Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

A reverse transcription-PCR (RT-PCR) was established to amplify a 379-bp cDNA fragment (nucleotides 747 to 1126, coding for amino acids 241 to 367) of the VP6 gene of group A rotaviruses associated with subgroup (SG) specificity. Thirty-eight human rotavirus strains characterized with SG-specific monoclonal antibodies were subjected to VP6-specific RT-PCR, and PCR amplicons were used for sequencing. Nucleic acid sequencing and phylogenetic analysis of the VP6 amplicons revealed two clusters, or genogroups. Two genetic lineages were distinguished within genogroup I, consisting of strains serologically characterized as SG I, and three genetic lineages were distinguished within genogroup II, composed of strains serologically characterized as SG II, SG I ؉ II, and SG non-I, non-II. Subgrouping of rotaviruses by means of serological methods may result in strains not being assigned the correct SG or in a failure of strains to subgroup. Molecular characterization of the SG-defining region of VP6 provided evidence for independent segregation of the rotavirus genes encoding VP4, VP6, and VP7.Rotaviruses are triple-layered particles of the Reoviridae family which are classified into groups (A to E) and subgroups (SG) according to the presence of epitopes on the middle-layer protein VP6 (16). VP6 is a trimeric protein that interacts with the inner-layer protein VP2 and with the two outer-layer proteins VP7 and VP4 (20). Most human rotavirus infections are caused by rotaviruses of group A. Within this group, SG I, II, I ϩ II, and non-I, non-II have been defined according to the presence or absence of two distinct epitopes reactive with one, both, or neither of the monoclonal antibodies (MAbs) 255/60 and 631/9 (11). Subgrouping enzyme-linked immunosorbent assays (ELISAs) have been used extensively in epidemiological studies. The rotaviruses most commonly found in humans belong to SG II (2,4,8,14,19), while SG I is common among animal rotaviruses (18,23).Previous studies have mapped SG I specificity to amino acid (aa) position 305 and the region between positions 296 and 299 and SG II specificity to residue 315 (18, 23). It is still unclear whether SG specificity is determined by linear or conformational epitopes, although there is some evidence that the epitopes recognized by SG-specific MAbs are conformational and are present only in the trimeric form of VP6 (9). Amino acid substitutions that are quite distant in the linear molecule may affect the conformation of the epitopes.Serological characterization of the surface proteins VP7 and VP4 has been shown to be unreliable due to antigenic drift through the accumulation of point mutations (7,12,21). It is possible that VP6 will undergo similar antigenic changes, resulting in poor reactivity in serological subgrouping assays.The aims of this work were to determine the SG of rotavirus strains on the basis of cDNA sequences of a fragment of the VP6 genes, to investigate correlations between molecular and serological subgrouping methods, and to study the variability of the VP6 ge...