MOUSE UBIQUITOUS MITOCHONDRIAL CREATINE-KINASE - GENE ORGANIZATION AND CONSEQUENCES FROM INACTIVATION IN MOUSE EMBRYONIC STEM-CELLS

Individual members of the creatine kinase isoenzyme family (CK; EC 2.7 .3.2), which play a prominent role in energy homeostasis, are encoded by four separate nuclear genes. We have isolated and characterized the complete mouse UbCKmit gene, the product of which is ubiquitously exp ressed and is located in the intermembrane space of mitochondria. Tran scription of this gene is initiated at multiple adjacent positions and the region immediately upstream of these sites shares many features w ith genes encoding housekeeping proteins. These include a high G/C con tent, absence of TATA and CCAAT motifs, and presence of SP1 and AP2 re cognition sequences. In addition, a binding site for HIP1, hormone-res ponsive elements, and three Mt-motifs, known as boxes shared between n uclear genes encoding mitochondrial proteins, were identified, To stud y the functional role of the UbCKmit protein, we have inactivated both UbCKmit alleles in mouse embryonic stem (ES) cells. UbCKmit-deficient cells, obtained by consecutive rounds of gene targeting using homolog ous recombination and drug selection-driven gene conversion events, sh ow no obvious growth disadvantage or abnormal differentiation potentia l. Activities of mitochondrial cytochrome c oxidase and citrate syntha se, as well as the rate of pyruvate oxidation, showed values equal to wild-type cells, indicating a normal aerobic metabolism. Mitochondria of in vivo differentiated knock-out cells were structurally intact, as demonstrated by electron microscopy. Approaches to study the role of the UbCKmit gene further are discussed.