TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells.

The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels.

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells.

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel.

Myosin-VIIb, a novel unconventional myosin, is a constituent of microvilli in transporting epithelia.

Mouse myosin-VIIb, a novel unconventional myosin, was cloned from the inner ear and kidney. The human myosin-VIIb (HGMW-approved symbol MYO7B) sequence and exon structure were then deduced from a human BAC clone. The mouse gene was mapped to chromosome 18, approximately 0.

Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin.

Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil.

BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels.

The recently defined DEG/ENaC superfamily of sodium channels includes subunits of the amiloride-sensitive epithelial sodium channel (ENaC) of vertebrate colon, lung, kidney, and tongue, a molluscan FMRFamide-gated channel (FaNaC), and the nematode degenerins, which are suspected mechanosensory channels. We have identified two new members of this superfamily (BNaC1 and BNaC2) in a human brain cDNA library. Phylogenetic analysis indicates they are equally divergent from all other members of the DEG/ENaC superfamily and form a new branch or family.

Ikaros, an early lymphoid-specific transcription factor and a putative mediator for T cell commitment.

In a screen for transcriptional regulators that control differentiation into the T cell lineage, a complementary DNA was isolated encoding a zinc finger protein (Ikaros) related to the Drosophila gap protein Hunchback. The Ikaros protein binds to and activates the enhancer of a gene encoding an early T cell differentiation antigen, CD3 delta. During development, Ikaros messenger RNA was first detected in the mouse fetal liver and the embryonic thymus when hematopoietic and lymphoid progenitors initially colonize these organs; no expression was observed in the spleen or the bone marrow.

Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16.

We described previously the isolation of a Saccharomyces cerevisiae 3-methyladenine (3-MeAde) DNA glycosylase repair gene (MAG) by its expression in glycosylase-deficient Escherichia coli alkA tag mutant cells and its ability to rescue these cells from the toxic effects of alkylating agents. Here we extend this cross-species functional complementation approach to the isolation of a full-length human 3-MeAde DNA glycosylase cDNA that rescues alkA tag E. coli from killing by methyl methanesulfonate, and we have mapped the gene to human chromosome 16.

Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.

We previously identified and characterized biochemically an O6-methylguanine (O6MeG) DNA repair methyltransferase (MTase) in the yeast Saccharomyces cerevisiae and showed that it recognizes both O6MeG and O4-methylthymine (O4MeT) in vitro. Here we characterize the cloned S. cerevisiae O6MeG DNA MTase gene (MGT1) and determine its in vivo role in protecting yeast from DNA alkylation damage.

Saccharomyces cerevisiae 3-methyladenine DNA glycosylase has homology to the AlkA glycosylase of E. coli and is induced in response to DNA alkylation damage.

We previously cloned a DNA fragment from Saccharomyces cerevisiae that suppressed the alkylation sensitivity of Escherichia coli glycosylase deficient mutants and we showed that it apparently contained a gene for 3-methyl-adenine DNA glycosylase (MAG). Here we establish the identity of the MAG gene by sequence analysis and describe its in vivo function and expression in yeast cells. The MAG DNA glycosylase specifically protects yeast cells against the killing effects of alkylating agents.

Cloning a eukaryotic DNA glycosylase repair gene by the suppression of a DNA repair defect in Escherichia coli.

If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes. We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents. The cloned yeast gene was then used to generate a mutant strain of S.

Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes.

The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents. We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer- HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents. One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa O6-methylguanine-DNA methyltransferase.