Nematode collagen genes

The collagen genes of nematodes encode proteins that have a diverse range of functions. Among their most abundant products are the cuticular collagens, which include about 80% of the proteins present in the nematode cuticle. The structures of these collagens have been found to be strikingly similar in the free-living and parasitic nematode species studied so far, and the genes that encode them appear to constitute a large multigene family whose expression is subject to developmental regulation. Collagen genes that may have a role in cell-cell interactions and collagen genes that correspond to the vertebrate type IV collagen genes have also been identified and studied in nematodes.     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.     

Pupal and larval cuticle proteins of Drosophila melanogaster

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.     

Obstructor-A is required for epithelial extracellular matrix dynamics, exoskeleton function, and tubulogenesis

The epidermis and internal tubular organs, such as gut and lungs, are exposed to a hostile environment. They form an extracellular matrix to provide epithelial integrity and to prevent contact with pathogens and toxins. In arthropods, the cuticle protects, shapes, and enables the functioning of organs. During development, cuticle matrix is shielded from premature degradation; however, underlying molecular mechanisms are poorly understood. Previously, we identified the conserved obstructor multigene-family, which encodes chitin-binding proteins. Here we show that Obstructor-A is required for extracellular matrix dynamics in cuticle forming organs. Loss of obstructor-A causes severe defects during cuticle molting, wound protection, tube expansion and larval growth control. We found that Obstructor-A interacts and forms a core complex with the polysaccharide chitin, the cuticle modifier Knickkopf and the chitin deacetylase Serpentine. Knickkopf protects chitin from chitinase-dependent degradation and deacetylase enzymes ensure extracellular matrix maturation. We provide evidence that Obstructor-A is required to control the presence of Knickkopf and Serpentine in the extracellular matrix. We propose a model suggesting that Obstructor-A coordinates the core complex for extracellular matrix protection from premature degradation. This mechanism enables exoskeletal molting, tube expansion, and epithelial integrity. The evolutionary conservation suggests a common role of Obstructor-A and homologs in coordinating extracellular matrix protection in epithelial tissues of chitinous invertebrates.     

Cuticular morphogenesis during continuous growth of the final instar larva of a moth

The larvae of the tobacco hornworm, Manduca sexta, grow continuously. During the feeding period of the fifth larval instar their weight increases ten-fold (ca. 1·2–12 g) accompanied by a four-fold expansion of the surface area of the abdominal cuticle. We have found that this cuticle contains structures which facilitate its expansion. Folds in the epicuticle (papillae) flatten as the cuticle expands. The endocuticle, in contrast, does not unfold but rather is plastically deformed. This plastic deformation is assisted by vertical structures in the cuticle (cuticular columns) which are more easily deformed than the surrounding lamellate cuticle. The head capsule cuticle, which does not expand as the larva grows, lacks papillae and cuticular columns. Thus, these are specialized structures that are reserved for cuticle that must expand as the larva grows.     

A large multigene family codes for the polypeptides of the crystalline trichocyst matrix in Paramecium.

The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.     

Linkage Disequilibrium in Human Ribosomal Genes: Implications for Multigene Family Evolution

Members of the rDNA multigene family within a species do not evolve independently, rather, they evolve together in a concerted fashion. Between species, however, each multigene family does evolve independently indicating that mechanisms exist which will amplify and fix new mutations both within populations and within species. In order to evaluate the possible mechanisms by which mutation, amplification and fixation occur we have determined the level of linkage disequilibrium between two polymorphic sites in human ribosomal genes in five racial groups and among individuals within two of these groups. The marked linkage disequilibrium we observe within individuals suggests that sister chromatid exchanges are much more important than homologous or nonhomologous recombination events in the concerted evolution of the rDNA family and further that recent models of molecular drive may not apply to the evolution of the rDNA multigene family.     

