Molecular characterization of double-stranded RNA segments encoding the major capsid proteins of a Palyam serogroup orbivirus that caused an epizootic of congenital abnormalities in cattle.

cDNA cloning of the double-stranded RNA genome of Chuzan virus, a member of the Palyam serogroup orbiviruses, was carried out and the complete nucleotide sequences of RNA segments 2, 3, 6 and 7, encoding the major capsid proteins VP2, VP3, VP5 and VP7, respectively, were determined. The individual segments had single open reading frames and short inverted repeats adjacent to the conserved terminal sequences. Comparative sequence analysis with other serogroups of the genus Orbivirus suggested that VP2 is the principal determinant of serotype specificity and the neutralizing antigen of the Palyam serogroup. VP5 is also considered to be associated with antigenic variability. Both VP3 and VP7 probably contain serogroup-specific epitopes. Phylogenetic profiles demonstrated that the Palyam serogroup virus is more closely related to African horsesickness virus than to bluetongue virus and epizootic haemorrhagic disease virus.

Pyrrole butyric acid derivatives as inhibitors of steroid 5 alpha-reductase.

A series of pyrrole butyric acid derivatives was synthesized and evaluated for inhibitory activity on human and rat steroid 5 alpha-reductase in vitro and ex vivo. 3-Benzoyl-4-alkylpyrrole-1-butyric acids and 1-methyl-2-alkyl-3-benzoylpyrrole-5-butyric acid derivatives were effective inhibitors. Structure activity relationships were evaluated among the 37 compounds synthesized. Compound 37 (HQL-1069) shows potent inhibitory activities against both rat and human 5 alpha-reductase.

gamma-Pyrones from Gonystylus keithii, as new inhibitors of parathyroid hormone (PTH)-induced Ca release from neonatal mouse calvaria.

New gamma-pyrones, 9'-oxopodopyrone (3) and 8-methyl-9'-oxopodopyrone (4) were isolated from the leaves of Gonystylus keithii, along with known gamma-pyrones, 10'-oxopodopyrone (1) and 8-methyl-10'-oxopodopyrone (2). These gamma-pyrones markedly inhibited the bovine parathyroid hormone (PTH)-induced Ca release from neonatal mouse calvaria in vitro. It is the first time that gamma-pyrones showed inhibitory effects on bone resorption, and these compounds may be seed compounds of new drugs for osteoporosis.

Transformation of pBR322-Derived Plasmids in PhytopathogenicPseudomonas avenae and Enhanced Transformation in ItsProline-Auxotrophic Mutant

Efficient transformation of pBR322 and its derivedplasmids, which have been widely used as cloning vectors in Escherichiacoli, was observed in Pseudomonas avenae (K1), the pathogen ofleaf blight disease in cereals. Moreover, there was a 10- to 50-foldtransformation efficiency (1.3-3.0 x 10(6)/&mgr;g DNA) in theproline-auxotrophic mutant (Pr47), whose virulence to rice seedlingsdecreased. Similar enhancement of the frequency of transfer by mobilizationof RSF1010, a broad host range plasmid, was observed in the recipient Pr47strain in mating with donor Pseudomonas syringae. The plasmidsharbored in these strains were maintained very stably after subcultures.Thus, a highly efficient transformation system with pBR322-derived plasmidsused as a vector and Pseudomonas as a host bacterium was developed.

Rice class III chitinase homologues isolated by random cloning of rice cDNAs.

We obtained seven kinds of cDNA clones putatively identified as encoding class III chitinases from cDNA libraries constructed from dichlorophenoxyacetic acid (2,4-D)- and benzyl adenine (BA)- treated rice callus. Putative amino acid sequences encoded in these cDNA clones were compared with those of known chitinases of other plants. Two clones coded for homologues that show high similarity to class III chitinases. These clones contained the common glutamic acid at the active site and were classified as true homologues. The other five clones, however, showed relatively low similarity to class III chitinases and their active sites contained aspartic acid instead of glutamic acid. These clones may correspond to relatives of a super family of class III chitinases. The location of the genes coding for these homologues on the rice genome has been determined by genetic linkage analysis with restriction fragment length polymorphism.

