Steven T. Case

DIAGNOSTIC AND MOLECULAR EVALUATION OF THREE IRIDOVIRUS-ASSOCIATED SALAMANDER MORTALITY EVENTS

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.

Effects of anti-C23 (Nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells☆

Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with 32P-labeled RNA precorsors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of 32P incorporation into 38S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of 32P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of 32P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. When protein extracts of Chironomus tentans salivary gland nuclei were probed on Western blots with anti-C23 antibody the predominant cross-reacting species was a 110-kDa polypeptide which had an electrophoretic mobility similar to that of protein C23. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

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.

A variant tandemly repeated nucleotide sequence in Balbiani ring 2 of chironomus tentans

pCtBR2-2 is a genomic clone from Chironomus tentans that hybridized in situ to Balbiani ring 2 (BR2) on salivary gland polytene chromosome IV. DNA sequencing indicated that the insert contained nearly four copies of a 180 bp tandemly repeated nucleotide sequence that was distinctly different from a previously reported BR2 repeat. Sequence titration experiments detected about 70 copies of the 180 bp repeat per haploid genome, which would correspond to approximately 34% of a 37 kb BR2 gene. Each 180 bp repeat included a conserved 90 bp segment whose sequence was internally nonrepeating (INR), and a variable 90 bp repeated (R) segment comprised of three 30 bp repeats that may have evolved from a 9 bp consensus sequence. Results presented here raise the distinct possibility that other BR genes may contain significantly different repeated sequences that have not been identified.

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, BR2α, BR2/β, 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 dotblot 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 BR2β repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2α repeats.

Individual variations in the content of giant secretory polypeptides in salivary glands of Chironomus.

Salivary glands in aquatic larvae of Chironomus are responsible for formation of a fiber that larvae use to construct feeding tubes. Major constituents of this fiber include a family (the sp-I family) of high Mr (1 × 106) secretory polypeptides. Because of our interest in the polypeptide composition and polymerization of the salivary fiber we conducted a survey of the electrophoretic pattern of sp-I components found in salivary glands obtained from individual larvae. The survey encompassed ten strains of Chironomus tentans, three strains of Chironomus pallidivittatus and four strains of Chironomus thummi. Salivary glands from C. tentans and C. pallidivittatus contained at least four sp-I components (sp-Ia, sp-Ib, sp-Ic and sp-Id) that behave identically with regard to their electrophoretic mobility and detectability when larvae were exposed to galactose or glycerol. Sp-I components in C. thummi were generally fewer and not directly comparable by electrophoretic mobility to sp-I components in the other two species. During this survey two important alterations were observed in the electrophoretic pattern of sp-I components obtained from C. tentans and C. pallidivittatus. First, all four sp-I components exhibited, with a low frequency, double bands that appeared as slow-versus-fast electrophoretic variants of a particular component. Secondly, the relative steady-state level of each sp-I component fluctuated in comparison to other sp-I components in the same extract. This fluctuation varied such that any one sp-I component might appear as a single prominent component. Sp-I components are encoded by a multigene family located in Balbiani rings (BRs). Results presented here, in conjunction with known nucleotide sequence data from BR genes, led us to the following conclusions. The slow and fast electrophoretic variants observed for each sp-I component suggest that each corresponding BR gene may be able to expand and/or contract in size. The observed degree of independent fluctuation in the steady-state level of each sp-I component suggests that each BR gene may be able to regulate its expression independently from the others. Finally, the observation that salivary glands sometimes contained only one prominent sp-I component led us to hypothesize that, whereas salivary fibers might typically be heteropolymers comprised of two or more types of sp-I components, homopolymers comprised of only one sp-I component may also exist.

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.

The 3′ ends of two genes in the Balbiani ring c locus ofChironomus thummi

SummaryThe 3′-end sequences of two nonallelic genes derived from the Balbiani ring c (BRc) locus ofChironomus thummi are described. Only one of the genes appears to be transcribed abundantly in normal late larval salivary glands. The two sequences are highly similar, even in the 3′ untranslated regions, but sharply diverge beyond the polyadenylation site. Together with evidence from the 3′ ends of BR1 and BR2 genes ofC. pallidivittatus andC. tentans, independently characterized by others, this result suggests the existence of a sequence-homogenization mechanism that operates across the 3′ ends of all BR genes characterized to date. The 3′-terminal coding region of each BRc gene is divided into two portions by a short intron. The upstream portion is homologous to and continuous with the tandem repeats that make up the internal core of each BR gene; however, that portion is variant in sequence relative to the core, and apparently is not subject to the homogenization process that operates on the core repeats. The portion downstream of the intron encodes a unique, 111-residue polypeptide highly different from the rest of the BRc product. The evolution of the various segments of the BRc genes is discussed.

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.

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.