过表达ZmSKIP基因提高烟草抗低温胁迫的能力

以野生型和过表达ZmSKIP基因烟草为试材, 研究了低温胁迫下过表达ZmSKIP对烟草抗氧化能力的影响。测定了不同低温处理时间下过表达ZmSKIP转基因烟草T3代植株和野生型植株抗氧化酶如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）活性和丙二醛（MDA）含量以及相对电导率, 结果表明, 低温下, 相对于野生型植株, 转基因烟草具有较高的抗氧化酶活性和较低的相对电导率和MDA含量, 说明过表达ZmSKIP提高了转基因植株的耐低温胁迫能力。     

过量表达GST基因对盐胁迫下转基因拟南芥氧化损伤的影响

利用模式生物拟南芥作为实验材料,通过测定谷胱甘肽-抗坏血酸代谢相关酶（GST、GPX、APX、GR、DHAR、MDHAR）的活性和GSH、ASA、MDA含量以及生物量等来研究过量表达具有过氧化物酶活性的盐地碱蓬谷胱甘肽转移酶基因（GST基因）对盐胁迫下转基因拟南芥氧化损伤的影响。结果显示,转基因拟南芥比野生型具有较高的GST、GPX以及MDHAR酶活性;前者还具有较多的还原型谷胱甘肽和抗坏血酸,并且谷胱甘肽库氧化水平较野生型高。盐胁迫不但部分抑制了野生型拟南芥的生长,同时也导致了大量脂质过氧化物的积累;而盐胁迫对转基因拟南芥的生长抑制不明显,也没有较多的脂质过氧化物的积累。结果表明,过量表达盐地碱蓬谷胱甘肽转移酶基因提高．广转基因拟南芥依赖于还原型谷胱甘肽的过氧化物清除途径,同时有可能改变了GSH和ASA的代谢途径,这两方面的作用导致了转基因拟南芥氧化损伤的降低,使转基因拟南芥在盐胁迫下保持较好的生长态势。     

西伯利亚蓼谷胱甘肽转移酶和半胱氨酸合成酶基因在酿酒酵母中的共表达

谷胱甘肽转移酶和半胱氨酸合成酶在清除活性氧（reactive oxygen species,ROS）中起重要作用。采用0．36mol·L^-1 NaHCO3对西伯利亚蓼（Polygonum sibiricum）进行胁迫处理,荧光定量PCR分析表明这2个基因的表达受盐胁迫强烈诱导。为了分析2个基因是否具有抗盐能力以及其相互协同能力,从cDNA文库中获得谷胱甘肽转移酶（GST）和半胱氨酸合成酶（Cs）2个基因,分别将GST、CS和GST＋CS转入酿酒酵母（Saccharomyces cerevisiae）中,并分别命名转基因酵母为ty-gst、tycs和ty-gc。在1mol·L^-1 Na2C03和5mol·L^-1 NaCl胁迫处理下,转基因酵母（ty-gst、ty-cs和ty-gc）的耐盐能力均明显高于野生型酵母（㈣,而三者之间并无显著差别。在0．4mol·L^-1 NaCl胁迫处理下,转基因酵母（ty-gst、ty-cs和ty-gc）的抗氧化酶类相关基因SOD1、SOD2、GPX1和GPX3的表达量均低于野生型酵母（对照）（wy）,而CTA7表达量均高于野生型酵母（对照）（wy）。转基因酵母ty-cs在0．4mol·L^-1 NaCl胁迫处理前后其超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）和谷胱甘肽过氧化物酶（glutathione peroxJdase,GPX）的活性均表现为最高。     

过量表达番茄类囊体膜抗坏血酸过氧化物酶基因（StAPX）提高了烟草种苗的抗氧化能力

为了探讨叶绿体类囊体膜抗坏血酸过氧化物酶（tAPX）与其抗氧化性的关系,从番茄叶片中分离了叶绿体类囊体膜抗坏血酸过氧化物酶基因（StA跚并转入到烟草中。以野生型（WT）、转正义StAPX烟草株系T3-3和T3-6为试材,测定了外源过氧化氢诱导的氧化胁迫条件下APX酶活性、过氧化氢酶（CAT）活性、过氧化氢（H2O2）含量、叶绿素荧光参数及叶绿素含量等。Northern杂交显示StAPX因的表达受外源H2O2氧化胁迫的诱导。氧化胁迫下转基因烟草的APX酶活性和清除H2O2的能力都显著高于野生型,并且转基因烟草比野生型具有更高的PSII最大光化学效率及叶绿素含量。结果表明,．刚尸舶勺过量表达有助于提高外源H2O2诱导的转基因烟草的抗氧化能力。     

胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析

胡杨(Populus euphratica Oliv.)具有极强抗盐碱能力。本实验室前期胡杨微阵列芯片数据结果显示:盐胁迫下,胡杨谷胱甘肽过氧化物酶基因(PeGPX)的转录上调,暗示该基因可能对胡杨耐盐性具有一定的作用。为分析 GPX 对植物耐盐性的贡献,本研究以胡杨为材料,利用 RT-PCR 方法克隆了胡杨谷胱甘肽过氧化物酶PeGPX基因,并在烟草中过量表达该基因,以分析谷胱甘肽过氧化物酶活性与植物耐盐性的关系。研究结果显示,实验中克隆的 cDNA (PeGPX)编码谷胱甘肽过氧化物酶,其 ORF 为 693 bp,其蛋白由 231 个氨基酸编码。过量表达 PeGPX 基因的烟草与野生型烟草的耐盐性实验结果显示,野生型烟草植株在加 NaCl(200 mmol/L)的 MS 培养基中生长 15 d 后,无明显的长高,且不长根;而转基因烟草在同样的加盐培养基上,生长基本没有受到抑制,植株生长状态良好,并且能够长根。光合数据显示,在盐胁迫下过量表达 PeGPX 基因烟草的净光合速率受到影响明显小于野生型烟草的净光合速率。酶活数据显示,转基因株系 GPX 酶活与野生型的相比在盐胁迫下活性有非常显著的提高。我们的研究结果说明:过表达 PeGPX 基因使得烟草的耐盐性得到显著提高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。     

过表达LeNLP4转录因子提高番茄抗低温胁迫能力

NAC（NAM-ATAF1,2-CUC2）转录因子在植物胁迫响应中起重要作用。为了探讨三舭丹基因在番茄抗低温胁迫中的功能,分离了番茄LeNLP4转录因子基因,并获得转正义LeNLP4基因番茄植株。荧光定量PCR分析表明,LeNLP4的表达受低温诱导。与野生型植株相比,在4℃胁迫下转基因植株具有较高的生长量和光系统II（PSH）最大光化学效率（Fv／Fm）、过氧化氢（H2O2）和超氧阴离子（O2-）清除速率、抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性,以及较低的丙二醛（MDA）含量和相对电导率（REC）。过表达株系中SICBF1的表达高于野生型。上述结果表明,LeNLP4的过表达提高了转基因番茄抗低温胁迫能力。     

过表达单脱氢抗坏血酸还原酶基因提高番茄抗UV-B胁迫能力

以野生型（WT）和转正义叶绿体单脱氢抗坏血酸还原酶基因（LeMDAR）番茄为试材,探讨了UV-B胁迫下过表达LeMDAR对番茄抗氧化能力的影响。测定了不同时间uV-B处理下番茄抗坏血酸（AsA）含量,脱氢抗坏血酸（DHA）含量,单脱氢抗坏血酸还原酶（MDAR）活性,光合速率和叶绿素荧光参数等。在UV-B处理下,转基因番茄植株的AsA含量、MDAR酶及抗坏血酸过氧化物酶（APx）活性、H：0：和超氧阴离子清除速率、净光合速率（只）高于野生型番茄。此外,紫外胁迫下,转基因株系丙二醛（MDA）含量和相对电导率（REC）较野生型增加的少。上述结果表明,MDAR对抗抗坏血酸再生具有重要作用,过表达LeMDAR提高了番茄植株抗氧化能力,对光合机构有保护作用。     

RNAi抑制萝卜过氧化物酶基因的效果受光照影响

农杆菌介导的RNAi技术已广泛应用于研究植物基因的功能.本实验应用小块萝卜肉质根体外培养,探讨光照对干扰萝卜过氧化物酶基因Rsprx1表达的影响.结果表明,干扰萝卜过氧化物酶基因Rsprx1表达后,抑制组中过氧化物酶活性显著低于对照组,光照减弱RNAi的抑制作用;抑制作用始于浸染后4 h, 过氧化物酶活性减低时,花青素含量增加,但光照增加花青素含量;HPLC结果显示,与对照组比,抑制组中花青素苷种类和含量有较大差异;花青素合成相关基因（RsCHS、RsDFR和RsLDOX）的mRNA水平在处理后明显上调.此外,过氧化氢酶活性和H2O2含量相应升高.由此表明,光照可影响农杆菌介导的RNAi效果,干扰萝卜过氧化物酶基因Rsprx1表达可以通过影响花青素合成相关基因的表达和过氧化氢含量,从而影响花青素代谢.     

