Recombination, RNA evolution, and bifunctional RNA molecules isolated through chimeric SELEX.

Exchange of RNA structural domains through recombination can be used to engineer RNAs with novel functions and may have played an important role in the early evolution of life. The degree of function an RNA element retains upon recombination into a new sequence context is a measure of how deleterious or beneficial recombination will be. When we fused pairs of aptamers previously selected to bind coenzyme A, chloramphenicol, or adenosine, the chimerae retained some ability to bind both targets, but with reduced binding activity both in solution and on affinity resins, probably due to misfolding. Complex populations of recombined RNAs gave similar results. Applying dual selection pressure to recombined populations yielded the combinations that were best suited to binding both targets. Most reselected RNAs folded into the active conformation more readily than chimerae built from arbitrarily chosen aptamers, as indicated both by solution Kd measurements and affinity resin binding activity. Deletion/selection experiments confirmed that the sequences required for binding are fully contained within the respective domains and not derived from interaction between the domains, consistent with the modular architecture of their original design. The combinatorial nature of the recombination methods presented here takes advantage of the full sequence diversity of the starting populations and yields large numbers of bifunctional molecules (10(6) to more than 1012). The method can be easily generalized and should be applicable to engineering dual-function RNAs for a wide variety of applications, including catalysis, novel therapeutics, and studies of long-range RNA structure.

A novel acidophilic RNA motif that recognizes coenzyme A.

Specific recognition of nucleotide cofactors by RNA may be important in engineering new RNA enzymes (ribozymes). Although in vitro selections (SELEX) have identified nucleic acid motifs ("aptamers") that bind a variety of adenosine cofactors, none of these recognizes coenzyme A (CoA), the primary biological cofactor used in acyltransfer reactions. We used SELEX experiments with two random RNA pools to identify aptamers that bind CoA. Functional boundary determination and extensive comparative sequence analysis (including reselection of a mutagenized, circularly permuted RNA) led to the identification of a 52 nucleotide minimal aptamer ("min52"). The RNA structural motif contains a large internal loop with 26 unpaired nucleotides flanked by helices of any base-paired sequence. Twenty loop nucleotides are specifically required for binding activity, 12 of which are derived from the original primer binding sequences. Specificity studies with CoA analogues demonstrated that the aptamer recognizes many adenosine analogues, including ATP, and that recognition is predominantly through the Höogsteen face of adenine. Binding activity is greatest at acidic pH (optimum near 5.0), in low or no monovalent salt, and at high concentrations of either Mg2+ or Mn2+. Strong binding activity (86% of maximum) is observed at pH 4.0, suggesting that at least some extreme conditions (acidic pH) may be compatible with RNA World theories of the origin and early evolution of life. In the presence of 10 mM Mg2+, binding is unaffected by the addition of 1 mM Ca2+, but it is mildly inhibited by 1 mM Zn2+ or Co2+ or by 0.1 mM Cu2+ or Ni2+. The dissociation constant (Kd) for the association of min52 RNA with ATP in solution was measured to be 2.4 +/- 0.4 microM under the conditions of the selection and 0.5 +/- 0.1 microM under optimized conditions. Finally, we show that the selected CoA aptamer populations contain other RNAs at low frequencies that preferentially recognize intact CoA and are not eluted from the resin by AMP alone.

RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX.

We used in vitro selection (SELEX) to isolate RNA 'aptamers' to S-adenosyl methionine (SAM). Individual aptamer sequences conform to the structural element noted previously for adenosine binding in selections for aptamers to ATP and NAD+. When we compare the patterns of sequence conservation among 65 adenosine-binding sequences to the published structure of the adenosine aptamer, we find that the most highly conserved nucleotides contact the bound adenosine directly, and that one conserved nucleotide outside the binding pocket is in position to stabilize nucleotides within the binding pocket. The aptamer's ability to bind diverse adenosine-containing cofactors is easily understood in terms of its mode of binding, which leaves the 5'position exposed to solvent. We propose that aptamers that bind their targets away from the reactive moiety may be particularly well suited for catalysis. Finally, we estimate that one sequence in 10(11) may be able to form this structural motif, and that there may be many other adenosine-binding motifs that have escaped detection because of their lower representation in the starting random pools.

