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。     

A152G突变对GH43家族麦氏交替单胞菌木聚糖酶XynZT-2的影响

前期研究发现GH43家族近缘物种相同位点存在天然突变.为了探究保守位点区域的基因突变对酶催化性能的影响,对来源于麦氏交替单胞菌(Alteromonas macleodii)的木聚糖酶XynZT-2利用软件计算、随机突变及天然存在的突变进行分子改造,定点突变第152位丙氨酸为甘氨酸(A152G),将原酶与突变酶基因转化到...     

碳源对manA基因突变大肠杆菌代谢的影响

ManA基因编码的甘露糖-6-磷酸异构酶在大肠杆菌中催化D-甘露糖和D-果糖的异构化,促进大肠杆菌对碳源的代谢吸收。本文通过研究manA基因突变大肠杆菌对碳源的利用和编码糖代谢基因情况,探讨甘露糖-6-磷酸异构酶对大肠杆菌糖代谢的影响。采用Ⅱ型内含子逆转录突变方法构建manA基因突变大肠杆菌,分析manA基因突变大肠杆菌对不同碳源的利用情况和manA基因突变对大肠杆菌糖代谢相关基因表达的影响,结果显示,大肠杆菌BL21(DE3)ΔmanA以甘露糖、果糖为碳源时,菌株生长受到显著抑制;以淀粉为碳源时,BL21(DE3)ΔmanA菌株的生长显著优于野生型大肠杆菌;以葡萄糖为碳源时,manA基因突变对大肠杆菌的生长无显著影响。通过基因表达分析,发现大肠杆菌BL21(DE3)ΔmanA中甘露糖代谢相关基因的表达显著性降低;果糖代谢途径中6-磷酸果糖激酶Ⅰ亚基的编码基因(pfkA)显著下调表达;水解淀粉的α-淀粉酶编码基因(malS)显著性上调表达。ManA基因突变影响大肠杆菌甘露糖、果糖和淀粉代谢途径中相关基因的表达,从而影响大肠杆菌对碳源的利用。     

通过修饰ALD6基因降低啤酒酵母产乙酸量

酿酒酵母中乙醛脱氢酶(ALD6)活性的高低控制着啤酒中乙酸含量的多少.通过醋酸锂一步转化法,将一段对遗传霉素有抗性的DNA片段转入酵母细胞中,与ALD6基因的ORF(open reading frames)进行同源重组,经过抗性筛选得到ald6基因突变菌.通过筛选、验证、驯化获得一株性能稳定的ald6基因缺陷型突变菌.     

沙门氏菌环丙沙星诱导株marR, soxR和acrR基因突变与多重耐药性的相关性研究

本文对11株动物源体外诱导的环丙沙星抗性沙门氏菌的mar操纵子基因marO, marR, marA, marB, soxR, soxS和acrR基因(包括启动子区)的点突变进行检测,结果显示在沙门氏菌诱导株中marR, soxR和acrR基因检测到新突变,而marO, marA, marB和soxS基因未发现突变.应用实时荧光定量PCR对诱导株的marA和soxS基因及外排泵基因acrA, acrB和tolC的mRNA表达水平进行检测,结果表明这些基因的表达量均显著增加,且marA, soxS和acrAB-tolC的mRNA表达水平与沙门氏菌诱导株的多重耐药表型(Mar表型)相关,环丙沙星诱导株对其它氟喹诺酮类药物和结构不相关的抗生素均产生耐药.本研究结果表明沙门氏菌诱导株的调控基因突变和mar操纵子介导的acrAB-tolC的表达上调均贡献了沙门氏菌诱导株的氟喹诺酮抗性和Mar表型,且诱导株的marR, soxR和acrR基因突变与氟喹诺酮抗性和Mar表型具有相关性.     

一种人类K-ras基因突变检测试剂盒的质量标准研究

目的 评价和分析一种新的人类K-ras基因突变检测试剂盒质量,确定该类试剂质量标准和评价方法.方法 利用实时荧光定量PCR方法,检测7种K-ras基因单一突变点质控品的准确性、特异性、最低检测量和重复性.分别检测7例肠癌患者K-ras基因突变阳性组织样本和10例阴性样本的准确性和特异性 .结果 试剂盒准确性、特异性、最低检出量和重复性均符合质量标准.能准确和特异的检出单一点突变,具有较好的灵敏度和重复性.结论 该试剂盒质量标准设定较合理,在指导K-ras基因突变检测方面具有较好的应用前景.     

