Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as “pan-genes,” being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.     

The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic,Mg2+-activated acetaldehyde dehydrogenase

The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg²⁺-activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg²⁺-activated ACDH activity had been lost from the mutants, although the mitochondrial, K⁺-activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205. © 1997 John Wiley & Sons, Ltd.     

18S rDNA和ITS相结合鉴定一株盐生海芦笋内生真菌的研究

对分离自盐生海芦笋的一株代号为Salicorn 8的真菌进行鉴定,提取基因组DNA后分别PCR扩增18S rDNA和ITS区域,基因测序并进行同源性分析,构建ITS基因系统发育树,结合形态学分析,将该菌鉴定为木贼镰刀菌(Fusarium equiseti)。     

Identification of a gene encoding a homocitrate synthase isoenzyme of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, most of the LYS structural genes have been identified except the genes encoding homocitrate synthase and α-aminoadipate aminotransferase. Expression of several LYS genes responds to an induction mechanism mediated by the product of LYS14 and an intermediate of the pathway, α-aminoadipate semialdehyde (αAASA) as an inducer. This activation is modulated by the presence of lysine in the growth medium leading to an apparent repression. Since the first enzyme of the pathway, homocitrate synthase, is feedback inhibited by lysine, it could be a major element in the control of αAASA supply. During the sequencing of chromosome IV of S. cerevisiae, the sequence of ORF D1298 showing a significant similarity with the nifV gene of Azotobacter vinelandii was reported. Disruption and overexpression of ORF D1298 demonstrate that this gene, named LYS20, encodes a homocitrate synthase. The disrupted segregants are able to grow on minimal medium and exhibit reduced but significant homocitrate synthase indicating that this activity is catalysed by at least two isoenzymes. We have also shown that the product of LYS20 is responsible for the greater part of the lysine production. The different isoforms are sensitive to inhibition by lysine but only the expression of LYS20 is strongly repressed by lysine. The N-terminal end of homocitrate synthase isoform coded by LYS20 contains no typical mitochondrial targeting sequence, suggesting that this enzyme is not located in the mitochondria.     

Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory-related yeast genes.     

基于vvhA基因检测创伤弧菌实时荧光PCR方法的建立

为建立创伤弧菌(VV)的快速检测方法,根据vvhA基因序列设计合成引物和探针,建立实时荧光PCR方法。结果显示:所建立的方法能特异扩增出VV标准阳性菌株,而对其它14种菌株没有扩增;该方法的灵敏度为15CFU/mL;稳定性和重复性试验结果表明,同一样品重复检测4次Ct值(循环阈值)的变异系数均小于2%;利用该检测方法对采集的156份样品进行检测,共计检出2份VV阳性样品,与行标法(SN/T 1870—2007)检测结果一致。该检测方法灵敏度高、特异性强,具有良好的实用性。     

一例点状掌跖角皮病大家系致病基因精细定位

目的在1例四代点状掌跖角皮病大家系中定位其致病基因所在区域。方法用覆盖8和15号染色体的微卫星标记对一例点状掌跖角皮病大家系进行致病基因的定位研究,用ABI3730测序仪进行微卫星标记的基因分型,利用Linkage软件(5.10 Version)和Cyrillic软件(2.01 Version)进行连锁和单倍型分析。结果该家系符合常染色体显性遗传模式,当外显率为100%时,排除了以前报道过的8q24.13-8q24.21区域,但在15号染色体上的微卫星标记D15S153处获得最大LOD值为5.38(重组率θ=0.00)。单倍型分析将该家系致病基因定位在微卫星标记D15S651和D15S988之间,该结果将国外定位的区域缩小至染色体15q22.2-15q22.31区域之间遗传距离为5.06cm内。结论染色体15q22.2-15q22.31区域存在该点状掌跖角皮病家系的致病基因,表明在中国人群中该病有遗传异质性存在。     