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles

The major histological components of the hair follicle are the hair cortex and cuticle. The hair cuticle cells encase and protect the cortex and undergo a different developmental program to that of the cortex. We report the molecular characterization of a set of evolutionarily conserved hair genes which are transcribed in the hair cuticle late in follicle development. Two genes were isolated and characterized, one expressed in the human follicle and one in the sheep follicle. Each gene encodes a small protein of 16 kD, containing greater than 50 cysteine residues, ranging from 31 to 36 mol% cysteine. Their high cysteine content and in vitro expression data identify them as ultra-high-sulfur (UHS) keratin proteins. The predicted proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Genomic blots reveal that the UHS keratin proteins are encoded by related multigene families in both the human and sheep genomes. Tissue in situ hybridization demonstrates that the expression of both genes is localized to the hair fiber cuticle and occurs at a late stage in fiber morphogenesis.     

Sequence comparisons among dispersed members of the Brassica S multigene family in an S 9 genome

Self-incompatibility (SI) systems prevent self-pollination and promote outbreeding. In Brassica, the SI genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are members of the S multigene family, which share the SLG-like domain (S domain), which encodes a putative receptor. We have cloned members of the S multigene family from the S9 haplotype of B. campestris (syn. rapa). In addition, eight distinct genomic regions harboring 10 SLG/SRK-like genes were characterized in the present study. Sequence analysis revealed two novel SRK-like genes, BcRK3 and BcRK6 (for B. campestris receptor kinases 3 and 6, respectively). Other genes that were characterized included SFR2 (for S gene family receptor 2), SLR2 (for S locus related gene 2), and a pseudogene. Based on phylogenetic analysis of the nucleotide sequences of the S domain regions, SLG and SRK appear to be distinct from other members of the S multigene family. Linkage analysis showed that most members of the S multigene family are dispersed in the Brassica genome, and that SLR1 (S locus related gene 1) is not linked to the SLR2 in B. campestris.     

Domain loss facilitates accelerated evolution and neofunctionalization of duplicate snake venom metalloproteinase toxin genes

Gene duplication is a key mechanism for the adaptive evolution and neofunctionalization of gene families. Large multigene families often exhibit complex evolutionary histories as a result of frequent gene duplication acting in concordance with positive selection pressures. Alterations in the domain structure of genes, causing changes in the molecular scaffold of proteins, can also result in a complex evolutionary history and has been observed in functionally diverse multigene toxin families. Here, we investigate the role alterations in domain structure have on the tempo of evolution and neofunctionalization of multigene families using the snake venom metalloproteinases (SVMPs) as a model system. Our results reveal that the evolutionary history of viperid (Serpentes: Viperidae) SVMPs is repeatedly punctuated by domain loss, with the single loss of the cysteine-rich domain, facilitating the formation of P-II class SVMPs, occurring prior to the convergent loss of the disintegrin domain to form multiple P-I SVMP structures. Notably, the majority of phylogenetic branches where domain loss was inferred to have occurred exhibited highly significant evidence of positive selection in surface-exposed amino acid residues, resulting in the neofunctionalization of P-II and P-I SVMP classes. These results provide a valuable insight into the mechanisms by which complex gene families evolve and detail how the loss of domain structures can catalyze the accelerated evolution of novel gene paralogues. The ensuing generation of differing molecular scaffolds encoded by the same multigene family facilitates gene neofunctionalization while presenting an evolutionary advantage through the retention of multiple genes capable of encoding functionally distinct proteins.     

