in oviducts following progesterone administration.     

Size, complexity and abundance of a specific poly(A)-containing RNA of liver from male Xenopus induced to vitellogenin synthesis by estrogen

Estrogen treatment of Xenopus males leads to the appearance of a new species of poly (A)-containing RNA in the liver, at a stage when large amounts of the estrogen-induced yolk precursor protein, vitellogenin, is produced. This estrogen-induced RNA sediments at 28 S and migrates on gels in aqueous solution with an apparent molecular weight of 2.0 X 10(6). Contour length measurements under denaturing conditions in the electron microscope reveal a molecular weight of 2.34 X 10(6) compared to the mouse 28-S rRNA. Labeling experiments show that the estrogen-induced RNA has a stability than the average liver poly(A)-containing RNA and represents 10-20% of the poly(a)-containing RNA in the cytoplasm after 24 h of labeling. Hybridization of complementary DNA, synthesized on the isolated estrogen-induced RNA, with its template suggests a complexity corresponding to a single species of poly(A)-containing RNA of such a high molecular weight. Hybridization of the complementary DNA with cytoplasmic poly (A)-containing RNA from estrogen-treated Xenopus males and control toads show that the estrogen-induced RNA constitutes 12-15% of all cytoplasmic poly(A)-containing RNA, and is at least 2000-fold less abundant in untreated males. Size, complexity and abundance of the estrogen-induced RNA are characteristics expected for a mRNA coding for vitellogenin.     

A quantitative analysis of cells in the ganglion cell layer of the chick retina

This study investigated the organization of cells in the ganglion cell layer (GCL) using Nissl staining, retrograde cell degeneration with axotomy of the optic nerve, and retrograde cell labeling by injections of horseradish peroxidase (HRP) into the optic nerve of chicks (posthatching day 1 and 8, P-1 and P-8). The total number of cells in the GCL was 6.1 x 10(6) (P-1) and 4.9 x 10(6) (P-8), and the cell density was 14,300 cells/mm2 (P-1) and 10,400 cells/ mm2 (P-8) on average. Two high-density areas, the central area (CA) and the dorsal area (DA), were observed in the central and dorsal retinas in both P-1 (22,000 cells/mm2 in CA, 19,000 cells/mm2 in DA) and P-8 chicks (19,000 cells/mm2 in CA, 12,800 cells/mm2 in DA). The cell densities in the temporal periphery (TP) and the nasal (NP) peripheral retinas were 7,800 cells/mm2 and 12,500 cells/mm2, respectively, in P-1 and 5,000 cells/ mm2 and 8,000 cells/mm2, respectively, in P-8 chicks. The cell density in the temporal periphery was 35% (P-8) lower than in the nasal periphery in both P-1 and P-8 chicks. Thirty percent (1.9 x 10(6) cells in P-1) of the total cells in the GCL were resistant to axotomy of the optic nerve. The distribution of the axotomy-resistant cells showed two high-density areas in the central and dorsal retinas, corresponding to the CA (5,800 cells/mm2) and the DA (3,200 cells/mm2). These cells also exhibited a center-peripheral increase (2,200 cells/mm2 in the TP) in P-1 chicks, but the high-density area was not found in the dorsal retina of P-8 chicks. From these data and the HRP study, the number of presumptive ganglion cells in P-8 chicks was estimated to be 4 x 10(6) (8,600 cells/mm2 on average), and the density in each area was 13,500 (CA), 10,200 (DA), and 4,300 (TP) cells/mm2. The peripheral/ center ratios of the density of ganglion cells were significantly different along the nasotemporal and dorsoventral axes. The density of ganglion cells decreased more rapidly toward the temporal periphery (TP/CA ratio: 0.47 in P-1 and 0.32 in P-8) than toward the nasal periphery (NP/CA ratio: 0.67 in P-1 and 0.52 in P-8). In contrast, there was no significant difference in the peripheral/center ratios between the dorsal retina (DP/CA ratio: 0.6 in P-1 and 0.56 in P-8) and ventral retina (VP/CA ratio: 0.58 in P-1 and 0.51 in P-8). A small peak in the density of the presumptive ganglion cells was detected in the dorsal retina of both P-1 chicks (10,800 cells/mm2) and P-8 chicks (10,200 cells/mm2). The HRP-labeled cells were small in the CA (M +/- SD: 35.7 +/- 9.1 microm2) and DA (40.0 +/- 11.3 microm2), and their sizes increased toward the periphery (63.4 +/- 29.7 microm2 in the TP) accompanied by a decrease in the cell density. However, the axotomy-resistant cells did not significantly increase in size toward the peripheral retina (12.2 +/- 2.2 microm2 in the CA, 15.2 +/- 3.2 microm2 in the DA, 15.1 +/- 3.8 microm2 in the TP). The characteristic distribution of ganglion cells could be related to visual behavior based upon the specialization of avian visual fields.     

