Secondary structure of ovalbumin messenger RNA.

The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.     

Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” seque?ce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.     

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M_r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA.The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes.The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.     

Purification and translation of ovalbumin,conalbumin, ovomucoid and lysozyme messenger RNA

Messenger RNAs for 4 egg white proteins (ovalbumin, conalbumin, ovomucoid and lysozyme) were assayed in a cell-free, protein-synthesizing system derived from rabbit reticulocytes. Each of these messengers was purified about 33-fold from hen oviduct polysomal RNA using oligo(dT) cellulose. The apparent minimal translational efficiencies varied from 22 translations for each ovalbumin mRNA to less than 1 for lysozyme mRNA. These messengers are not polycistronic with each other since they have distinct sedimentation values of: lysozyme, 8.5S; ovomucoid, 11S; ovalbumin, 15S; and conalbumin 18S. Several aspects of this system indicate that it will be valuable for dissecting the fine control of mRNA metabolism.     

Non-parallel kinetics and the role of tissue-specific factors in the secretion of chicken ovalbumin and lysozyme from Xenopus oocytes

Ovalbumin and lysozyme made in Xenopus oocytes under the direction of injected chicken oviduct messenger RNA accumulate at different rates in the surrounding culture medium. Pulse-chase experiments confirm that the intrinsic rate of lysozyme secretion from oocytes is 12 times that of ovalbumin. This slower rate of ovalbumin export is maintained following injection of either diluted oviduct RNA or purified ovalbumin messenger, the latter having been obtained by hybridization to cloned ovalbumin complementary DNA. These results suggest that the differential rates of transport observed in oocytes are not the consequence of competition for amphibian or avian factors and show that oviduct-specific proteins are not required for ovalbumin secretion.     

Organization of coding and intervening sequences in the chicken ovalbumin split gene

The interruptions in the chicken ovalbumin gene which were reported previously (Breathnach, Mandel and Chambon, 1977) are shown to be due to the presence of intervening sequences which separate the messenger-coding sequences. We present evidence for an additional interruption of the gene, which, together with those reported earlier and by Garapin et al. (1978b), make a total of six intervening sequences. All of these intervening sequences are located in the DNA region that corresponds to the part of the ov mRNA which codes for amino acids. The seven coding fragments of the split ovalbumin gene are arranged in the same order and relative orientation as in the ovalbumin double-stranded cDNA. All the sequences coding for ov mRNA are contained in a chromosomal DNA region of 6000 bp, which is more than 3 times longer than ov mRNA. The general organization of the ovalbumin split gene is discussed.     

Partial purification of the ovalbumin gene

Cellulose-bound DNA complementary to ovalbumin mRNA was used in a continuous hybridization system to isolate single-stranded DNA molecules containing the ovalbumin gene. Fragmented DNA segments containing the ovalbumin gene were enriched 300–350 fold in one cycle of purification. Two cycles of purification resulted in a DNA fraction which was enriched 2300 fold in the ovalbumin sequence. The method is suitable for purification of the ovalbumin sequence from both sheared DNA fragments, as well as larger molecular weight DNA containing more than twice the number of nucleotides necessary to code for ovalbumin mRNA. The chromatographic procedures were specific and reproducible. In addition, the recovery of ovalbumin DNA was essentially quantitative (80–100%), even when large amounts of starting DNA (70–75 mg) were used. This purification scheme should also be useful for the enrichment of other unique sequence genes from eucaryotic DNA.     

The 5'-end structure of ovalbumin mRNA in isolated nuclei and polysomes.

We used the chemical reagents dimethylsulfate and 4'-aminomethyl-4,5',8-trimethylpsoralen and the enzyme T1 ribonuclease to compare the 5'-end structure of ovalbumin mRNA in situ in purified hen oviduct nuclei and polysomes with that of the isolated mRNA. The qualitative pattern of structure-dependent base modifications and T1 ribonuclease cleavage sites in intranuclear and polysomal ovalbumin mRNAs was found to be nearly identical to those in isolated ovalbumin mRNA. These structural data are consistent with the presence of a trigonal stem-loop structure at the 5'-end of ovalbumin mRNA (hairpin-1) in nuclei and polysomes. Similar results were obtained for a coding region structure (hairpin-3) in intranuclear ovalbumin mRNA. We have recently shown that hairpin-1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eucaryotic initiation factor-2 (eIF-2). The presence of hairpin-1 in ovalbumin mRNA in both a pretranslation state (nuclei) and active translation state (polysomes) is consistent with its hypothesized biological function as an intracellular initiation signal that facilitates the translation of this mRNA.     

Effect of estrogen on ovalbumin gene expression in differentiated nontarget tissues

By use of cloned DNA fragments as probes, low levels of ovalbumin RNA sequences (structural and intervening sequences) were detected in nuclear RNA extracts of nontarget tissues, such as liver, spleen, brain, and heart of chicks. The expression of the ovalbumin gene sequences was hormone dependent. In estrogen-stimulated chicks, a low level of ovalbumin RNA sequences, ranging from 0.2 to 0.7 molecule per cell, was present in nontarget tissues while less than 0.01 molecule per cell could be found in the same tissues of unstimulated chicks. A significant amount of the ovalbumin mRNA sequences was also found in polysomes of liver and brain. The ovalbumin mRNA sequences could be translated into proteins which were only localized in a few cells among the entire population of liver cells as determined by an immunocytochemical assay. These results suggest that there are some cells in liver, spleen, heart, and brain which can respond to hormone stimulation and produce ovalbumin mRNA and its translational product.     

Cytochalasin B selectively releases ovalbumin mRNA precursors but not the mature ovalbumin mRNA from hen oviduct nuclear matrix

Hen oviduct nuclear matrix-bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP-dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP, but inhibited the ATP-induced detachment of mature mRNA. RNA release was also achieved with a monoclonal antibody against actin but not with monoclonal antibodies against tubulin and intermediate filaments. These results suggest that actin-containing filaments are involved in the restriction of immature messengers to the cell nucleus.     

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.     

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.