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Cloning and expression of human NF-YC
M Bellorini1, K Zemzoumi, A Farina
1Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, Milan, Italy.
Gene
|July 1, 1997
Summary
The human NF-YC gene was cloned, revealing high conservation with rat sequences. A functional CCAAT-binding mini-NF-Y complex was constructed, with NF-YC mRNA levels varying across tissues, notably in the testis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The CCAAT box is a crucial eukaryotic promoter element.
- NF-Y (CBF) is a conserved heterotrimeric protein that binds to the CCAAT box.
- NF-YB and NF-YC subunits possess a histone-like motif.
Purpose of the Study:
- To clone the complete cDNA for the human NF-YC gene.
- To characterize the human NF-YC protein and its role in CCAAT binding.
- To investigate the expression patterns of NF-YC in human tissues.
Main Methods:
- Cloning of the human NF-YC gene.
- Expression and purification of the yeast homology domain of NF-YC in bacteria.
- Electrophoretic mobility shift assay (EMSA) to form a mini-NF-Y complex.
- Quantitative analysis of NF-YC mRNA levels in human tissues.
Main Results:
- The human NF-YC ORF encodes a 335 amino acid protein with high sequence identity to the rat ortholog.
- A functional CCAAT-binding mini-NF-Y complex was successfully constructed using bacterial expressed domains of NF-YA, NF-YB, and NF-YC.
- NF-YC mRNA expression was found to be similar across most human tissues, with notable exceptions in the testis.
Conclusions:
- The human NF-YC gene sequence is highly conserved across species.
- The histone-like domains of NF-YA, NF-YB, and NF-YC are sufficient for CCAAT binding.
- Tissue-specific expression of NF-YC suggests potential specialized roles in certain organs, like the testis.