Analysis of human Per4.

The molecular mechanism of the circadian pacemaker depends on the oscillatory expression of clock gene constituents. The Drosophila period gene is central to the clock mechanism in these animals. Three homologs of this gene identified in mice (mPer1-3) and humans (hPer1-3) display rhythmic expression and are important for normal clock function. Recently, analysis of the draft sequence of the human genome has revealed the presence of a fourth Per gene family member. Surprisingly, the deduced hPer4 cDNA has no open reading frame encoding a full-length PER-like protein. This sequence is characterized by numerous deletions, insertions, frame shifts and base pair changes, and its genomic structure is devoid of introns. The presence of an MER-2 mobile element fossil within the Per4 locus predicted that this gene would also be present in non-human primates. Rhesus monkey Per4 displays similar sequence anomalies and is 92.8% identical to hPer4. Sequence comparisons indicate that Per4 originated from a Per3 predecessor and that it is relatively new to the Period gene family. We conclude that hPer4 and RmPer4 are pseudogenes and descended from the retrotransposition of an ancestral Per3 gene.

A time-less function for mouse timeless.

The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.

Sex-linked period genes in the silkmoth, Antheraea pernyi: implications for circadian clock regulation and the evolution of sex chromosomes.

Regulation of the period (per) gene is a critical feature of circadian clock function in insects. Here, we show that per is sex-linked in the silkmoth, Antheraea per-nyi. The previously described silkmoth per gene is found on the Z chromosome. Silkmoth per is not dosage compensated at either the RNA or the protein level. Although earlier studies showed the presence of an oscillating endogenous antisense per transcript, we show that this transcript comes from a locus on the female-specific W chromosome. We further demonstrate the presence of a homolog of per on W that encodes a truncated protein. Rhythmicity of male (ZZ) moths demonstrates that neither of the W-linked per-like genes is essential for clock function. The presence of a per allele with duplications on W provides insight into the evolution of the sex chromosomes.

Electrophorus electricus as a model system for the study of membrane excitability.

The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus electricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorus have provided a unique model system that is both specialized and appropriate for the study of excitable cell membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by means of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secretory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these other excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas other excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofilament cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions and the proteins associated with them, it provides a highly specialized membrane model system. This review examines the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane proteins involved. The valuable contributions that electric tissue continues to make toward the understanding of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a model system for the study of the regulation of membrane excitability by second messengers and signal transduction pathways.

A major second messenger mediator of Electrophorus electricus electric tissue is CaM kinase II.

Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca2+ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca(2+)-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca2+/calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca(2+)-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes.

Annexin IV inhibits calmodulin-dependent protein kinase II-activated chloride conductance. A novel mechanism for ion channel regulation.

Ca(2+)-activated Cl- current (ICl,Ca) in colonic T84 cells is inhibited by the specific peptide inhibitor of Ca2+/calmodulin-dependent kinase II (CaM KII). Annexin IV, a Ca(2+)-dependent phospholipid binding protein also inhibits Ca(2+)-dependent anion current activation (Kaetzel, M.A., Chan, H.-C., Dubinsky, W.P., Dedman, J.R., and Nelson, D.J. (1994) J. Biol. Chem. 269, 5297-5302). Intracellular injection of antibodies against annexin IV enhances current activation; this activation is inhibited by the peptide inhibitor of CaM KII. Intracellular application of autonomously active CaM KII in the presence of ATP induced a Cl- current similar to that activated by the Ca2+ ionophore A23187. Current activation by the exogenous kinase was completely inhibited in the presence of purified annexin IV. In vitro, annexin IV does not inhibit CaM KII activity nor does it act as a substrate for CaM KII. Thus, it appears that annexin IV inhibits phosphorylation-dependent anion conductance activation by preventing CaM KII-ion channel interaction rather than by direct interaction with the enzyme itself. These findings suggest a novel mechanism by which Ca(2+)-dependent membrane binding proteins, cytoplasmic kinases, and ion channels interact to regulate membrane conductance. The characterization of unique channel regulatory pathways in Cl- transporting epithelia may identify potential avenues of alternate therapy to compensate for the loss of functional Cl- channels in the disease of cystic fibrosis.