Expression of symbiotic and nonsymbiotic globin genes responding to microsymbionts on Lotus japonicus

Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.     

The PIWI‐Interacting RNA Molecular Pathway: Insights From Cultured Silkworm Germline Cells

The PIWI‐interacting RNA (piRNA) pathway, one of the major eukaryotic small RNA silencing pathways, is a genome surveillance system that silences selfish genes in animal gonads. piRNAs guide PIWI protein to target genes through Watson–Crick RNA–RNA base‐parings. Loss of piRNA function causes genome instability, inducing failure in gametogenesis and infertility. Studies using fruit flies and mice as key experimental models have resulted in tremendous progress in understanding the mechanism underlying the piRNA pathway. Recent work using cultured silkworm germline cells has also expanded our knowledge of piRNA biogenesis in particular, since these silkworm cells are the only cells of germline origin that can be cultured. In this review, we describe elucidation of the piRNA pathway using cultured silkworm cells as an experimental model by focusing on recent work in biochemistry and structural biology. Earlier studies that made important contributions to the field are also described.     

A locus controlling the activity of UDP-galactose:GM2(NeuGc) galactosyltransferase in mouse liver is linked to the H-2 complex

Genetic polymorphism in the expression of the G_M1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of G_M1(NeuGc) synthetase, UDP-galactose:G_M2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2^d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F₁ mice, and the female F₁ mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.Abbreviations NeuGc N-glycolylneuraminic acid The ganglioside nomenclature is based on the system of Svennerholm, J Neurochem (1963) 10:613-23. The sialic acid species present is shown in parentheses after the ganglioside abbreviation.     

Overexpression of CAF1 encoding a novel Ca2+-binding protein stimulates the transition of Dictyostelium cells from growth to differentiation

Among the expressed genes associated with the switch-over of Dictyostelium cells from cell proliferation to differentiation, the Calfumirin-1 ( CAF1 ) gene has been shown to be preferentially expressed at the initial step of differentiation, encoding a novel Ca²⁺-binding protein (Abe & Maeda 1995). To analyze precisely the function of CAF1 , transformants overexpressing the CAF1 mRNA at the vegetative growth phase and also CAF1 -null mutants were prepared, and their developmental features were compared with those of parental wild-type cells. As a result, the CAF1 -overexpression was found to promote cell differentiation, possibly through prompt induction of the cAMP receptor 1 ( CAR1 ) gene expression. In addition, the CAF1 -overexpressing cells were able to differentiate even under low external Ca²⁺ ([Ca²⁺]_e) conditions around 10⁻⁶mol/L at which non-transformed wild-type cells never differentiated. Unexpectedly, however, the CAF1 -null mutant produced by homologous recombination exhibited apparently normal development to form fruiting bodies on non-nutrient agar. These results seem to indicate that CAF1 -overexpression has a stimulatory effect on differentiation, but that the CAF1 protein is not necessarily required for the phase-shift of cells from growth to differentiation.     

Epstein-Barr virus nuclear antigen leader protein induces expression of thymus- and activation-regulated chemokine in B cells

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.     

Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection

The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.     

CLUMSY VEIN,the Arabidopsis DEAH‐box Prp16 ortholog,is required for auxin‐mediated development

Pre‐messenger RNA (pre‐mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAH‐box RNA‐dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre‐mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre‐mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin‐mediated development including male‐gametophyte transmission, apical–basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv‐1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv‐1 mutation does not have an equal influence on pre‐mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin‐mediated development.