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cDNA cloning of a murine homologue of Drosophila single-minded, its mRNA expression in mouse development, and
1Department of Chemistry, Faculty of Science, Tohoku University, Sendai, Japan.
Abstract:
A combination of the RT-PCR method and subsequent screening of the cDNA library of mouse skeletal muscle with the cDNA isolated by RT-PCR used as a probe led to isolation of cDNAs encoding a polypeptide (mSim) with bHLH and PAS domains which show high similarity to the corresponding regions of Drosophila Sim, a master regulator in neurogenesis. Experiments using a GST-fusion protein demonstrated that mSim heterodimerizes with Arnt (Ah receptor nuclear translocator), even more efficiently than AhR (Ah receptor) does with Arnt. RNA blot analysis using RNAs from various tissues of mice indicated that mSim transcript is expressed in several limited tissues such as muscle, kidney and lung of adult animals. Distribution of mSim mRNA was always accompanied with that of Arnt. All the results suggest a regulatory role of mSim in partnership with Arnt. Chromosomal location of the mSim gene was determined by fluorescent in situ hybridization to be localized on the C3.3-C4 band of mouse chromosome 16 which is syntenic with the human chromosome 21q22 carrying the Down syndrome critical region, where a gene highly homologous to the Drosophila sim was localized. Whole mount in situ hybridization using a unique part of mSim cDNA sequence showed that mSim mRNA was expressed in the ventral diencephalon, branchial arches and limbs. These findings will provide an approach to the cause of the Down syndrome as well as the elucidation of the functional roles of mSim in animal development.
Insights
Researchers identified a novel mouse protein, mSim, with bHLH and PAS domains, crucial for development. mSim partners with Arnt and its gene is located on chromosome 16, offering insights into Down syndrome.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- The Drosophila Sim protein is a key regulator in neurogenesis.
- Understanding novel transcription factors and their roles in mammalian development is crucial.
Purpose of the Study:
- To isolate and characterize novel genes involved in mammalian development.
- To investigate the function and interactions of a newly identified mouse protein, mSim.
- To explore the potential link between mSim and developmental disorders.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) and cDNA library screening.
- GST-fusion protein experiments to assess protein-protein interactions.
- RNA blot analysis and whole mount in situ hybridization for gene expression analysis.
- Fluorescent in situ hybridization (FISH) for chromosomal localization.
Main Results:
- Isolation of cDNAs encoding mSim, a protein with bHLH and PAS domains homologous to Drosophila Sim.
- mSim efficiently heterodimerizes with Arnt, a known transcription factor.
- mSim transcripts are detected in specific adult mouse tissues (muscle, kidney, lung) and during embryonic development (ventral diencephalon, branchial arches, limbs).
- The mSim gene is located on mouse chromosome 16, syntenic with the human chromosome 21q22 (Down syndrome critical region).
Conclusions:
- mSim plays a regulatory role in partnership with Arnt.
- The localization of the mSim gene provides a potential link to Down syndrome.
- mSim is a significant factor in mammalian development, particularly in neurogenesis and organogenesis.