儿童感染肺炎链球菌的 青霉素结合蛋白基因突变分析

目的分析儿童感染肺炎链球菌的青霉素结合蛋白基因突变与青霉素耐药水平之间的关系。方法自2012年1月至2014年12月期间分离的1 317株肺炎链球菌中随机抽取出青霉素MIC=2.0μg/mL、4.0μg/mL、≥8.0μg/mL各20株共60株作为实验菌株,采用PCR方法对实验菌株进行青霉素结合蛋白PBP1a、PBP1b、PBP2a、PBP2b、PBP2x、PBP3的基因扩增,扩增产物进一步纯化和测序,测序结果与青霉素敏感肺炎链球菌R6就国际上公认的PBPs保守序列进行比对分析。结果 60株肺炎链球菌的PBP2b、PBP1a、PBP2x、PBP2a基因的保守区或保守区附件均发现氨基酸突变,未发现PBP3与PBP1b突变。中介与耐药菌株基因突变位点存在重合,主要出现在单一的PBP1a序列的370STMK模体元件内Thr371Ser置换突变或伴有PBP2b序列的Thr451Ala/Ser和Ala624Gly置换突变,同时PBP2a序列的465SLN模体元件前置位发生Ser461Ala的置换突变。结论肺炎链球菌对青霉素中、高水平耐药菌株绝大部分合并有不同PBP序列中4~6个氨基酸的置换突变,但合并多个氨基酸置换突变并非就必然引起耐药水平的相应升高。中、高水平耐药与PBP1a、PBP2b、PBP2a的变异关系密切,其中PBP1a的STMK保守区域Thr371Ser置换是引起耐药的主要因素之一。     

耐β-内酰胺类抗生素肺炎链球菌青霉素结合蛋白PBPs的氨基酸基因变异

目的 通过对台州地区耐β-内酰胺类抗生素肺炎链球菌青霉素结合蛋白基因PBPs的研究,分析肺炎链球菌的耐药机制.方法 收集2009年至2010年浙江台州地区63例肺炎链球菌临床分离菌株,进行培养、鉴定及药敏试验,PCR扩增耐药株青霉素结合蛋白PBPs基因并测序分析结果.结果 PBPs基因的变异位点主要集中在保守序列STMK,SSN区域及其附近.与以往报道相一致.结论 肺炎链球菌的PBPs基因变异与β-内酰胺类抗生素耐药性密切相关.     

肺炎链球菌pbp2b和pbp1a基因突变与青霉素耐药的相关性研究

目的探讨耐青霉素肺炎链球菌pbp2b和pbp1 a基因的突变与青霉素耐药的关系,为明了肺炎链球菌的耐药性变异机制,防治其感染提供实验依据。方法从呼吸道感染患儿痰标本中分离肺炎链球菌163株,液体培养基连续稀释法测定其对青霉素的最小抑菌浓度(M IC),套式聚合酶链反应(nPCR)扩增pbp2b和pbp1 a基因,扩增产物直接DNA测序,所测序列与青霉素敏感株(SPN R6)的基因序列进行比较,并分析其氨基酸结构的改变。结果 163株肺炎链球菌中检出青霉素敏感菌75株,中度敏感17株,青霉素耐药菌71株(44%)。耐药菌中58株存在pbp2b突变(81.7%),其中,56株为点突变,2株为CCT插入突变;在27株有pbp2b基因突变的B型和C型耐药菌中,21株出现了不同程度的pbp1 a基因突变。PBP2B氨基酸结构改变以苏氨酸变为丙氨酸、精氨酸变为赖氨酸为主,PBP1A以丙氨酸变为苏氨酸、谷氨酸变为天门冬氨酸为主。结论肺炎链球菌的pbp2b和pbp1 a基因突变与对青霉素的耐药性密切相关,PBP2b突变导致低水平耐药;PBP2b和PBP1A突变导致高水平耐药。     

