Developmentally regulated expression of a Balbiani ring 1 gene for a 180-kD secretory polypeptide in Chironomus tentans salivary glands before larval/pupal ecdysis.

The expression of a Balbiani ring 1 gene that codes for a salivary gland-specific 180-kD secretory polypeptide (sp180) is regulated developmentally. Immunoblots of salivary gland protein incubated with an affinity-purified nonapeptide-reactive antibody demonstrated that the salivary gland content of sp180 increases as much as 10-fold between stages 8 and 10 of the fourth larval instar. Hybridization of RNA dot-blots with an oligonucleotide probe indicated that the observed increase in sp180 was preceded by a parallel 20-fold increase in the steady state level of its mRNA beginning between stages 7 and 8. In vitro nuclear transcription experiments demonstrated that there was a 10-fold acceleration in the rate of sp180 gene transcription between stages 6 and 10. The limited period of expression of the sp180 gene contrasted dramatically with the expression of Balbiani ring genes BR1, BR2 alpha, BR2 beta, and BR6, which code for the sp-I family of fibrous secretory polypeptides. The appearance of sp180 in secretion coincided with microscopically visible changes in the bundling of these fibrous polypeptides. At the same time, we noticed changes in the appearance and consistency of feeding tubes that larvae construct with this secretion. These results lead us to propose that sp180 may modify the structure or utilization of fibrous secretory polypeptides specifically for the assembly of pupation tubes necessary for larval/pupal ecdysis.

A novel giant secretion polypeptide in Chironomus salivary glands: implications for another Balbiani ring gene.

Chironomus salivary glands contain a family of high Mr (approximately 1,000 X 10(3)) secretion polypeptides thought to consist of three components: sp-Ia, sp-Ib, and sp-Ic. The use of a new extraction protocol revealed a novel high Mr component, sp-Id. Results of a survey of individual salivary glands indicated that sp-Id was widespread in more than a dozen strains of C. tentans and C. pallidivittatus. Sp-Id was phosphorylated at Ser residues, and a comparison of cyanogen bromide and tryptic peptide maps of 32P-labeled polypeptides suggested that sp-Ia, sp-Ib, and sp-Id are comprised of similar but nonidentical tandemly repeated amino acid sequences. We concluded that sp-Id is encoded by an mRNA whose size and nucleotide sequence organization are similar to Balbiani ring (BR) mRNAs that code for the other sp-I components. Furthermore, parallel repression of sp-Ib and sp-Id synthesis by galactose led us to hypothesize that both of their genes exist within Balbiani ring 2.

Secondary structure of synthetic peptides derived from the repeating unit of a giant secretory protein from Chironomus tentans.

The secretory proteins of Chironomus tentans larvae, which are used to construct underwater feeding and pupation tubes, assemble into complexes in vitro. Members of a family of 1000 kDa proteins, the spIs, appear to form the fibrous backbone of the assembled complexes. The spIs consist of a core of tandemly repeating units of 60 to 90 amino acids that can be subdivided into two regions: the subrepeat region, made up of short internal repeats, and the constant region, which lacks simple subrepeats. We have synthesized peptides representative of the constant and subrepeat regions of one of the spIs, and have examined their secondary structure using Fourier transform IR and CD spectroscopy. The IR spectrum of the constant peptide indicates that this peptide has alpha-helical regions and beta-turns. The CD spectrum confirms this. The IR spectrum of the subrepeat peptide is similar to that of the poly(Gly)II helix, and also may indicate the presence of beta-turns. The CD spectrum is consistent with this helical structure. Extrapolation of these results to intact spIs is in agreement with secondary structure prediction and modeling studies. Our results indicate that the alpha-helices and poly(Gly)II-like helices are not arranged as coiled-coils, which are often found in fibrous proteins. We suggest that these structural elements may be in an unusual arrangement in the spIs, organized as alternating alpha-helices and poly(Gly)II or collagen-like helices, interspersed with beta-turns.

Single-stranded regions in DNA isolated from different developmental stages of the sea urchin.