Retention of functional characteristics by bovine oviduct and uterine epithelia in vitro

The composition of fluids within the bovine oviduct and uterine lumen, important in fertilisation and early embryonic development, is ultimately determined by the transport properties of the epithelial cells which line the lumen. A preparation has therefore been devised to study the role of these cells in oviduct and uterine fluid formation. Pure preparations of epithelial cells, as judged immunocytochemically, were isolated by enzyme digestion, and grown on collagen filters in primary culture. The cells re-establish intercellular junctions to form a confluent epithelial layer. Serial samples from the apical and basal media were analysed for K+, Na+, Ca2+, glucose and lactate. Bovine oviduct epithelial cells maintained gradients of K+ and Ca2+ (apical > basal) for up to 14 days after confluence, while bovine uterine epithelial cells maintained apical > basal gradients of K+. Both types of epithelium exhibited a small transepithelial electrical potential difference and a higher uptake of glucose and production of lactate in the basal, as opposed to apical medium. There were no consistent differences in any of these parameters with the stage of the oestrous cycle at which the cells were removed. The data indicate that bovine oviduct and uterine epithelia may be isolated and grown as polarised layers in primary culture. The preparations will now enable the mechanisms underlying the secretion of ions and non-electrolytes to be determined.     

Cloning of total mRNA populations from adult and embryonic mice

Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated approximately 400,000 for the 13th day embryo and neonatal mouse and approximately 610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the "G-C tailing" method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by "nick translation" using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back-hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs.     

Replication of semliki forest virus: polyadenylate in plus-strand RNA and polyuridylate in minus-strand RNA

The 42S RNA from Semliki Forest virus contains a polyadenylate [poly(A)] sequence that is 80 to 90 residues long and is the 3'-terminus of the virion RNA. A poly(A) sequence of the same length was found in the plus strand of the replicative forms (RFs) and replicative intermediates (RIs) isolated 2 h after infection. In addition, both RFs and RIs contained a polyuridylate [poly(U)] sequence. No poly(U) was found in virion RNA, and thus the poly(U) sequence is in minus-strand RNA. The poly(U) from RFs was on the average 60 residues long, whereas that isolated from the RIs was 80 residues long. Poly(U) sequences isolated from RFs and RIs by digestion with RNase T1 contained 5'-phosphorylated pUp and ppUp residues, indicating that the poly(U) sequence was the 5'-terminus of the minus-strand RNA. The poly(U) sequence in RFs or RIs was free to bind to poly(A)-Sepharose only after denaturation of the RNAs, indicating that the poly(U) was hydrogen bonded to the poly(A) at the 3'-terminus of the plus-strand RNA in these molecules. When treated with 0.02 mug of RNase A per ml, both RFs and RIs yielded the same distribution of the three cores, RFI, RFII, and RFIII. The minus-strand RNA of both RFI and RFIII contained a poly(U) sequence. That from RFII did not. It is known that RFI is the double-stranded form of the 42S plus-strand RNA and that RFIII is the experimetnally derived double-stranded form of 26S mRNA. The poly(A) sequences in each are most likely transcribed directly from the poly(U) at the 5'-end of the 42S minus-strand RNA. The 26S mRNA thus represents the nucleotide sequence in that one-third of the 42S plus-strand RNA that includes its 3'-terminus.