Generation of a YAC contig encompassing the extra glume gene, eg, in rice.

We have used DNA markers from a high density molecular map of rice (Oryza sativa) to tag a single gene expressed as a flower morphogenesis mutation, extra glume (eg). Using an F2 population segregating for eg, obtained from a cross between IR24 and F136 (eg/eg), we constructed a partial molecular map and located eg relative to restriction fragment length polymorphism markers. The region between two markers appears to span the eg locus on rice chromosome 1 and extends to a genetic length of 3.8 cM. The yeast artificial chromosome (YAC) library obtained from rice variety 'Nipponbare', which carries the wild-type allele of eg, was screened to completely cover the locus by overlapping YAC clones. The eg allele should be contained in two overlapping YACs. YAC size determination by pulsed-field gel electrophoresis indicated that this region has a physical length of approximately 400 kb. We anticipate that the tagging of eg in a relatively short stretch of DNA will allow a molecular characterization of this gene through map-based cloning. Key words : rice, gene tagging, YAC contig, flower morphogenesis, extra glume.

An ordered yeast artificial chromosome library covering over half of rice chromosome 6.

Yeast artificial chromosome (YAC) clones carrying DNA marker sequences located on the rice genetic map of chromosome 6 were ordered for physical mapping. A total of 122 restriction fragment length polymorphism markers, 16 sequence-tagged site markers, and five random amplified polymorphic DNA markers located, on average, at 0.9-cM intervals, were used for YAC clone screening by colony/Southern hybridization and PCR screening, respectively. A total of 216 individual YACs were selected from our YAC library of 7000 clones covering six genome equivalents. Each DNA marker could select, on average, 4.8 YAC clones, with 11 clones being the maximum. The YACs localized to the corresponding linkage map positions form 43 contigs and encompass about 60% of rice chromosome 6. This is the first step in constructing a physical map covering the whole rice genome by chromosome landing with YAC clones. These YACs and data will be used soon to isolate phenotypical trait genes by map-based cloning.

Comparison off genetic distance and order off DNA markers in five populations of rice.

A group of about 300 evenly distributed DNA markers from a high density RFLP linkage map of rice constructed using an F2 population derived from a japonica variety, Nipponbare, and an indica variety, Kasalath, were used to evaluate gene order and genetic distance in four other rice mapping populations. The purpose of this study was to determine the degree to which information gained from the high density linkage map could be applied to other mapping populations, particularly with regard to its utility in bridging quantitative traits and molecular and physical mapping information. The mapping populations consisted of two F2 populations derived from Dao Ren Qiao/Fl-1084 and Kinandangputi/Fl-1007, recombinant inbred lines from Asominori/IR24, and a backcross population from Sasanishiki/Habataki//Sasanishiki. All DNA markers commonly mapped in the four populations showed the same linkage groups as in the Nipponbare/Kasalath linkage map with conserved linkage order. The genetic distance between markers among the different populations did not vary to a significant level in any of the 12 chromosomes. The differences in some markers could be attributed to the size of the population used in the construction of the linkage maps. Furthermore, the conservation of linkage order found in the distal region of chromosomes 11 and 12 was also confirmed in the RFLP maps based on the four populations of rice. These suggest that any major genetic information from the Nipponbare/Kasalath map can be expected to be approximately the same in other crosses or populations. This high density RFLP linkage map, which is being utilized in constructing a physical map of rice, can be very useful in interpreting genome structure with great accuracy in other populations. Key words : linkage map, japonica, indica, gene order, genetic distance.

Physical mapping of rice chromosome 1 with yeast artificial chromosomes (YACs).

