Ovalbumin RNA Research Articles

Gene expression profile analysis in experimental asthma

Nives Zimmermann, Elizabeth A Moulton, Bruce J Aronow, Emily E Muntel, ME Rothenberg Children's Hospital of Cincinnati, Cincinnati, OH Asthma is a complex chronic inflammatory disorder that involves coordinated actions of multiple pathways. In order to elucidate the interplay of these pathways, we studied gene expression profiles in a murine model of asthma using DNA microarray analysis with an Affymetrix gene chip. Sets of mice were sensitized and challenged with ovalbumin and lung RNA was hybridized to the microarray. Affymetrix and Genespring software programs were used to analyze genes that significantly changed in response to allergen. Overall, more genes had increased expression rather than decreased expression; however, only a select number of the total genes significantly changed. Analysis of 12,423 genes revealed a two-fold or greater change in only 3.41% of the total genes; 2.74% of genes increased and 0.67% decreased. Functional classification of differentially expressed genes showed immune-related genes predominantly in the increased group (39%) compared to the decreased group (7%). In contrast, genes involved in development and homeostasis composed the majority of decreased genes (59%), while only comprising 18% of the increased genes. Many of the dysregulated genes were already known to have a role in allergic responses (e.g. ehemokines), thus validating the experimental analysis. Interestingly, genes involved in pathways not previously implicated in allergic diseases, as well as unknown genes, were also significantly upregulated. Northern blot analysis was performed with representative genes identified as allergen-induced by microarray analysis (e.g. eotaxin-1, Adam-8, Cdc-2a and Bcl-3) and provided further verification of the utility of the experimental approach. Kinetic analysis was performed in which gene expression was studied at early (3hrs) and late (18hrs) time points following one or two allergen challenges. Distinct sets of genes were dysregulated at the different time points and there was only partial overlap of the identified genes. In summary, wide spectrum genomic analysis has identified a discrete set of genes involved in experimental asthma. Interestingly, the majority have not been previously implicated in asthma, providing important insight into the pathophysiology of allergic airway inflammation.

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.

Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.

Hybrid selection with cDNA-silica

A partial length ovalbumin cDNA-silica was produced using primer extension of (dT)18-silica with annealed partial ovalbumin RNA and reverse transcriptase. This cDNA-silica was used to test whether full-length ovalbumin RNA could be selectively purified in the presence of a large excess of other (mouse muscle) RNA. The cDNA-silica synthesized had minimally 60 pmol cDNA per gram silica and had a capacity for full-length ovalbumin RNA of minimally 38 μg/g. Even when other RNA was present in greater than 1000-fold excess, ovalbumin RNA was selectively retained by the cDNA-silica and was eluted in yields of 43% with an enrichment which varied over the range of 29–162-fold in various experiments. These results show that even rare RNAs can be selectively purified in high yield using cDNA-silica. The importance of these results to hybrid selection and subtractive library preparation is discussed.

Differential polyadenylation pattern of ovalbumin precursor RNAs during development.

The expression of the ovalbumin gene encoding for the major hen oviduct protein slows down with age. Analysis of Northern blots of electrophoretically separated total and poly(A) + RNA from oviducts of hens of different age with an ovalbumin-specific probe (nick-translated 9.5 kb ovalbumin gene DNA cloned into pBR322) revealed that the largest high molecular weight ovalbumin RNA precursor (7.9 kb band, representing the putative primary transcript of the ovalbumin gene) was most intense if total RNA from non-egg-laying old hen oviduct was checked as compared to that from egg-laying mature animals. On the other side, the 7.9 kb RNA precursor band was readily detected in the poly(A) + RNA from mature hen oviduct whereas it was invisible in the old hen oviduct poly(A) + RNA fraction. The lack of detection of the 7.9 kb RNA species within the poly(A) + RNA fraction and its increased concentration within the total oviduct RNA, both from old animals, suggest that age-dependent impairment of ovalbumin mRNA processing may be caused by altered polyadenylation of distinct RNA precursors.

Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription.

