Chorion mRNA Research Articles

Early establishment and autonomous implementation of a developmental program controlling silkmoth chorion gene expression

To address the question of whether prechoriogenic follicles of the silkmoth Bombyx mori have the capacity to enter choriogenesis in organ culture and define the stage at which choriogenesis becomes established as a follicle-autonomous program, we have cultured immature ovarioles dissected from developing pupae and examined the protein synthetic profiles of follicular cells of individual follicles at the end of the culture period. The protein synthetic profiles of the cultured follicles were also correlated with corresponding profiles of chorion mRNA accumulation. Our results demonstrate that the last 17 (±2) vitellogenic follicles of Day 5 to 7 pupae are capable of initiating choriogenesis in organ culture. The earliest vitellogenic stage to enter choriogenesis in vitro does so after 34 (±4) hr in culture and follicles entering choriogenesis in vitro are capable of proceeding through all choriogenic stages at a speed comparable to that occurring in vivo. Therefore, once the choriogenic program becomes established in follicular cells, it can be implemented autonomously in the absence of extrafollicular factors. Earlier vitellogenic stages lack this capacity, presumably because they require additional hemolymph factors to establish the choriogenic potential. Our results demonstrate that the choriogenic potential of cultured vitellogenic follicles cannot be influenced by addition of 20-hydroxyecdysone to the culture medium.

EM analysis of Drosophila chorion genes: Amplification, transcription termination and RNA splicing

We have used the electron microscope to examine ultrastructurally several events occurring during the biogenesis of two very abundant chorion (eggshell) mRNA molecules in the follicle cells of Drosophila melanogaster--namely, selective gene amplification, transcription initiation and termination, and RNA processing. We find that the highly transcribed s36 and s38 genes are positioned in the central region of large, multi-forked amplified DNA structures. Transcript morphology is consistent with the known presence of a small intron at the 5' end of each gene. Mature transcripts are associated with spliceosomes, demonstrating that splice site selection occurs co-transcriptionally but that splicing is completed after transcript release from the template. We have also mapped the termination sites for the genes. The two genes exhibit efficient termination very near their poly(A) sites--within a 210 bp region for s36 and a 360 bp region for s38.

Silkmoth chorion antisense RNA: Structural characterization, developmental regulation and evolutionary conservation

Choriogenic follicular cells of the silkmoth Bombyx mori contain significant quantities of antisense RNA transcribed from chorion genes. Antisense RNA derived from a chorion gene with a high content of cysteine, HcB.12, was characterized in detail. The antisense transcripts are initiated downstream from the 3' end of HcB.12 mRNA and extend over 75% of the length of the gene, comprising its entire second exon and part of its intervening sequence. The antisense RNA is devoid of any significant open reading frames and is not polyadenylated. These features, combined with the presence of specific sequence motifs within its transcribed and upstream region, suggest that antisense RNA may be transcribed by RNA polymerase III. Chorion antisense RNA is detectable only in choriogenic follicular cells and appears to be co-ordinately regulated with chorion mRNA. Its cytoplasmic accumulation during choriogenesis parallels that of the corresponding mRNA. Although chorion mRNA is at least five times more abundant than antisense RNA, the latter is present as a single-stranded entity in follicular cytoplasm but can form perfect duplexes with its mRNA complement upon annealing in vitro. The possible involvement of antisense RNA transcription in the pathway that controls the programmed expression of chorion genes at the level of transcription initiation or post-transcriptional processing is discussed.

Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection.

Recombinant baculoviruses as vectors for identifying proteins encoded by intron-containing members of complex multigene families.

Using a transfer vector derived from Bombyx mori nuclear polyhedrosis virus (BmNPV), we have constructed recombinant baculoviruses that contain complete silk moth chorion chromosomal genes encoding high-cysteine proteins under the control of the polyhedrin promoter. Silk moth tissue culture cells infected with these recombinant viruses were found to contain abundant RNA sequences of sizes similar to those of the authentic chorion mRNAs. Chorion transcripts present in infected cells were initiated almost exclusively at the cap site of the polyhedrin start site. Primer extension and RNase protection experiments revealed that a considerable proportion of the resultant transcripts were spliced at the same sites as those utilized in follicular cells for the production of functional chorion mRNA. Electrophoretic analysis and immunoprecipitation of the proteins of host cells infected with the recombinant viruses revealed the presence of the corresponding chorion proteins. We conclude that baculovirus vectors can be used for expressing efficiently not only cDNAs or simple genes devoid of intervening sequences but also intron-containing chromosomal genes. Thus, recombinant baculoviruses offer a powerful alternative to hybrid-selected translation, particularly when the identification of proteins encoded by members of complex multigene families is required.