烟草NtNAC1基因的克隆及其在烟草中的抗旱功能分析

NAC转录因子在植物信号转导及非生物损伤过程中起重要作用。本实验从烟草c DNA文库中克隆了NtNAC1基因,c DNA编码区全长861 bp,编码286个氨基酸。进化树分析结果显示,Nt NAC1基因编码的氨基酸序列与马铃薯同源性最高。农杆菌介导的遗传转化获得37株转基因烟草,用20%PEG6000处理转基因和野生型植株7 d。结果显示,转基因植株的超氧化物歧化酶(SOD)、过氧化物酶(POD)的酶活性都高于野生型,丙二醛(MDA)和脯氨酸(Pro)含量都低于野生型。RT-PCR分析结果显示,Nt NAC1基因以及Nt NAC基因表达高于野生型,脯氨酸合成的2个关键酶吡咯啉-5-羧酸合成酶(P5CS)和鸟氨酸-δ-氨基转移酶(δ-OAT)基因表达低于野生型。选取T_0代转基因和野生型种子,对苗期根系进行耐旱性分析。结果发现,在300 mmol·L~(-1)甘露醇胁迫下,转基因根系比野生型长,野生型根系的生长明显受到抑制。这表明转Nt NAC1基因的表达可能提高了植株的抗旱能力。     

西伯利亚蓼谷胱甘肽转移酶和半胱氨酸合成酶基因在酿酒酵母中的共表达

谷胱甘肽转移酶和半胱氨酸合成酶在清除活性氧(reactive oxygen species,ROS)中起重要作用。采用0.36 mol.L-1NaHCO3对西伯利亚蓼(Polygonum sibiricum)进行胁迫处理, 荧光定量PCR分析表明这2个基因的表达受盐胁迫强烈诱导。为了分析2个基因是否具有抗盐能力以及其相互协同能力, 从cDNA文库中获得谷胱甘肽转移酶(GST)和半胱氨酸合成酶(CS)2个基因, 分别将GST、CS和GST+CS转入酿酒酵母(Saccharomyces cerevisiae)中, 并分别命名转基因酵母为ty-gst、tycs和ty-gc。在1 mol.L-1 Na2CO3和5 mol.L-1 NaCl胁迫处理下, 转基因酵母(ty-gst、ty-cs和ty-gc)的耐盐能力均明显高于野生型酵母(wy), 而三者之间并无显著差别。在0.4 mol.L-1 NaCl胁迫处理下, 转基因酵母(ty-gst、ty-cs和ty-gc)的抗氧化酶类相关基因SOD1、SOD2、GPX1和GPX3的表达量均低于野生型酵母(对照)(wy), 而CTA1表达量均高于野生型酵母(对照)(wy)。转基因酵母ty-cs在0.4 mol.L-1 NaCl胁迫处理前后其超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)和谷胱甘肽过氧化物酶(glutathione peroxidase, GPX)的活性均表现为最高。     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid-k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFalpha1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long-term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid-containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid-k locus was observed. (c) 1993 John Wiley & Sons, Inc.     

Application of comparative proteome analysis to reveal influence of cultivation conditions on asymmetric bioreduction of β-keto ester by Saccharomyces cerevisiae

Industrial bakers' yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and D: -arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

Effect of altered sterol composition on the salt tolerance of Zygosaccharomyces rouxii

To obtain mutants containing altered sterol composition and sterol contents, nystatin-resistant mutants were isolated in Zygosaccharomyces rouxii. Two of nine mutants isolated were resistant toward 20 μg of nystatin per ml, while the other seven showed resistance toward 50 μg per ml. However, the seven mutants could not grow at 35°C. TN5, a mutant of the first group, showed the same sterol composition as the wild type strain, with ergosterol and zymosterol as major sterols, whereas it contained free sterols about 70% of those of the wild type. TN1 and TN3, representative mutants of the second group, had altered sterol compositions, containing three major sterols, zymosterol, ergosta-5,7,24-trienol, and an unidentified sterol. TN1 and TN3 could not grow in YPD medium containing more than 8% NaCl, whereas TN5 grew in the same medium containing 15% NaCl after a longer lag phase than the wild type strain. TN1 and TN3, in particular TN3, when incubated in YPD medium containing 15% NaCl, leaked significant amounts of glycerol. Protoplasts of these mutants were more labile than those of the wild-type cells. These facts suggest that the amount and kind of ergosterol in the cell membrane might be concerned with the salt tolerance of Z. rouxii.     