儿童感染青霉素耐药肺炎链球菌的 多位点序列分型

目的对儿童感染的青霉素耐药肺炎链球菌进行多位点序列分型,了解厦门地区肺炎链球菌青霉素耐药菌株遗传背景。方法采用多位点序列分型法对2012年1月至2014年12月期间分离的60株青霉素耐药肺炎链球菌进行分子分型。结果 60株青霉素耐药肺炎链球菌MLST法共检出24个ST型,其中发现6个新的ST型,分别被命名为ST10004、ST10005、ST10006、ST10007、ST10008和ST10009。存在一个优势型别ST271,占31.7%(19/60),发现了4个克隆群和20种单一克隆,其中主要克隆群为国际流行耐药克隆群Taiwan19F-14,占41.7%(25/60)。结论本地区分离的儿童青霉素耐药肺炎链球菌主要以ST271型为主,属国际流行耐药克隆群Taiwan19F-14,是引起儿童呼吸道感染肺炎链球菌多重耐药的主要原因。     

耐青霉素肺炎链球菌研究进展

肺炎链球菌 (Streptococcus Pneumoniae,SP)是一种常见病原菌 ,可引起细菌性肺炎、脑膜炎、心内膜炎、中耳炎、鼻窦炎、败血症等。 196 7年澳大利亚的 Hansan和 Bullen首次报道了从 1例低丙种球蛋白血症的支气管扩张症患者痰液中分离出耐青霉素肺炎链球菌 (penicillin- resistantstreptococcus pneum oniae,PRSP) [1 ] ,1977年南非亦报道了耐青霉素的肺炎链球菌 [2 ] ,此后许多国家和地区有关于PRSP散在发病和流行的报道 ,引起人们重视。现就耐青霉肺炎链球菌的研究现状作一综述。1　耐青霉素肺炎链球菌的流行病学目前全世界几乎所有…     

应用分子生物学方法鉴定6群肺炎链球菌血清型

目的利用分子生物学方法,鉴定26株6群肺炎链球菌的血清型。方法利用6群肺炎链球菌荚膜多糖相关基因设计合成6对特异性引物,PCR扩增26株肺炎链球菌,并对PCR产物进行基因序列的测定及分析。结果26株肺炎链球菌cpsD蛋白229个氨基酸中有7个突变点,第220~222位均没有缺失;其中,19株肺炎链球菌具有与wci Nα蛋白氨基酸序列完全一致的氨基酸组成,第150位均为Ala,第38位均为Asp,其余7株与wciN_α蛋白氨基酸序列没有相似性,但与wciN_β蛋白氨基酸序列相似性超过99%;15株wciP蛋白第195位为Ser,11株wci P蛋白第195位为Asn。综合分析比对后,26株6群肺炎链球菌中,11株属于6A型肺炎链球菌,8株属于6B型肺炎链球菌,4株属于6C型肺炎链球菌,3株属于6D型肺炎链球菌。结论分子生物学方法可用于6群肺炎链球菌血清型的鉴定,为完善6群肺炎链球菌的菌种档案提供了实验依据。     

儿科痰培养肺炎链球菌耐药性分析

目的了解厦门市妇幼保健院儿科患者下呼吸道感染肺炎链球菌的耐药情况。方法对厦门市妇幼保健院2007年9月至2009年12月从儿科分离的95株肺炎链球菌用K-B纸片法检测其对青霉素等5种抗菌药物的敏感度,对耐苯唑西林的菌株用E-test法检测青霉素和头孢曲松的MIC值。结果95株肺炎链球菌对红霉素、克林霉素高度耐药,耐药率分别为97.9%、96.8%,对左氧氟沙星几乎敏感,未检出耐万古霉素菌株。E-test法测试青霉素不敏感率为58.9%（56株）,头孢曲松敏感率为93.7%（89株）。ermB因介导的红霉素耐药菌株92株,占98.9%,mefE基因介导的红霉素耐药仅1株（1.1%）。结论厦门市妇幼保健院分离的肺炎链球菌耐药严重,PNSP检出率较高（58.9%）,对红霉素、克林霉素高度耐药,临床应尽量减少此类药物的经验性用药,依据药敏结果选择抗菌药物进行治疗。     

