Discontinuity of homology of Escherichia coli and Salmonella typhimurium DNA in the lac region

Summary Partial homology of Salmonella typhimurium DNA to Escherichia coli DNA was demonstrated by Southern hybridization blots to exist on either side of the lac operon of E. coli but no homology was detected between S. typhimurium DNA and about 12 kb of E. coli DNA including the lac genes as well as about 5 kb of E. coli DNA between lac and proC. Thus portions of DNA seem to have been either added to the E. coli genome or deleted from the S. typhimurium genome since their divergence from a common ancestor. Although an IS1 element was located near the lac operon of E. coli, the insertional element was shown not to be near any of the junctures of discontinuity of E. coli - S. typhimurium homology near lac.     

Experimental and molecular docking investigation on DNA interaction of N‐substituted phthalimides: antibacterial,antioxidant and hemolytic activities

A series of Schiff base molecules derived from a phthalimide scaffold was investigated as efficient antibacterial, antioxidant and DNA‐interacting agents. The spectroscopic characterization of these derivatives was studied in detail using elemental analysis and spectroscopic techniques. The DNA‐binding profile of title molecules against Ct‐DNA (calf thymus) was investigated by absorbance, fluorescence, hydrodynamics and thermal denaturation investigations. The bacterial inhibition potential of these molecules was investigated against Escherichia coli and Staphylococcus aureus. Molecule 3c emerged as the most active against S. aureus (IC₅₀: 14.8 μg/mL), whereas compounds 3a and 3b displayed potential antibacterial activities against E. coli (IC₅₀: 49.7 and 67.6 μg/mL). Molecular docking studies of these compounds against GlcN‐6‐P synthase were carried out to rationalize antibacterial efficiency of these molecules. These newly synthesized molecules were screened for their scavenging capacity against 2,2‐diphenyl‐1‐picryl‐hydrazyl (DPPH) and H₂O₂ free radicals and the results were compared with ascorbic acid as synthetic antioxidant. The title molecules 3a, 3b and 3e showed less than 20% hemolysis, which indicated their significant non‐toxic behavior.     

Structure and function of 5S RNA: the role of the 3'' terminus in 5S RNA function

Summary 5S RNA from B. stearothermophilus and E. coli was reacted with NaIO₄ and aniline to remove their 3 terminal nucleoside. These modified 5S RNA molecules were then incorporated in B. stearothermophilus 50 S ribosomal subunits and tested for biological activities. 50 S ribosomes containing the modified 5S RNAs exhibited full activity and we therefore conclude, that the 3 terminus of 5S RNA does not play an active role in protein synthesis.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Isolation of Escherichia coli mRNA and Comparison of Expression Using mRNA and Total RNA on DNA Microarrays

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3′-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Comparison of RNA sequences from widely divergent species

Summary Tests that take into account the effects of gaps have been applied to 5S ribosomal RNA sequences from the bacteria,E. coli andP. fluorescens, and from KB carcinoma cells. The 5S RNAs from KB andP. fluorescens, when compared to that ofE. coli are shown to be more similar than random sequences of the same composition. Intrasequential analyses of 5S RNAs give some evidence for partial gene duplication or repetitive subsequences, but the proposed duplication of Brownlee, Sanger and Barrell (1968) is not supported by our data.     

Phylogeny of the 5S ribosomal RNA fromSynechococcus lividus II: The cyanobacterial/chloroplast 5S RNAs form a common structural class

Summary The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacteriumSynechococcus lividus II has been determined. The sequence is 5-UGCCUAGUGUUUAUGGCGCG-GUGGAACCACGCUGAUCCAUCCCGAACUC-AGAGGUGAAACAUCGCAGCGGUGAAGAU-AGUUGGAGGGUAGCCUCCUGCAAAAAUA-GCUCAAUGCUAGGCA_OH-3. This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA ofS. lividus II has 27 base differences compared with the 5S RNA of the related strainS. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs fromS. lividus II,S. lividus III, and spinach chloroplasts are identical, but differ considerably from that ofEscherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA ofE. coli and these cyanobacterial and chloroplast 5S RNAs.     

Small stable RNA of Neurospora crassa.

