OsSEND-1: a new RAD2 nuclease family member in higher plants

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H₂O₂, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.     

The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family

The deployment of broad-spectrum resistance genes is the most effective and economic means of controlling blast in rice. The cultivar Er-Ba-Zhan (EBZ) is a widely used donor of blast resistance in South China, with many cultivars derived from it displaying broad-spectrum resistance against blast. Mapping in a set of recombinant inbred lines bred from the cross between EBZ and the highly blast-susceptible cultivar Liangjiangxintuanheigu (LTH) identified in EBZ a blast resistance gene on each of chromosomes 1 (Pish), 6 (Pi2/Pi9) and 12 (Pita/Pita-2). The resistance spectrum and race specificity of the allele at Pi2/Pi9 were both different from those present in other known Pi2/Pi9 carriers. Fine-scale mapping based on a large number of susceptible EBZ?×?LTH F(2) and EBZ?×?LTH BC(1)F(2) segregants placed the gene within a 53-kb segment, which includes Pi2/Pi9. Sequence comparisons of the LRR motifs of the four functional NBS-LRR genes within Pi2/Pi9 revealed that the EBZ allele is distinct from other known Pi2/Pi9 alleles. As a result, the gene has been given the designation Pi50(t).     

Raver2, a new member of the hnRNP family

Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo-cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB-mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio-temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.     

Genetic analysis and molecular mapping of a nuclear recessive male sterility gene, ms91(t), in rice.

Mutations that result in plant male sterility provide means not only to probe reproductive development but also to facilitate commercial heterosis application and hybrid seed production. In this study, we report a novel male sterility gene, ms91(t), in a spontaneous mutant line (SH38) from a Chinese rice cultivar (Oryza sativa subsp. japonica 'Jijing14'). The sterility of SH38 was studied by examining its progenies derived from crosses with 6 japonica cultivars. Corresponding F2 populations were obtained by selfing each of the 6 F1s and a backcross population was produced by crossing SH38 to the F1 of SH38 x C18. Our results revealed that SH38 has normal agronomic traits but produces no pollen grains. Segregations of male-sterile and male-fertile progenies in the F2 and backcross populations fit well with ratios of 3:1 and 1:1, respectively, indicating that ms91(t) is a single recessive gene. Amplified fragment length polymorphism (AFLP) analysis of SH38 and Jijing14 plants showed the presence of a unique band in SH38. Simple sequence repeat (SSR) analysis of the bulked and individual progenies of the F2 population of SH38 x C18 showed linkage of ms91(t) with the SSR marker RM5853 on chromosome 1. Subsequently, ms91(t) was fine-mapped to the interval between markers RM7075 (3.75 cM) and RM5638 (3.57 cM). Our results would facilitate the isolation of ms91(t) and male sterility in heterosis application.     

Dissection of the functional domains of the Leishmania surface membrane 3'-nucleotidase/nuclease, a unique member of the class I nuclease family

Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family.     

A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450

We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.     

Zebrafish tenascin-W,a new member of the tenascin family

A cDNA clone encoding tenascin-W, a novel member of the tenascin family, was isolated from a 20- to 28-h postfertilization (hpf) zebrafish cDNA library on the basis of the conserved epidermal growth factor-like domains represented in all tenascin molecules. An open reading frame of 2796 base pairs encodes a mature protein consisting of heptad repeats, a cysteine-rich amino terminal region, 3.5 epidermal growth factor-like repeats, five fibronectin type III homologous repeats, and a domain homologous to fibrinogen. These domains are the typical modular elements of molecules of the tenascin family. Sequence comparison demonstrated that TN-W shares homologies with the members of the tenascin family but is not a species homolog of any identified tenascin. The expression pattern of tn-w was analyzed by in situ hybridization in 1-day-old embryos, in 3-day-old larvae, and in juvenile zebrafish. At 24–25 hpf, tn-w mRNA was expressed in the lateral plate mesoderm, most conspicuously in the presumptive sclerotome. Migrating cells of sclerotomal and neural crest origins also showed high levels of expression. At 3 days, expression by sclerotomal and neural crest cells continued to be observed while expression in the somitic mesoderm was decreased. In juvenile fish, tn-w was expressed weakly by cells in the myosepta and, more strongly, by presumably nonneuronal cells in the dorsal root ganglia. In these tissues and at the same developmental stages, the expression of tn-w partially overlapped with the distribution of tn-c mRNA. In addition, tn-c was expressed in the central nervous system (CNS) and in the axial mesoderm, neither of which expressed tn-w at any of the age stages examined. The expression pattern of tn-w suggests an involvement in neural crest and sclerotome cell migration and in the formation of the skeleton. Similar and possibly overlapping functions could also be performed by tn-c, which appears to have additional functions during the development of the CNS. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 1–16, 1998     