低蛋白酶A啤酒酵母突变株及其基因突变机理和中试、生产规模的试验研究

本研究采用优化的NTG和EMS条件进行连续诱变,结合酸变性血红蛋白平板的筛选条件,快速而高效地选育低产蛋白酶A啤酒酵母突变菌株.突变菌株啤酒发酵所分泌的蛋白酶A稳定并显著低于出发菌株,说明突变菌株经过诱变后,编码蛋白酶A的基因序列可能发生突变,导致分泌蛋白酶A活力下降.通过设计编码蛋白酶A的PEP4基因特征引物,采用PCR技术扩增其PEP4基因,并通过测序得出其基因序列,与出发酵母菌株序列和标准酵母菌株相应基金序列比对,并研究其突变位点.结果表明发生突变的氨基酸位于N端的第134位,发生突变的氨基酸为天冬氨酸,即由天冬氨酸突变为天冬酰胺.PEP4基因相应N端的第134位天冬氨酸是蛋白酶A酶活中心的三个氨基酸之一,由此从基因水平解释了突变菌株产生低蛋白酶A活力的分子机理.突变菌株经过实验室发酵评价后应用于中试和大生产,其试验结果表明:突变菌株和出发菌株酿造性能基本一致、对应的风味骨架成分和相应口感比较接近,而蛋白酶A酶活降低30%以上.生产规模上突变菌株生产的纯生啤酒保存6个月后泡持性仍达210秒以上,泡沫衰减率5%～7%,比对照样低50%.本研究通过诱变育种选育得到低蛋白酶A、各项发酵性能指标良好的酵母突变菌株,经过大生产规模化试验验证该突变菌株适用于纯生啤酒的生产,能明显改善纯生啤酒泡沫稳定性,为彻底解决目前啤酒行业普遍存在的纯生啤酒泡沫衰减问题奠定了基础.     

高分泌型无赖百当烟草8306突变材料的创制与分析

8306是具有晾晒烟风格的烤烟育种材料.在前期试验中创制了NtCPS2基因突变株系8306-1,抑制了晾晒烟风格代表性物质赖百当二萜的合成.为进一步提高8306-1株系中西柏烷二萜和蔗糖酯含量(质量分数),采用基因编辑技术对8306-1株系的NtCycB2基因进行敲除,获得了纯合突变株系8306-2.与8306、830...     

沙门菌传代中(氟)喹诺酮类抗生素筛选株药敏性及相关基因突变和表达

目的研究在不同浓度(氟)喹诺酮类抗生素存在条件下,沙门菌在传代时得到的筛选株中与耐药性相关部分基因的变异和表达水平变化规律及其与耐药性间的关联性。方法使用含有一定浓度(氟)喹诺酮类抗生素的肉汤培养基对沙门菌进行培养,在含有相同浓度同类抗生素的平板上划线筛选突变株,微量肉汤稀释法测定筛选株的药敏性,聚合酶链式反应(PCR)结合DNA测序确定喹诺酮耐药决定区(quinolone resistance-determining region,QRDR)基因突变,实时荧光定量PCR(real-time qPCR)法检测外排泵AcrAB-TolC编码基因表达水平。结果沙门菌(ATCC 14028s)在含有不同代、不同浓度(氟)喹诺酮类抗生素的LB培养基中培养、筛选后,筛选株对抗生素耐受性均有不同程度增加。萘啶酮酸第5～7代筛选株gyrA突变,发生Asp87Tyr变异;环丙沙星第4～7代筛选株gyrA突变,发生Asp87Asn变异;加替沙星、左氧氟沙星和德拉沙星第1～7代筛选株gyrA中均未检出核苷酸突变。随着抗生素浓度增加,各抗生素相应筛选株中外排泵AcrAB-TolC编码基因表达水平较原始菌株增加,差异有统计学意义(P0.05),第7代菌株acrAB-tolC表达量间差异无统计学意义(P0.05)。(氟)喹诺酮类抗生素对不同代筛选株的最小抑菌浓度(MIC)与其对沙门菌(ATCC 14028s)的MIC值比值、gyrA突变、acrAB-tolC表达水平与抗生素作用浓度和筛选代数间呈正相关。结论在(氟)喹诺酮类抗生素作用下,沙门菌可通过QRDR基因突变及增加acrAB-tolC表达适应抗生素胁迫环境;新一代(氟)喹诺酮类抗生素作用于沙门菌时,菌株QRDR靶位点突变概率降低;多次重复作用后,菌株acrAB-tolC表达量虽然增加,但表达量间差异无统计学意义(P0.05),避免了因突变导致耐药性的进一步增强。     