We have constructed a physical map of rice chromosome 1 using yeast artificial chromosomes (YACs). A YAC library of 350 kb average insert size, covering about 6 rice haploid genome equivalents, was screened using 182 DNA markers which we had previously located on chromosome 1, by colony hybridization and polymerase chain reaction (PCR) amplification. One hundred and sixty-two DNA markers identified at least one YAC each carrying one, two or more marker sequences, for a total of 476 clones. Of these identified YACs, 284 were located in their original positions on chromosome 1. These 284 YACs defined 69 YAC contigs or islands which are estimated to cover more than 60% of the total chromosome length. The use of mapped DNA markers in constructing a physical map facilitates the integration of genetic and physical maps, as well as fine ordering of the DNA markers, especially at sites where the markers are clustered tightly on the genetic map. Our high density molecular map has been proven, by chromosome landing with YACs using mapped DNA markers, to cover more than half of the entire length of chromosome 1. The remaining 192 YACs were selected by other copies of DNA markers that mapped on chromosome 1. This description of the YAC contigs formed on chromosome 1 constitutes the second report of rice physical mapping, following that for chromosome 6.

Characterization and mapping of cDNA encoding aspartate aminotransferase in rice, Oryza sativa L.

Fifteen cDNA clones, putatively identified as encoding aspartate aminotransferase (AST, EC 2.6.1.1.), were isolated and partially sequenced. Together with six previously isolated clones putatively identified to encode ASTs (Sasaki, et al. 1994, Plant Journal 6, 615-624), their sequences were characterized and classified into 4 cDNA species. Two of the isolated clones, C60213 and C2079, were full-length cDNAs, and their complete nucleotide sequences were determined. C60213 was 1612 bp long and its deduced amino acid sequence showed 88% homology with that of Panicum miliaceum L. mitochondrial AST. The C60213-encoded protein had an N-terminal amino acid sequence that was characteristic of a mitochondrial transit peptide. On the other hand, C2079 was 1546 bp long and had 91% amino acid sequence homology with P. miliaceum L. cytosolic AST but lacked in the transit peptide sequence. The homologies of nucleotide sequences and deduced amino acid sequences of C2079 and C60213 were 54% and 52%, respectively. C2079 and C60213 were mapped on chromosomes 1 and 6, respectively, by restriction fragment length polymorphism linkage analysis. Northern blot analysis using C2079 as a probe revealed much higher transcript levels in callus and root than in green and etiolated shoots, suggesting tissue-specific variations of AST gene expression.

Characterization and genetic mapping of simple sequence repeats in the rice genome.

We searched partial sequences of over 22,706 rice cDNA and 1220 genomic DNA clones to find and characterize simple sequence repeats (SSRs) in the rice genome. The most frequently found repeated SSR motif in both cDNA and genomic DNA sequences was d(CCG/CGG)n. The second most frequently found SSR was d(AG/CT)n. In contrast with mammalian genomes, in which d(AC/GT)n sequences are the most abundant, d(AG/GT)n sequences were not frequently observed in rice. Sequences containing d(CCG/CGG)n, d(AG/CT)n repeats, and other SSRs were chosen for polymorphism detection. It was predicted that 17 of 20 SSRs in cDNA sequences were located in 5'-untranslated regions near initiation codons. Twenty-two loci can be mapped on our RFLP linkage map by these SSRs. Six markers were tested with 16 japonica rice varieties as templates for PCR. Two markers exhibited amplified fragment length polymorphism among these rice varieties, implying that SSRs are polymorphic among rice varieties which have similar genetic backgrounds. Even these polymorphic SSRs are located within or around genes which code ubiquitous proteins.

Effect of mutations on the intracellular localization of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin.

