Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.     

Specific expression of pfkfb4 gene in spermatogonia germ cells and analysis of its 5'-flanking region

The results presented demonstrate the expression of pfkfb4 gene in adult testis and in a mouse spermatogonia germ cell line (GC-1spg). The genomic organization of the human pfkfb4 gene shows the existence of 14 exons and 13 introns, spanning 45 kb. A detailed analysis of the 5'-flanking region by transient transfection assays with different 5'-deletion promoter constructs in GC-1spg and mouse sertoli cells (TM-4), allows us to define the minimal promoter unit, containing several GC-rich and ETF sequences along the first -141 nucleotides involved in basal expression. This gene is activated by serum and chemical hypoxia (CoCl(2) treatment) whereas beta-estradiol decreases its expression.