Comparative studies using 125I- and 111In-labeled monoclonal antibodies

HDP-1 monoclonal antibody was labeled with ¹¹¹In using deferoxamine, diethylenetriaminepentaacetic acid or 1-(para-bromoacetamidobenzyl)-EDTA as chelating agents or with ¹²⁵I. The in vitro binding capacity and stability of the labeled molecules were evaluated using affinity chromatography. The biodistribution and imaging capabilities were compared using an animal model system that does not involve the use of tumors. Similar studies were done using the corresponding labeled F(ab′)₂ and Fab′ fragments. All labeled molecules, except those treated with deferoxamine, were stable in vitro. When tested in vivo, all retained their capacity to localize in the target tissue (lung). The lung %ID/g levels for the ¹¹¹In-labeled molecules were, however, slightly lower than those observed for the corresponding ¹²⁵I-labeled molecules. High uptake was also observed in the liver or kidneys when the ¹¹¹In-labeled molecules were used; no such results were obtained with the ¹²⁵I-labeled molecules. More work appears to be necessary before the use of bifunctional chelates becomes the optimal method for radiolabeling monoclonal antibodies for use in tumor imaging.     

Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT)

Although Leishmania parasites have been shown to modulate their host cell''s responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.     

Synthesis and evaluation of anthranilic acid-based transthyretin amyloid fibril inhibitors

Eight small molecules were synthesized to evaluate the structure activity relationships (SAR) of N-substituted anthranilic acids. The molecules were synthesized by benzylation or arylation of methyl anthranilate. A light scattering-based amyloid fibril formation assay was used to evaluate potential inhibitors of transthyretin (TTR) amyloid fibril formation in vitro. The m-carboxyphenylated and o-trifluoromethylphenylated anthranilic acids are potent inhibitors that will be subjected to further SAR and structural analysis.     

Decreased thermal stability of collagens containing analogs of proline or lysine

Fibroblasts were incubated with analogs of proline or lysine and the thermal stability of procollagen molecules containing the analogs was investigated using pepsin digestion at different temperatures as an enzymatic probe of conformation. The procollagens containing either 4-cis-hydroxy-l-proline, 3,4-dehydroproline, or 4,5-trans-dehydrolysine were less stable than normal procollagen and these abnormal collagens were largely in a non-triplehelical conformation within the cells at 37 °C. These results support the idea that procollagen molecules which are not in a triple-helical conformation are not secreted at a normal rate. Procollagens containing both 4,5-trans-dehydrolysine and a proline analog were much less stable than molecules containing a single type of analog. This result suggests that simultaneous administration of both types of analogs may have a greater effect on collagen accumulation in whole-animal experiments than administration of a single analog.     

Physical mapping and characterization of Chlamydomonas mitochondrial DNA molecules: Their unique ends,sequence homogeneity,and conservation

Sixteen site-specific endonucleases were used to characterize the mitochondrial (mt)-DNA of Chlamydomonas reinhardtii. Recognition sites for SmaI, XhoI, and BglII were absent in the mtDNA. mtDNA fragments appeared in stoichiometric proportions in every nuclease digest indicating that C. reinhardtii mtDNA consists of a homogenous population of molecules devoid of either inter- or intramolecular heterogeneity. Six DNA fragment maps were derived for those endonucleases that produced discrete and readily measurable DNA fragments. These maps, which exhibited marked internal consistency, also suggested that the linear mtDNA molecules possessed unique ends. This was subsequently confirmed by in vitro 5′-end labeling of mtDNA molecules prior to endonuclease digestion. These results indicate that (1) the linear mtDNA isolated under our experimental conditions possessed not only unique ends but also a nonpermuted gene sequence and (2) such mtDNA molecules were generated by a site-specific cleavage of the closed circular mtDNA molecules shown to exist in vivo. mtDNA sequence conservation in Chlamydomonas is quite striking. No difference in endonuclease cleavage pattern has yet been detected among a number of C. reinhardtii strains or between mating types.     