We have already cloned the polyhedrin genes of the wild-type strain H Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and its mutant, strain A. In this work, polyhedrin genes of mutant BmCPV strains C1 and C2 were cloned and their nucleotide sequences were determined. The polyhedrin amino acid sequences of strains C1 and C2 were compared with that of strain H. Strains C1 and C2 contained two and three sites of mutation in their polyhedrin genes, respectively. Four amino acids (249RLLV) were added at the carboxy terminus of the polyhedrin of strain A, C1 and C2 and the corresponding polyhedrin genes were introduced into a baculovirus expression vector. Intracellular localization of expressed polyhedrin as well as the morphology and localization of polyhedra were investigated by Western blot and microscopy analysis. Recombinant baculovirus containing the polyhedrin gene of strain H produced hexahedral polyhedra in both the cytoplasm and the nucleus. However, the hexahedral polyhedra of strain A were localized only in the nucleus. Normal polyhedra were not observed in cells infected with recombinant baculoviruses expressing strain C1 or C2 polyhedrin genes, but amorphous structures were found in infected cells. Results of expression of a chimaeric luciferase-containing carboxyl-terminal sequence of strain A demonstrated that this sequence was responsible for the nuclear localization. We suggest that a mutation at the carboxy terminus of BmCPV polyhedrin led to nuclear localization of polyhedrin and that several other mutations were responsible for modification of the crystallization pattern of polyhedrin.

Characterization of cDNA encoding for phosphoglucose isomerase of rice (Oryza sativa L.).

Two types of genes (Pgi-a and Pgi-b) encoding phosphoglucose isomerase (PGI; EC 5.3.1.9) were cloned from cDNA libraries of rice cultured cells (Oryza sativa L.). Pgi-a and Pgi-b consisted of 2132 and 2030 nucleotides, respectively. The homology between these genes was 93.0% at nucleotide level. The homology scores between these genes in protein coding region and 3' non-coding region were 95.6% and 79.4%, respectively. PGI proteins encoded by Pgi-a and Pgi-b consisted of 567 and 568 amino acid, respectively, sharing 95.8% homology at amino acid sequences. Of 11 PGI genes from other plant species and organisms whose amino acid sequences had been determined, a dicotyledonous plant Clarkia lewisii PGI showed the highest homology (about 80%) with rice PGIs. GC contents at the third position of rice PGI genes were about 40%. In order to confirm the enzyme activity of the protein encoded by the rice cDNA, Pgi-a was subcloned into an expression vector, pBluescript II SKp, which was introduced into Escherichia coli. The transformant had an additional PGI activity from Pgi-a.

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice.

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F2 population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08 750 was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).

Detection of segregation distortions in an indica-japonica rice cross using a high-resolution molecular map.

We have constructed a high-resolution rice genetic map containing 1383 DNA markers covering 1575 cM on the 12 linkage groups of rice using 186 F2 progeny from a cross between a japonica variety, 'Nipponbare', and an indica variety, 'Kasalath'. Using this high-resolution molecular linkage map, we detected segregation distortion in a single wide cross of rice. The frequencies of genotypes for 1181 markers with more than 176 genotype data were plotted along this map to detect segregation distortion. Several types of distorted segregation were observed on 6 of the chromosomes. We could detect 11 major segregation distortions at ten positions on chromosomes 1, 3, 6, 8, 9, and 10. The strongest segregation distortion was at 107.2 cM on chromosome 3 and may be the gametophyte gene 2 (ga-2). The 'Kasalath' genotype at this position was transmitted to the progeny with about a 95% probability through the pollen gamete. At least 8 out of the 11 segregation distortions detected here are new. The use of the high-resolution molecular linkage map for improving our understanding of the genetic nature and cause of these segregation distortions is discussed.

Genomic organization of 57 ribosomal protein genes in rice (Oryza sativa L.) through RFLP mapping.