The role of the eucaryotic T-A-T-A box (Hogness box) homology sequence located approximately 30 base pairs upstream from the RNA initiation site has been further examined by oligodeoxynucleotide-directed site-specific mutagenesis. A method was employed which allows insertion of nucleotide-specific mutations into virtually any double-stranded, recombinant plasmid DNA. A synthetic mixed oligonucleotide, bearing defined multiple nucleotide substitutions at a single site, was used both as a specific mutagen during primed DNA repair synthesis in vitro, as well as a highly sensitive hybridization probe for the identification of the mutated cloned DNA. Using this methodology, an A leads to G transition mutation was introduced into the second position of the ovalbumin gene T-[A]-T-A box, and the effect of modifying this highly conserved nucleotide on the expression of the mutant DNA was analyzed in a cell-free transcription system. Comparison of two allelic ovalbumin genes, slightly divergent upstream from the TATA box, resulted in identical in vitro transcription efficiencies. While correct initiation of ovalbumin RNA transcripts was not affected, the efficiency of gene expression using the mutant template, compared to either the corresponding wild-type sequence or the allelic gene, was markedly reduced. These results suggest that it is the T-A-T-A box sequence which plays a role in the efficient initiation of RNA transcription in vitro and further supports the implication that this region may serve a promoter-related function in eucaryotic transcription.

Transcription factors from oviduct and HeLa cells are similar.

Total HeLa cell extract was separated into multiple components using first DEAE-Sephadex and then phosphocellulose column chromatography. Four major fractions, DE175, DE500, P100, and P1000, from HeLa cells are found to be essential for accurate and efficient transcription of cloned ovalbumin DNA fragments in the reconstituted system. DE175 serves as the source for RNA polymerase II and DE500 minimizes the synthesis of small molecular size RNA. P100 enhances the levels of specific transcription, while P1000 is absolutely required for correct initiation. Chick oviduct crude extract was also fractionated into multiple components according to the same procedure. Similar efficiency of specific initiation could be obtained when an individual fraction (DE175, DE500, P100, or P1000) from oviduct cells was exchanged for a fraction from HeLa cells. These results indicate that chick oviduct tissue also contains general transcription factors like that of HeLa cells and these factors can be fractionated according to the same procedure. In this fractionation scheme, we were able to separate the bulk of RNase activity into the P350 fraction which was not required for initiation of ovalbumin RNA transcription. Thus, this reconstituted system is suitable for studying in vitro transcription in a homologous system derived from tissue extracts, even though a substantial amount of cellular ribonuclease may be associated with it.

Processing of high molecular weight ovalbumin and ovomucoid precursor RNAs to messenger RNA

The existence of poly(A) sequences in the multiple high molecular weight forms of ovalbumin and ovomucoid nuclear RNA has been determined. The results indicate that all the bands observed in the total nuclear RNA including the largest (7.8 kb) were also detected in the poly(A) RNA. Kinetic labeling and chase experiments in oviduct tissue-suspension system indicated that the ovalbumin and ovomucoid high molecular weight RNAs which were labeled with a short-time incubation can be chased by cold nucleosides and actinomycin D into mature mRNAs. The largest RNAs labeled in this oviduct suspension system have a size of 7.8 kb for ovalbumin and 5.5 kb for ovomucoid, which correspond respectively to the “a” bands of steady state RNA. The processing of the precursors to mature mRNA was also examined by electron microscopic analysis, by hybridizing oviduct nuclear RNA to probes which were isolated from different segments of intervening DNA sequences and by measuring the turnover kinetics of specific intervening sequences present in cellular poly(A) RNA. The results indicate that the seven intervening sequence regions of the ovomucoid precursor are removed in a preferred but not necessarily an obligatory order.

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. coli RNA polymerase to transcribe specifically the conalbumin and ovalbumin genes 8 1/2 h after hormone administration when transcription of these genes by endogenous RNA polymerase was elevated 5- and 30-fold, respectively. Furthermore, we were unable to detect any significant effect of either of these hormones on the total number of E. coli RNA polymerase binding sites in oviduct nuclei or chromatin. In contrast, after several days of hormone administration, we detected an apparent preferential ovalbumin RNA synthesis by E. coli RNA polymerase and this effect could be transferred to unstimulated nuclei by a 0.35 M salt extract of active nuclei. However, further experiments revealed that this preferential ovalbumin RNA synthesis is an artifact produced by transcription from contaminating ovalbumin mRNA. We conclude that E. coli RNA polymerase does not recognize steroid hormone-induced changes in oviduct chromatin.

Levels of ovalbumin messenger RNA sequences in nonoviduct tissues of the chicken

The levels of ovalbumin mRNA sequence in nonoviduct tissues of the chicken were examined in nuclear and cytoplasmic fractions of hen liver, spleen, estrogen-stimulated chick liver, and 10-day-old embryo using RNA-cDNA hybridization. In general, the tissues studied revealed little or no detectable ovalbumin RNA, at the limit of detection of some 10 −5% of total RNA. The results suggest that the ovalbumin gene is either transcriptionally inactive or transcribed only very infrequently in nonoviduct tissues, and that the different target tissue responses of liver and oviduct to estrogen stimulation resides largely in the control of transcription.