First report on exploring structural requirements of alpha and beta thymidine analogs for PfTMPK inhibitory activity using in silico studies

With the emergence of multi-drug resistance of the currently available antimalarial drugs including the “magic bullet” artemisinin derivatives in the market, there is an urgent need for discovery and development of new potent antimalarial molecules. The present work deals with quantitative structure–activity relationship (QSAR) modeling, pharmacophore mapping and docking studies of a series of 35 thymidine analogs as inhibitors of Plasmodium falciparum thymidylate kinase (PfTMPK), an enzyme that catalyzes phosphorylation of thymidine monophosphate (TMP) to thymidine diphosphate (TDP). The models were validated both internally and externally and significant statistical results were obtained, indicating the robustness and reliability of the developed models. The docking study was performed using the LigandFit option of receptor–ligand interactions protocol section available in Discovery Studio 2.1 where lower RMSD values (0.6931 Å) between the co-crystallized ligand and re-docked ligand assured that the ligand was bound in the same binding pocket. The QSAR, pharmacophore mapping and docking studies provide an understanding of important structural requirements or essential molecular properties, or features of molecules, and important binding interactions, and provide an important guidance for the chemist to synthesis of new molecules with improved PfTMPK inhibitory activity profile. This work revealed the importance of –NH-fragment, electrophilicity of the molecules and the number of oxygen atom towards the PfTMPK inhibitory activity of the molecules. To the best of our knowledge, this work presents the first QSAR and pharmacophore report for thymidine analogs which may serve as an efficient tool for the design and synthesis of potent molecules as PfTMPK inhibitors to address the increasing threat of multi-drug resistance against P. falciparum.     

Molecular modeling,synthesis, antibacterial and cytotoxicity evaluation of sulfonamide derivatives of benzimidazole,indazole, benzothiazole and thiazole

A new series of heterocyclic molecules bearing sulfonamide linkage has been synthesized and screened for antibacterial activity. During antibacterial screening using broath dilution method, molecules were found to be highly active (MIC value 50–3.1?µg/mL) against different human pathogens, namely B. cerus, S. aureus, E. coli and P. aeruginosa, and most effective against E. coli. A great synergistic effect was observed during determination of FIC where molecules were used in combination with reference drugs chloramphenicol and sulfamethoxazole. The MIC value of the combination – varying concentration of test compounds and ½ MIC of reference drugs or varying concentration of reference drugs and ½ MIC of test compounds, was reduced up to ¹/₄ or ¹/₃₂ of the original value, indicating thereby the combination was 4–32 times more potent than the test molecule. The molecules also showed low degree of cytotoxicity against PBM, CEM and VERO cell lines. The results positively indicated towards the development of lead antibacterials using the combination approach.     

Exploring the impact of nucleic acids on protein stability in bacterial cell lysate

- Addition of salt enhanced thermal stability of model substrate proteins by reducing electrostatic repulsion between protein molecules.- However, the opposite effect was observed with bacterial cell lysate, indicating that certain molecules within the lysate could enhance protein stability via electrostatic interactions.- Such molecules present in cell lysate were found to be nucleic acids known to have a potent anti-aggregation activity toward proteins involving electrostatic interactions.- Nucleic acids showed chaperone activity in physiological salt concentration within cells and in buffer or medium commonly used in experiments.- The chaperone activity of nucleic acids should be taken into account when performing various in vitro assays using cell lysate or samples containing nucleic acids.     

Evidence for the presence of ribonucleotides at the 5' termini of some DNA molecules isolated from Escherichia coli polAex2.