RNA aptamers to the peptidyl transferase inhibitor chloramphenicol.

BACKGROUND: The problem of how macromolecules adopt specific shapes to recognize small molecules in their environment is readily addressed through in vitro selections (the SELEX protocol). RNA-antibiotic interactions are particularly attractive systems for study because they provide an opportunity to expand our understanding of molecular recognition by RNA and to facilitate ribosomal modeling. Specifically, the antibiotic chloramphenicol (Cam) naturally binds bacterial ribosomes in the 'peptidyl transferase loop' of 23S ribosomal RNA to inhibit peptide bond formation. RESULTS: We identified Cam-binding RNA molecules ('aptamers') from two independent initial random RNA populations. Boundary determinations, ribonuclease S1 sensitivity analyses and the activity of truncated minimal RNAs identified a structural motif that is shared by sequences from both selections. The pseudosymmetric motif consists of a highly conserved central helix of five to six base pairs flanked by A-rich bulges and additional helices. Addition of Cam prior to ribonuclease S1 protected nucleotides in the conserved cores from cleavage. Reselection from a pool of mutated variants of the minimal aptamer further refined the sequence requirements for binding. Finally, we used proton nuclear magnetic resonance (NMR) to establish a 1:1 RNA: Cam stoichiometry of the complex. Both the protection and NMR data both show that Cam stabilizes the active fold of this aptamer. CONCLUSIONS: There are many different RNA sequences that can bind Cam. The Cam aptamers that we examined have a well-defined secondary structure with a binding pocket that appears to be stabilized by Cam. This RNA motif superficially resembles the Cam-binding site in 23S rRNA, although further work is needed to establish the significance of these similarities.

Bent pseudoknots and novel RNA inhibitors of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase.

The reverse transcriptase (RT) of the human immunodeficiency virus (HIV) is a proven target for therapeutic intervention of HIV infections. We have found several new RNA inhibitors of HIV-1 RT that differ significantly from the pseudoknot ligands found previously, along with a wide variety of pseudoknot variants. One pseudoknot variant and three novel ligands were studied in more detail. Each specifically inhibits DNA polymerization by HIV RT (half-maximal inhibition at 0.3 to 20 nM inhibitor), but not that of RTs derived from MMLV or AMV. The minimal binding element of each isolate was determined by deletion analysis and by gel electrophoresis of protein-bound, partially alkaline-hydrolyzed RNA. Truncations of three of the isolates bound nearly as well as (or better than) the parental sequences, while most deletions in the fourth caused substantial disruption of binding. The truncated versions of two isolates were subjected to six rounds of secondary SELEX after resynthesizing them mutagenically. Patterns of conserved and covarying nucleotides yielded structural models consistent with 5' and 3' boundary determinations for these molecules. Among the four isolates studied in detail, the first is confirmed as being a pseudoknot, albeit with substantial structural differences as compared to the canonical pseudoknots identified previously. The second forms a stem-loop structure with additional flanking sequences required for binding. Tentative structural models for the other two isolates are presented. The minimal fully active truncations of each of these four isolates compete with each other and with a classical RNA pseudoknot for binding to HIV RT, suggesting that they all recognize the same or overlapping sites on the protein, in spite of their apparently dissimilar structures. We model their interactions with RT as mimicking the 40 to 45 degrees bend in dsDNA co-crystallized with RT.

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli.

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, beta-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for beta-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.

Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure.

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Early evolution of photosynthesis: clues from nitrogenase and chlorophyll iron proteins.

