Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.

A high-density genetic map is essential for comparative genomic studies and fine mapping of QTL, and can also facilitate genome sequence assembly. Here, a high density genetic map of Asian seabass was constructed with 3321 SNPs generated by sequencing 144 individuals in a F2 family. The length of the map was 1577.67 cM with an average marker interval of 0.52 cM. A high level of genomic synteny among Asian seabass, European seabass, Nile tilapia and stickleback was detected. Using this map, one genome-wide significant and five suggestive QTL for growth traits were detected in six linkage groups (i.e. LG4, LG5, LG11, LG13, LG14 and LG15). These QTL explained 10.5–16.0% of phenotypic variance. A candidate gene, ACOX1 within the significant QTL on LG5 was identified. The gene was differentially expressed between fast- and slow-growing Asian seabass. The high-density SNP-based map provides an important tool for fine mapping QTL in molecular breeding and comparative genome analysis.

The extreme halophile Halobacterium halobium synthesizes intracellular gas-filled vesicles that confer buoyancy. A cluster of 13 genes on the 200-kb endogenous plasmid pNRC100 has been implicated in the biosynthesis of gas vesicles. Here, we show that two gas vesicle proteins are encoded by genes in the rightward operon, gvpA and gvpC, by Western blotting (immunoblotting) analysis with antibodies directed against LacZ-GvpA and LacZ-GvpC fusion proteins. Our results are consistent with previous data showing that the gvpA gene product is the major gas vesicle protein and demonstrate for the first time that thegvpC gene product is also present in H. halobium gas vesicles. Northern (RNA) blotting analysis showed two RNA species, an abundant 0.35-kb transcript of gvpA and a minor 2.5-kb transcript of gvpAC, and a third gene 3' to gvpAC, named gvpN. The gvpN gene encodes a hypothetical acidic protein with a molecular weight of 39,000 and a nucleotide binding motif. We used a site-directed mutagenesis method involving double recombination in Escherichia coli to insert a kanamycin resistance cassette just beyond the stop codon of gvpN. Introduction of the mutated gene cluster into an H. halobium mutant with a deletion of the entire gas vesicle gene cluster resulted in gas vesicle-positive transformants; this result suggests that gvpN is the last gene of the rightward gas vesicle transcription unit. We discuss the design and utility of the kanamycin resistance cassette for the mutagenesis of other genes in large operons.Many aquatic bacteria, such as the extreme halophile Halobacterium halobium, produce intracellular gas-filled vesicles that provide buoyancy and allow cells to float at the surface of liquid cultures (3, 27). The vesicles contain ambient gas and are surrounded by a rigid membrane composed of protein with little or no lipids. The vesicle shape is generally cylindrical in the midsection and conical at the ends. There are two types of gas vesicles in wild-type H. halobium NRC-1; the vast majority (99%) are lemon or spindle shaped, and a few (1%) are more elongated or cylindrical. Studies on cyanobacterial vesicles have suggested that vesicle biosynthesis is initiated with the cones and proceeds by the addition of protein subunits to the central cylindrical region (18,31). The accumulation of gases within vesicles is thought to result from the differential permeability of the membrane to gases and water (34).Our initial interest in gas vesicles resulted from the observation that H. halobium gas vesicle-deficient (Vac-) mutants arise at an extremely high frequency, about 1%. Cloning of the first gas vesicle protein gene, gvpA, opened the way to an investigation of the genetic basis of the mutation (6, 29). The H. halobium gvpA gene was found to map to a 200-kb plasmid, pNRC100, which suffers rearrangements, such as insertions, deletions, and inversions, at a high frequency.

To study the functions of the 13 gyp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the gyp gene cluster and introduced a kanamycin resistance (K) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gyp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the c cassette in any of the gyp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the K cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the K insertions resulted in phenotypic reversion. Electron microscopic analysis of the K mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicles than in the wild type, while interruptions ofgvpF, -G, -H, -I, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate that gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL,