The ALS1 (agglutinin-like sequence) gene of Candida albicans encodes a protein similar to alpha-agglutinin, a cell-surface adhesion glycoprotein of Saccharomyces cerevisiae (Hoyer et al. 1995). A central domain of a tandemly repeated 108-bp sequence is found in the ALS1 coding region. This tandem-repeat motif hybridizes to multiple C. albicans genomic DNA fragments, indicating the possibility of other ALS1-like genes in C. albicans (Hoyer et al. 1995). To determine if these fragments constitute a gene family, tandem-repeat-hybridizing genomic fragments were isolated from a fosmid library by PCR screening using primers based on the consensus tandem-repeat sequence of ALS1 (Hoyer et al. 1995). One group of fosmids, designated ALS3, encodes a gene with 81% identity to ALS1. The sequences of ALS1 and ALS3 are most conserved in the tandem-repeat domain and in the region 5' of the tandem repeats. Northern-blot analysis using unique probes from the 3' end of each gene demonstrated that ALS1 expression varies, depending on which C. albicans strain is examined, and that ALS3 is hyphal-specific. Both genes are found in a variety of C. albicans and C. stellatoidea strains examined. The predicted Als1p and Als3p exhibit features suggesting that both are cell-surface glycoproteins. Southern blots probed with conserved sequences from the region 5' of the tandem repeats suggest that other ALS-like sequences are present in the C. albicans genome and that the ALS family may be larger than originally estimated.
Additional genes in the growing ALS family ofCandida albicans were isolated by PCR screening of a genomic fosmid library with primers designed from the consensus tandem-repeat sequence of ALS1. This procedure yielded fosmids encoding ALS2 and ALS4. ALS2 andALS4 conformed to the three-domain structure ofALS genes, which consists of a central domain of tandemly repeated copies of a 108-bp motif, an upstream domain of highly conserved sequences, and a domain of divergent sequences 3′ of the tandem repeats. Alignment of five predicted Als protein sequences indicated conservation of N- and C-terminal hydrophobic regions which have the hallmarks of secretory signal sequences and glycosylphosphatidylinositol addition sites, respectively. Heterologous expression of an N-terminal fragment of Als1p in Saccharomyces cerevisiae demonstrated function of the putative signal sequence with cleavage following Ala17. This signal sequence cleavage site was conserved in the four other Als proteins analyzed, suggesting identical processing of each protein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluorescence with an anti-Als antiserum. Staining was observed on mother yeasts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other ALS genes, ALS2 andALS4 were differentially regulated. ALS4expression was correlated with the growth phase of the culture;ALS2 expression was not observed under many different in vitro growth conditions. The data presented here demonstrate thatALS genes encode cell-surface proteins and support the conclusion that the size and number of Als proteins on theC. albicans cell surface vary with strain and growth conditions.
The novel type I TGFbeta family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.
We have recently identified, in zebrafish, a novel type I receptor of the TGFbeta family, alk8, that participates in Bmp signaling pathways to mediate early dorsoventral patterning of neurectodermal and mesendodermal tissues. Since Bmps play significant roles in tooth specification, initiation, and differentiation, we hypothesized that alk8 may play a role in directing the Bmp-mediated epithelial mesenchymal cell interactions regulating tooth development. Immunohistochemical analysis demonstrates that Alk8 is expressed in developing zebrafish and mouse teeth. Examination of tooth development in zebrafish with disrupted alk8 signaling revealed specific defects in tooth development. Ectopic expression of constitutively active Alk8 results in the formation of elongated tooth structures, while expression of dominant-negative Alk8 results in arrested tooth development at the bud stage. These results are consistent with the established requirements for Bmp signaling in tooth development and demonstrate that Alk8 is a key regulator of tooth development.
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