Definition of the 5′ and 3′ ends of transcripts of the ovalbumin gene

We present data which map the 5′ and 3′ ends of transcripts of the natural ovalbumin gene. First, hybridization of radiolabeled 5′ and 3′ fragments of the gene to oviduct nuclear RNA which had been electrophoresed in agarose gels and transferred to DBM paper localized the 5′ and 3′ ends of precursor molecules and demonstrated that the largest ovalbumin RNA molecules detectable by this method (∼7.8 kb) were similar in size to the ovalbumin natural gene. Second, hybridization of end-labeled probes to oviduct nuclear RNA followed by digestion with S1 nuclease and analysis on polyacrylamide gels mapped more precisely the 5′ and 3′ ends of the precursor molecules, and these termini were coincident with the beginning and end of the structural sequence of the natural gene. These results suggest that the 7.8 kb precursor is likely to be the primary transcript of the ovalbumin gene.

Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene

The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [(3)H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [(3)H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.

Identification of potential ovomucoid mRNA precursors in chick oviduct nuclei

Denaturing gel electrophoresis of chick oviduct nuclear RNA reveals multiple species of RNA that are 1.5-5 times larger than ovomucoid mRNA. By analogy with ovalbumin RNA processing, these data suggest that ovomucoid mRNA is derived from a primary transcript that contains intervening sequences.

Transcription of a cloned ovalbumin ds-cDNA in [formula omitted] oocytes

A recombinant plasmid containing a double-stranded DNA copy of the ovalbumin messenger RNA (pOV230 DNA) was successfully transcribed by injecting the purified DNA into Xenopus laevis oocyte nuclei. Injection of pOV230 DNA resulted in the synthesis of plasmid sequences and ovalbumin messenger RNA sequences while injection of plasmid DNA resulted in plasmid RNA transcription only. 21–38% of the total [ 3H]RNA isolated from pOV230 DNA injected oocytes was capable of hybridizing to pOV230 DNA. Strand selectivity experiments revealed that 80–100% of the ovalbumin RNA synthesized was coding strand RNA.

The ovalbumin gene: transcriptional regulation by estrogen.

De novo synthesis of RNA sequences corresponding to intervening as well as to structural sequences of the ovalbumin gene have been detected in isolated oviduct nuclei. Their presence in the nuclear transcripts and their time course of induction support the hypothesis that transcription of structural and intervening sequences of the natural ovalbumin gene are regulated by steroid hormones. These results are in agreement with out previous demonstration of high-molecular-weight species of ovalbumin RNA in nuclei that contain structural as well as intervening RNA sequences and are thus likely precursors to mature cytoplasmic mRNAov. Analysis of the size of in vivo nuclear RNA by gel electrophoresis under denaturing conditions, revealed that withdrawal of hormone depletes the level of high molecular weight ovalbumin RNA as well as that of nature mRNAov and that readministration of estrogen induces the accumulation of both species. These results are consistent also with transcriptional regulation of the ovalbumin gene. In addition, they rule out the possibility that the rapid accumulation of mature mRNAov after secondard stimulation results from processing of ovalbumin RNA precursors that might have been stored in the withdrawn oviduct. We conclude that steroid hormones exert a primary effect at the level of gene transcription. Following this event, a series of coordinated cellular responses may occur which involve RNA processing, mRNA transport to the cytoplasm, protein synthesis and mRNA degradation. The final consequence of this network of molecular reactions is the induced cellular function inherent to a specific steroid hormone.

No more than seven interruptions in the ovalbumin gene: comparison of genomic and double-stranded cDNA sequences.

We have determined the sequence of ovalbumin RNA (ov-mRNA) using a double-stranded cDNA (dscDNA) plasmid. We have also determined the sequence of the previously characterized exonic regions of the chicken ovalbumin gene. The comparison of these various sequences has shown that there are no additional interruptions in the mRNA-coding sequences above those 7 already characterized. There is only one single base discrepancy between the two mRNA sequences determined using the dscDNA or the genomic clones. This demonstrates the accuracy and reproducibility of the cloning and sequencing techniques. The ovalbumin mRNA sequence was found to be 1872 nucleotides in length, 13 nucleotides larger than the previous value reported by McReynolds et al. [Nature 273, 723-728 (1978)].

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydryl-agarose and hybridized to radioactive ovalbumin cDNA. (ii) [3H]UTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of alpha-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vitro RNA synthesis is retained in isolated nuclei.

