Effect of the pleiotropic GrB mutation of Bombyx mori on chorion protein synthesis

The Gr^B mutation has a profound pleiotropic effect, leading in the homozygous state to the absence or extreme reduction of a substantial number of chorion proteins. The effect shows developmental specificity: most of the proteins normally synthesized beginning with stage III of choriogenesis or later, but possibly none of these normally beginning with stage II, are eliminated in the mutant. More subtle quantitative effects on certain other proteins are also observed, including prolongation of synthesis of some proteins which normally terminate at stage VIII. The proteins eliminated in the mutant are present in the heterozygote at intermediate levels, quantitatively close to those in the wild-type. The differences in chorion protein composition result from correspondingly altered protein synthesis rather than from post-translational degradation or modification. The missing proteins also fall to be synthesized in vitro when total RNA from mutant follicles is translated in the wheat germ system. It appears that as a consequence of the mutation, a set of mRNAs fails to be synthesized or accumulated. These results are consistent with the possibility that Gr^B is a regulatory mutation, or a deletion eliminating multiple chorion genes, clustered predominantly according to the developmental period of their expression.     

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 stage-specific 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.     

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.     

Specific protein synthesis in cellular differentiation: V. A secretory defect of chorion formation in the Grcol mutant of Bombyx mori

When homozygous, the Gr^col mutation of Bombyx mori causes the production of an eggshell in which most proteins are underrepresented to varying degrees. Neither the relative rates nor the timing of chorion protein synthesis appear to be affected; instead, the mutant phenotype results from the post-translational loss of normally synthesized proteins. The extent of loss of each protein correlates with its developmental timing, being maximal at early to middle stages. At the same stages, secretion appears to be deficient: chorion proteins overaccumulate within mutant cells, and slowly disappear. A preliminary electron microscopic examination has revealed the presence of mutant-specific cytoplasmic vesicles. The deficient complement of secreted proteins fails to form the highly ordered structure characteristic of normal chorion.     

Evolution of chorion structural genes and regulatory mechanisms in two wild silkmoths: A preliminary analysis

Summary We report a preliminary analysis of structural and regulatory evolution of the A and B chorion gene families in two wild silkmoths,Antheraea pernyi andAntheraea polyphemus. Homospecific and heterospecific dot hybridizations were performed between previously characterizedA. polyphemus complementary DNA clones and total or stage-specific follicular mRNAs from the two species. The hybridization patterns indicated substantial interspecies changes in the abundance of corresponding mRNA sequences (heteroposic evolution) without substantial changes in their developmental specificities (heterochronic evolution). In addition, the proteins encoded in the two species by corresponding mRNAs were determined by hybrid-selected translation followed by electrophoretic analysis. The results suggested that the proteins evolve in size, presumably through internal deletions and duplications.     

Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum

A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.     

Synthesis and stability of developmentally regulated dictyostelium mRNAs are affected by cell-cell contact and cAMP

Postaggregation Dictyostelium discoideum cells contain 3000 mRNA species that are absent from preaggregation cells; these aggregation-dependent sequences comprise 30% of the mass of mRNA in these cells. We show that the synthesis and stability of these regulated mRNA sequences are affected by both cell-cell contact and cAMP. Three independent assays are used to quantitate these mRNAs: in vitro translation followed by two-dimensional gel analysis of the protein products; hybridization of gel-separated RNAs to cloned DNAs; and hybridization of mRNA to a cDNA probe specific for the population of regulated sequences. In postag-gregation cells, the half-life of both the developmentally regulated mRNAs and the constitutive mRNAs present throughout growth and differentiation is the same—about 4 hr. Following disaggregation, all of the late mRNA sequences are degraded and decay with a half-life of 25 to 45 min. The constitutive species are unaffected; 2.5 hr after disaggregation, the ratio of late to constitutive mRNAs is about 6% that of normal plated cells. Addition of cAMP to cells that have been disaggregated for 2.5 hr (or longer) restores the level of most late mRNAs within 3 hr. We conclude that cAMP stimulates the synthesis of these mRNAs and may also act to stabilize them in the cytoplasm. This effect of cAMP is dependent on the cells having been in contact with other cells; cAMP has no effect on the levels of mRNA in suspension-starved, aggregation-competent cells that have never formed cell-cell aggregates.     

Selection and sequence analysis of a cDNA clone encoding a known chorion protein of the A family.

Using as criteria the size, abundance and developmental specificity of hybridizing mRNA sequences, we have selected from our chorion cDNA library a clone corresponding to a specific chorion protein, A4--cl. Comparison between the clone sequence and the largely known sequence of A4--cl validates the use of the cDNA library for sequence analysis of the chorion multigene families. The two major chorion protein families, A and B, share certain structural similarities.     

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3′ untranslated regions (3′-UTR). There are some similarities between the 3′-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3′-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments. Sequence data from this article were deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession Nos. Genomic sequences of pea apyrase: AB023621, AB030444, AB030445, AB038554, AB038555. cDNA sequences of pea apyrase: AB022319, AB027614, AB038668, AB038669.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.     

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.     

Unmasking the role of the 3′ UTR in the cytoplasmic polyadenylation and translational regulation of maternal mRNAs

The poly(A)-dependent translational regulation of maternal mRNAs is an important mechanism to execute stage-specific programs of protein synthesis during early development. This control underlies many crucial developmental events including the meiotic maturation of oocytes and activation of the mitotic cell cycle at fertilization. A recent report⁽¹⁾ demonstrates that the 3′ untranslated region of the cyclin A1, B1, B2 and c-mos mRNAs determines the timing and extent of their cytoplasmic polyadenylation and translational activation during Xenopus oocyte maturation. These studies further establish that protein synthesis can be temporally and quantitatively controlled by developmentally regulated changes in the polyadenylation of maternal mRNAs.     

Distribution of root mRNA species in other vegetative organs of pea (Pisum sativum L.)

Summary A cDNA library was prepared from, poly(A)⁺ RNA from roots of pea (Pisum sativum L.). Twenty five clones were selected by use of random numbers and used as probes on Northern blots to analyse the distribution of their corresponding mRNA species in other vegetative pea organs: leaf, stem and developing cotyledon. Fifteen cDNA inserts hybridised to single mRNA species, five hybridised to two mRNA species and one hybridised to five homologous mRNAs. Four cDNA clones (16% of those selected) gave no hybridization signals, indicating that the steady state levels of mRNAs were below the detection limit (i.e.less than 2.5 x 10^-5% of poly(A)⁺ RNA). Most of the root mRNAs were represented in all four pea organs as sequences of low and medium abundance. All but two cDNAs encoded mRNA species enhanced in root. However, cDNA clones appeared not to encode mRNA species expressed in a strictly organ-specific manner, as no mRNA unique to root was found. Thus, if organ-unique mRNA species are present, they are only present at a very low level of abundance in the poly(A)⁺RNA population.