Summary Small stable RNAs in wild-type Neurospora crassa were investigated by analyzing the cell contents of long term ³²P_i labeled cultures in thin slab polyacrylamide gels. Because of the rigid fungal cell wall and the potency of nucleases the degradation of RNA in opening the cells was rather extensive. Some of these degradation problems were circumvented by using a slime strain of N. crassa which lacks a rigid cell wall. Our findings show that N. crassa. like many other eukaryotes, contains a number of small stable RNA molecules. We also found that the ribosomal RNA, the so called 5.8S, migrates slower on polyacrylamide gels than the 6S RNA of E. coli, which contains 184 nucleotides. The relative migration of the molecules was not changed when the samples were denatured prior to electrophoresis. The mobility of the Neurospora rRNA molecule suggested a chain length of 220 nucleotides. Fingerprinting of a T₁ ribonuclease digest indicated a chain length of 212 nucleotides. Because of the unusually large size of the so-called 5.8S rRNA we found it more appropriate to refer to this molecule as a 7S rRNA. It seems that the N. crassa 7S rRNA is the largest low molecular weight ribosomal RNA studied thus far.     

Expression ofStreptomyces melC andchoA genes by a clonedStreptococcus thermophilus promoter STP2201

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected inEscherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone ST_P2201 as a strong promoter inE. coli. Subcloning of a ST_P2201-melC DNA fragment into the pMEU-seriesS. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel⁺ phenotype toE. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg^–1 protein) inE. coli, and to produce an apparently inactivemelC gene product that reacts with anti-tyrosinase antiserum inS. thermophilus. SubstitutingmelC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to anE. coli transformant at a level of (1.06±0.15)×10^–7 units mg^–1 protein. Introduction of this plasmid intoS. thermophilus by electrotransformation yielded ChoA transformant that produced the enzyme at about 25% of the level found inE. coli.     

Analysis of ribosomal inter‐subunit sites as targets for complementary oligonucleotides

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2′‐O‐methyl oligoribonucleotides (2′‐OMe RNAs). Further, the inhibition efficiencies of the designed ribosome‐interfering 2′‐OMe RNAs were tested using a β‐galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC₅₀ values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2′‐OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double‐filter retention assay. Further, we applied heat‐shock chemical transformation to introduce 2′‐OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC₅₀ in the cell‐free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter‐subunit bridges can help design efficient antisense oligomers to probe the ribosome function.     

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su⁺ 3 tRNA₁^Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.     

Organization of ribosomal protein genes in Escherichia coli: I. Physical structure of DNA from transducing λ phages carrying genes from the aroE-str region

The physical structures of the genomes of five transducing bacteriophages (λaroE, λtrkA, λspc1, λspc2, and λfus2) carrying various portions of the aroE-trkA-spc-str segment of the Escherichia coli chromosome have been determined. Two methods were used: (a) heteroduplex analysis of DNA molecules from these phages, and (b) analysis of fragments obtained from digestion of the DNA by restriction endonucleases EcoRI and HindIII. In λaroE, λtrkA, λspc1 and λspc2, whose genome lengths vary from about 75% to about 104% of the λpapa genome, the right arm of λ DNA is present, whereas various portions of the left arm have been replaced by E. coli DNA. In λfus2, however, about 93% of the λ DNA molecule is replaced by E. coli DNA, the resultant genome being 103.5 %λ units long (Figs 1 and 2). All five phages contain an identical λ-E. coli junction at 1.9 %λ units from the left λ terminus, and there is complete homology between the common portions of the inserted E. coli DNA. Since these phages were independently isolated, we believe that the genetic organization of the E. coli DNA carried by these phages probably reflects the organization of the relevant segments of the E. coli chromosome. Comparison of the physical and genetic maps of these transducing phages has allowed us to assign a physical position to the ribosomal and neighbouring genes, including those coding for the α subunit of RNA polymerase and the elongation factors G and Tu, on the bacterial DNA.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Organization of Lrp-binding sites upstream of ilvlH in Salmonella typhimurium

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvlH. Lrp binds to six sites upstream of ilvlH, and Lrp binding is required for ilvlH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvlH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvlH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.     