Characterization and mapping of a new male sterility mutant of anther advanced dehiscence （t） in rice

Anther dehiscence is very important for pollen maturation and release.The mutants of anther dehiscence in rice (Oryza sativa L.) arefew,and related research remains poor.A male sterility mutant of anther dehiscence in advance,add(t),has been found in Minghui 63 and its sterility is not sensitive to thermo-photo.To learn the character of sterilization and the function of the add(t) gene,the morphological and cytological studies on the anther and pollen,the ability of the pistil being fertilized,inheritance of the mutant,and mapping of add(t)gene have been conducted.The anther size is normal but the color is white in the mutant against the natural yellow in the wild-type.The pollen is malformed,unstained,and small in the KI-I2 solution.The anther dehiscence is in advance at the bicellular pollen stage.A crossing test indicated that the grain setting ratio of the add(t) is significantly lower than that of the CMS line 2085A.The ability of the pistil being fertilized is most probably decreased by the add(t) gene.The male sterility is controlled by a single recessive gene of add(t).This gene is mapped between the markers of R02004 (InDel) and RM300 (SSR) on chromosome 2,and the genetic distance from the add(t) gene to these markers is 0.78 cM and 4.66 cM,respectively.     

Phenotypic characterization, genetic analysis, and molecular mapping of a new mutant gene for male sterility in rice.

xs1 is a male-sterile rice mutant derived from a spontaneous mutation. The floret of the mutant, consisting of 6 stamens and 1 pistil, looks the same as that of the wild type except that the filaments are long and thin and the anthers are withered in white transparence. It is confirmed that xs1 is a no-pollen type of male-sterile mutant, for no pollen grains can be stained with I(2)-KI solution and the anther locules are always hollow. Anther transverse sections indicate that the mutant microspores are abnormally condensed and agglomerated to form a deeply stained cluster at the late microspore stage, which results in cessation of the vacuolation process of microspores, and, therefore, the mutant forms no functional pollens for reproduction. Genetic analysis of 4 F(2) populations and 3 BC(1)F(1) populations revealed that the mutation is controlled by a single recessive gene, termed VR1 (Vacuolation retardation 1). Screening of 432 F(2) mutant individuals derived from the cross of xs1 x G603 with simple sequence repeat markers revealed that VR1 is located between the molecular markers RM17411 and RM5030, at distances of 0.7 and 1.5 cM, respectively, on chromosome 4. VR1 is a new male fertility controlling gene located on chromosome 4 in rice.     

Identification of active transposon dTok, a member of the hAT family, in rice

Recent completion of the sequencing of the rice genome has revealed that it contains >40% repetitive sequences, most of which are related to inactive transposable elements. During the molecular analysis of the floral organ number 1/multiple pistil 2 (fon1/mp2) mutant, we identified an active transposable element dTok0 that was inserted at the kinase domain of FON1, a homolog of CLAVATA1. Insertion of the element into FON1 generated an 8 bp duplication of its target sites, which is one of the major characteristics of the hAT family of transposons. The dTok0 element was actively transposed out of the FON1 gene, leaving 5-8 bp footprints. Reinsertion into a new location was observed at a low frequency. Analysis of the genome sequence showed that the rice cultivar 'Nipponbare' contains 25 copies of dTok elements; similar numbers were present in all the Oryza species examined. Because dTok0 does not encode a transposase, enzyme activity should be provided in trans. We identified a putative autonomous transposon, Tok1 that contains an intact open reading frame of the Ac-like transposase.     

STAM2, a new member of the STAM family, binding to the Janus kinases

We here cloned a cDNA encoding STAM2, a new member of the STAM family, which contains an SH3 domain and ITAM. STAM2 like STAM1 is associated with Jak2 and Jak3, and involved in the signaling for DNA synthesis and c-myc induction mediated by IL-2 and GM-CSF. Co-expression of the SH3 deletion mutants of STAM1 and STAM2 induces an additive effect on suppressing DNA synthesis upon stimulation with IL-2 and GM-CSF, suggesting that STAM1 and STAM2 exhibit compensatory effects on the signaling pathways downstream of Jak2 and Jak3 upon stimulation with GM-SCF and IL-2, respectively.