Conservation of the Caenorhabditis elegans timing gene clk-1 from
yeast to human: a gene required for ubiquinone biosynthesis with
potential implications for aging
*† Lynn M. King,
* Tanya Jonassen,
Douglas J. Wilkin,
Catherine F. Clarke,
Clair A. Francomano
Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 10 Center Dr. MSC 1852, Bldg. 10, Room
10C101, Bethesda, Maryland 20892-1852, USA
Medical Genetics Service, Hospital Necker, INSERM U-393, 149 rue de Sevres, Paris 75743, France
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California–Los Angeles, Los Angeles, California
Received: 15 March 1999 / Accepted 11 June 1999
Abstract. Mutations in the Caenorhabditis elegans gene clk-1
have a major effect on slowing development and increasing life
span. The Saccharomyces cerevisiae homolog COQ7 encodes a
mitochondrial protein involved in ubiquinone biosynthesis and,
hence, is required for respiration and gluconeogenesis. In this
study, RT-PCR and 5Ј RACE were used to isolate both human and
mouse clk-1/COQ7 homologs. Human CLK-1 was mapped to Chr
16(p12–13.1) by Radiation Hybrid (RH) and fluorescence in situ
hybridization (FISH) methods. The number and location of human
CLK1 introns were determined, and the location of introns II and
IV are the same as in C. elegans. Northern blot analysis showed
that three different isoforms of CLK-1 mRNA are present in sev-
eral tissues and that the isoforms differ in the amount of expres-
sion. The functional equivalence of human CLK-1 to the yeast
COQ7 homolog was tested by introducing either a single or mul-
ticopy plasmid containing human CLK-1 cDNA into yeast coq7
deletion strains and assaying for growth on a nonfermentable car-
bon source. The human CLK-1 gene was able to functionally
complement yeast coq7 deletion mutants. The protein similarities
and the conservation of function of the CLK-1/clk-1/COQ7 gene
products suggest a potential link between the production of ubi-
quinone and aging.
A role for genetics in the aging process has long been proposed. In
the nematode Caenorhabditis elegans, mutations in the timing
gene clock-1 (clk-1) have been identified that increase longevity
(Wong et al. 1995; Lakowski and Hekimi 1996; Ewbank et al.
1997). The phenotype of the clk-1 mutants is pleiotropic, and the
reported mutations slow the rate of embryonic and postembryonic
development, cell cycle duration, and growth rate, and also
lengthen the timing of adult behaviors including defecation, swim-
ming, and pharyngeal pumping (Wong et al. 1995). From sequence
comparisons, it has been proposed that clk-1 is conserved in eu-
karyotes including yeast, rat, mouse, and human, although only a
partial sequence was compared for the latter two species in the
initial report (Ewbank et al. 1997).
In the yeast Saccharomyces cerevisiae, COQ7 (Marbois and
Clarke 1996), which is identical to the independently isolated
CAT5 gene (Proft et al. 1995), has been identified as the clk-1
homolog (Ewbank et al. 1997). COQ7 is required for the synthesis
of ubiquinone (coenzyme Q, or Q) and influences respiration
(Marbois and Clarke 1996). Q is a lipid soluble component of
mitochondria that functions in respiration by transporting electrons
in the inner mitochondrial membrane of eukaryotes (Brandt and
Trumpower 1994). COQ7 (CAT5) also functions in the derepres-
sion of gluconeogenic enzymes that accompanies the transfer of
yeast from glucose to a nonfermentable carbon source such as
glycerol or ethanol (Proft et al. 1995). Recently, it has been shown
that the yeast Coq7p is a mitochondrial protein directly involved in
Q synthesis, and its effect on gluconeogenesis is a secondary con-
sequence of the defect in respiration (Jonassen et al. 1998). The C.
elegans clk-1 and rat Coq7 genes rescue coq7 deletion mutants of
yeast by allowing growth on a nonfermentable carbon source, thus
demonstrating a conserved biochemical function of COQ7/clk-1/
Coq7 in Q biosynthesis (Jonassen et al. 1996; Ewbank et al. 1997).
This report describes the full-length human and mouse clk-1
cDNAs, demonstrates the functional equivalence of human CLK-1
to yeast COQ7, provides a physical map location and genomic
organization for the human CLK-1 gene, and shows the levels of
human CLK-1 mRNA in several tissues. The data show great
nucleotide sequence conservation of human, mouse, and rat CLK-
1/Coq7 homologs. In addition, the functional complementation
studies of yeast coq7 deletion mutants by human, nematode, and
rat CLK-1/COQ7 shows conservation of function in Q biosynthe-
sis across phylogenetic boundaries.
Materials and methods
Isolation and sequencing of human CLK-1 and mouse Coq7 cDNA.
We used the reverse-transcription polymerase chain reaction (RT-PCR)
and the 5Ј and 3Ј rapid amplification of cDNA ends (RACE) procedures to
obtain the full-length human CLK-1 and mouse Coq7 cDNA sequences.
Total RNA from human heart (Clontech, Palo Alto, CA) or liver [isolated
by standard methods (Chomczynski and Sacchi 1987)] was used to gen-
erate reverse transcribed cDNA for PCR amplification (RT-PCR). Mouse
RNA was isolated from heart tissue by a standard method (Chomczynski
and Sacchi 1987). RT-PCR was performed with either the Smart PCR
cDNA synthesis kit (Clontech), the SuperScript Preamplification System
kit (Gibco BRL, Rockville, MD), or the Genamp kit (Perkin Elmer, Foster
City, CA). Oligonucleotide primers for amplifying human CLK-1 were
designed based on rat Coq7 (GenBank accession number U46149) and
partial human CLK-1 (GenBank accession number U81276) cDNA se-
quences. Oligonucleotide primers for amplifying mouse Coq7 were de-
signed based on a partial sequence (GenBank accession number U81277).
Kits for 5Ј and 3Ј RACE (Gibco, BRL) were used to amplify 5Ј and 3Ј
* The first two authors contributed equally to this work.
† Present address: Department of Endocrinology and Medicine, V.A.
Medical Center, 650 E. Indian School Road, Phoenix, AZ 85012, USA.
Correspondence to: C.A. Francomano
Mammalian Genome 10, 1000–1004 (1999).
© Springer-Verlag New York Inc. 1999