We have purified a set of small DNA molecules from various strains of exponentially growing Escherichia coli, including E. coli polAex2. This material included very short molecules (2 S), the nascent DNA (“Okazaki fragments”) and some longer molecules. Most of the [³H]thymidine incorporated during a brief period of labeling was found in the 5 S to 15 S Okazaki fragments. There was a large number of the 2 S molecules in the cell. The properties of the 5′ ends of these molecules were investigated using three procedures. (1) The DNA preparation, pulse-labeled with [³H]thymidine, was reacted with polynucleotide kinase and ATP to insure that all 5′ ends were phosphorylated. After subjection of the DNA to alkaline hydrolysis, the proportion of incorporated ³H pulse-label that became susceptible to digestion by spleen exonuclease was determined. In different experiments there was an increment of up to 20% in the amount of pulse-labeled E. coli polAex2 DNA that could be hydrolyzed by the exonuclease after treatment with alkali. (2) As in the preceding protocol, phosphorylation of the 5′ ends was assured by reaction with kinase and ATP; the preparation was then treated with alkali and the number of 5′-OH ends generated that could be labeled with ³²P using [γ-³²P]ATP and kinase in a second reaction was determined. The data indicated that 3 to 30% of the molecules could be labeled after alkali digestion, but not before. (3) The DNA molecules were reacted with kinase and [γ-³²P]ATP after having been exposed previously to alkaline phosphatase. The end-labeled molecules were then subjected to an alkaline hydrolysis and the resulting hydrolysate chromatographed on a polyethyleneimine-cellulose thinlayer plate. Alkali treatment was found to release 2′(3′),5′-ribonucleoside diphosphates from 1 to 30% of the molecules; pAp and pGp predominated. Control experiments showed that these ribonucleotides were covalently linked to the 5′ ends of polydeoxyribonucleotides. Curiously, the smaller the DNA molecule the less likely it was to possess a 5′-terminal ribonucleotide. Very few apparent RNA/DNA molecules were observed in the non-polAex2 strains tested. These observations are in part in agreement with previous reports, and we infer that at least some of the nascent E. coli polAex2 DNA molecules are initiated in vivo with a ribonucleotide primer. The relatively smaller proportion of molecules with apparent 5′-terminal ribonucleotides among the smaller DNA molecules and in strains other than E. coli polAex2 suggests to us that there may exist a mechanism for initiating DNA molecules that does not require an RNA primer.     

The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation.

The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus laevis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon filters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafiltrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus laevis are regulated by a G₁ kidney chalone of less than 10,000 MW.     

Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy

The domainal substructure and molecular conformation of human fibrinogen have been investigated by evaluating scanning transmission electron microscopic images of freeze-dried or negatively contrasted native fibrinogen (fractions I-4 and I-9), glutaraldehyde-treated fibrinogen, or plasmic core fragments D₁ and E₂. Although some unstained freeze-dried native or glutaraldehyde-treated fibrinogen molecules were relatively compact and even occasionally spheroidal, typical images were elongated symmetrical tridomainal structures 460 Å ± 20 Å in length; frequently they were bent into a variety of elongated though non-linear arrangements. Their identification as monomolecular forms of fibrinogen by scanning transmission electron microscopic mass measurements resolved uncertainties relating to the identity of such objects as single molecules. The central domains of fraction I-4 molecules had a greater mass than those of fraction I-9 (1.01 × 10⁵M_rversus 7.5 × 10 M_r, respectively). This difference accounted for the observed mass difference between fraction I-4 and fraction I-9 molecules (i.e. 3.27 × 10⁵M_rversus 2.97 × 10⁵M_r, respectively) and suggested that the COOH-terminal region of the Aα chain (major portions of which are always absent from fraction I-9 molecules) is situated within the mass integration radius for the central domain. When the COOH-terminal region of the Aα chain was present it appeared in negative stain as a thread-like structure originating between the middle and outer domains and extending toward the central domain, sometimes appearing to wind around the long axis.The outer domains of negatively stained molecules resembled negatively stained images of fragment D₁ and could frequently be resolved into at least two discrete subdomains, forming an oblong structure usually canted at an angle of ～120 ° to 150 ° relative to the long axis. Our findings are consistent with prevailing tridomainal structural models of fibrinogen and suggest that these molecules are flexible and may exist in unfolded configurations, or as relatively compact, partially or completely folded forms.     

Production of sophorolipids with eicosapentaenoic acid and docosahexaenoic acid from Wickerhamiella domercqiae var. sophorolipid using fish oil as a hydrophobic carbon source

Sophorolipids (SLs) were synthesized by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 grown on fish oil and glucose. They were purified using preparative HPLC and their structures were identified by MS/MS. The yields of total and lactonic SLs were 47 and 19 g l^?1 in shake-flasks when fish oil 4 % (v/v) was used with glucose in the medium. The composition of SL mixture contained more than 20 SL molecules. Several unconventional SL molecules with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) including zero-, mono- and di-acetylated acidic SLs with EPA and a di-acetylated acidic SL with DHA were obtained. Two unconventional lactonic SL molecules, non-acetylated lactonic SL with 22:3 and non-acetylated lactonic SL with 20:0, were also obtained.     