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%（118/231）和45.9%（106/231）;gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。     

临床肺炎链球菌常见序列型青霉素耐药性的流行病学研究

从菌种的水平研究和阐述临床分离的肺炎链球菌青霉素耐药机制存在一定的局限。为探讨以序列型(Sequence type,ST)为基础研究肺炎链球菌青霉素耐药机制的可行性,本研究分析了1997~2014年间北京常见STs肺炎链球菌488株和2015年重庆酉阳县、四川中江县常见STs菌株88株的青霉素最低抑菌浓度(Minimun inhibitory concentration,MIC)的分布及年份分布。结果显示北京分离株中除了ST342外,属于某一种ST的所有分离株的青霉素MIC值具有一定分布范围,或者<0.25 mg/L,或者≥0.25 mg/L。青霉素MIC <0.25 mg/L的分离株多分布于2001年以前,此年份后≥0.25 mg/L的分离株出现,并逐渐成为主要种群。但这个年份分布规律对于某一种ST并不明显,某一种ST在最初发现的几个年份中就具有不同青霉素MIC水平的分离株。重庆酉阳县和四川中江县常见STs型青霉素MIC分布于0.25~2.0 mg/L(≥0.25 mg/L),包括ST271、ST320和ST81。本研究从流行病学角度揭示了肺炎链球菌临床分离株常见STs的青霉素MIC值分布规律,支持以STs为基础研究其青霉素耐药机制。     

白鱀豚MHC基因类DQB1座位第二外元的序列变异分析

测定了 4 5个克隆的白豚 (Lipotesvexillifer)MHCⅡ类基因DQB座位第二外元 172bp的核苷酸序列 ,共获得 15种序列 ,发现了 2 2个变异位点。核苷酸的非同义替换明显多于同义替换 ,并造成了 15个氨基酸的改变。氨基酸的替换趋于集中在假定的与抗原的选择性识别相关的位点附近。白豚DQB基因的核苷酸和氨基酸序列与文献报道的白鲸 (Delphinapterusleucas)和一角鲸 (Monodonmonoceros)DQB1序列具有较高的同源性。氨基酸序列不具备人及其它一些灵长类动物DQB2基因所共有的基序 (Motif) ,而与牛DQB1基因的基序相近 ,说明本研究得到的白豚MHC序列应属于类DQB1基因。同一个体出现了多种序列的情况 ,提示白豚的DQB基因可能存在着座位重复。白豚的类DQB1座位的序列中存在多种基序的不同组合 ,推测是由于基因转换造成的.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

Genomic organization of the para-sodium channel alpha-subunit genes from the pyrethroid-resistant and -susceptible strains of the diamondback moth

We examined the genomic organization of the para-sodium channel alpha-subunit gene of the diamondback moth, Plutella xylostella (L.). The nucleotide sequence contained 34 putative exons, which covered almost the entire coding region of the gene producing 1,889 amino acid residues. Deduced amino acid identity to the hscp locus of Heliothis virescens was 84%. Comparison of deduced amino acid sequences of the permethrin-resistant and -susceptible strains showed two substitutions other than kdr and super-kdr-like substitutions. They were Ala to Thr (A1060T) and Pro to Ser (P1836S) at the linker region of the domains II-III and the carboxyl terminus, respectively. Furthermore, we developed PCR amplification protocols for the rapid detection of both substitutions.     

Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae

Clinical isolates of Streptococcus pneumoniae that have greatly increased levels of resistance to penicillin (greater than 1000-fold) have been reported from South Africa during the last ten years. Penicillin resistance in these strains is entirely due to the development of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. We have cloned and sequenced the coding region for the transpeptidase domain of penicillin-binding protein 2B from three penicillin-sensitive strains of S. pneumoniae and from a penicillin-resistant South African strain. The amino acid sequences of the transpeptidase domains of PBP2B of the three penicillin-sensitive strains were identical and there were only between one and four differences in the nucleotide sequences of their coding regions. The corresponding region of the PBP2B gene from the penicillin-resistant strain differed by 74 nucleotide substitutions which resulted in 17 alterations in the amino acid sequence of PBP2B. The most remarkable alteration that has occurred during the development of the 'penicillin-resistant' form of PBP2B is the substitution of seven consecutive residues in a region that is predicted to form a loop at the bottom of the penicillin-binding site.     

Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori

The correlation between the substitutions of penicillin-binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillinresistant (n=3), low-susceptible (n=3), and susceptible (n=13) H. pylori isolates, the substitution Asn562-->Tyr, which is adjacent to KTG motif (555-557), was common and specific to amoxicillin-resistant H. pylori. Additionally, all amoxicillin-resistant isolates had multiple substitutions such as Ser414-->Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin-resistant H. pylori isolates had multiple substitutions including Asn562-->Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

The Mycobacterium avium-intracellulare complex dnaB locus and protein intein splicing

Intein is a protein sequence mebedded in-frame within a precursor protein and is posttranslationally excised by a self-catalytic protein splicing process. Protein splicing is believed to follow a pathway requiring Cys, Ser, or Thr residues at the intein N-terminus and substitutions other than Cys, Ser, or Thr residues prevent splicing. We show that the dnaB locus in some strains of M. avium-intracellulare complex (MAC) contains intein and that the intein N-terminal amino acid is Ala [Ala-type]. We demonstrate that the M. avium DnaB precursor protein undergoes posttranslational proteolytic processing producing proteins corresponding to the sizes of the DnaB and intein. Further, by Western analysis we detect a protein corresponding to the size of the spliced DnaB protein in MAC cell extracts. Together, these results indicate that the Ala-type MAC DnaB inteins can splice and provide another example that points to an interesting alternative splicing mechanism (Southworth, M. W., Benner, J., and Perler, F. B., EMBO J. 19, 5019-5026, 2000).     

Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations.

Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.     

Polyomavirus middle T-antigen NPTY mutants.

A polyomavirus middle T-antigen (MTAg) mutant containing a substitution of Leu for Pro at amino acid 248 has previously been described as completely transformation defective (B. J. Druker, L. Ling, B. Cohen, T. M. Roberts, and B. S. Schaffhausen, J. Virol. 64:4454-4461, 1990). This mutant had no alterations in associated proteins or associated kinase activities compared with wild-type MTAg. Pro-248 lies in a tetrameric sequence, NPTY, which is reminiscent of the so-called NPXY sequence in the low-density-lipoprotein receptor. In the low-density-lipoprotein receptor, mutations in the NPXY motif but not in the surrounding amino acids abolish receptor function, apparently by decreasing receptor internalization (W. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265:3116-3123, 1990). To determine whether this sequence represents a functional motif in MTAg as well, a series of single amino acid substitutions was constructed in this region of MTAg. All of the mutations of N, P, T, or Y, including the relatively conservative substitution of Ser for Thr at amino acid 249, resulted in a transformation-defective MTAg, whereas mutations outside of this sequence allowed mutants to retain near-wild-type transformation capabilities. Transformation-defective mutants with mutations in the NPTY region behaved similarly to the mutant with the original Pro-248-to-Leu-248 mutation when assayed for associated proteins and activities in vitro; that is, they retained a full complement of wild-type activities and associated proteins. Further, insertion of the tetrameric sequence NPTY downstream of the mutated motif restored transforming abilities to these mutants. Thus, the tetrameric sequence NPTY in MTAg appears to represent a well-defined functional motif of MTAg.     

Random mntagenesis of the gene for bacteriophage T7 RNA polymerase

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.