Chlorophyll (Chl) is often viewed as having preceded bacteriochlorophyll (BChl) as the primary photoreceptor pigment in early photosynthetic systems because synthesis of Chl requires one fewer enzymatic reduction than does synthesis of BChl. We have conducted statistical DNA sequence analyses of the two reductases involved in Chl and BChl synthesis, protochlorophyllide reductase and chlorin reductase. Both are three-subunit enzymes in which each subunit from one reductase shares significant amino acid identity with a subunit of the other, indicating that the two enzymes are derived from a common three-subunit ancestral reductase. The "chlorophyll iron protein" subunits, encoded by the bchL and bchX genes in the purple bacterium Rhodobacter capsulatus, also share amino acid sequence identity with the nitrogenase iron protein, encoded by nifH. When nitrogenase iron proteins are used as outgroups, the chlorophyll iron protein tree is rooted on the chlorine reductase lineage. This rooting suggests that the last common ancestor of all extant photosynthetic eubacteria contained BChl, not Chl, in its reaction center, and implies that Chl-containing reaction centers were a late invention unique to the cyanobacteria/chloroplast lineage.

Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

The Rhodobacter capsulatus chlorin reductase-encoding locus, bchA, consists of three genes, bchX, bchY, and bchZ.

The bchA locus of Rhodobacter capsulatus codes for the chlorin reductase enzyme in the bacteriochlorophyll synthesis pathway. Previous work has suggested that this locus might encompass a single gene. We have sequenced the bchA locus and found it to contain three coding segments, which we designate bchX, bchY, and bchZ. Each coding segment contains its own translational initiation sequence and follows codon utilization patterns consistent with those of previously published R. capsulatus genes. When various regions of the bchA locus and flanking sequences were subcloned into an expression vector and expressed in Escherichia coli, the three coding segments were all expressed as separate peptides. Finally, conservation of amino acid sequences between bchX and a subunit of the protochlorophyllide reductase (bchL, 34% identity) and the nitrogenase Fe protein (nifH, 30 to 37% identity) suggests structural and mechanistic commonalities among all three proteins.

bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.

Cyproheptadine antagonism of p-chloroamphetamine hypothermia in mice.

Serotonin and 5-hydroxyindoleacetic acid levels were measured in the brains of mice following intraperitoneal administration of 10 mg/kg p-chloramphetamine (PCA). Indolyl levels were decreased at the 60 min interval, but were unchanged 30 min following PCA injection. PCA-induced hypothermia was found to be dependent upon dose and ambient temperature. Hypothermia was attenuated by cyproheptadine and slightly enhanced in the presence of fluoxetine. Mechanisms other than intraneuronal release and serotonin depletion appear to mediate PCA-induced hypothermia.

An integrated medical record system.

Clear, well-organized, thorough medical records are fundamental to the success of a quality healthcare organization. This article specifically addresses the problem of integrating multiple records from several group and individual practices into a single common medical record for each patient. All aspects of the record system are described--from initial development, record protocol, and start-up and operational costs, to implementation, quality control, and results of the new system. The information set forth is valuable to all healthcare administrations interested in establishing or maintaining integrated medical record systems in their group practices.

Cobaltous chloride-induced hypothermia in mice III: effect of pretreatment with 5-hydroxytryptaminergic agents.

The influence of various 5-hydroxytryptaminergic agonist and antagonist drugs on body-temperature response to cobaltous chloride in mice was noted. Pretreatment with p-chloroamphetamine, p-chlorophenylalanine, and p-iodoamphetamine antagonized the body-temperature response to cobalt. p-Chloroamphetamine and p-chlorophenylalanine reduced, while p-iodoamphetamine elevated, brain serotonin levels. The uptake inhibitor agents, fluoxetine and nisoxetine, failed to modify the ability of p-chloroamphetamine to antagonize cobalt hypothermia. Cyproheptadine, methergoline, and xylamidine pretreatment enhanced rather than antagonized body-temperature depression by cobalt. Tryptophan hydroxylase inhibitors antagonized cobalt hypothermia, but receptor-blocking agents were without influence, indicating that antagonism was mediated through mechanisms other than the depletion of serotonin. Elevation rather than depletion of brain serotonin by p-iodoamphetamine and failure of uptake inhibitors to modify p-chloroamphetamine antagonism of cobalt hypothermia lend further support for a nonserotonergic role of these amines in their ability to antagonize body-temperature depression by cobaltous chloride in mice.