鞘氨醇单胞菌属菊粉酶基因lev的克隆、表达及酶学性质的研究

基于454高通量测序细菌JB13全基因组,获得一个编码菊粉酶的基因lev,该基因全长为1518bp,编码505个氨基酸组成的多肽,成熟蛋白的理论分子量为55.70ku。将基因lev克隆到表达载体pET28a(+),并在大肠杆菌BL21(DE3)中表达,表达产物具有菊粉酶活性,并初步研究了该酶的酶学性质。结果表明:酶作用的最适pH为5.5,最适温度为50℃,该研究为鞘氨醇单胞菌属来源菊粉酶的首次报道。     

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.     

一株耐高乙醇和低pH己酸产生菌Lysinibacillus fusiformis- pBE3-ep-SigB的构建

以Lysinibacillus fusiformis为出发菌,分别转染SigA和SigB突变基因,获得耐高乙醇和低pH胁迫的优良己酸产生菌。以pET32a（+）-SigA/SigB为模板,采用均匀试验优化SigA/SigB易错聚合酶链式反应（ep PCR）扩增体系中的Mn2+和Mg2+浓度,通过ep PCR获得ep-SigA/SigB突变基因,与载体pBE3-MCS连接后,构建L. fusiformis-pBE3-ep-SigA/SigB突变菌体库,将两种菌体库在pH值2～5和乙醇含量2%～12%条件下进行筛选。结果表明,L. fusiformis-pBE3-ep-SigB重组菌株耐受最高乙醇含量为8%左右,最低耐受pH值为2.5左右;当乙醇含量为8%、pH值为3.5时,L. fusiformis-pBE3-ep-SigB重组菌株的己酸产量达到（254.35±39.74） mg/100 mL,显著高于工程菌株L. fusiformis-pBE3-ep-SigA的己酸产量（P＜0.01）,且为出发菌L. fusiformis己酸产量（93.62±10.33） mg/100 mL的271.68%,表明L. fusiformis-pBE3-ep-SigB重组菌株具有较强的抗高乙醇浓度和低pH值胁迫产己酸的能力。     

XV. Yeast sequencing reports. Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes,a putative riboflavin synthase beta chain gene,and four new open reading frames

The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains. The nucleotide sequence reported here has been submitted to the EMBL data library under the Accession Number X84036.     

遗传性对称性色素异常症:一例家系报道及DSRAD基因突变的研究

目的报道1例遗传性对称性色素异常症家系,并对家系成员的DSRAD基因突变进行检测。方法PCR扩增该家系患者和健康对照个体DSRAD基因的全部外显子,并进行DNA测序,以100例无亲缘关系的正常人作对照。结果该家系患者DSRAD基因的第14外显子3388碱基发生了一个T→C的杂合突变,导致第1130位半胱氨酸被精氨酸替代,该区域可能存在DSRAD基因的脱氨基酶。家系中健康对照个体及无亲缘关系的正常人均未发现该突变。结论该遗传性对称性色素异常症家系中存在DSRAD基因的特异性突变,该突变引起编码蛋白功能缺陷,导致出现皮肤色素异常的临床表型。     

Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms

The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of α_S1-casein, β-casein, κ-casein, and β-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the κ-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr₁₃₆ and Ala₁₄₈. This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.     

深海古菌Thermococcus siculi HJ21高温普鲁兰酶基因的克隆及表达

根据NCBI 上公布的普鲁兰酶基因的保守序列设计简并引物,以Thermococcus siculi HJ21 基因组DNA 为模板进行PCR,得到T. siculi HJ21 普鲁兰酶的基因,测序后通过Blast(NCBI)数据库比对和分析表明扩增基因有一个4056bp、编码1351 个氨基酸的开放阅读框,为一新的普鲁兰酶基因。将该基因插入表达载体pET28a,并转化Escherichia coli BL21(DE3),经IPTG 诱导,测定普鲁兰酶比活力。重组转化子的细胞破碎液有高温普鲁兰酶活性,SDS-PAGE 电泳结果显示出分子质量约为150kD 的特异性条带。     

