Sphingolipids Influence the Sensitivity of Lipid Bilayers to Fungicide,Syringomycin E

Sphingolipids with long chain bases hydroxylated at the C4 position are a requisite for the yeast, Saccharomyces cerevisia, to be sensitive to the ion channel forming antifungal agent, syringomycin E (SRE). A mutant S. cerevisiae strain, Δsyr2, having sphingolipids with a sphingoid base devoid of C4-hydroxylation, is resistant to SRE. To explore the mechanism of this resistance, we investigated the channel forming activity of SRE in lipid bilayers of varying composition. We found that the addition of sphingolipid-rich fraction from Δsyr2 to the membrane-forming solution (DOPS/DOPE/ergosterol) resulted in lipid bilayers with lower sensitivity to SRE compared with those containing sphingolipid fraction from wild-type S. cerevisiae. Other conditions being equal, the rate of increase of bilayer conductance was about 40 times slower, and the number of SRE channels was about 40 times less, with membranes containing Δsyr2 versus wild-type sphingolipids. Δsyr2 sphingolipids altered neither SRE single channel conductance nor the gating charge but the ability of SRE channels to open synchronously was diminished. The results suggest that the resistance of the Δsyr2 mutant to SRE may be partly due to the ability of sphingolipids without the C4 hydroxyl group to decrease the channel forming activity of SRE.     

Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1) in Cryptococcus neoformans reveals unique biochemical features

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1) from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasites which hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin (SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulate substrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcal enzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phytoceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environments is required for C. neoformans to cause disease, this study has important implications for understanding of C. neoformans pathogenicity.     

The Sphingenine/Sphinganine Ratio in Sphingolipids of Transplantable Rat Tumors Depends on a Transplantation Organ

The content of sphingenine (sphingosine) and sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.     

Mutations that are synthetically lethal with a<Emphasis Type="Italic"> gas1Δ</Emphasis> allele cause defects in the cell wall of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The GAS1 -related genes of fungi encode GPI-anchored proteins with -1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1 allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1 cells confirmed the important role of -1,6-glucan in cells that are defective in the processing of -1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1 defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1 mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2 mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1 cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.Communicated by C. P. Hollenberg     

AtBS14a and AtBS14b, two Bet1/Sft1-like SNAREs from Arabidopsis thaliana that complement mutations in the yeast SFT1 gene

SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells. Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity. Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Δ cells but not bet1Δ cells. These data together with structure–function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.     

Actinotrichia collagens and their role in fin formation

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

Glutathione S-Transferase M1, T1, P1 Genotypes and Risk for Development of Colorectal Cancer

The glutathione S-transferase (GST) supergene family is an important part of cellular enzyme defense against endogenous and exogenous chemicals, many of which have carcinogenic potential. The present investigation was conducted to detect a possible association between polymorphisms at the GSTM1, GSTT1, and GSTP1 genes and the interaction with cigarette smoking and colorectal cancer incidence. We examined 181 patients with colorectal cancer and 204 controls. DNA was extracted from whole blood, and the GSTM1, GSTT1, and GSTP1 polymorphisms were determined using a real-time polymerase chain reaction and fluorescence resonance energy transfer with a Light-Cycler instrument. Associations between specific genotypes and the development of colorectal cancer were examined by use of logistic regression analysis to calculate odds ratios (OR) and 95% confidence intervals (CI). The GSTM1 polymorphism was associated with an increased risk of developing colorectal cancer (OR = 1.62, 95% CI: 1.06–2.46). Also the risk of colorectal cancer associated with the GSTT1 null genotype was 1.64 (95% CI: 1.10–2.59). Statistically no differences were found between patients with colorectal cancer and control groups for the GSTP1 Ile/Ile, Ile/Val and Val/Val genotypes. In addition, the frequencies of the GSTM1 and GSTT1 deletion genotypes differed significantly between the cases and controls for current smokers; the GSTT1 null genotype especially is associated with a greater risk of colorectal cancer (OR = 2.44, 95% CI: 1.24–4.81). The GSTM1 and GSTT1 deletions were associated with an increased risk of developing a transverse or rectal tumor (OR = 1.86, 95% CI: 1.15–3.00; OR = 1.70, 95% CI: 1.02–2.84; respectively). The glutathione S-transferase polymorphisms were not associated with risk in patients stratified by age. The risk of colorectal cancer increased as putative high-risk genotypes increased for the combined genotypes of GSTM1 null, GSTT1 null, and either GSTP1 valine heterozygosity or GSTP1 valine homozygosity (OR = 2.69, 95% CI: 1.02–7.11). In conclusion, the results obtained in this study clearly suggest that those susceptibility factors related to different GST polymorphic enzymes are predisposing for colorectal cancer.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Two translesion synthesis DNA polymerase genes, AtPOLH and AtREV1, are involved in development and UV light resistance in Arabidopsis

Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.     

The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase

The Caenorhabditis elegans gene laf-1 is critical for both embryonic development and sex determination. Laf-1 is thought to promote male cell fates by negatively regulating expression of tra-2 in both hermaphrodites and males. We cloned laf-1 and established that it encodes a putative DEAD-box RNA helicase related to Saccharomyces cerevisiae Ded1p and Drosophila Vasa. Three sequenced laf-1 mutations are missense alleles affecting a small region of the protein in or near helicase motif III. We demonstrate that the phenotypes resulting from laf-1 mutations are due to loss or reduction of laf-1 function, and that both laf-1 and a related helicase vbh-1 function in germline sex determination. Laf-1 mRNA is expressed in both males and hermaphrodites and in both the germline and soma of hermaphrodites. It is expressed at all developmental stages and is most abundant in embryos. LAF-1 is predominantly, if not exclusively, cytoplasmic and colocalizes with PGL-1 in P granules of germline precursor cells. Previous results suggest that laf-1 functions to negatively regulate expression of the sex determination protein TRA-2, and we find that the abundance of TRA-2 is modestly elevated in laf-1/+ females. We discuss potential functions of LAF-1 as a helicase and its roles in sex determination.     

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the MC1R gene

Variations in vertebrate skin and hair color are due to varied amounts of eumelanin (brown/black) and phaeomelanin (red/yellow) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates. We have sequenced the entire coding region of the MC1R gene in Black-boned, Nanping indigenous and Romney Marsh sheep populations and found two silent mutation sites of A12G and G144C, respectively. PCR-RFLP of G144C showed that frequency of allele G in Black-boned, Nanping indigenous and Romney Marsh sheep was 0.818, 0.894 and 0, respectively. Sheep with GG genotype had significantly higher (P < 0.05) tyrosinase activity than sheep with CC genotype in the all investigated samples. Moreover, there was significant effect of MC1R genotype on coat color, suggesting that MC1R gene could affect coat color but not black traits. There would be merit in further studies using molecular techniques to elucidate the cause of black traits in these Black-boned sheep.     

Effects of constant light on circadian rhythmicity in mice lacking functional <Emphasis Type="Italic">cry</Emphasis> genes: dissimilar from <Emphasis Type="Italic">per</Emphasis> mutants

Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116, 2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL) in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1 ^−/− and mCry2 ^−/− knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination, rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209, 2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation.     

