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Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library.

ABSTRACT: To identify the protein-tyrosine kinases that are expressed during chicken embryonic development, a 10-day chicken embryo cDNA expression library was screened with anti-phosphotyrosine antibodies. Of the positive clones isolated, many encoded the same protein-tyrosine kinase, which we designate Cek1 (chicken embryo kinase 1). Its amino acid sequence suggests that the Cek1 protein is a transmembrane tyrosine kinase and presumably the receptor for an unknown ligand. Antibodies prepared to the cloned Cek1 kinase recognize, in immunoblotting experiments, two protein bands with apparent molecular weights of 100,000 and 110,000. The Cek1 protein was detected in many chicken embryonic tissues, but not in the corresponding adult tissues (with the exception of brain). The Cek1 kinase appears to be very closely related to two protein-tyrosine kinases whose partial sequences have been recently determined, human Flg and mouse Bek. Cloning using anti-phosphotyrosine antibodies has allowed us to detect, in addition to Cek1, several other protein-tyrosine kinases that are expressed during chicken embryonic development, some of which have not been previously identified.

SUBMITTER: Pasquale EB

PROVIDER: S-EPMC297640 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library.

Pasquale E B EB Singer S J SJ

Proceedings of the National Academy of Sciences of the United States of America 19890701 14

To identify the protein-tyrosine kinases that are expressed during chicken embryonic development, a 10-day chicken embryo cDNA expression library was screened with anti-phosphotyrosine antibodies. Of the positive clones isolated, many encoded the same protein-tyrosine kinase, which we designate Cek1 (chicken embryo kinase 1). Its amino acid sequence suggests that the Cek1 protein is a transmembrane tyrosine kinase and presumably the receptor for an unknown ligand. Antibodies prepared to the clon ...[more]

PMID: 2473471

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Project description:BackgroundThe sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation.ResultsWe report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity.ConclusionAerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.

Project description:BackgroundEimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.MethodsWith the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.ResultsAdopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.ConclusionsOur results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

Dataset Information

Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library.

Publications

Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library.

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