Identification of low-dye-binding (ldb) mutants of Saccharomyces cerevisiae

We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-dye-binding) mutants. This was done by complementation of the mutant's phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.     

保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌(Lactobacillus delbrueckii subsp.bulgaricus)L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中,构建重组表达载体pET28aldb0120和pET28aldb0094,并转化到大肠埃希菌(Escherichia coli) BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定,结果显示,LDB0120和LDB0094的比活力分别为0和25 U/mg,表明LDB0094是具有低活性的L-乳酸脱氢酶,而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵,重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸,浓度分别为41.9和227.9 mg/L,而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合...     

Effect of aPMR1 disruption on the processing of heterologous glycoproteins secreted in the yeastSaccharomyces cerevisiae

TheSaccharomyces cerevisiae PMR1 gene encodes a Ca²⁺-ATPase localized in the Golgi. We have investigated the effects ofPMR1 disruption inS. cerevisiae on the glycosylation and secretion of three heterologous glycoproteins, human α₁-antitrypsin (α₁-AT), human antithrombin III (ATHIII), andAspergillus niger glucose oxidase (GOD). Thepmr1 null mutant strain secreted larger amounts of ATHIII and GOD proteins per a unit cell mass than the wild type strain. Despite a lower growth rate of thepmr1 mutant, two-fold higher level of human ATHIII was detected in the culture supernatant from thepmr1 mutant compared to that of the wild-type strain. Thepmr1 mutant strain secreted α₁-AT and the GOD proteins mostly as core-glycosylated forms, in contrast to the hyperglycosylated proteins secreted in the wild-type strain. Furthermore, the core-glycosylated forms secreted in thepmr1 mutant migrated slightly faster on SDS-PAGE than those secreted in themnn9 deletion mutant and the wild type strains. Analysis of the recombinant GOD with anti-α1,3-mannose antibody revealed that GOD secreted in thepmr1 mutant did not have terminal α1,3-linked mannoses unlike those secreted in themnn9 mutant and the wild type strains. The present results indicate that thepmr1 mutant, with the super-secretion phenotype, is useful as a host system to produce recombinant glycoproteins lacking high-mannose outer chains.     

The ectopically parting cells 1-2 (epc1-2) mutant exhibits an exaggerated response to abscisic acid

The ECTOPICALLY PARTING CELLS 1 (EPC1) gene encodes a putative retaining glycosyltransferase of the GT64 family, and epc1-1 mutant plants have a severely dwarfed phenotype. A new mutant allele of this gene, epc1-2, has been isolated. Reduced cell adhesion that has previously been reported for the epc1-1 mutant was not observed for either the epc1-1 or epc1-2 mutants grown in our conditions, suggesting that EPC1 does not affect cell adhesion but is involved in some other process affecting plant growth and development. It is shown that the epc1-2 mutant exhibits hypersensitivity to the phytohormone abscisic acid in germination and root elongation assays, however it shows an unaltered response to gibberellin, epi-brassinosteroid, auxin, or ethylene. An EPC1:YFP fusion protein is localized to small motile structures within the cytosol that are similar in size and number to the Golgi apparatus. Analysis of cell wall pectins revealed that levels of beta-(1,4)-galactan in the epc1-2 mutant are reduced by 50%, whilst other pectic polysaccharides (homogalacturonan, arabinan, and rhamnogalacturonan II) are unchanged.     

The podocyte-specific inactivation of Lmx1b, Ldb1 and E2a yields new insight into a transcriptional network in podocytes

Patients with nail-patella syndrome, which among other symptoms also includes podocyte-associated renal failure, suffer from mutations in the LMX1B gene. The disease severity among patients is quite variable and has given rise to speculations on the presence of modifier genes. Promising candidates for modifier proteins are the proteins interacting with LMX1B, such as LDB1 and E47. Since human kidney samples from patients are difficult to obtain, conventional Lmx1b knock-out mice have been extremely valuable to study the role of Lmx1b in podocyte differentiation. In contrast to findings in these mice, however, in which a downregulation of the Col4a3, Col4a4 and Nphs2 genes has been described, no such changes have been detected in kidney biopsies from patients. We now report on our results on the characterization of constitutive podocyte-specific Lmx1b, Ldb1 and E2a knock-out mice. Constitutive podocyte-specific Lmx1b knock-out mice survive for approximately 2 weeks after birth and do not present with a downregulation of the Col4a3, Col4a4 and Nphs2 genes, therefore they mimic the human disease more closely. The podocyte-specific Ldb1 knock-out mice survive longer, but then also succumb to renal failure, whereas the E2a knock-out mice show no renal symptoms for at least 6 months after birth. We conclude that LDB1, but not E2A is a promising candidate as a modifier gene in patients with nail-patella syndrome.     