Constitutive expression of the SAP1 gene from willow (Salix discolor) causes early flowering in Arabidopsis thaliana

SAP1-1 and SAP1-2 were isolated from the male reproductive buds of willow (Salix discolor, clone S365). SAP1-1 differs from SAP1-2 based on a few nucleotide substitutions, but the sizes of their full-length cDNAs are identical. The deduced amino acid sequences of SAP1-1 and SAP1-2 were 98% similar and contain the same C-terminal amino acid motif “GYGA” like that of PTAP1-2 from Populus trichocarpa. The expression patterns of SAP1 in various parts of the male reproductive buds of S. discolor implicate this gene in the formation of the inflorescence meristems, bracts, and floral meristems. To characterize the functions of SAP1, we assessed Arabidopsis thaliana transformed with 35S∷SAP1-1. A total of 52 transgenic T₁ lines were obtained, and a 3:1 segregation ratio was obtained in the T₂ generation of each line. In the T₃ generation, five homozygous transgenic lines were obtained, which were used for further analysis. Screening of transgenic lines was greatly facilitated by the detection of GFP expression starting with germinating seeds. Phenotypes of the homozygous transgenic lines included early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, and formation of flowers with greater number of petals, stamens, and pistils. Northern analysis showed similar expression levels in all five lines. This study provides the first functional analysis of an APETALA1 (AP1)/SQUAMOSA (SQUA) homolog from a dioecious species and suggests that SAP1 is a homolog of the AP1/SQUA gene.     

对增强辣椒疫霉菌抗性的研究

病原物诱导型启动子能精确控制抗病基因在侵染位点的表达,是抗病基因工程的有效工具。prp1-1是来自马铃薯谷胱甘肽巯基转移酶基因启动子的一个273bp的片段,能够快速准确地启动被侵染位点抗病基因的表达;Rs-AFP2是具有对致病性丝状真菌的广谱抗性。该研究构建prp1-1调控Rs-AFP2基因表达的载体,经农杆菌介导转化法导入辣椒。逆转录PCR检测发现,转基因辣椒只在受到疫霉菌孢子侵染时,才由prp1-1启动Rs-AFP2基因的转录。用疫霉菌孢子灌根接种转基因辣椒T1代植株,35株T1代辣椒中有29株表现出明显的疫霉菌抗性。另将23株T1代辣椒种于人工气候箱,发现其形态和发育特征与相同条件下的非转基因植株无明显区别。研究表明,prp1-1调控Rs-AFP2的诱导表达达到了增强辣椒疫霉菌抗性的目的,而且避免了负面效应的发生。     

Cooperative functions of Hes/Hey genes in auditory hair cell and supporting cell development

Notch-mediated lateral inhibition has been reported to regulate auditory hair cell and supporting cell development from common precursors. While the Notch effector genes Hes1, Hes5 and Hey1 are expressed in the developing cochlea, inactivation of either of them causes only mild abnormality, suggesting their functional redundancy. To explore the roles of Hes/Hey genes in cochlear development, we examined compound heterozygous or homozygous mutant mice that lacked Hes1, Hes5 and Hey1 alleles. We found that a reduction in Hes/Hey gene dosage led to graded increase of hair cell formation. However, if at least one allele of Hes1, Hes5 or Hey1 was intact, excessive hair cells were accompanied by overproduction of supporting cells, suggesting that the hair cell increase does not occur at the expense of supporting cells, and that each Hes/Hey gene functions to induce supporting cells. By contrast, when all alleles of Hes1, Hes5 and Hey1 were inactivated, the number of hair cells increased more drastically, whereas that of supporting cells was unchanged compared with control, suggesting that supporting cell formation was balanced by their overproduction and fate conversion into hair cells. The increase of the cell numbers seemed to occur after the prosensory domain formation in the mutants because the proliferation state and the size of the prosensory domain were not affected. Thus, Hes1, Hes5 and Hey1 cooperatively inhibit hair cell formation, and one allele of Hes1, Hes5 or Hey1 is sufficient for supporting cell production probably by lateral inhibition in the sensory epithelium. Strikingly, Hes/Hey mutations lead to disorganized cell alignment and polarity and to hearing loss despite hair cell overproduction. These results suggest that Hes/Hey gene dosage is essential not only for generation of appropriate numbers of hair cells and supporting cells by controlling cell proliferation and lateral inhibition but also for the hearing ability by regulating the cell alignment and polarity.