Effects of <Emphasis Type="Italic">Escherichia coli</Emphasis> RraA Orthologs of <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> on the Ribonucleolytic Activity of RNase E In Vivo

RraA is a recently discovered protein inhibitor of RNase E that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli. In the genome of Vibrio vulnificus, two open reading frames that potentially encode proteins homologous to E. coli, RraA-designated RraAV1 and RraAV2, have respectively 80.1% and 59.0% amino acid identity to RraA. The authors report that coexpression of RraAV1 protein in E. coli cells overproducing RNase E rescued these cells from growth arrest and restored their normal growth, whereas coexpression of RraAV2 protein further inhibited the growth of E. coli cells, whose growth was already impaired by overproduction of RNase E. Analyses of the steady-state level of various RNase E substrates indicated that the coexpression of RraAV1 more efficiently inhibited RNase E action than coexpression of RraA, and consequently resulted in the more increased abundance of each RNA species tested in vivo. The inhibitory effect by RraAV2 coexpression on RNase E was observed only in the case of trpA mRNA, indicating the possibility of RNA substrate-dependent inhibition of RraAV2 on RNase E. The findings suggest that these regulators of ribonuclease activity have both a conserved inhibitory function and a differential inhibitory activity on RNase E-like enzymes across the species.     

抗菌肽P7抑制大肠杆菌的非膜作用机制北大核心CSCD

【目的】研究抗菌肽P7抑制大肠杆菌的非膜作用机制。【方法】P7与溴化乙锭竞争结合大肠杆菌基因组DNA的荧光光谱,分析P7与DNA的结合方式;流式细胞术分析P7与大肠杆菌基因组DNA结合对细菌细胞周期的影响;采用磁珠富集和PCR扩增相结合的方法分析P7特异结合的DNA序列;通过实时荧光定量PCR分析P7对大肠杆菌DNA复制和SOS损伤修复基因表达的影响;核酸染料的荧光分析研究P7对大肠杆菌DNA和RNA合成的影响。【结果】P7以嵌插的方式作用于大肠杆菌基因组DNA碱基对并形成肽-DNA复合物,使溴化乙锭-DNA复合体系的荧光强度减弱。P7可以显著增加大肠杆菌细胞周期中S期细胞数目,抑制大肠杆菌DNA复制。P7特异性结合rnh A使该基因表达水平显著下调2.24倍。同时,在肽的影响下参与大肠杆菌DNA复制相关的ssb、dna G、lig B和rnh A基因的表达水平显著下调(P<0.05),DNA损伤修复的rec A和rec N基因显著上调(P<0.05)。P7可降低大肠杆菌DNA和RNA的合成。【结论】P7特异性地结合rnh A序列引起大肠杆菌DNA的损伤并抑制大肠杆菌的DNA复制。在P7的影响下,参与大肠杆菌DNA复制相关的基因的表达水平下调,DNA损伤修复基因显著上调,同时抑制大肠杆菌DNA和RNA的合成。     

Conditions and mutations affecting the maintenance and replication of plasmid DNA in Escherichia coli 15

Summary An Escherichia coli 15 strain has been constructed which contains, in addition to the plasmids inherent to E. coli 15 (P 1-like DNA and minicircular DNA), the colicinogenic factor E₁ (Col E₁). Whereas the P 1-like DNA of E. coli 15 is unaffected by the uptake of the colicin plasmid, the number of copies of minicircular DNA of E. coli 15 decreases and an equivalent amount of Col E₁ DNA becomes established in the cell. The ratio between these two small plasmids is dependent on the growth temperature. The mode of replication of minicircular DNA and Col E₁ DNA is very similar, but is different in various respects from that of the P 1-like plasmid: 1. Both small plasmids continue to replicate in the presence of chloramphenicol, whereas the replication of P 1-like DNA stops like the chromosomal DNA. 2. Rifampicin inhibits the synthesis of both small plasmids rather rapidly. The replication of P 1-like DNA continues during the remaining replication cycle of the chromosome in the presence of rifampicin. 3. The replication of Col E₁ DNA and of the minicircular DNA still proceeds at elevated temperatures (45–50°C), whereas little or no incorporation of ³H-thymidine into P 1-like DNA is observed at these temperatures. 4. Mutants have been obtained, which show altered properties in the maintenance and replication of the plasmids without being affected in the replication of the chromosomal DNA. In all these mutants the replication and (or) maintenance of the minicircular DNA of E. coli 15 and Col E₁ DNA is affected in the same way, but not that of the P 1-like plasmid.