Regulated expression of ras gene constructs in Dictyostelium transformants

• Published in: Proceedings of the National Academy of Sciences - PNAS 1985-10, Vol.82 (20), p.7005-7009
• Main Authors: Reymond, C.D, Nellen, W, Firtel, R.A
• Format: Article
• Language: English
• Subjects: Base Composition; Base Sequence; Biological and medical sciences; Complementary DNA; CONTROL GENETICO; Cultured cells; DICTYOSTELIUM; Dictyostelium - genetics; DNA; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; GENE; Gene expression; Gene expression regulation; GENES; GENETIC CONTROL; Genetic Vectors; LUTTE GENETIQUE; Messenger RNA; Molecular and cellular biology; Molecular genetics; Oncogenes; ras genes; ras Proteins; RNA; RNA, Messenger - genetics; Somatic cells; Transcription, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.82.20.7005

Constructs were made in which ≈ 500 base pairs of the 5′flanking region of the ras gene of Dictyostelium discoideum and variable amounts of the coding region were linked to a ras cDNA in a transformation vector. These constructs then were used to transform Dictyostelium cells and their regulation was examined. In Dictyostelium transformants, transcripts from the ras gene constructs were found at high levels in cells in fast-shaking cultures containing cAMP, whereas transcripts were either not detectable or present at very low levels in cultures lacking exogenous cAMP. In slow-shaking culture, a significantly lower level of ras RNA was detected. When normal developing aggregates were dissociated, RNA from the ras constructs decreased rapidly but then reaccumulated in the presence of cAMP. These results show that the sequences necessary for the response to external cAMP are present within an ≈ 650-base-pair region upstream from the ATG start codon and/or within portions of the protein-coding region. Moreover, the proper regulation of ras gene expression in high-copy-number transformants suggests that trans-acting factors which may control transcription are not limiting. Vector constructs were also examined in which the cDNA was present in the opposite orientation compared to the gene fragment (antisense orientation). When these were transfected into cells, no transformants were obtained, suggesting that expression of the ras gene is essential for vegetative growth.