Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome.

While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. EF-G's indispensability is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of E. coli EF-G induced by mutations that closely aligned with eEF2's Thr56 residue. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNAPhe-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which wasn't observed when the mutants interact solely with the sarcin/ricin loop. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.

In Vivo Production of Small Recombinant RNAs Embedded in 5S rRNA-Derived Protective Scaffold.

Preparative synthesis of RNA is a challenging task that is usually accomplished by either chemical or enzymatic polymerization of ribonucleotides in vitro. Herein, we describe an alternative approach in which RNAs of interest are expressed as a fusion with a 5S rRNA-derived scaffold. The scaffold provides protection against cellular ribonucleases resulting in cellular accumulations comparable to those of regular ribosomal RNAs. After isolation of the chimeric RNA from the cells, the scaffold can be removed, if necessary, by deoxyribozyme-catalyzed cleavage followed by preparative electrophoretic separation of the reaction products. The protocol is designed for sustained production of high quality RNA on the milligram scale.

Expansion segments in bacterial and archaeal 5S ribosomal RNAs.

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.

Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces.

BACKGROUND: The International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth. RESULTS: The cultivable bacterial and fungal population ranged from 104 to 109 CFU/m2 depending on location and consisted of various bacterial (Actinobacteria, Firmicutes, and Proteobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Amplicon sequencing detected more bacterial phyla when compared to the culture-based analyses, but both methods identified similar numbers of fungal phyla. Changes in bacterial and fungal load (by culture and qPCR) were observed over time but not across locations. Bacterial community composition changed over time, but not across locations, while fungal community remained the same between samplings and locations. There were no significant differences in community composition and richness after propidium monoazide sample treatment, suggesting that the analyzed DNA was extracted from intact/viable organisms. Moreover, approximately 46% of intact/viable bacteria and 40% of intact/viable fungi could be cultured. CONCLUSIONS: The results reveal a diverse population of bacteria and fungi on ISS environmental surfaces that changed over time but remained similar between locations. The dominant organisms are associated with the human microbiome and may include opportunistic pathogens. This study provides the first comprehensive catalog of both total and intact/viable bacteria and fungi found on surfaces in closed space systems and can be used to help develop safety measures that meet NASA requirements for deep space human habitation. The results of this study can have significant impact on our understanding of other confined built environments on the Earth such as clean rooms used in the pharmaceutical and medical industries.

Evaluation of Acquired Antibiotic Resistance in Escherichia coli Exposed to Long-Term Low-Shear Modeled Microgravity and Background Antibiotic Exposure.

The long-term response of microbial communities to the microgravity environment of space is not yet fully understood. Of special interest is the possibility that members of these communities may acquire antibiotic resistance. In this study, Escherichia coli cells were grown under low-shear modeled microgravity (LSMMG) conditions for over 1,000 generations (1000G) using chloramphenicol treatment between cycles to prevent contamination. The results were compared with data from an earlier control study done under identical conditions using steam sterilization between cycles rather than chloramphenicol. The sensitivity of the final 1000G-adapted strain to a variety of antibiotics was determined using Vitek analysis. In addition to resistance to chloramphenicol, the adapted strain acquired resistance to cefalotin, cefuroxime, cefuroxime axetil, cefoxitin, and tetracycline. In fact, the resistance to chloramphenicol and cefalotin persisted for over 110 generations despite the removal of both LSMMG conditions and trace antibiotic exposure. Genome sequencing of the adapted strain revealed 22 major changes, including 3 transposon-mediated rearrangements (TMRs). Two TMRs disrupted coding genes (involved in bacterial adhesion), while the third resulted in the deletion of an entire segment (14,314 bp) of the genome, which includes 14 genes involved with motility and chemotaxis. These results are in stark contrast with data from our earlier control study in which cells grown under the identical conditions without antibiotic exposure never acquired antibiotic resistance. Overall, LSMMG does not appear to alter the antibiotic stress resistance seen in microbial ecosystems not exposed to microgravity.IMPORTANCE Stress factors experienced during space include microgravity, sleep deprivation, radiation, isolation, and microbial contamination, all of which can promote immune suppression (1, 2). Under these conditions, the risk of infection from opportunistic pathogens increases significantly, particularly during long-term missions (3). If infection occurs, it is important that the infectious agent should not be antibiotic resistant. Minimizing the occurrence of antibiotic resistance is, therefore, highly desirable. To facilitate this, it is important to better understand the long-term response of bacteria to the microgravity environment. This study demonstrated that the use of antibiotics as a preventive measure could be counterproductive and would likely result in persistent resistance to that antibiotic. In addition, unintended resistance to other antimicrobials might also occur as well as permanent genome changes that might have other unanticipated and undesirable consequences.

