Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel.

Human TWIK-1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK-1, a mammalian TWIK-1-related K+ channel. Despite a low amino acid identity between TWIK-1 and TREK-1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore-forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK-1 are inwardly rectifying while K+ currents generated by TREK-1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK-1 currents are insensitive to pharmacological agents that block TWIK-1 activity such as quinine and quinidine. Extensive inhibitions of TREK-1 activity are observed after activation of protein kinases A and C. TREK-1 currents are sensitive to extracellular K+ and Na+. TREK-1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK-1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.     

Human Microtubule-Associated Protein 1a (MAP1A) Gene: Genomic Organization, cDNA Sequence, and Developmental- and Tissue-Specific Expression

Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity. MAP1a (HGMW-approved symbol MAP1A) stabilizes microtubules in postnatal axons. We describe human MAP1a's genomic organization and deduced cDNA and amino acid sequences. MAP1a is a single-copy gene spanning 10.5 kb. MAP1a coding sequence is contained in five exons. Translation begins in exon 3. Human MAP1a contains 2805 amino acids (predicted molecular weight 306.5 kDa) and is slightly larger than rat MAP1a (2774 amino acids). Like rat and bovine MAP1a, human MAP1a contains conserved tubulin binding motifs in the amino-terminal region. The carboxy-terminal portion contains a conserved pentadecapeptide that is present in the light chain portion of rat and bovine MAP1a/LC2 polyprotein. We show that human MAP1a gene expression occurs almost exclusively in the brain and that there is approximately 10-fold greater gene expression in adult brain compared to fetal brain. Strong, interspecies conservation between human and rat MAP1a cDNA and amino acid sequences indicates important relationships between MAP1a's function and its primary amino acid sequence.     

POLLEN DISCOUNTING AND THE SPREAD OF A SELFING VARIANT IN TRISTYLOUS EICHHORNIA PANICULATA: EVIDENCE FROM EXPERIMENTAL POPULATIONS

Floral traits that increase self-fertilization are expected to spread unless countered by the effects of inbreeding depression, pollen discounting (reduced outcross pollen success by individuals with increased rates of self-fertilization), or both. Few studies have attempted to measure pollen discounting because to do so requires estimating the male outcrossing success of plants that differ in selfing rate. In natural populations of tristylous Eichhornia paniculata, selfing variants of the mid-styled morph are usually absent from populations containing all three style morphs but often predominate in nontrimorphic populations. We used experimental garden populations of genetically marked plants to investigate whether the effects of population morph structure on relative gamete transmission by unmodified (M) and selfing variants (M‘) of the mid-styled morph could explain their observed distribution. Transmission through ovules and self and outcross pollen by plants of the M and M’ morphs were compared under trimorphic, dimorphic (S morph absent), and monomorphic (L and S morphs absent) population structures. Neither population structure nor floral morphology affected female reproductive success, but both had strong effects on the relative transmission of male gametes. The frequency of self-fertilization in the M' morph was consistently higher than that of the M morph under all morph structures, and the frequency of self-fertilization by both morphs increased as morph diversity of experimental populations declined. In trimorphic populations, total transmission by the M and M' morphs did not differ. The small, nonsignificant increase in selfing by the M' relative to the M morph was balanced by decreased outcross siring success, particularly on the S morph. In populations lacking the S morph, male gamete transmission by the M' morph was approximately 1.5 times greater than that by the M morph because of both increased selfing and increased success through outcross pollen donation. Therefore, gamete transmission strongly favored the M' morph only in the absence of the S morph, a result consistent with the distribution of the M' morph in nature. This study indicates that floral traits that alter the selfing rate can have large and context-dependent influences on outcross pollen donation.     

A lipoprotein signal peptide encoded by the staphylococcal conjugative plasmid pSK41 exhibits an activity resembling that of Enterococcus faecalis pheromone cAD1.

The traH gene of the staphylococcal conjugative plasmid pSK41 has recently been shown to encode a lipoprotein (N. Firth, K. P. Ridgway, M. E. Byrne, P. D. Fink, L. Johnson, I. T. Paulsen, and R. A. Skurray, Gene 136:13-25, 1993). Here we report that traH encodes a product recognized as a pheromone by Enterococcus faecalis cells harboring the conjugative plasmid pAD1. The mature traH product is not essential for this phenomenon, as expression of pheromone-like activity was found to require sequences encoding only the pro-TraH signal peptide.     

Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape.     

Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.     

Analysis of human extrachromosomal DNA elements originating from different β-satellite subfamilies

By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of -satellite DNA. Further experiments have shown that the three sequences belong to at least two different -satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that -satellite sequences obtained from the spcDNA of human cells have been assigned to -satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that -satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.This paper is dedicated to Prof. Dr. Ulrich Wolf on his 60th birthday, January 1993     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.