Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 nontransposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

viruses were injected to follicles on both wings for later studies. Chickens were raised in cages and observed on a daily basis over a two-month period. The regenerated feathers were plucked and examined with a dissection or scanning electron micrograph microscope for abnormalities compared with normal primary remiges. Histology and in situ hybridizationParaffin sections (5 mm) were stained with haematoxylin and eosin or prepared for in situ hybridization following routine procedures 26 . Cryostat sections (10 mm) were stained with X-gal. TUNEL staining was performed using a kit (Roche). Nonradioactive wholemount or section in situ hybridization or section in situ hybridization was performed according to the protocol described 22,26 . After hybridization, sections were incubated with an antidigoxigenin Fab conjugated to alkaline phosphatase (Boehringer Mannheim). Colour was detected by incubating with a Boehringer Mannheim purple substrate (Roche).

To determine the chromosomal positions of expressed rice genes, we have performed an expressed sequence tag (EST) mapping project by polymerase chain reaction-based yeast artificial chromosome (YAC) screening. Specific primers designed from 6713 unique EST sequences derived from 19 cDNA libraries were screened on 4387 YAC clones and used for map construction in combination with genetic analysis. Here, we describe the establishment of a comprehensive YAC-based rice transcript map that contains 6591 EST sites and covers 80.8% of the rice genome. Chromosomes 1, 2, and 3 have relatively high EST densities, approximately twice those of chromosomes 11 and 12, and contain 41% of the total EST sites on the map. Most of the EST-dense regions are distributed on the distal regions of each chromosome arm. Genomic regions flanking the centromeres for most of the chromosomes have lower EST density. Recombination frequency in these regions is suppressed significantly. Our EST mapping also shows that 40% of the assigned ESTs occupy only approximately 21% of the entire genome. The rice transcript map has been a valuable resource for genetic study, gene isolation, and genome sequencing at the Rice Genome Research Program and should become an important tool for comparative analysis of chromosome structure and evolution among the cereals.

Background: ␣-L-Rhamnosidase hydrolyzes ␣-linked L-rhamnose from rhamnoglycosides or polysaccharides. Results: The crystal structure of Streptomyces avermitilis ␣-L-rhamnosidase belonging to glycoside hydrolase family 78 was determined. Conclusion:The L-rhamnose complexed structure revealed the catalytic mechanism of the enzyme and a calcium-dependent carbohydrate-binding module. Significance: Efficient catalysis of an exo-rhamnosidase requires a novel carbohydrate-binding module that binds terminal L-rhamnose sugars.

Background: Glycoside hydrolase family 62 ␣-L-arabinofuranosidases specifically release L-arabinose from arabinoxylan. Results: The crystal structure of glycoside hydrolase family 62 ␣-L-arabinofuranosidase from Streptomyces coelicolor was determined. Conclusion: L-Arabinose and xylohexaose complexed structures revealed the mechanism of substrate specificity of this enzyme. Significance: Efficient catalysis by glycoside hydrolase family 62 ␣-L-arabinofuranosidase requires its binding to terminal xylose sugars at the substrate-binding cleft.

Exo-1,5-␣-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the ␣-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-␣-L-arabinofuranosidase. The catalytic module is composed of a 5-bladed ␤-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a ␤-trefoil-fold. A sugar complex structure with ␣-1,5-L-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with ␣-L-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite ؊1, formed by the flexible loop region Tyr-281-Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.L-Arabinose residues are widely distributed in plant cell walls, where they are present in polymers such as arabinans, arabinoxylans, arabinogalactans, and arabinogalactan proteins (1). Research on plant cell walls is becoming a necessity because worldwide attention has now focused on bioethanol production to combat global warming and to improve global energy security. Because of competition between food and fuel, lignocellulose is expected to be used as a material for fuel ethanol production in the future. Generally, lignocellulose contains cellulose, which makes up ϳ40% of the total amount of cell wall components, together with ϳ20% hemicellulose, which is mainly composed of pentoses such as xylose and arabinose (2). Hemicelluloses often become bad factors in bioethanol production because the efficiency of ethanol conversion from pentoses is significantly lower than that from hexoses (3, 4).In contrast, L-arabinose is used as a functional sugar in the food industry. This sugar has a sweet taste and selectively inhibits intestinal sucrase activity in a noncompetitive manner and consequently suppresses plasma glucose increase due to sucrose ingestion (5-7). Therefore, L-arabinose may also be useful in preventing excess sucrose utilization.Because the structure of L-arabinose-containing polysaccharides is highly variable and complex, a wide variety of ␣-L-arabinofuranosidases (EC 3.2.1.55) that have various substrate specificities are necessary for the hydrolysis of such polysaccharides and for the production of L-arabinose. We have previously purified some ␣-L-arabi...

Plasmid pL32 from the Natto strain of Bacillus subtilis belongs to a group of low-copy-number plasmids in gram-positive bacteria that replicate via a theta mechanism of replication. We studied the DNA region encoding the replication protein, RepN, of pLS32, and obtained the following results. Transcription of the repN gene starts 167 nucleotides upstream from the translational start site of repN. The copy number of repN-coding plasmid pHDCS2, in which the repN gene was placed downstream of the IPTG (isopropyl-1-thio-␤-D-galactopyranoside)-inducible Pspac promoter, was increased 100 fold by the addition of IPTG. Histidine-tagged RepN bound to a specific region in the repN gene containing five 22-bp tandem repeats (iterons) with partial mismatches, as shown by gel retardation and foot printing analyses. Sequence alterations in the first three iterons resulted in an increase in plasmid copy number, whereas those in either the forth or fifth iteron resulted in the failure of plasmid replication. The iterons expressed various degrees of incompatibility with an incoming repN-driven replicon pSEQ243, with the first three showing the strongest incompatibility. Finally, by using a plasmid, pHDMAEC21, carrying the sequence alterations in all the five iterons in repN and thus unable to replicate but encoding intact RepN, the region necessary for replication was confined to a 96-bp sequence spanning the 3-terminal half of the fourth iteron to an A؉T-rich region located downstream of the fifth iteron. From these results, we conclude that the iterons in repN are involved in both the control of plasmid copy number and incompatibility, and we suggest that the binding of RepN to the last two iterons triggers replication by melting the A؉T-rich DNA sequence.Circular plasmids can be grouped into two classes by the mode of replication. One group replicates via a rolling-circle intermediate, while the other uses the theta-type intermediate. In the case of plasmids from natural isolates of Bacillus subtilis, it has been demonstrated that the sizes of the plasmids belonging to the first group are small, while those belonging to the second group are large (37). The small plasmids of B. subtilis are further grouped into seven classes based on their sizes and restriction patterns and show a replication function related to that of pC194 derived from Staphylococcus aureus (26). It remains unknown why the only pC194-type replicon is found in B. subtilis among rolling-circle replicons with different types of replication functions (13,18,20). On the other hand, plasmids that replicate via a theta mechanism of replication are classified into six groups (36); in addition, a recently reported plasmid, pBS72, carries a replicon of a new type (37). The large plasmids found in natural isolates of B. subtilis show diverse modes of theta replication, as exemplified by pLS20 (25), pLS32 (36), and pBS72 (37). Whereas the replication functions are unique for pLS20 and pBS72, several plasmids, including pLS32 and their relatives (see below), show amino a...

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