Long-term labeled sea urchin embryo (Strongylocentrotus purpuratus) DNAs were examined for size of recovered pieces, single-strandedness, and length of continuous double-stranded regions. Sizing on neutral sucrose gradients indicates that morula stage DNA sediments predominantly at 31 S, blastula stage DNA at 27 S, and gastrula stage DNA as a broad range of sizes of greater than 29 S. Treatment of [3H]thymidine-labeled DNA with Aspergillus oryzae S1 nuclease removes 19% of the 3H from morula stage DNA, 4% of the 3H from blastula stage DNA, and less than 0.1% of the 3H from gastrula stage DNA. Sedimentation of S1 nuclease treated [3H]DNAs on alkaline sucrose gradients indicates that in native morula stage DNA there is a nick or gap in one strand approximately every 9700 base pairs, in native blastula stage DNA about every 3300 base pairs, and very few nicks or gaps in native gastrula stage DNA.

Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.

We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.

Selective deletion of large segments of Balbiani ring DNA during molecular cloning.

Transcription units in Balbiani ring 1 (BR1) and Balbiani ring 2 (BR2) of Chironomus tenans salivary glands are comprised of about 40 kb of repetitive DNA sequences organized in a satellite-like array. Because of this sequence organization, it was possible to prepare 30 to 40-kb target DNA fragments for cloning by performing limit restriction endonuclease digestion of high-Mr genomic DNA. These fragments were ligated to cohesive termini of the linearized cosmid, pHC79, packaged in vitro, and used to transduce Escherichia coli. Alternatively, target fragments were randomly sheared to a mean length of 8-10 kb, annealed to plasmid pBR322 using homopolymeric tails, and used for bacterial transformation. Recombinant cosmids and plasmids generally contained inserts which were proportional to the length of target fragments used in ligation reactions. However, the subset of recombinants that hybridized to 32P-labeled 75S RNA (highly enriched in BR1 + BR2 sequences) had disproportionately smaller inserts. With the exception of one metastable clone with a 2.1-kb insert, all others had inserts of 0.8 or 0.4 kb. Similar results were obtained in host cells that were recA- or recBC-. The most likely conclusion is that repetitive BR sequences are highly unstable during replication in E. coli and are selectively deleted.

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar.

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.

Characterization of a cloned, moderately repeated sequence from Balbiani ring 2 in Chironomus tentans.

pCtBR2-1 is a recombinant plasmid with a 750-bp insert of Chironomus tentans genomic DNA. When pCtBR2-1 was hybridized in situ to salivary gland polytene chromosomes, it hybridized exclusively to Balbiani ring 2 (BR2), a giant chromosomal puff. It was also shown that the insert contained four tandemly repeated sequences that were delineated by HinfI sites which occurred every 190 bp. The purified insert reassociated to C. tentans DNA with a C0t1/2 = 0.48 indicating that the sequence was moderately repeated within the genome. Hybridization of radioactive pCtBR2-1 to nitrocellulose blots containing partial HinfI digests of genomic DNA revealed that the 190-bp repeats were organized into one or more blocks of 11 to 12 copies in tandem. Hybridization of the recombinant plasmid to limit digests of genomic DNA also demonstrated that repeated sequences in BR2 were not homogeneous. As much as 70% of BR2 appeared to be represented by a 26-kb HhaI-resistant core, while the remaining 30% may have HhaI sites at 190-bp intervals, similar to pCtBR2-1.

A peptide-reactive antibody to a Balbiani ring gene product: immunological evidence that a 6.5-kb RNA in Chironomus tentans salivary glands is mRNA for a 180-kDa nonfibrous component of larval secretion.

An immunological approach was utilized to demonstrate that a tissue-specific Balbiani ring (BR) transcript in Chironomus tentans is the mRNA for a secreted 180-kDa polypeptide. Balbiani ring 1 (BR1) on the polytene chromosome IV of larval salivary glands contains a gene comprised of tandemly duplicated nucleotide sequences that are transcribed into a salivary gland-specific, 6.5-kb poly(A)+RNA for which a partial cDNA sequence exists [Dreesen et al., J. Biol. Chem. 260 (1985) 11824-11830]. A nonapeptide was synthesized so that its amino acid sequence corresponded to an open reading frame in the cDNA. This peptide was used to raise rabbit polyclonal antisera and to purify the peptide-reactive antibody by affinity chromatography. The affinity-purified antibody bound specifically to a 180-kDa polypeptide on Western blots containing extracts of total salivary gland protein. Western blot analysis of microdissected cellular vs. lumenal fractions of salivary glands indicated that this 180-kDa polypeptide was primarily localized in the lumen. Consequently, this polypeptide was designated a secretory polypeptide (sp180). Finally, the peptide-reactive antibody was used to localize sp180 in a nonfibrous component of salivary gland secretion by indirect immunofluorescence microscopy.