Birth-and-death evolution of the internalin multigene family in Listeria

The birth-and-death model of multigene family evolution describes patterns of gene origination, diversification and loss within multigene families. Since it was first developed in the 1990s, the model has been found to characterize a large number of eukaryotic multigene families. In this paper, we report for the first time a bacterial multigene family that undergoes birth-and-death evolution. By analyzing the evolutionary relationships among internalins, a relatively large and diverse family of genes associated with key virulence functions in Listeria, we demonstrate the importance of birth-and-death evolution in the diversification of this important bacterial pathogen. We also detected two instances of lateral gene transfer within the internalins, but the estimated frequency would have been much higher had it not been analyzed within the context of birth-and-death evolutionary dynamics and a phenomenon that we term "paralog-sorting", which involves the unequal transmittal of gene duplicates during or subsequent to the speciation process. As such, in addition to providing the first demonstration of birth-and-death evolution within a bacterial multigene family, our results indicate that the extent of lateral transfer in bacterial multigene families should be re-examined in the light of birth-and-death evolution.     

A computer simulation of evolutionary forces controlling the size of a multigene family

Summary A Monte Carlo-type simulation of the evolution of a multigene family was performed. The model was designed to study the selective forces which may control the size of a multigene family. As expected, we find that direct selection on the size of the multigene family can control its size. More important, we find that selection acting upon the family as a single functional unit, in conjunction with homologous but unequal crossing over, can also control the size of a multigene family.     

A diverse family of phenylalanine ammonia-lyase genes expressed in pine trees and cell cultures

Using degenerate PCR primers that target evolutionarily conserved sequences in pal genes, we show that in the gymnosperm, Pinus banksiana, phenylalanine ammonia-lyase (PAL) is encoded by a multigene family of at least eight to ten loci. Five classes of pal sequence were easily distinguished among 28 clones sequenced from the products of PCR amplification of haploid genomic DNA. The dominant sequence from each class was named, yielding pal1 to pal5 loci. These genes shared 68.8% to 94.0% nucleotide identity over the 366 bp region compared. All of pal1 to pal5 were expressed in cell suspension cultures treated with a fungal elicitor and all but pal3 were expressed in differentiating xylem tissue of a mature tree. Only pal1 was expressed in unelicited cell cultures. While these P. banksiana genes are quite divergent, they are still more similar to each other than to any angiosperm pal gene cloned to date. For its roles in development and defense, PAL production in P. banksiana is coordinated from a large, diverse multigene family. We discuss evidence suggesting that other pines have similar pal gene family structures.     

The coalescent and infinite-site model of a small multigene family

The infinite-site model of a small multigene family with two duplicated genes is studied. The expectations of the amounts of nucleotide variation within and between two genes and linkage disequilibrium are obtained, and a coalescent-based method for simulating patterns of polymorphism in a small multigene family is developed. The pattern of DNA variation is much more complicated than that in a single-copy gene, which can be simulated by the standard coalescent. Using the coalescent simulation of duplicated genes, the applicability of statistical tests of neutrality to multigene families is considered.     

Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference

Insect chitinases are a multigene family that is encoded by a rather large and diverse group of genes. The main function of chitinases is to digest the chitin contained in tissues such as the cuticles and gut lining during molting. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) during the pupation and eclosion stages of Spodoptera exigua. First, efficient silencing of the SeChi and SeChi-h genes through specific double-stranded RNA (dsRNA) injection led to a significant reduction in the mRNA levels of SeChi and SeChi-h. Additionally, different phenotypic defects were observed at the pupal and adult stages after injection of the SeChi and SeChi-h dsRNAs. After injecting SeChi dsRNA in the pupal stage, the cuticle of the head split open and the pupal cuticle was visible under the old larval cuticle. However, after injecting the SeChi-h dsRNA, animals died without exhibiting any special phenotypes. At the adult death stage, animals injected with dsSeChi could not shed their pupal shell completely, and their old cuticles remained attached to their head or chest. However, the main lethal phenotype was that insects did not emerge after dsSeChi-h injection. Additionally, the average survival rates of S. exigua were 52.02% and 40.38% at the pupal and adult stages, respectively, after injection with SeChi dsRNA. For the insects injected with SeChi-h dsRNA, the survival rates were 72.38% and 48.52%, respectively. These results suggest that SeChi and SeChi-h may have different biologic functions during the pupal-adult molting.