Four hundred cDNA clones from rice (Oryza sativa L.) callus and root cDNA libraries, with a high similarity to about 70 kinds of ribosomal proteins (r-protein) in eukaryotic as well as procaryotic organisms, were identified by their deduced amino acid sequences. Southern hybridization of 114 independent cDNA clones with total rice genomic DNA showed 77 distinct and specific hybridization patterns. Of the 77 clones representing the above hybridization patterns, copies of 67 clones corresponding to 57 r-proteins could be estimated and, among these, only 6 clones were single copy, indicating that almost 90% of these r-proteins in rice were encoded by small multigene families. Loci of 36 r-protein genes could be mapped on the rice linkage map by using 30 full-length cDNA clone sequences from specific RELP bands. Another 21 expressed gene loci were mapped using 3' untranslated region specific cDNA probes amplified from the multicopy cDNA clones representing 17 of the r-protein multicopy gene families. The above 57 loci were mapped from 51 cDNA clones and 41 of these r-protein genes mapped to regions that did not show any clustering, while in 5 cases, pairs of r-protein genes cosegregated or linked closely. The r-protein genes in rice were located throughout the 12 chromosomes and it was found that more than one copy within a multigene family may be expressed simultaneously.

Conservation of fine-scale DNA marker order in the genomes of rice and the Triticeae.

DNA markers distribute over large chromosomal regions exhibit conservation of order (collinearity) in different cereal species, but it is not known whether this is maintained on a finer scale, i.e. < or = 2 cM. To address this, sets of two or more genetically linked DNA markers were localised to yeast artificial chromosomes containing rice DNA inserts. Linkage analysis of these DNA markers in barley revealed complete correspondence with their genetic order in rice, the distance between linked sequences on rice chromosomes being < 1.6 cM or < or = 1 + 10(6) bp (1 Mb). Thus, DNA markers separated in this range are collinear in rice, barley and, by inference, other members of the Triticeae. These results are discussed with respect to the use of rice as a key system for the isolation of cereal genes.

Screening of RAPD markers linked to the photoperiod-sensitivity gene in rice chromosome 6 using bulked segregant analysis.

Bulked segregant analysis was used to determine randomly amplified polymorphic DNA (RAPD) markers in a specific interval in the middle of chromosome 6 of rice for tagging the photoperiod sensitivity gene. Two pools of F2 individuals (japonica cv. Nipponbare and indica cv. Kasalath) were constructed according to the genotypes of three restriction fragment length polymorphism (RFLP) markers located at both ends and the middle of the targeted interval. Then another pair of pools were constructed based on the "graphical genotype," which was made with our high density linkage map. RAPD analysis was performed using these DNA pools as templates, and polymorphic fragments were detected and mapped. Using 80 primers, either singly or pairwise, we tested 2,404 primer pairs and established 14 markers tightly linked to the photoperiod sensitivity gene. The obtained RAPD markers were converted into sequence-tagged sites by cloning and sequencing of the polymorphic fragments and they can be used directly for construction of physical maps. This bulked segregant method can be applied for any species and any region of interest in which detailed linkage maps or physical maps are needed.

cDNA sequences of three kinds of beta-tubulins from rice.

Complete nucleotide sequences of three kinds of rice beta-tubulin cDNA clones (pTUB22, R1623 and R2242) were determined. Southern hybridization indicated that these beta-tubulins consist of one gene family. Using RFLP mapping, these three beta-tubulin cDNAs were mapped to different chromosomes indicating at least three loci for the beta-tubulin gene. The deduced amino acid sequences of these cDNAs showed a high similarity to other plant beta-tubulins. The asparagine residue located at the 100th amino acid from the N-terminus of plant beta-tubulins was also conserved with these three beta-tubulins. This asparagine is thought to be responsible for the sensitivity against rhizoxin, the toxin of the pathogen of rice seedling blight, Rhizopus sp. a soil-borne microorganism. Expression of the three beta-tubulin genes was analyzed by Northern blotting and all three clones were expressed in root, the possible target tissue of rhizoxin. These results suggest that these clones are candidates of beta-tubulins targeted by rhizoxin.