Effect of various stimulus parameters on electrically-induced catecholamine secretion by thin slices of bovine adrenal medulla.

We examined the role of various stimulus parameters in electrically-induced catecholamine secretion by thin slices of bovine adrenal medulla. The stimulus parameters examined were voltage, duration, pulse width, and frequency for square-wave monophasic pulses. As each was examined it was held constant at a selected value for the evaluation of subsequent stimulus characteristics. For 16 mm2 tissue slices, catecholamine secretion was approximately linearly related to stimulus voltage over the range 20--80 volts, with a threshold of 20 V. Increasing the voltage beyond 80 V did not enhance secretion. Similarly, catecholamine secretion was dependent upon the frequency of stimulation. For stimuli delivered at 50 V for a 10-sec interval there was a four-fold increase in secretion over the frequency range 10--100/sec. Increasing pulse width at a constant voltage (50 V) over the range 0.4--2.0 msec resulted in a four-fold increase in catecholamine secretion. For pulses of 50 V, 50/sec and 0.8 msec pulse width, secretion was dependent upon the duration of the stimulus. Enhanced secretion was evident for times as short as 2 sec; between 5 and 15 sec of stimulation catecholamine secretion was linearly related to stimulus duration. Over the range 2--15 sec there was a five-fold enhancement of secretion. Electrically-induced catecholamine secretion by slices was markedly dependent upon stimulus parameters. In general, it was enhanced by increasing voltage, stimulus duration, pulse width and frequency. For most experiments a good choice of stimulus parameters appears to be 50 V 10 sec duration, 0.8 msec pulse width delivered at a frequency of 50/sec. Maximizing all stimulus parameters resulted in a 17-fold enhancement of secretion.

p-Chloroamphetamine antagonism of cobaltous chloride-induced hypothermia in mice.

Serotonin levels were measured in the brains of mice at 2 h and 10 days following the administration of a single dose of 10 mg/kg, i.p. of p-chloroamphetamine HCl (PCA). A 44% reduction in the concentration of brain serotonin was noted 2 h after PCA injection. No significant change in brain serotonin was observed 10 days following PCA administration. The time course of the body temperature response to cobaltous chloride (25 mg/kg, i.p.) was recorded 60 min following pretreatment with PCA (10 mg/kg, i.p.). PCA reduced the hypothermic response by approximately 60%, presumably through the depletion of central 5-hydroxytryptamine.

Cobaltous chloride-induced hypothermia. II: Pretreatment with sympathoplegics, antihistamines, and narcotic antagonists.

Body temperature depression was noted in rats, mice, and hamsters following intraperitoneal cobaltous chloride administration (25 mg/kg). Intracerebral cobalt injection elicited hypotermia in rats and mice but not in hamsters. Body temperature depression appeared to be centrally mediated in rats and mice and peripherally mediated in hamsters. The effect of intraperitoneal and intracerebral pretreatment with phentolamine, diphenhydramine, propranolol, cimetidine, and naloxone on the mouse rectal temperature response to cobalt (25 mg/kg ip) was noted. Systemic phentolamine injection (intraperitoneal) did not alter the cobalt response, whereas intracerebral administration partially antagonized cobalt-induced hypothermia, indicating that antagonism was mediated centrally. Pretreatment with propranolol and cimetidine failed to modify the temperature response. Intracerebral diphenhydramine did not influence cobalt hypothermia. However, this agent reduced the cobalt response when given intraperitoneally, presumably through a peripheral inhibitory mechanism. The intracerebral injection of naloxone 30 min prior to cobalt slightly enhanced hypothermia, apparently through a central action. Intracerebral 6-hydroxydopamine injection depleted brain norepinephrine and dopamine but exhibited no apparent influence on cobalt-induced hypothermia.