Patterns of region-specific chorion gene expression in the silkmoth and identification of shared 5′ flanking genomic elements

Time-dependent and cell-specific changes in concentrations of total RNA, of poly A+ RNA, and of specific mRNAs have been measured throughout silkmoth choriogenesis. Levels of total RNA and of poly A+ RNA are maintained throughout much of choriogenesis, but decrease at least fourfold during the very late period, in parallel with a decrease in overall protein synthesis. Very late period changes in total RNA and in poly A+ RNA are less pronounced in the aeropyle crown region, where a subset of chorion proteins is preferentially synthesized, than in the flat region. Maximal accumulation of the E1 and E2 chorion mRNAs occurs preferentially in the aeropyle crown region during the very late period. Uridine pulse-labeling studies suggest that E1 and E2 transcription is similarly aeropyle crown region-specific and immediately precedes the time of maximal E1 and E2 RNA accumulation and protein synthesis. Sequences from the 5′ flanking regions of E1 and E2 genes have been compared. Several oligonucleotide sequences are present in both genes, and some are duplicated. These are potential cis-acting, regulatory elements.

Developmentally regulated expression of Drosophila chorion genes introduced at diverse chromosomal positions

Drosophila chorion genes are organized into two clusters that are selectively amplified in the ovarian follicle cells. During oogenesis the transcription of individual genes is temporally regulated, resulting in distinct, stage-specific profiles of chorion mRNA accumulation. P element-mediated gene transfer was used to study the regulation of genes encoding the major chorion proteins s15-1 and s38-1. Transformed chorion genes integrated at diverse chromosomal locations exhibited proper tissue-specific and stage-specific expression, despite separation from the gene clusters. Qualitatively normal expression was not dependent on the ability of the inserted DNA to undergo amplification. However, chromosome position quantitatively influenced the RNA produced by the transformed genes. The level of RNA per gene copy produced by individual transformed genes varied approximately tenfold, after correction for differences in gene dosage due to the amplification of some inserted sequences. Transformation experiments with an s38-1-lacZ fusion gene demonstrated that cis-regulatory sequences sufficient for the stage-specific program of s38-1 expression were confined to a 1.3 × 10 3 base-pair segment between −748 and +573 relative to the s38-1 initiation site. Finally, egg chamber-specific amplification was induced at the site of two s38-1 insertions, suggesting that an amplification control element resides near this gene.

Mutants suppressing in trans chorion gene amplification in Drosophila.

Two recessive female-sterile mutants, K451 and K1214 , disrupt chorion formation by causing underproduction of all major chorion proteins. We present evidence that this effect is due to underaccumulation of the chorion mRNAs and that, in turn, this is caused by a substantial reduction in the level of chorion gene amplification. The mutants are X-linked but located at two sites far from the chorion gene cluster at 7F1 -2; their effect is even more pronounced on the third chromosome chorion gene cluster, and thus the wild type gene must act in trans. The time course of amplification in mutant and wild-type follicles is documented.

Differential patterns in the temporal experession ofBombyx mori chorion genes.

cDNA clones for two distinct families ofBombyx mori chorion protiens, Class A (m2774) and Class Hc (m5000), were used to study the developmental regulation of specific chorion mRNAs. Individual follicles from maturing ovarioles were assayed by Northern blotting techniques and amino acid pulse-labeling to determine concomitant RNA accumulation and protein synthesis patterns with a temporal resolution of 2.2-2.6 h of developmental age. RNAs hybridizing to the two cloned chorion sequences at high criterion showed distinctive, non-overlapping patterns of accumulation during the middle-late and very late stages of choriogenesis. Moreover, the periods of expression of these RNAs coincided exactly with the synthetic periods of two distinct subsets of chorion proteins, one containing Class A and B components, and the other containing Class Hc components alone. These results suggest that chorion gene transcription and translation are tightly coupled.

Regulatory effects of purine and pyrimidine nucleoside 5'-di(tri)-phosphate-3'-diphosphates on eucaryote protein synthesis.

Adenosine-, guanosine-, cytidine- and uridine-5'-di(tri)-phosphate-3'-diphosphates were enzymatically prepared by the use of Streptomyces adephospholyticus ATP nucleotide pyrophosphokinase (E.C. 2.7.6.4). Their regulatory effects were then investigated on translation of rat liver, rabbit globin and silkworm pupae chorion mRNAsby the wheat germ lysate system in vitro. They were found to exert base-specific and mRNA species-specific stimulatory or inhibitory effects, respectively. In particular, cytidine 5'-diphosphate-3'-diphosphate stimulated chorion mRNA translation 3-fold. The significance and limitations of the findings were discussed in relation to natural occurrence and possible regulatory functions of the nucleotides in eucaryotes.

Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.

Molecular analysis of the Gr B mutation in Bombyx mori through the use of a chorion cDNA library

To study the molecular basis of the Gr B mutation, which prevents the synthesis of many stage-specific chorion proteins, a cDNA library has been constructed from wild-type chorion mRNA of Bombyx mori strain 703. By differential screening of the library with +/+ and B/B mRNAs, under appropriately stringent conditions to minimize cross hybridizations of related chorion sequences, we have selected several distinct clones corresponding to RNA sequences which are affected by the mutation (that is, are represented only in +/+ mRNA) or are unaffected (that is, are represented in both +/+ and B/B mRNAs). We show by Southern analysis that, whereas unaffected gene sequences are represented in both +/+ and B/B chromosomal DNA, affected sequences have been deleted from B/B DNA. The organization and regulation of developmental stage-specific chorion genes are discussed in light of these findings and the known effects of Gr B on stage-specific protein synthesis.

Coordinately expressed members of two chorion multi-gene families are clustered, alternating and divergently orientated.

The chorion (eggshell) of silkmoths consists of more than 100 distinct proteins. Individual components are synthesised in characteristic and overlapping sequence, at early, middle, late or very late developmental periods of choriogenesis (which lasts 2 days in total)1,2. Protein sequencing3,4 indicates that components which belong to two size classes, A and B, are encoded by two respective families of evolutionarily homologous genes (multi-gene families). Genetic analysis in Bombyx mori5,6 has shown that chorion genes are tightly linked in three clusters on a single chromosome (n = 28). Total chorion mRNA of the wild silkworm, Antheraea polyphemus, has been copied into double-stranded cDNA and cloned by recombinant DNA procedures7. Many distinct cDNA clones have been selected and identified as members of the A and B multi-gene families by detailed cross-hybridisation7 and sequencing8 analysis. Stringent conditions which minimise cross-hybridisation permit use of these clones as specific probes. In many cases, we know the protein encoded and the developmental period when the clone sequence can be found in cytoplasmic RNA7,8. Thus, we may now use these cDNA clones as probes to determine how genes of known developmental properties and evolutionary history are arranged within the chorion chromosomal region. We report here that two cloned, 14-kilobase chromosomal DNA segments contain multiple chorion genes. In each segment, the genes represent both A and B families, but only one developmental class: one segment contains only middle-period A and B genes and the other only late-period A and B genes. In both segments, members of the two families are arranged in alternating sequence and divergent orientation.

Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The cross-hybridization analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stagespecific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families.

Antigody-nucleic acid complexes. Inhibition of translation of silkmoth chorion messenger ribonucleic acid with antibodies specific for 7-methylguanosine.

Antibodies specific for 7-methylguanosine (m7G) were evaluated for their ability to inhibit the translation of chorion mRNA in a wheat germ, cell-free amino acid incorporating system. Results obtained with antibody concentrations of 0.5--1.5 microM revealed dose-dependent inhibition of [3H]-labeled amino acid incorporation into acid-insoluble radioactivity. Inhibition of translation was attributed to the interaction of anti-m7G antibodies with the 5' termini of chorion mRNAs on the basis that (a) anti-m7G antibodies coupled to Sepharose (anti-m7G-Sepharose) immunospecifically retained 5'-terminal cap structures of chorion mRNAs, i.e., m7G (5')ppp(5')Nm, (b) significant inhibition of translation required a 2-h preincubation of anti-m7G antibodies with mRNA, and (c) similar preincubation periods with anti-m7G antibodies in the presence of the competing nucleoside hapten (m7G) obviated the inhibitory effect of the antibody. The nature of the anti-m7G antibody-mRNA complex was examined by digesting chorion mRNA with nuclease P1 before (predigested) and after (postdigested) immunospecific adsorption to anti-m7G-Sepharose adsorbent. Whereas predigested preparations yielded a single cap structure of the type m7G(5')ppp(5')N, the predominating cap in the postdigested sample was m7G(5')ppp(5')NpNpN. These latter data revealed that the nucleotide sequence adjacent to the cap was not significantly masked by the antibody and suggest the utility of anti-m7G antibody as a site-specific probe.

