The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

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Dorsal and ventral aspects of the eye are distinct from the early stages of development. The developing eye cup grows dorsally, and the choroidal fissure is formed on its ventral side. Retinal axons from the dorsal and ventral retina project to the ventral and dorsal tectum, respectively. Misexpression of the Tbx5 gene induced dorsalization of the ventral side of the eye and altered projections of retinal ganglion cell axons. Thus, Tbx5 is involved in eye morphogenesis and is a topographic determinant of the visual projections between retina and tectum.

Much progress has been made in understanding limb development. Most genes are expressed equally and in the same pattern in the fore- and hindlimbs, which nevertheless develop into distinct structures. The T-box genes Tbx5 and Tbx4, on the other hand, are expressed differently in chick wing (Tbx5) and leg (Tbx4) buds. Molecular analysis of the optomotor blind gene, which belongs to the same family of transcription factors, has revealed that this gene is involved in the transdetermination of Drosophila wing and leg imaginal discs. In addition, expression of Tbx5 and Tbx4 correlates well with the identity of ectopic limb buds induced by fibroblast growth factor. Thus, it is thought that Tbx5 and Tbx4 might be involved in determining limb identity. Another candidate is the Pitx1 gene, which encodes a bicoid-type homeodomain transcription factor that is expressed in leg buds. Here we determine the importance of these factors in establishing limb identity.

A tight loop between members of the fibroblast growth factor and the Wnt families plays a key role in the initiation of vertebrate limb development. We show for the first time that Tbx5 and Tbx4 are directly involved in this process. When dominant-negative forms of these Tbx genes were misexpressed in the chick prospective limb fields, a limbless phenotype arose with repression of both Wnt and Fgf genes By contrast, when Tbx5 and Tbx4 were misexpressed in the flank, an additional wing-like and an additional leg-like limbs were induced, respectively. This additional limb formation was accompanied by the induction of both Wnt and Fgf genes These results highlight the pivotal roles of Tbx5 and Tbx4 during limb initiation, specification of forelimb/hindlimb and evolution of tetrapod limbs, placing Tbx genes at the center of a highly conserved genetic program.

The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.

Extensive misexpression studies were carried out to explore the roles played by Tbx5, the expression of which is excluded from the right ventricle (RV) during cardiogenesis. When Tbx5 was misexpressed ubiquitously,ventricular septum was not formed, resulting in a single ventricle. In such heart, left ventricle (LV)-specific ANF gene was induced. In search of the putative RV factor(s), we have found that chick Tbx20 is expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene was repressed. When misexpression was spatially partial, leaving small Tbx5-negative area in the right ventricle,ventricular septum was shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induced an ectopic boundary of Tbx5-positive and-negative regions in the right ventricle, at which an additional septum was formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND were changed with definitive cardiac abnormalities. Furthermore, we report that human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation was abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, our data indicate that Tbx5 specifies the identity of LV through tight interactions among several heart-specific factors, and highlight the essential roles of Tbx5 in cardiac development.

Cloning of the gene for undecaprenyl diphosphate synthase was successful, providing the first primary structure for any prenyltransferase that catalyzes Zprenyl chain elongation. A genomic DNA library of Micrococcus luteus B-P 26 was constructed in Escherichia coli, and the recombinant clones were grown on nylon membranes. The membrane was incubated directly by floating it on a reaction mixture containing radiolabeled isopentenyl diphosphate, nonlabeled farnesyl diphosphate, and Mg 2؉ . Only the clones harboring plasmids encoding prenyltransferases could take up the substrates to synthesize and accumulate radiolabeled products inside the cells in amounts large enough to be detectable by autoradiography. Four positive colonies were found among about 4,000 bacterial colonies of the genomic DNA library. Two of them carried the gene for undecaprenyl diphosphate synthase, which catalyzes the Z-prenyl chain elongation, and the others carried the (all-E)-hexaprenyl diphosphate synthase genes (hexs-a and hexs-b; Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Bacteriol. 180, 1578 -1581). The undecaprenyl diphosphate synthase, which had a predicted molecular mass of 28.9 kDa, was overproduced in E. coli cells by applying a soluble expression system, and it was purified to near homogeneity. The deduced primary structure of the Z-prenyl chain-elongating enzyme is totally different from those of E-prenyl chain-elongating enzymes, which have characteristic conserved regions, including aspartate-rich motifs.In the biosynthesis of isoprenoid compounds, including sterols, respiratory quinones, carotenoids, glycosyl carrier lipids, natural rubber, and prenyl proteins, all of these compounds are derived from linear prenyl diphosphates, which are synthesized by sequential condensations of isopentenyl diphosphate (IPP) 1 with allylic prenyl diphosphates. These condensations are catalyzed by a family of prenyltransferases.During the last decade the structural genes for many kinds of prenyltransferases that catalyze E-type prenyl chain elongation have been cloned and characterized (1). Multiple alignments of the deduced amino acid sequences of the E-type prenyl chain-elongating enzymes, including farnesyl diphosphate synthase (FPS), geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase (HexPS), heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase, and decaprenyl diphosphate synthase, have shown the presence of two characteristic aspartate-rich DDXXD motifs and several other conserved regions in the primary structures of prenyltransferases (2-4). X-ray crystallography (5) and site-directed mutagenesis studies of FPS (6 -15) and geranylgeranyl diphosphate synthase (16,17) have revealed the precise mechanisms of prenyl chain elongation and determination of the product chain length.On the other hand, nothing is known about the structure of Z-prenyl chain-elongating enzymes. Consequently, only limited information is available about the molecular mechanism of enzymatic Z-prenyl chain elongation.In bacteria, und...