Structure and polymorphism of the Chironomus thummi gene encoding special lobe-specific silk protein, ssp160.

cDNA encoding Chironomus thummi ssp160 was used to isolate a genomic clone that hybridized in situ to band A2b on polytene chromosome IV, the site of the ssp160 gene. DNA sequencing, primer extension and gene/cDNA nucleotide sequence alignment revealed the gene contains six exons and five introns; 70% of ssp160 is encoded in exon 3. Variations between cDNA and gene sequences led to the design of a polymerase chain reaction, restriction fragment length polymorphism assay that was subsequently used to demonstrate the existence of polymorphic alleles whose distribution varied between geographically separated populations of larvae. The polymorphism is associated with codon deletions in a six-amino-acid repeat containing an N-linked glycosylation motif. These deletions may have resulted from slipped-strand mispairing during DNA replication.

Identification of divergent homologs of Chironomus tentans sp185 and its Balbiani ring 3 gene in Australasian species of Chironomus and Kiefferulus.

A 185-kDa silk protein (sp185) from Chironomus tentans, present in both larval and prepupal silks, contains a striking amino acid sequence motif, Cys-X-Cys-X-Cys, which occurs about every 22-26 residues. Homologous proteins have been found in Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220), which apparently differ in size but are very similar in overall composition and sequence. While surveying Australasian species of Chironomus and Kefferulus we obtained evidence for immunologically related silk protein having similar size and amino acid composition, but noticeably less Cys. Interspecies in situ hybridization to polytene chromosomes with C. tentans and C. pallidivittatus cDNA probes indicated that each species had a related gene. One pair of C. tentans cDNA-derived primers enabled polymerase chain reaction amplification of a discrete fragment of this gene from Kiefferulus 'cornishi'. Preliminary sequence information for this fragment confirmed the presence of an encoded Cys-X-Cys-X-Cys motif in what appeared to be a similar protein region containing less Cys. We conclude that homologs of C. tentans sp185 and its gene have been identified which may contain significant deviations in structure. Once suitable libraries are available, probes described here will be useful for selecting cDNA and genomic clones for detailed study.

High molecular mass complexes of aquatic silk proteins.

Little is known about specific protein protein associations that take place during formation of Chironomus tentans silk. The aim of this study was to learn if C. tentans salivary glands contain biochemically discrete silk protein complexes. Examination of native extracts by non-denaturing agarose gel electrophoresis and immunoblotting revealed two SDS-resistant complexes: C1a, nominally containing silk proteins spIa, sp185 and sp140, and C1b, containing spIb, sp185 and sp140. The data also implied that C1a and C1b can further associate into SDS-sensitive homo- or hetero-oligomers. Sedimentation of extracts in preparative glycerol gradients resulted in a heterogeneous distribution of C1a and C1b centered near 30S. Examination of gradient fractions by denaturing polyacrylamide gel electrophoresis and immunoblotting indicated that C1a and C1b co-sediment with spIs, sp185, and sp140; however, these fractions also contained sp40, sp17 and sp12. In contrast, two other silk proteins sedimented throughout the gradient. Electron micrographs of a complex-containing fraction showed discrete, sometimes oligomeric lattice-like structures that, over time, assembled in vitro into multistranded beaded fibers. It is proposed that C1a and C1b are quaternary structures that are intermediates in the assembly pathway of C. tentans silk.

Laser-Raman and FT-IR spectroscopic studies of peptide-analogues of silkmoth chorion protein segments.

Silkmoth chorion, the proteinaceous major component of the eggshell, with extraordinary mechanical and physiological properties, consists of a complex set of proteins, which have a tripartite structure: a central, evolutionarily conserved, domain and two more variable 'arms'. Peptide-analogues of silkmoth chorion protein central domain segments have been synthesized. Laser-Raman and infrared spectroscopic studies suggest the preponderance of antiparallel beta-pleated sheet structure for these peptides, both in solution and in the solid state.

Spectroscopic studies of Manduca sexta and Sesamia nonagrioides chorion protein structure.

