Boron Plays an Important Role in the Regulation of Plant Cell Growth

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GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin

Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. It has been postulated that plants have both membrane-bound and soluble GA receptors; however, no GA receptors have yet been identified. Here we report the isolation and characterization of a new GA-insensitive dwarf mutant of rice, gid1. The GID1 gene encodes an unknown protein with similarity to the hormone-sensitive lipases, and we observed preferential localization of a GID1-green fluorescent protein (GFP) signal in nuclei. Recombinant glutathione S-transferase (GST)-GID1 had a high affinity only for biologically active GAs, whereas mutated GST-GID1 corresponding to three gid1 alleles had no GA-binding affinity. The dissociation constant for GA4 was estimated to be around 10(-7) M, enough to account for the GA dependency of shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA-dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Together, our results indicate that GID1 is a soluble receptor mediating GA signalling in rice.     

Mitoferrin is essential for erythroid iron assimilation

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.     

Identification of a host protein essential for assembly of immature HIV-1 capsids.

To form an immature HIV-1 capsid, 1,500 HIV-1 Gag (p55) polypeptides must assemble properly along the host cell plasma membrane. Insect cells and many higher eukaryotic cell types support efficient capsid assembly, but yeast and murine cells do not, indicating that host machinery is required for immature HIV-1 capsid formation. Additionally, in a cell-free system that reconstitutes HIV-1 capsid formation, post-translational assembly events require ATP and a subcellular fraction, suggesting a requirement for a cellular ATP-binding protein. Here we identify such a protein (HP68), described previously as an RNase L inhibitor, and demonstrate that it associates post-translationally with HIV-1 Gag in a cell-free system and human T cells infected with HIV-1. Using a dominant negative mutant of HP68 in mammalian cells and depletion-reconstitution experiments in the cell-free system, we demonstrate that HP68 is essential for post-translational events in immature HIV-1 capsid assembly. Furthermore, in cells the HP68-Gag complex is associated with HIV-1 Vif, which is involved in virion morphogenesis and infectivity. These findings support a critical role for HP68 in post-translational events of HIV-1 assembly and reveal a previously unappreciated dimension of host-viral interaction.     

An essential role for a phospholipid transfer protein in yeast Golgi function

Progression of proteins through the secretory pathway of eukaryotic cells involves a continuous rearrangement of macromolecular structures made up of proteins and phospholipids. The protein SEC14p is essential for transport of proteins from the yeast Golgi complex. Independent characterization of the SEC14 gene and the PIT1 gene, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein in yeast, indicated that these two genes are identical. Phospholipid transfer proteins are a class of cytosolic proteins that are ubiquitous among eukaryotic cells and are distinguished by their ability to catalyse the exchange of phospholipids between membranes in vitro. We show here that the SEC14 and PIT1 genes are indeed identical and that the growth phenotype of a sec14-1ts mutant extends to the inability of its transfer protein to effect phospholipid transfer in vitro. These results therefore establish for the first time an in vivo function for a phospholipid transfer protein, namely a role in the compartment-specific stimulation of protein secretion.     

Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake

Frequently, crop plants do not take up adequate amounts of iron from the soil, leading to chlorosis, poor yield and decreased nutritional quality. Extremely limited soil bioavailability of iron has led plants to evolve two distinct uptake strategies: chelation, which is used by the world's principal grain crops; and reduction, which is used by other plant groups. The chelation strategy involves extrusion of low-molecular-mass secondary amino acids (mugineic acids) known as 'phytosiderophores' which chelate sparingly soluble iron. The Fe(III)-phytosiderophore complex is then taken up by an unknown transporter at the root surface. The maize yellow stripe1 (ys1) mutant is deficient in Fe(III)-phytosiderophore uptake, therefore YS1 has been suggested to be the Fe(III)-phytosiderophore transporter. Here we show that ys1 is a membrane protein that mediates iron uptake. Expression of YS1 in a yeast iron uptake mutant restores growth specifically on Fe(III)-phytosiderophore media. Under iron-deficient conditions, ys1 messenger RNA levels increase in both roots and shoots. Cloning of ys1 is an important step in understanding iron uptake in grasses, and has implications for mechanisms controlling iron homeostasis in all plants.     

Isolation and characterization of the gene for a yeast mitochondrial import receptor

We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria.     

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.     

葡萄糖在细胞吸收硼酸及取代硼酸中的作用研究

硼中子俘获治疗(boron neutron capture therapy, BNCT)是肿瘤治疗的一种二元体系，利用含10B药物对肿瘤细胞的高选择性、在肿瘤细胞中有足够的富集量和滞留时间，用具有合适能量的超热中子束照射肿瘤部位，发生中子俘获反应10B(n，α)7Li，反应释放出的高传能线密度α粒子和7Li，在一个细胞大小范围杀死肿瘤细胞．因此，含10B药物的高效、高选择性对肿瘤细胞的靶向输送是BNCT有效性提高的关键．为了提高肿瘤细胞对含硼化合物的吸收，本文基于糖类物质与硼酸及其衍生物的相互作用，以及糖类具有在肿瘤细胞中富集的性质，研究了葡萄糖对正常细胞和肿瘤细胞吸收硼酸及其衍生物的影响，探讨了利用糖类物质增加肿瘤细胞中10B含量的可能性．研究结果表明，随着葡萄糖含量的增加，肿瘤细胞对硼酸及取代硼酸的吸收增加，而正常细胞的吸收增加不明显．此外，葡萄糖对硼化合物在细胞中的滞留无明显的影响．      

Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.     