Bacillus safensis FO-36b and Bacillus pumilus SAFR-032: a whole genome comparison of two spacecraft assembly facility isolates.

BACKGROUND: Bacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B. pumilus SAFR-032, and the draft genomes of B. safensis JPL-MERTA-8-2 and the type strain B. pumilus ATCC7061T. Additional comparisons were made to 61 draft genomes that have been mostly identified as strains of B. pumilus or B. safensis. RESULTS: The FO-36b gene order is essentially the same as that in SAFR-032 and other B. pumilus strains. The annotated genome has 3850 open reading frames and 40 noncoding RNAs and riboswitches. Of these, 307 are not shared by SAFR-032, and 65 are also not shared by MERTA and ATCC7061T. The FO-36b genome has ten unique open reading frames and two phage-like regions, homologous to the Bacillus bacteriophage SPP1 and Brevibacillus phage Jimmer1. Differing remnants of the Jimmer1 phage are found in essentially all B. safensis / B. pumilus strains. Seven unique genes are part of these phage elements. Whole Genome Phylogenetic Analysis of the B. pumilus, B. safensis and other Firmicutes genomes, separate them into three distinct clusters. Two clusters are subgroups of B. pumilus while one houses all the B. safensis strains. The Genome-genome distance analysis and a phylogenetic analysis of gyrA sequences corroborated these results. CONCLUSIONS: It is not immediately obvious that the presence or absence of any specific gene or combination of genes is responsible for the variations in resistance seen. It is quite possible that distinctions in gene regulation can alter the expression levels of key proteins thereby changing the organism's resistance properties without gain or loss of a particular gene. What is clear is that phage elements contribute significantly to genome variability. Multiple genome comparison indicates that many strains named as B. pumilus likely belong to the B. safensis group.

Draft Genome Sequence of Marinobacter vinifirmus Type Strain FB1.

The gammaproteobacterium Marinobacter vinifirmus is associated with moderately saline environments and is often found in marine ecosystems. Here, we report the draft genome sequence of M. vinifirmus type strain FB1 (3.8 Mbp, 3,588 predicted genes). The presented sequence will improve our understanding of the taxonomy and evolution of the genus Marinobacter.

Draft Genome Sequences from a Novel Clade of Bacillus cereus Sensu Lato Strains, Isolated from the International Space Station.

The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three strains), and Russian Segment Zvezda Module (two strains).

The adaptation of Escherichia coli cells grown in simulated microgravity for an extended period is both phenotypic and genomic.

Microorganisms impact spaceflight in a variety of ways. They play a positive role in biological systems, such as waste water treatment but can be problematic through buildups of biofilms that can affect advanced life support. Of special concern is the possibility that during extended missions, the microgravity environment will provide positive selection for undesirable genomic changes. Such changes could affect microbial antibiotic sensitivity and possibly pathogenicity. To evaluate this possibility, Escherichia coli (lac plus) cells were grown for over 1000 generations on Luria Broth medium under low-shear modeled microgravity conditions in a high aspect rotating vessel. This is the first study of its kind to grow bacteria for multiple generations over an extended period under low-shear modeled microgravity. Comparisons were made to a non-adaptive control strain using growth competitions. After 1000 generations, the final low-shear modeled microgravity-adapted strain readily outcompeted the unadapted lac minus strain. A portion of this advantage was maintained when the low-shear modeled microgravity strain was first grown in a shake flask environment for 10, 20, or 30 generations of growth. Genomic sequencing of the 1000 generation strain revealed 16 mutations. Of the five changes affecting codons, none were neutral. It is not clear how significant these mutations are as individual changes or as a group. It is concluded that part of the long-term adaptation to low-shear modeled microgravity is likely genomic. The strain was monitored for acquisition of antibiotic resistance by VITEK analysis throughout the adaptation period. Despite the evidence of genomic adaptation, resistance to a variety of antibiotics was never observed.

Bacillus pumilus SAFR-032 Genome Revisited: Sequence Update and Re-Annotation.