Plasmid R6K Contains Two FunctionaloriTswhich can Assemble Simultaneously in Relaxosomesin vivo

Plasmid R6K contains two functional origins or transfer (oriT), in contrast to previously characterized conjugative plasmids. TheoriTsare formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the α and β origins of replication. The gene for R6KoriT-nickase,taxC, was identified by transposon mutagenesis and sequenced, revealing that TaxC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stranded DNA containing its target sequence. Identification of thenicsites suggested that the R6KoriTsbelong to the RP4/VirD2oriTfamily. Cleavage was highly specific and did not occur with oligonucleotides cleaved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid.niccleavage ofin vivopre- assembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphodiesterase to produce double strand cleavages at thenicsites. 35% of the molecules were cleaved simultaneously at bothnicsites, both in the case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum percentage of individual R6K molecules containing two pre-assembled relaxation complexes.     

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

ATP-DnaA binds to multiple DnaA boxes in the Escherichia coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg-285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that had alternative DNA binding sequence specificity and chimeric oriC molecules bearing the alternative DnaA binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg-285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg-285 fingers face inward to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.     

Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Kinetoplast DNA (? = 1.703 g/ml.) was isolated by preparative cesium chloride ultracentrifugation in a fixed-angle rotor from total cell DNA of Leishmania tarentolae and examined in terms of sedimentation properties, melting characteristics, and appearance in the electron microscope. It consisted of several molecular types, either free or bound together in associations of variable size: minicircles (molecular weight = 0.56 ± 0.03 × 10⁶), catenated minicircles, “figure 8” molecules, and long molecules. The associations seem to be held together by the long molecules threading through the smaller circles and catenanes. The large associations could be broken down by sonication, DNase II-treatment, or shear forces. Minicircles, catenated dimers, trimers, and small linear fragments were separated on preparative sucrose gradients of sonicated DNA, and S_20,w values were assigned to each molecular type by band sedimentation in the analytical ultracentrifuge.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). I. Undamaged covalent circular lambda DNA molecules in uvrA+ recA+ lysogenic host cells are cut following superinfection with psoralen-damaged lambda phages.

When λ bacteriophages were treated with a photosensitizing agent, psoralen or khellin, and 360 nm light, monoadducts and interstrand crosslinks were produced in the phage DNA. The DNA from the treated phages was injected normally into Escherichia coli uvrA^? (λ) cells and it was converted to the covalent circular form in yields similar to those obtained in experiments with undamaged λ phages. In excision-proficient host cells, however, there was a dose-dependent reduction in the yield of rapidly sedimenting molecules, and a corresponding increase in slow sedimenting material, the extent of this conversion corresponding to about one cut per two crosslinks. Presumably, the damaged λ DNA molecules were cut by the uvrA endonuclease of the host cell, but were not restored to the original covalent circular form.The presence of psoralen damage in λ phage DNA greatly increased the frequency of genetic exchanges in λ phage-prophage crosses in homoimmune lysogens (Lin et al., 1977). As genetic recombination is thought to depend on cutting and joining in DNA molecules, experiments were performed to test whether psoralen-damaged λ DNA would cause other λ DNA in the same cell to be cut. E. coli (λ) host cells were infected with ³²P-labeled λ phages and incubated to permit the labeled DNA to form covalent circles. When these host cells were superinfected with untreated λ phages, there was no effect upon the circular DNA. When superinfected with λ phages that had been treated with psoralen and light, however, many of the covalent circular molecules were cut. The cutting of undamaged molecules in response to the damaged DNA was referred to as “cutting in trans”. It required the uvrA⁺ and recA⁺ host gene functions, but neither recB⁺ nor any phage gene functions. It occurred normally in non-lysogenic hosts treated with chloramphenicol before infection. Cutting in trans may be one of the steps in recA-controlled recombination between psoralen crosslinked phage λ DNA and its homologs.     

The Escherichia coli C homoprotocatechuate degradative operon: hpc gene order,direction of transcription and control of expression

The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of nonreplicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of β-glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, β-galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.     

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.     

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.