Drosophila bearing the ocelliless mutation underproduce two major chorion proteins both of which map near this gene

Two bands of putative Drosophila chorion mRNA, E3 and E4, have been shown to hybridize in situ near 7E11 ( Spradling and Mahowald, 1979) within a region known to contain a gene, ocelliless, which may be involved in chorion production ( Johnson and King, 1974). We have investigated the synthesis of “chorion mRNAs” and chorion proteins in flies carrying this mutation. A reduction of labeling of both E3 and E4 was observed in stage 12 egg chambers from homozygous ocelliless females. In addition, they produce mature oocytes which contain greatly reduced amounts of several major chorion proteins, including those normally produced in stage 12, c36 and c38. To investigate whether the reduction was due to a direct effect of the mutation, the genes for these proteins were mapped. Recombination analysis using electrophoretic variants of c36 and c38 showed that both proteins are coded on the X chromosome at a site between crossveinless and vermillion. Further mapping with the deficiency chromosomes Df(1)KA14 and Df(1)RA2 narrowed the region containing the structural genes to a 16 band region between 7E10 and 8A4. The ocelliless gene, as well as the site of in situ hybridization, are located within this same interval. In normal ovarian follicles, both c36 and c38 are produced in equal amounts and with the same developmental specificity ( Waring and Mahowald, 1979). In the mutant, both are reduced to a similar extent. In oc +/ oc heterozygotes, both the c36 and c38 genes on the mutant chromosome produce much less product than the corresponding genes on the oc + chromosome. The cis-acting nature of the ocelliless mutation suggests that it may disrupt sequences involved in controlling the expression of the structural information for these proteins.

The control of chorion protein synthesis in silkmoths: mRNA production parallels protein synthesis

During a defined developmental period, the follicular cells of silkmoths devote nearly all of their protein synthetic capacity to production of the chorion proteins. In this study we show that synthesis and accumulation of chorion mRNAs as a class correlate with the period of chorion protein synthesis. Prior to the onset of choriogenesis, no newly synthesized mature size 8–10S polyadenylated chorion mRNA is detectable in the cytoplasm, and no translatable chorion mRNA accumulates anywhere in the cell. During choriogenesis, mRNA transcription is sustained. The evidence suggests that mRNA production is an important element in the regulation of choriogenesis, although resolution of the mRNAs is insufficient to test whether the details of choriogenesis are under mRNA control. The synthesis of rRNA in the follicular cells begins much earlier, reaches a maximum with the onset of choriogenesis, and declines precipitously thereafter. Thus, the kinetics of chorion mRNA synthesis and accumulation are unrelated to the rRNA metabolism.

Electrophoretic patterns of deadenylylated chorion and globin mRNAs.

Treatment of silkmoth chorion mRNAs with calf thymus RNase H (EC 3.1.4.34; RNA-DNA-hybrid ribonucleotidohydrolase) in the presence of oligo(dT) specifically and effectively removes the 3'-terminal poly(A) sequences. Excision of non-poly(A) fragments cannot be detected. Under these conditions, RNase H leads to increased electrophoretic homogeneity of rabbit globin mRNA, presumably as a result of removal of poly(A) sequences that are inherently variable in length. Treatment with RNase H converts the three diffuse zones of messages for the several chorion proteins into multiple sharp bands.

Full length and discrete partial reverse transcripts of globin and chorion mRNAs

Rabbit globin mRNA was copied by AMV reverse transcriptase in the presence of various concentrations of deoxyribonucleotides (dNTPs). The cDNAs were analyzed by electrophoresis under denaturing conditions in formamide-polyacrylamide gels. Discrete size products were detected, ranging from 65 to 650 nucleotides—that is, up to the full length of the mRNA template. Increasing the concentrations of all four dNTPs stimulated formation of full-length transcripts and made the incomplete copies less abundant. Hybridization and nuclease digestion experiments indicated that the full-size product is indeed a complete transcript of globin mRNA. Similar results were obtained with chorion mRNAs. The possible usefulness of the discrete partial transcripts is discussed.

Purification of a family of specific messenger ribonucleic acids from moth follicular cells.

When moth follicular cells synthesize the characteristic, low-molecular-weight chorion (eggshell) proteins, they contain at least two groups of 8-9S RNAs which appear to be chorion mRNA. This class of RNA yields a characteristic radiolabeled profile on polyacrylamide slab gels. The same profile is obtained irrespective of whether lebeling is performed in vivo or in organ culture, and whether the RNAs are purified from whole cells or specifically released from polysomes. Polysomes contain putative chorion mRNA only when the cells actually synthesize chorion protein. The RNA size is correlated with the size of the chorion proteins: two month species differing in the size distribution of their chorion proteins show a corresponding difference in the size distribution of the RNAs.