皮状丝孢酵母脂肪酸CoA连接酶 (Facl)基因的克隆与分析

从皮状丝孢酵母(Trichosporon cutaneum)中克隆了Facl基因及其5'-上游序列,并对Facl基因及其5'-上游序列进行了分析。根据前期的基因组文库测序结果,获得了898 bp DNA序列。以此为基础,2次使用染色体步移(Genome walking)方法,分别获得了854 bp和1 236 bp 5'-DNA序列。根据获得的5'和3'端序列,合成引物用于扩增Facl基因的全长DNA和c DNA序列。分析发现,皮状丝孢酵母Facl基因c DNA包含一个1 662 bp的开放读码框,DNA不含内含子序列。同时获得了482 bp的5'-上游序列。在5'-上游序列区域存在一系列预测的顺式作用元件。相似性分析的结果表明T.cutaneum和Rhodococcus erythropolis的Facl氨基酸序列相似性最高。该研究旨在为后继的Facl基因的功能和表达的研究、油脂代谢的遗传改造奠定基础。     

Construction of a Single PEP4 Allele Deletion in Saccharomyces carlsbergensis and a Preliminary Evaluation of Its Brewing Performance

The secretion of proteinase A (encoded by PEP4) from brewer's yeast is detrimental to the foam stability of unpasteurized beer. The aim of this study was to construct mutants of the allopolyploid Saccharomyces carlsbergensis strain TT, which were partially or completely deficient in proteinase A activity. Allelic PEP4 genes were consecutively disrupted by using the Cre‐loxP recombination system combined with PCR‐mediated gene disruption. A single PEP4 deletion mutant TT‐M was successfully constructed. However, no viable mutant could be obtained when the second allelic PEP4 gene was deleted. The brewing performances of the parent strain and the modified strain were compared on a 100 L pilot fermenter scale. Proteinase A activity in fermented wort brewed with mutant strain TT‐M was significantly lower (p<0.05) than that of the parent strain TT, whereas no significant difference on either maltose or maltotriose assimilation (p>0.05) was found. The mutant TT‐M remained genetically stable, as shown by diagnostic PCR, after re‐streaking for 20 generations. The flavor and taste of the final fermented wort, brewed with the mutant strain TT‐M, was evaluated by the Tsingtao expert sensory panel, and found to be comparable to that of the parent strain and exhibited no distinct defects. The flavor component profiles of these two finished products were also comparable. The study demonstrated allelic genes in polyploid industrial yeasts could be efficiently and consecutively deleted by the retractive primer disruption strategy, and the mutant of Saccharomyces carlsbergensis partially deficient in proteinase A contributed to an improvement in foam stability.     

The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae

The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.     

一起空肠弯曲菌引起食源性疾病事件的病原学研究及溯源分析

目的 对一起食源性疾病事件进行病原菌检测，了解病原菌毒力基因携带情况并进行溯源分析。方法 对事件采集的样本经FilmArray多重PCR系统进行快速初筛，同时进行细菌分离培养鉴定。使用PCR检测技术对分离菌株进行毒力基因检测，采用16S rRNA基因序列分析与PFGE分型方法对分离菌株进行同源性分析。结果 2份患者肛拭子样本和4份食堂厨工肛拭子样本检出空肠弯曲菌，检出菌株均携带flaA、cadF、imaA、cdtA、cdtB、cdtC等毒力基因。16S rRNA基因序列分析表明6株分离菌株均为空肠弯曲菌，1株菌株与其他5株菌株分子发育距离稍远。6株菌株经PFGE分型可分为3种带型，3株菌和2株菌分别呈现同一带型，2种带型相似性为52.2%；另1株菌为另一带型，与其他菌株带型相似性仅为26.7%。结论 实验室结果表明这是一起由不同克隆株的空肠弯曲菌感染引起的食源性疾病事件。