PCR-Targeting体系构建棒状链霉菌基因突变株

棒状链霉菌(Streptomyces clavuligerus)可产生多种β-内酰胺类化合物,其中包括克拉维酸。利用聚合酶链式反应(PCR)-Targeting体系构建棒状链霉菌突变株是获得克拉维酸高产菌株的又一可行方法。采用该体系对棒状链霉菌的lat基因进行了敲除,获得了没有抗性标记的lat基因被敲除的突变株S.clavuligerus lat::scar,其克拉维酸产量是出发菌株的2.8倍。由于最终构建的突变株没有抗性标记,因此可以将该方法应用于突变棒状链霉菌的其他基因,实现用一种抗性标记突变同一菌株不同基因的目的,为链霉菌的基因突变提供了又一种有效的方法。     

Detailed analysis of mortality rates in the female progeny of 1,001 Holstein bulls allows the discovery of new dominant genetic defects

Reducing juvenile mortality in cattle is important for both economic and animal welfare reasons. Previous studies have revealed a large variability in mortality rates between breeds and sire progeny groups, with some extreme cases due to dominant mutations causing various syndromes among the descendants of mosaic bulls. The purpose of this study was to monitor sire-family calf mortality within the French and Walloon Holstein populations, and to use this information to detect genetic defects that might have been overlooked by lack of specific symptoms. In a population of heifers born from 1,001 bulls between 2017 and 2020, the average sire-family mortality rates were of 11.8% from birth to 1 year of age and of 4.2, 2.9, 3.1, and 3.2% for the perinatal, postnatal, preweaning, and postweaning subperiods, respectively. After outlining the 5 worst bulls per category, we paid particular attention to the bulls Mo and Pa, because they were half-brothers. Using a battery of approaches, including necropsies, karyotyping, genetic mapping, and whole-genome sequencing, we described 2 new independent genetic defects in their progeny and their molecular etiology. Mo was found to carry a de novo reciprocal translocation between chromosomes BTA26 and BTA29, leading to increased embryonic and juvenile mortality because of aneuploidy. Clinical examination of 2 calves that were monosomic for a large proportion of BTA29, including an orthologous segment deleted in human Jacobsen syndrome, revealed symptoms shared between species. In contrast, Pa was found to be mosaic for a dominant de novo nonsense mutation of GATA 6 binding protein (GATA6), causing severe cardiac malformations. In conclusion, our results highlight the power of monitoring juvenile mortality to identify dominant genetic defects due to de novo mutation events.     

全基因组测序和real-time PCR法检测食源性沙门氏菌parC、gyrA基因突变特征

以45 株食源性沙门氏菌喹诺酮耐药株为对象,采取全基因组测序和实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）法检测parC、gyrA基因突变位点,并对检测方法可靠性、耐药基因突变特征进行评估和分析。首先将4 株沙门氏菌进行二代全基因组测序,根据测序数据分析结果,建立了一种real-time PCR法检测gyrA Asp87Tyr、gyrA Asp87Asn、parC Thr57Ser和parC Ser80Ile这4 个突变位点。将沙门氏菌进行qnrS、qnrA、qnrB的real-time PCR检测,发现有31 株菌未检出qnr基因。以这31 株菌为对象,采取real-time PCR法筛查基因突变位点,结果发现parC Thr57Ser和gyrA Asp87Asn型突变最常见。将real-time PCR阳性的10 株菌扩增parC、gyrA基因全长并测序,real-time PCR检测和测序结果完全吻合,说明了real-time PCR检测的可靠性。全基因组测序和real-time PCR法相结合的方法用于耐药基因突变筛查,既可以发现新的基因突变,又可以快速筛查大样本的主要突变类型,可作为沙门氏菌耐药性研究的一种可靠手段。     

利用HRM技术快速筛选低镉烟草突变体

低镉烟草种质资源对烟草育种及烟叶安全具有重要意义。本研究利用EMS处理贵烟1号获得诱变群体，按株系种植2020个M₂株系，每株系种植10株，株系内3株叶片混样提取DNA，并采用HRM技术对样本池筛选烟草镉转运基因NtHMA4突变体，共获得49份疑似突变材料。DNA测序鉴定发现，其中21个株系发生SNP突变，其中15个为错义突变，其余为同义突变。对突变体与和野生型材料叶片的镉含量分析发现，有8份突变体的镉含量较野生型显著下降（16.19%~42.08%）。利用HRM技术跟踪检测10份种子可正常萌发的错义突变体M₃家系，结果表明，HRM技术不仅可快速筛选烟草EMS诱变群体中的突变材料，结合DNA测序也可进一步对突变杂合体后代进行快速、准确的基因分型。      

