Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.     

Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins.

Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vp1, carry individual nuclear targeting signals, Vp3(198-206) (Vp2(316-324) and Vp1(1-8), respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vp1 or Vp2/3, nuclear targeting-defective mutants thereof, Vp2/3(202T) and Vp1 delta N5, or nonoverlapping SV40 variants in which the genes for Vp1 and Vp2/3 are separated, and mutant derivatives of the gene carrying either one or both mutations. Nuclear targeting was assessed immunocytochemically following nuclear microinjection of the variant DNAs. When Vp2/3 and Vp1 mutants with defects in the nuclear targeting signals were expressed individually, the mutant proteins localized mostly to the cytoplasm. However, when mutant Vp2/3(202T) was coexpressed in the same cell along with wild-type Vp1, the mutant protein was effectively targeted to the nucleus. Likewise, the Vp1 delta N5 mutant protein was transported into the nucleus when wild-type Vp2/3 was expressed in the same cells. These results suggest that while Vp1 and Vp2/3 have independent nuclear targeting signals, additional signals, such as those defining protein-protein interactions, play a concerted role in nuclear localization along with the nuclear targeting signals of the individual proteins.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

The Presequence of a Precursor to the {delta}-Subunit of Sweet Potato Mitochondrial F1ATPase is not Sufficient for the Transport of {beta}-Glucuronidase (GUS) into Mitochondria of Tobacco, Rice and Yeast Cells

A precursor to the     

Functions of milk protein gene 5′ flanking regions on human growth hormone gene

Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S₁ casein (bαS₁CN), beta casein (bβCN), kappa casein (b_kCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS₁CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS₁CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS₁CN was shown most suitable, because the bαS₁CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc.     

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Differential regulation of IgA production by TGF-beta and IL-5: TGF-beta induces surface IgA-positive cells bearing IL-5 receptor, whereas IL-5 promotes their survival and maturation into IgA-secreting cells.

Transforming growth factor beta (TGF-beta) and IL-5 have been shown to augment IgA production by LPS-stimulated murine B cells. We investigated the effect of TGF-beta on the expression of surface Ig-isotype and IL-5 receptor on LPS-stimulated B cells. TGF-beta increased the proportion of both surface IgA-positive (sIgA+) B cells and sIgG2b+ B cells and enhanced IgA and IgG2b production by LPS-stimulated B cells. TGF-beta synergized with IL-5 only for IgA production of the seven Ig-isotypes and in combination with IL-5 caused a significant increase in the proportion of sIgA+ B cells up to 17.4%. In contrast, IL-5 decreased the proportion of sIgG2b+ B cells and sIgG3+ B cells and inhibited the production of IgG2b and IgG3 by LPS-stimulated B cells. About 50% of sIgA+ cells induced by TGF-beta expressed IL-5 receptor. They secreted peak levels of IgA and seemed to maintain long viability in the presence of IL-5; whereas TGF-beta had the opposite effects on sIgA+ B cells and down-regulated the IL-5 receptor expression. These results indicate that TGF-beta increases the number of sIgA(+)- and IL-5 receptor-positive B cells which respond to IL-5 giving rise to IgA-secreting cells and also support the notions that TGF-beta preferentially induces switching to sIgA+ B cells and IL-5 induces the maturation of postswitch sIgA+ B cells into IgA-secreting cells in a stepwise fashion.