To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.

The outer membrane of mitochondria contains channels called VDAC (mitochondrial porin), which are formed by a single 30-kDa protein. Cysteine residues introduced by site-directed mutagenesis at sites throughout Neurospora crassa VDAC (naturally devoid of cysteine) were specifically biotinylated prior to reconstitution into planar phospholipid membranes. From previous studies, binding of streptavidin to single biotinylated sites results in one of two effects: reduced single-channel conductance without blockage of voltage gating (type 1) or locking of the channels in a closed conformation (type 2). All sites react with streptavidin only from one side of the membrane. Here, we extend this approach to VDAC molecules containing two cysteines and determine the location of each biotinylated residue with respect to the other within the membrane. When a combination of a type 1 and a type 2 site was used, each site could be observed to react with streptavidin. Two sets of sites located on opposite surfaces of the membrane were identified, thereby establishing the transmembrane topology of VDAC. A revised folding pattern for VDAC, consisting of 1 ␣ helix and 13 ␤ strands, is proposed by combining these results with previously obtained information on which sites are lining the aqueous pore.VDAC channels are found in the mitochondrial outer membrane of cells from all eukaryotic kingdoms (1). They are the major pathways by which metabolites, including ATP, pass through this outer membrane (2, 3) and, therefore, are likely to play important roles in the regulation of mitochondrial functions.Each VDAC channel consisting of a single 30-kDa polypeptide (4, 5) forms an aqueous pore ϳ3 nm in diameter (6, 7). Detailed information on the voltage-gating properties of VDAC and the associated conformational rearrangements have been obtained from studies of channels reconstituted into planar phospholipid membranes. These studies have demonstrated that, in response to transmembrane potentials of 30 mV or above, part of the channel wall, which serves as the voltage sensor, moves out of the membrane resulting in channel closure (8 -10). These closed channels are essentially impermeable to ATP and have a reduced permeability to organic anions (2, 11). However, they are still permeable to small non-electrolytes (up to 1.8 nm in diameter) and to small monovalent cations (12). VDAC can close in response to transmembrane potentials of both polarities, due to the presence of two different gating processes.The transmembrane topology of VDAC molecules has yet to be firmly established. Theoretical considerations predicted a "␤-barrel" structure (13,14). "Sided" ␤ strands that would be appropriate to separate an apolar from a polar environment, were tested by the use of site-directed mutations (15). If engineered charge changes in proposed transmembrane segments affected channel selectivity, the associated protein segment was proposed to be a transmembrane strand. A lack of effect resulted in assignment of the segment to the membrane surface. In thes...

The motion of the sensor regions in a mitochondrial voltage-gated channel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin. Site-directed mutagenesis was used to introduce single cysteine residues into Neurospora crassa VDAC (naturally lacks cysteine). These were chemically biotinylated and reconstituted into planar phospholipid membranes. In the 19 sites examined, only two types of results were observed upon streptavidin addition: in type 1, channel conductance was reduced, but voltage gating could proceed; in type 2, channels were locked in a closed state. The result at type 1 sites is interpreted as streptavidin binding to sites in static regions close to the channel opening. The binding sterically interferes with ion flow. The result at type 2 sites indicates that these are located on a mobile domain and coincide with the previously identified sensor regions. The findings are consistent with closure resulting from the movement of a domain from within the transmembrane regions to the membrane surface. No single site was accessible to streptavidin from both membrane surfaces, indicating that the motion is limited. From the streptavidin-induced reduction in conductance at type 1 sites, structural information was obtained about the location of these sites.

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We have characterized the 5' and 3' ends of the rat beta 1-adrenergic receptor transcript using RNase protection assays and have used transient transfection analysis to identify regions of the beta 1-adrenergic gene 5'-flanking sequences which are important for expression. The transcript has multiple start sites, occurring primarily in two clusters at bases -250 and -280, relative to the first base of the initiation codon. Two potential polyadenylation signals at +2450 and +2732 are both functional, although the site at +2732 is preferred both in C6 glioma cells and in heart tissue. Characterization of the gene by transient transfection analysis has identified a region between bases -389 and -325 which is necessary for expression. The specific deletion of a potentially functional inverted CCAAT sequence within this region does not significantly alter activity. In addition to the region from -389 and -325, deletion of the bases between -1 and -159 and between -186 and -211 significantly alters expression. Both of these regions are down-stream from the beta 1-adrenergic receptor gene start sites and may function either through regulation of transcription or through alteration of the transcript structure.