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 expressed and is located in the intermembrane space of mitochondria. Transcription of this gene is initiated at multiple adjacent positions and the region immediately upstream of these sites shares many features with genes encoding housekeeping proteins. These include a high G/C content, absence of TATA and CCAAT motifs, and presence of SP1 and AP2 recognition sequences. In addition, a binding site for HIP1, hormone-responsive elements, and three Mt-motifs, known as boxes shared between nuclear genes encoding mitochondrial proteins, were identified. To study 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 homologous recombination and drug selection-driven gene conversion events, show no obvious growth disadvantage or abnormal differentiation potential. Activities of mitochondrial cytochrome c oxidase and citrate synthase, 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.
Steeghs
K
K
Department of Cell Biology and Histology, Faculty of Medical Sciences, University of Nijmegen, The Netherlands.
Peters
W
W
Brückwilder
M
M
Croes
H
H
Van Alewijk
D
D
Wieringa
B
B
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000235:genetics* / Q000378:metabolism; D005314:Embryonic and Fetal Development; D004789:Enzyme Activation; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D013234:Stem Cells / Q000201:enzymology*
D003402:Creatine Kinase