Bacillus pumilus strain SAFR-032 is a non-pathogenic spore-forming bacterium exhibiting an anomalously high persistence in bactericidal environments. In its dormant state, it is capable of withstanding doses of ultraviolet (UV) radiation or hydrogen peroxide, which are lethal for the vast majority of microorganisms. This unusual resistance profile has made SAFR-032 a reference strain for studies of bacterial spore resistance. The complete genome sequence of B. pumilus SAFR-032 was published in 2007 early in the genomics era. Since then, the SAFR-032 strain has frequently been used as a source of genetic/genomic information that was regarded as representative of the entire B. pumilus species group. Recently, our ongoing studies of conservation of gene distribution patterns in the complete genomes of various B. pumilus strains revealed indications of misassembly in the B. pumilus SAFR-032 genome. Synteny-driven local genome resequencing confirmed that the original SAFR-032 sequence contained assembly errors associated with long sequence repeats. The genome sequence was corrected according to the new findings. In addition, a significantly improved annotation is now available. Gene orders were compared and portions of the genome arrangement were found to be similar in a wide spectrum of Bacillus strains.

Draft Genome Sequence of Marinobacter sp. Strain P4B1, an Electrogenic Perchlorate-Reducing Strain Isolated from a Long-Term Mixed Enrichment Culture of Marine Bacteria.

The perchlorate-reducing strain Marinobacter sp. strain P4B1 was isolated from a long-term perchlorate-degrading enrichment culture seeded with marine sediment. The draft genome of Marinobacter sp. P4B1 is comprised of the bacterial chromosome (3.60 Mbp, G+C 58.51%, 3,269 predicted genes) and its associated plasmid pMARS01 (0.14 Mbp, G+C 52.95%, 165 predicted genes).

Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities.

BACKGROUND: The International Space Station (ISS) is a unique built environment due to the effects of microgravity, space radiation, elevated carbon dioxide levels, and especially continuous human habitation. Understanding the composition of the ISS microbial community will facilitate further development of safety and maintenance practices. The primary goal of this study was to characterize the viable microbiome of the ISS-built environment. A second objective was to determine if the built environments of Earth-based cleanrooms associated with space exploration are an appropriate model of the ISS environment. RESULTS: Samples collected from the ISS and two cleanrooms at the Jet Propulsion Laboratory (JPL, Pasadena, CA) were analyzed by traditional cultivation, adenosine triphosphate (ATP), and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assays to estimate viable microbial populations. The 16S rRNA gene Illumina iTag sequencing was used to elucidate microbial diversity and explore differences between ISS and cleanroom microbiomes. Statistical analyses showed that members of the phyla Actinobacteria, Firmicutes, and Proteobacteria were dominant in the samples examined but varied in abundance. Actinobacteria were predominant in the ISS samples whereas Proteobacteria, least abundant in the ISS, dominated in the cleanroom samples. The viable bacterial populations seen by PMA treatment were greatly decreased. However, the treatment did not appear to have an effect on the bacterial composition (diversity) associated with each sampling site. CONCLUSIONS: The results of this study provide strong evidence that specific human skin-associated microorganisms make a substantial contribution to the ISS microbiome, which is not the case in Earth-based cleanrooms. For example, Corynebacterium and Propionibacterium (Actinobacteria) but not Staphylococcus (Firmicutes) species are dominant on the ISS in terms of viable and total bacterial community composition. The results obtained will facilitate future studies to determine how stable the ISS environment is over time. The present results also demonstrate the value of measuring viable cell diversity and population size at any sampling site. This information can be used to identify sites that can be targeted for more stringent cleaning. Finally, the results will allow comparisons with other built sites and facilitate future improvements on the ISS that will ensure astronaut health.

In Vivo Production of Small Recombinant RNAs Embedded in a 5S rRNA-Derived Protective Scaffold.

Preparative synthesis of RNA is a challenging task that is usually accomplished using either chemical or enzymatic polymerization of ribonucleotides in vitro. Herein, we describe an alternative approach in which RNAs of interest are expressed as a fusion with a 5S rRNA-derived scaffold. The scaffold provides protection against cellular ribonucleases resulting in cellular accumulations comparable to those of regular ribosomal RNAs. After isolation of the chimeric RNA from the cells, the scaffold can be removed if necessary by deoxyribozyme-catalyzed cleavage followed by preparative electrophoretic separation of the cleavage reaction products. The protocol is designed for sustained production of high quality RNA on the milligram scale.

The presence of nitrate dramatically changed the predominant microbial community in perchlorate degrading cultures under saline conditions.

