A Drosophila derailed homolog, doughnut, expressed in invaginating cells during embryogenesis.

Members of the RYK family of receptors are homologous to tyrosine kinases but do not exhibit kinase activity in vitro. We describe a new member of this family in Drosophila, which we call Doughnut (DNT). The protein product was found to be 70% identical to the Drosophila Derailed (DRL) protein and 35-40% identical to the mammalian RYK proteins. During Drosophila embryogenesis, DNT was found to be expressed in a highly dynamic pattern, including many invaginating cells. Many aspects of the expression pattern resembled that of unpaired, a gene that encodes a secreted protein that stimulates the Drosophila JAK/STAT signaling pathway. RYK proteins contain amino acid substitutions at residues that are highly conserved amongst proteins that exhibit kinase activity. Therefore, it has been unclear whether RYK family members are catalytically active or, if they are not, how they might transduce a signal. When expressed in cell culture DNT became phosphorylated on tyrosine, as did a mutant form of the receptor, containing an arginine residue in place of lysine within the predicted nucleotide binding site. These results suggest that DNT associates with a catalytically active kinase, but may not be capable of autophosphorylation.

Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway.

In vertebrates, many cytokines and growth factors have been identified as activators of the JAK/STAT signaling pathway. In Drosophila, JAK and STAT molecules have been isolated, but no ligands or receptors capable of activating the pathway have been described. We have characterized the unpaired (upd) gene, which displays the same distinctive embryonic mutant defects as mutations in the Drosophila JAK (hopscotch) and STAT (stat92E) genes. Upd is a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. We propose that Upd is a ligand that relies on JAK signaling to stimulate transcription of pair-rule genes in a segmentally restricted manner in the early Drosophila embryo.

Sea urchin FGFR muscle-specific expression: posttranscriptional regulation in embryos and adults.

We have shown previously by in situ hybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoon et al., J. Biol. Chem. 271, 20119-20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find that SpFGFR transcripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence of SpFGFR transcripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive, SpFGFR transcripts in lantern muscle. Together, these results suggest that only a minority of SpFGFR transcripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, IgS ligand binding domain sequences.

SpFGFR, a new member of the fibroblast growth factor receptor family, is developmentally regulated during early sea urchin development.

We describe the cloning of a new fibroblast growth factor receptor, SpFGFR1, that is differentially regulated at the level of transcript abundance during sea urchin embryogenesis. Sequence representing the conserved tyrosine kinase domain was obtained by reverse transcription-polymerase chain reaction using degenerate primers, and the entire open reading frame was obtained by standard cDNA library screening methods. SpFGFR contains a series of domains characteristic of FGFRs: three immunoglobulin-like motifs, an acid box, a transmembrane domain, a relatively long juxtamembrane sequence, a split tyrosine kinase domain, and two conserved intracellular tyrosine residues. Alternative splicing of SpFGFR generates two variants (Ig3L and Ig3S), which differ by insertion in the center of the Ig3 domain of 34 extra amino acids, encoded by an additional exon. Transcripts encoding both variants accumulate when morphogenesis begins with mesenchyme cell ingression and gastrulation. SpFGFR transcripts accumulate in all cell types of the embryo, although in situ hybridization shows that they are somewhat enriched in cells of oral ectoderm and endoderm. Transcripts encoding the Ig3S variant, whose structure resembles more closely that of vertebrate receptors, are enriched in endomesoderm, suggesting that the SpFGFR variants could play distinct roles in the sea urchin embryo.

Intraepithelial anchoring fibril components.

Cultured human keratinocytes and cultured human cervical carcinoma cells (ME-180) contained intracellular pools of antigens that reacted with the anchoring fibril antibodies AF1 and AF2. In keratinocytes, these antigens formed a basement membrane-like structure near the apical portions of the cells. Using flow cytometric techniques, pretreatment of the ME-180 cells with acetone revealed large intracellular pools of antigen. The intracellular epitope was calcium sensitive. Some forms of recessive dystrophic epidermolysis bullosa have retention of intracellular portions of the anchoring fibril suggesting a relation of the intracellular anchoring fibril antigens to that disease.

Monoclonal antibodies to two different epitopes in a 30-kD CNBr peptide of the K1 and K2 keratins.

Two anti-keratin monoclonal antibodies, Kab-2 and Kab-3, with specificities for different epitopes of type II (basic) human epidermal keratins, were produced. These antibodies had different immunofluorescent staining patterns on human fetal epidermis. Western blots and solid phase RIA showed both antibodies bound to 65-67-kD basic keratins (K1 and K2) extracted from foreskin epidermis. Competitive binding studies with the two Kab antibodies and other anti-keratin monoclonal antibodies showed that Kab-2 and Kab-3 recognized related epitopes, distinct from the epitopes recognized by other anti-keratin antibodies AE-1, 2, and 3. Kab-2 and Kab-3 epitopes were distinguished by differences in their reactivity with peptides generated by Staphylococcus aureus V8 protease digestion of the K1 keratin; the antibodies recognized both common and unique peptides. Western blots of cyanogen bromide digests of the K1 keratin showed that both Kab antibodies reacted with a 30-kD fragment of the molecule presumed to be the N-terminal CNBr peptide. We interpret these data to indicate that in tissues, portions of the N-terminal region of the K1 keratin are differentially available for reaction with these monoclonal antibodies and that morphologic differences in staining with monoclonal antibodies to the same molecule can reflect epitope specificity or epitope availability related to supramolecular organization.

Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids. Construction of the HIV-1 tat gene.

The construction of the HIV-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described. IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication. The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (greater than 100 bases) within the plasmid by oligonucleotide-directed mutagenesis. IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene. Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression. Using this approach, the 261 bp tat gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125. The 15 kD tat protein was produced from this synthetic gene in E. coli upon IPTG induction. However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.

Monoclonal antibody to a 35 kD epidermal protein induces cell detachment.

A murine monoclonal antibody (ECS-1) was prepared from BALB/c mice immunized with trypsinized cultured human foreskin keratinocytes. The antibody showed a pattern suggestive of intercellular staining on the nucleated layers of normal human epidermis, adult palm, mouse lip epidermis, and cultured human keratinocytes. ECS-1 stained human fetal skin by 9 weeks estimated gestational age. ECS-1 reacted with a 35 kD protein extracted from neonatal foreskin epidermis and cultured human keratinocytes. The protein required Nonidet P-40 or sodium dodecyl sulfate and mercaptoethanol for solubilization. ECS-1 induced epidermal cell detachment which was enhanced by complement. ECS-1 shares characteristics with human pemphigus antibodies.