Transcription of the chicken ovalbumin and conalbumin gene during early secondary induction with estrogens.

Estrogens stimulate the rate of transcription of the ovalbumin and conalbumin gene in the chicken oviduct. The synthesis of ovalbumin and conalbumin mRNA was studied in isolated nuclei. RNA synthesized in vitro was distinguished from preexisting nuclear RNA by affinity labeling the in vitro products with a mercurated nucleotide and subsequent purification of the Hg-RNA on SH-agarose. The content of ovalbumin and conalbumin mRNA sequences in the in vitro transcripts was determined by hybridization to cDNA. After the withdrawal of implanted hormones from chickens, the synthesis of conalbumin and ovalbumin RNA increased 2.5- and at least 20-fold, respectively, by treatment with estrogens. The maximal rate of transcription of the conalbumin gene is achieved within 2 h after estrogen induction, whereas the rate of transcription of the ovalbumin gene becomes maximal after a lag of several hours. These results demonstrate that estrogens affect two genes in the same target cell differently.

Effect of estrogen on gene expression in the chick oviduct.

Mercurated UTP was used as a substrate for RNA polymerases in the in vitro transcription of chromatin so that newly synthesized RNA could be efficiently separated from endogenous chromatin RNA by means of sulfhydryl-Sepharose affinity chromatography. Utilizing this technique, it was possible to examine the effect of varying enzyme to DNA ratios on the transcription of specific genes from chromatin. For both Escherichia coli RNA polymerase and wheat germ RNA polymerase II, lowering the enzyme to DNA ration resulted in an increase in the percentage of ovalbumin mRNA sequences transcribed from chick oviduct chromatin. Similar results were also obtained for the transcription of the globin gene from chick reticulocyte chromatin. On the other hand, transcription of the globin gene from oviduct chromatin or the ovalbumin gene from reticulocyte chromatin or deproteinized chick DNA was not significantly affected by varying enzyme to DNA ratios. These results indicate that preferential transcription of certain chromatin genes relative to total RNA synthesis can occur and that this process is dependent on the presence of chromosomal proteins. Utilizing a cDNA probe complementary to the anticoding strand of the ovalbumin gene, the degree of asymmetry of the in vitro transcription of this gene was also examined. The percentage of ovalbumin RNA sequences homologous to the anticoding strand was not significantly affected when the enzyme to DNA ratio was lowered 16-fold. Since the percentage of coding ovalbumin mRNA sequences increased more than 6-fold over the same range, the percentage of asymmetric transcription of this gene increased. At the lowest enzyme to DNA ratio tested, the transcription of the ovalbumin gene from oviduct chromatin was almost totally asymmetric and, thus, closely resembled the pattern of gene transcription characteristic of the in vivo state.

Ovalbumin Messenger Ribonucleic Acid Translation: COMPARABLE RATES OF POLYPEPTIDE INITIATION AND ELONGATION ON OVALBUMIN AND GLOBIN MESSENGER RIBONUCLEIC ACID IN A RABBIT RETICULOCYTE LYSATE

Abstract Optimum conditions for translating ovalbumin messenger RNA in a cell-free, protein-synthesizing system derived from rabbit reticulocytes were similar to those for endogenous globin messenger RNA. At suboptimal conditions the translation of ovalbumin messenger RNA was usually inhibited more than globin messenger RNA. Hemin prolonged the synthesis of both proteins, and Q, an inhibitor of initiation formed during incubation, inhibited the translation of both messengers. The rates of elongation on ovalbumin and globin messenger RNA were nearly identical at 0.86 amino acid per s at 26°, and were maintained for at least an hour. After 10 min of incubation, ovalbumin was synthesized on polysomes composed of 9 ribosomes (75% of the maximum size possible), corresponding to a rate of initiation of about 1.1 ribosomes per min at 26°. The size of ovalbumin-synthesizing polysomes gradually fell to 3 during a 70-min incubation. The rate of initiation on globin messenger RNA was about 30% faster than on ovalbumin messenger RNA, but there was a corresponding decrease in this rate during incubation. Ovalbumin messenger RNA was not degraded in the proteinsynthesizing system. These results suggest that protein synthesis stops in this system due to a failure of polypeptide initiation. The efficiency of messenger RNA translation was estimated by two independent methods. Both methods indicate that each globin messenger RNA was translated about 70 times during a 90-min incubation. Each ovalbumin messenger RNA was translated about 45 times according to one method, but only up to 18 times by the other. The ratio of the values obtained by these two methods is a means of estimating the percentage of active messenger in an RNA preparation.