BACKGROUND: Perchlorate contamination has been detected in both ground water and drinking water. An attractive treatment option is the use of ion-exchange to remove and concentrate perchlorate in brine. Biological treatment can subsequently remove the perchlorate from the brine. When nitrate is present, it will also be concentrated in the brine and must also be removed by biological treatment. The primary objective was to obtain an in-depth characterization of the microbial populations of two salt-tolerant cultures each of which is capable of metabolizing perchlorate. The cultures were derived from a single ancestral culture and have been maintained in the laboratory for more than 10 years. One culture was fed perchlorate only, while the other was fed both perchlorate and nitrate. RESULTS: A metagenomic characterization was performed using Illumina DNA sequencing technology, and the 16S rDNA of several pure strains isolated from the mixed cultures were sequenced. In the absence of nitrate, members of the Rhodobacteraceae constituted the prevailing taxonomic group. Second in abundance were the Rhodocyclaceae. In the nitrate fed culture, the Rhodobacteraceae are essentially absent. They are replaced by a major expansion of the Rhodocyclaceae and the emergence of the Alteromonadaceae as a significant community member. Gene sequences exhibiting significant homology to known perchlorate and nitrate reduction enzymes were found in both cultures. CONCLUSIONS: The structure of the two microbial ecosystems of interest has been established and some representative strains obtained in pure culture. The results illustrate that under favorable conditions a group of organisms can readily dominate an ecosystem and yet be effectively eliminated when their advantage is lost. Almost all known perchlorate-reducing organisms can also effectively reduce nitrate. This is certainly not the case for the Rhodobacteraceae that were found to dominate in the absence of nitrate, but effectively disappeared in its presence. This study is significant in that it reveals the existence of a novel group of organisms that play a role in the reduction of perchlorate under saline conditions. These Rhodobacteraceae especially, as well as other organisms present in these communities may be a promising source of unique salt-tolerant enzymes for perchlorate reduction.

Evolution of the ribosome at atomic resolution.

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call "insertion fingerprints." Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

Draft Genome Sequence of Deinococcus phoenicis, a Novel Strain Isolated during the Phoenix Lander Spacecraft Assembly.

Deinococcus phoenicis strain 1P10ME(T) is a radiation- and desiccation-resistant bacterium isolated from a cleanroom facility where the Phoenix Lander spacecraft was assembled. In order to facilitate investigations of the nature of the extreme resistance of D. phoenicis to bactericidal factors, a draft genome sequence of D. phoenicis was determined.

Secondary structure and domain architecture of the 23S and 5S rRNAs.

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided (http://apollo.chemistry.gatech.edu/RibosomeGallery).

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.

BACKGROUND: Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. RESULTS: Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. CONCLUSIONS: The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

Stress-driven in vivo selection of a functional mini-gene from a randomized DNA library expressing combinatorial peptides in Escherichia coli.

A plasmid-borne randomized mini-gene library expressing a population of combinatorial 20-mer peptides with no bias toward any biological function was used as an initial source of genetic variance during stress-driven evolution of Escherichia coli. The transformed bacteria were evolved under multiple rounds of selective pressure imposed by nearly lethal concentrations of NiCl(2), AgNO(3), or K(2)TeO(3). At the final stage, clones conferring resistance to NiCl(2) were found to carry identical functional mini-genes, which conferred significant nickel tolerance on the host cells. Expression of the mini-gene markedly improved growth parameters of the evolved clones at subinhibitory concentrations of NiCl(2) while being slightly detrimental in the absence of the stress. This substantial increase in resistance, as compared with control cultures adapted in the absence of the mini-gene, is shown to be largely due to coadaptation with changes elsewhere in the E. coli genome. Clones resistant to AgNO(3) and K(2)TeO(3) harbored plasmid variants with an inactive mini-gene and with a deleted mini-gene operon, respectively. In those cases, an exploration of the mini-gene sequence space apparently was fruitless, and the developed toxicity tolerance was likely to be exclusively associated with acquired adaptive mutations. Overall, the results demonstrate a very natural outcome in which the mini-genes were expected to be either successfully integrated into the bacterial genetic network or rejected depending on their effect on host fitness. This approach is immediately useful as a laboratory model to study the dynamics of bacterial adaptive evolution at the molecular level and is especially relevant as a rapid method to study cellular response to recently acquired genetic material.

Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations.

A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases. The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants. No expensive reagents or equipment were required. Plasmid quality was evaluated by gel electrophoresis and restriction analysis. The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.