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。     

酿酒酵母遗传缺陷型菌株化学转化方法研究

目的:通过改善转化条件,提高缺陷型酿酒酵母DNA转化效率。方法:以醋酸锂化学转化法为基础,以酿酒酵母菌株fab1::KAN作为遗传转化受体,以敲除MZM1基因为转化目的,对影响遗传缺陷型菌株转化效率的参数(醋酸锂前处理、热激时间及转化后复苏时间)进行优化,确定适合缺陷型酵母DNA转化的最佳方法。结果:不同时间(30 min,60 min和120 min)醋酸锂的前处理均可以提高其转化效率,热激前使用醋酸锂处理酵母30 min转化效率最高;热激10 min是转化效率由高到低的转折点,30 min热激明显降低了转化效率;YPD培养基用于转化后酵母的复苏,随培养时间(30 min,60 min和120 min)的延长,酵母转化效率逐步增加,120 min作为热敏性菌株转化后的复苏时间较佳。结论:与野生型菌株类似,通过转化条件的优化,遗传缺陷型菌株的转化效率也可以满足大多数试验要求。     

Catalase gene disruptant of the human pathogenic yeast Candida albicans is defective in hyphal growth, and a catalase-specific inhibitor can suppress hyphal growth of wild-type cells

Although the catalase gene (CAT1) disruptant of the human pathogenic yeast Candida albicans was viable under ordinary growth conditions, we previously found that it could not grow on YPD (yeast extract/peptone/dextrose) containing SDS or at higher growth temperatures. To investigate the pleiotrophic nature of the disruptant, we examined the effect of the catalase inhibitor 3-AT on the growth of wild-type strains. Surprisingly, the addition of 3-AT and SDS caused the wild-type cells to be non-viable on YPD plates. We found an additional phenotype of the catalase gene disruptant: it did not produce normal hyphae on Spider medium. Hyphal growth was observed in a CAP1 (Candida AP-1-like protein gene) disruptant, a HOG1 (high-osmolarity glycerol signaling pathway gene) disruptant, and the double CAP1/HOG1 disruptant, suggesting that the defect in hyphal formation by the catalase disruptant was independent of these genes. Addition of 3-AT and SDS to hyphae-inducing media suppressed growth of normal hyphae in the wild-type strain. The potential necessity for catalase action upon exposure to hyphae-inducing conditions was confirmed by the immediate elevation of the catalase gene message. In spite of the requirement for catalase during hyphal growth, the catalase gene disruptant was capable of forming germ tubes in medium containing serum.     

Wall mannoproteins in cells from colonial phenotypic variants of Candida albicans

Candida albicans ATCC 26555 switched at high frequency (10(-1) to 10(-3)) between several phenotypes identified by colony morphology on a defined mineral amino-acid-containing agar medium supplemented with arginine and zinc (LAZ medium). When cells taken from colonies exhibiting distinct morphologies were plated directly onto LAZ agar, spontaneous conversion to all the variant phenotypes occurred at combined frequencies of 2.1 x 10(-1) to 9.5 x 10(-3). However, when cells taken from the different colonial phenotypes were plated directly onto an undefined medium (yeast extract/peptone/dextrose; YPD medium), or first incubated in liquid YPD medium and then cloned on YPD agar, all colonies observed exhibited the same phenotype (smooth-white). When cells from the smooth-white colonies were plated as clones on LAZ agar, the original switch phenotype reappeared. These results suggest that environmental conditions such as the growth medium (and possibly the temperature) influence switching by suppressing phenotype expression, but have no effect on genotype. The variant colony morphologies also appeared to be associated with differences in the relative proportions of yeast and mycelial cells. Zymolyase digests of wall preparations obtained from cells belonging to different colonial phenotypes were analysed by SDS-PAGE. After blotting to nitrocellulose paper, the mannoproteins were stained with Concanavalin A, with a polyclonal antiserum enriched in antibodies against mycelium-specific wall components, and with a monoclonal antibody raised against a high-molecular-mass mannoprotein band (260 kDa) specific to the walls of mycelial cells. The results suggest that phenotypic switching might be associated with changes in the degree of glycosylation in high-molecular-mass mannoproteins, or in the way these mannoproteins are bound to other cell wall components.