一种自剪切内含肽重组酿酒酵母表达优化研究

自剪切内含肽纯化系统在大肠杆菌中已经得到很好的应用。为了在真核生物中应用该系统,以酿酒酵母为宿主,p HR质粒为表达载体,构建微型内含肽ΔI-CM intein酿酒酵母表达系统。以猪免疫球蛋白Ig G的Fc domain为亲和标签,绿色荧光蛋白gfp为报告蛋白,在酿酒酵母GPD强启动子作用下,表达出融合的Fc-intein-gfp蛋白。检测发现融合序列在起始密码子ATG前加入一段Kozak序列有利于gfp表达。在此基础上,将微型内含肽ΔI-CM intein序列替换成密码子优化的ΔI-CMintein-O序列,显著促进gfp蛋白的表达量,初步实现了ΔI-CM intein融合蛋白的高效表达。为新型自剪切内含肽酿酒酵母表达系统的建立和重组蛋白高效纯化的实现奠定了基础。     

The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19.

Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.     

绿色荧光蛋白基因转化大岩桐的研究

利用农杆菌介导法 ,用含有绿色荧光蛋白基因的二元双价表达载体pBINm -gfp5 -ER转化大岩桐 ,并得到卡那霉素 (Kanamycin ,Kan)抗性再生植株 .对其进行初步PCR检测 ,结果表明 ,K2 0 0 (含Kan 2 0 0mg/L)培养基上的绿苗中有 3株PCR结果呈阳性 .对PCR阳性的植株进行了点杂交分析 ,均表现出较强的杂交信号 ,这说明外源基因已整合转入到大岩桐基因组中 .在荧光显微镜下观察转基因大岩桐 ,发现部分花、叶细胞均发出一定强度的绿色荧光     

Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination.

The repair of DNA double-strand breaks is essential for cells to maintain their genomic integrity. Two major mechanisms are responsible for repairing these breaks in mammalian cells, non-homologous end-joining (NHEJ) and homologous recombination (HR): the importance of the former in mammalian cells is well established, whereas the role of the latter is just emerging. Homologous recombination is presumably promoted by an evolutionarily conserved group of genes termed the Rad52 epistasis group. An essential component of the HR pathway is the strand-exchange protein, known as RecA in bacteria or Rad51 in yeast. Several mammalian genes have been implicated in repair by homologous recombination on the basis of their sequence homology to yeast Rad51: one of these is human XRCC2. Here we show that XRCC2 is essential for the efficient repair of DNA double-strand breaks by homologous recombination between sister chromatids. We find that hamster cells deficient in XRCC2 show more than a 100-fold decrease in HR induced by double-strand breaks compared with the parental cell line. This defect is corrected to almost wild-type levels by transient transfection with a plasmid expressing XRCC2. The repair defect in XRCC2 mutant cells appears to be restricted to recombinational repair because NHEJ is normal. We conclude that XRCC2 is involved in the repair of DNA double-strand breaks by homologous recombination.     

Chromosomal localization of a novel retinoic acid induced geneRA28 and protein distribution of its encoded protein

GeneRA28 is a retinoic acid induced novel gene isolated in our laboratory previously. All-trans retinoic acid (ATRA) was used to induce lung adenocarcinoma cell line GLC-82, andRA28 was obtained by subtractive hybridization. Green fluorescent protein (GFP) has emerged as a unique tool for examining introcellular phenomena in living cells. GFP possesses an intrinsic fluorescence at 488 nm that does not require other co-factors. In this report, an eukaryotic expression plasmid pEGFP-C1-RA28 was constructed and transfected with parental cell line GLC-82 to analyze protein expression and its distribution in living cells. Moreover, radiation hybrid (RH) technique was used to localizeRA28 to the chromosome. The results show that geneRA28 is mapped to the chromosome 19q13.1 region, its encoded protein is distributed on cell membrane. All the results further demonstrate that GFP and RH techniques are accurate, fast, repetitive, and will be powerful methods for investigating the gene and protein localization.     

A proteoglycan mediates inductive interaction during plant vascular development

Inductive cell-cell interactions are essential for controlling cell fate determination in both plants and animals; however, the chemical basis of inductive signals in plants remains little understood. A proteoglycan-like factor named xylogen mediates local and inductive cell-cell interactions required for xylem differentiation in Zinnia cells cultured in vitro. Here we describe the purification of xylogen and cloning of its complementary DNA, and present evidence for its role in planta. The polypeptide backbone of xylogen is a hybrid-type molecule with properties of both arabinogalactan proteins and nonspecific lipid-transfer proteins. Xylogen predominantly accumulates in the meristem, procambium and xylem. In the xylem, xylogen has a polar localization in the cell walls of differentiating tracheary elements. Double knockouts of Arabidopsis lacking both genes that encode xylogen proteins show defects in vascular development: discontinuous veins, improperly interconnected vessel elements and simplified venation. Our results suggest that the polar secretion of xylogen draws neighbouring cells into the pathway of vascular differentiation to direct continuous vascular development, thereby identifying a molecule that mediates an inductive cell-cell interaction involved in plant tissue differentiation.     

Wee1(+)-like gene in human cells.

The wee1+ gene is a mitotic inhibitor controlling the G2 to M transition of the fission yeast Schizosaccharomyces pombe and encodes a protein kinase with both serine- and tyrosine-phosphorylating activities. We have cloned a human gene (WEE1Hu) similar to wee1+ by transcomplementation of a yeast mutant. WEE1Hu encodes a protein homologous to the S. pombe wee1+ and mik1+ (a functionally redundant sibling of wee1+) kinases and effectively rescues a wee1 mutation. We report here that overexpression of WEE1Hu in fission yeast generates very elongated cells as a result of inhibition of the G2-M transition in the cell cycle. In addition, we detected a 3-kilobase-long WEE1Hu messenger RNA in all the human cell lines we examined. We conclude that a wee1(+)-like gene exists and is expressed in human cells.