The secondary structure of Manduca sexta and Sesamia nonagrioides chorion proteins has been studied in intact chorions using laser-Raman and Fourier transform infra-red (FTIR) spectroscopy and in a solution containing extracted and reassembled chorion proteins using circular dichroism (CD) spectroscopy. Laser-Raman and IR spectra suggest the predominance of antiparallel beta-pleated sheet structure in intact chorion proteins of both Lepidoptera species. The bands at 1673, 1674 cm-1 (amide I) and 1234-1238 cm-1 (amide III) in the laser-Raman spectra can best be interpreted as resulting from abundant antiparallel beta-pleated sheet structure. Analysis of the amide I band suggests that chorion proteins consist of 60-70% antiparallel beta-pleated sheet and 30-40% beta-turns. Supporting evidence for the prevalence of antiparallel beta-pleated sheet in chorion proteins was supplied using FTIR spectroscopy by the observation of a very intense absorption band at 1635 cm-1 (amide I) and of a weak band at 1530, 1525 cm-1 (amide II) from chorions of both species. Surprisingly, analysis of the CD spectra of extracted and reassembled chorion proteins suggests that, in solution, they retain a regular secondary structure most probably dominated by beta-pleated sheet. We therefore suggest that the prominent regular beta-sheet structure of chorion proteins may exist in solution and dictate the aggregation and polymerization process in vivo.

Correlated changes in steady-state levels of Balbiani ring mRNAs and secretory polypeptides in salivary glands of Chironomus tentans.

Balbiani rings (BRs) on polytenized chromosomes in Chironomid salivary glands contain members of a homologous multigene family that encodes a family (the sp-I family) of high Mr secretory polypeptides. Each of these BR genes is comprised largely of tandemly duplicated core repeat sequences consisting of related constant (C) regions and intergenically divergent subrepeat (SR) regions. A set of oligodeoxyribonucleotide probes were synthesized that correspond to the transcribed strand of the SR region of BR1, BR2 alpha, BR2 beta, and BR6 core repeats. Under a defined set of conditions, it was possible to show that each oligonucleotide probe hybridized exclusively to its cognate repeat type without hybridization to other repeat types in cloned DNA templates. These BR probes were then used in dot-blot hybridization experiments to simultaneously follow alterations in the steady-state level of BR mRNAs in response to prolonged exposure of larvae to galactose. The results indicated that the relative amounts of these four BR mRNAs may change in a noncoordinate manner. These BR probes were also used in experiments to compare simultaneously the salivary gland content of sp-I components and specific BR mRNAs in larvae that exhibited naturally occurring or induced alterations in BR gene expression. A correlation was found which suggested that sp-Ia is encoded in a gene comprised of BR1 repeats, sp-Ib is encoded by BR2 beta repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2 alpha repeats.

Secretory proteins of Chironomus salivary glands: structural motifs and assembly characteristics of a novel biopolymer.

Salivary glands of Chironomus synthesize a family of at least ten secretory proteins that can be grouped into three size classes: the large (about 1000 kDa), intermediate (100- to 200 kDa), and small (less than 100 kDa). After synthesis, secretory proteins undergo a dramatic transformation to form a novel biopolymer. Secretory proteins accumulate in the central lumen of the gland, forming dissociable complexes that appear as a network of smooth fibrils and multistranded beaded fibers. When secretory protein complexes are extruded through the secretory duct, the fibers become oriented in parallel arrays; when these parallel arrays of fibers emerge from the mouth of larvae they are an insoluble, silk-like thread. Regulation of secretory protein-coding gene expression determines which secretory proteins are synthesized, thus, the composition of silk threads. At least two types of threads are produced: larval silk is used to construct tubes for protective housing and assist with feeding; prepupal silk is used to construct tubes for larval/pupal ecdysis (pupation). Variations in composition presumably contribute to different mechanical properties of larval and prepupal silk threads. Since the macroscopic physical properties of polymerized silk most likely reflect the microscopic structure and interaction of secretory proteins, it becomes important to learn the principles which govern secretory protein assembly at the molecular level. Which secretory proteins interact and what are the sites used for intraportein and protein-protein interactions during the assembly of this biopolymer? All eight secretory proteins characterized thus far contain tandemly repeated peptide sequences (ranging from 14-90 amino acids in length) and/or a periodic distribution of Cys residues. These motifs appear to be unique; no other biopolymer has either the repeated peptide sequences or composite structure of chironomid silk threads. The evolutionary conservation of motifs within repeats and among different secretory proteins suggests that the sequences and three-dimensional structures of the motifs may be important for assembly of secretory proteins into complexes, oriented fibers, and silk threads. Further study of secretory protein assembly will bring us closer to understanding how this silk assembles in vivo. By learning principles that nature employs to construct such a novel composite biopolymer, it may become feasible to design and produce new classes of fibers or biomolecular materials with distinctive properties that are currently unavailable.