The Effect of a Suppressed rad52 Mutation on the Suppression of rad6 by srs2

We have cloned a suppressor of a temperature-sensitive rad52 allele and found it to be a mutation in PUP1, a gene encoding a protease subunit of the 20s proteasome. This identity prompted us to examine the interrelationship among PUP1, RAD52, SRS2 and RAD6 because srs2 mutations not only suppress some rad52 mutations but also suppress deletions of rad6, a gene encoding a protein in the ubiquination-dependent proteolysis pathway. We have found that while srs2 suppresses the UV sensitivity of rad6 in the presence of RAD52, srs2 cannot suppress rad6 when the temperature-sensitive allele of rad52 is present. This inability of srs2 to suppress rad6 is irrespective of the incubation temperature or whether pup1 is suppressing the temperature-sensitive rad52 mutation. © 1997 by John Wiley & Sons, Ltd.     

Yeast mutants with increased bacterial transposon Tn5 excision

Five complementing recessive mutations that exhibit increased bacterial transposon Tn5 precise excision in yeast Saccharomyces cerevisiae were obtained by ethylmethanesulfonate treatment. One of these mutations (tex1) was submitted to extensive genetic analysis. tex1 is a recessive temperature-sensitive mutation resulting in a 20-100-fold increase in Tn5 excision. It also has increased frequencies of ochre mutation reversion, of forward mutation to canavanine resistance, and loss of chromosome III or its right arm. The possible mechanism of tex1 effects is discussed.     

Two mutations in Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae)

Clear eye (c) and dark body colour (d) mutations in Oryzaephilus surinamensis (L.) are described. The c mutation is phenotypically identical to “pearl” (Blackman, 1966), lacking pigment in the compound eyes and also in the larval ocelli and Malpighian tubules. These structures are deeply pigmented in the wild type. The d mutation appears as a blackish-brown coloration of the adult, pupa and larva; the wild type body colour is chestnut brown. Crosses made between clear-eyed and dark insects and also crosses of individuals, showing both mutations, with wild type insects showed that the mutations were characterised by recessive autosomal inheritance and were not linked. There was some evidence that the viability of insects homozygous for both mutations was slightly reduced.     

Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance

Sixteen Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4096 μg/mL). Among them, 4 Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5 μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), and single mutation in parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70 ~ 100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.     

Isolation and characterization of a phosphoprotein phosphatase-deficient mutant in yeast

The ppd1 mutant of yeast, Saccharomyces cerevisiae, was isolated as a suppressor of the cyr2 mutation which caused alteration of the catalytic subunit of cAMP-dependent protein kinase. Three peaks of phosphoprotein phosphatase activity (peak I, II and III) were identified by DEAE-Sephacel chromatography of crude extracts of the wild-type strain. The ppd1 mutant was deficient in peak III phosphoprotein phosphatase activity. The peak III enzyme efficiently utilized the phosphorylated forms of NAD-dependent glutamate dehydrogenase and trehalase as substrate. The ppd1 mutation did not suppress the cyr1, CYR3 or ras1 ras2 mutations. The ppd1 locus was located on chromosome II and had identical characteristics with glc1. The ppd1 mutation suppressed the G1 arrest caused by nutritional limitation, but maintained sensitivity to mating pheromone. In diploids homozygous for the ppd1 mutation, no premeiotic DNA replication and commitment to intragenic recombination occurred and no spores were formed, suggesting that the accumulation of phosphorylated proteins in the absence of one of the phosphoprotein phosphatases is required for mitosis but not for the initiation of meiosis.     

IV. Yeast sequencing reports. Nucleotide sequence of the SAC2 gene of Saccharomyces cerevisiae

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC2 gene. A cloned genomic DNA fragment that complements the cold-sensitive growth phenotype associated with such a suppressor mutation (sac2-1) was sequenced. The fragment contained an open reading frame that encodes a 641 amino acid predicted hydrophilic protein with a molecular weight of 74 445. No sequences with significant similarity to SAC2 were found in the GenBank and EMBL databases. A SAC2 disruption mutation was constructed which had phenotypes similar to the sac2-1 point mutation. A haploid SAC2 disruption strain failed to grow at low temperature and the disruption allele suppressed the temperature-sensitive act1-1 growth defect. The suppression phenotype was dependent on the strain background. The SAC2 sequence has been submitted to the EMBL data library (Accession Number Z29988).