Packing interactions between transmembrane helices alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.     

Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast

Inosine (I) at position 34 (wobble position) of tRNA is formed by the hydrolytic deamination of a genomically encoded adenosine (A). The enzyme catalyzing this reaction, termed tRNA A:34 deaminase, is the heterodimeric Tad2p/ADAT2.Tad3p/ADAT3 complex in eukaryotes. In budding yeast, deletion of each subunit is lethal, indicating that the wobble inosine tRNA modification is essential for viability; however, most of its physiological roles remain unknown. To identify novel cell cycle mutants in fission yeast, we isolated the tad3-1 mutant that is allelic to the tad3(+) gene encoding a homolog of budding yeast Tad3p. Interestingly, the tad3-1 mutant cells principally exhibited cell cycle-specific phenotype, namely temperature-sensitive and irreversible cell cycle arrest both in G(1) and G(2). Further analyses revealed that in the tad3-1 mutant cells, the S257N mutation that occurred in the catalytically inactive Tad3 subunit affected its association with catalytically active Tad2 subunit, leading to an impairment in the A to I conversion at position 34 of tRNA. In tad3-1 mutant cells, the overexpression of the tad3(+) gene completely suppressed the decreased tRNA inosine content. Notably, the overexpression of the tad2(+) gene partially suppressed the temperature-sensitive phenotype and the decreased tRNA inosine content, indicating that the tad3-1 mutant phenotype is because of the insufficient I(34) formation of tRNA. These results suggest that the wobble inosine tRNA modification is essential for cell cycle progression in the G(1)/S and G(2)/M transitions in fission yeast.     

Hailey-Hailey disease as an orthodisease of PMR1 deficiency in Saccharomyces cerevisiae

The term orthodisease has recently been introduced to define human disorders in which the pathogenic gene has orthologs in model organism genomes. Here, we describe Hailey-Hailey disease (HHD), a blistering skin disorder caused by haploinsufficiency of ATP2C1 as an orthodisease from a Saccharomyces cerevisiae perspective. ATP2C1 encodes the human secretory pathway Ca(2+)/Mn(2+) ATPase hSPCA1 and is orthologous to the PMR1 gene in S. cerevisiae. hSPCA1 fully complements PMR1 deficiency in yeast and pmr1DeltaS. cerevisiae has proved to be a valuable tool to screen ATP2C1 mutations and address potential pathogenic/pharmacologic mechanisms in HHD. Consequently, this human skin disorder is an ideal example of an orthodisease.     

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides

We studied the phosphorylation of the inner core region of N-linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N-oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.-K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648-13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A(3) (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C(1)) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9. (see structure below)     

水稻msp1-4突变体的鉴定及其UDT1和GAMYB基因的表达分析

通过对粳稻‘9522’辐射诱变,得到一隐性雄性不育突变体msp1-4（MULTIPLE SPOROCYTE）,用遗传定位方法将该基因座位定位在分子标记WY-4和WY-8之间,相距0．8cM,物理距离247kb。测序分析证明这247kb区间中的MSP1基因的编码区在第758bp到767bp之间发生了10个碱基的缺失。形态学观察结果表明该突变体和已经报告过的msp1突变体的表型基本一致。为分析水稻其它与花药发育相关的基因在msp1-4中的表达变化,用半定量RT-PCR技术检测到影响绒毡层和花粉发育的重要基因UDT1和GAMYB的表达在突变体中比在野生型水稻中低,说明这2个基因可能位于MSP1基因的下游。     

Mutant HNF-1alpha and mutant HNF-1beta identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1alpha and mutant HNF-1beta in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1alpha and 13 mutant HNF-1alpha, as well as wild HNF-1beta and 2 mutant HNF-1beta, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1alpha and wild HNF-1beta significantly transactivated DPP-IV promoter, but mutant HNF-1alpha and mutant HNF-1beta exhibited low transactivation activity. Moreover, to study whether mutant HNF-1alpha and mutant HNF-1beta change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1alpha or wild HNF-1beta, or else respective dominant-negative mutant HNF-1alphaT539fsdelC or dominant-negative mutant HNF-1betaR177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1alpha cells and wild HNF-1beta cells, whereas they decreased in HNF-1alphaT539fsdelC cells and HNF-1betaR177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1alpha and wild HNF-1beta have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1alpha and mutant HNF-1beta attenuate the stimulatory effect.