Biomedical subjects
Publications and source records attributed to L Gold.
The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.
SELEX is a technology for the identification of high affinity oligonucleotide ligands. Large libraries of random sequence single-stranded oligonucleotides, whether RNA or DNA, can be thought of conformationally not as short strings but rather as sequence dependent folded structures with high degrees of molecular rigidity in solution. This conformational complexity means that such a library is a source of high affinity ligands for a surprising variety of molecular targets, including nucleic acid binding proteins such as polymerases and transcription factors, non-nucleic acid binding proteins such as cytokins and growth factors, as well as small organic molecules such as ATP and theophylline. The range of applications of this technology for new discovery extends from basic research reagents to the identification of novel diagnostic and therapeutic reagents. Examples of these applications are described along with a discussion of underlying principles and future developments expected to further the utility of SELEX.
Vascular remodeling in adult atherosclerotic pulmonary arteries is characterized by discrete areas of neointimal smooth muscle cell extracellular matrix gene expression in close proximity to non-foamy macrophages, suggesting regulation by local macrophage-associated factors. The purpose of these studies was to begin addressing the role of putative macrophage-associated factors such as transforming growth factor-beta (TGF-beta), by determining the spatial relationship between TGF-beta and neointimal matrix gene expression in human atherosclerotic pulmonary arteries. For example, the participation of TGF-beta in vascular remodeling could be inferred by its colocalization with non-foamy macrophages in areas of active matrix synthesis. In situ hybridization and immunohistochemistry demonstrated focal neointimal procollagen gene expression in close association with non-foamy but not foamy macrophages. Immunohistochemistry with isoform-specific anti-TGF-beta antibodies demonstrated all three isoforms of TGF-beta associated with non-foamy macrophages, but foamy macrophages were not immunoreactive. Neointimal and medial smooth muscle cells stained lightly. In contrast, intense TGF-beta immunoreactivity was also associated with medial smooth muscle cells in normal nonremodeling vessels. Immunohistochemistry with antibodies specific for latent TGF-beta was similar to immunohistochemistry for mature TGF-beta in both remodeling and nonremodeling vessels. Finally, using an antibody specific for active TGF-beta 1, immunoreactivity was only seen in non-foamy neointimal macrophages but not in foamy macrophages or medial smooth muscle cells from hypertensive or normal vessels. These observations suggest non-foamy macrophages may participate in modulating matrix gene expression in atherosclerotic remodeling via a TGF-beta-dependent mechanism.
Vast libraries of oligonucleotide sequences can be made. Such libraries have been screened and/or selected to identify individual molecules showing various properties, including high affinity/high specificity binding (to something) or new enzymatic activity. The SELEX protocol is used to identify these interesting oligonucleotides; two recent reviews of the SELEX protocol and its power have appeared recently. Oligonucleotides derived through the SELEX process have applications in diagnostics and therapeutics, and have additional value as research reagents and compounds that provide chemical plausibility to some ideas about evolution of the biosphere. Oligonucleotide conformations are shockingly robust and potent.
RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.
Infection of Escherichia coli by bacteriophage T4 induces a mRNA ribonuclease activity that shows specificity for cleavage within the sequence GGAG. Substrates of the activity in vivo include a number of phage mRNAs that are cleaved at GGAGs within the Shine/Dalgarno domains of their translation initiation regions. Induction of the ribonuclease depends on the product of the T4 gene regB. We describe here the overproduction and extensive purification of the RegB protein. RegB precisely co-purifies with an activity that cleaves within the sequence GGAG in oligonucleotide and polynucleotide RNAs and is therefore likely to constitute the sequence-specific catalytic component of the observed activity. We further report that the low cleavage rate observed with our preparations of purified RegB is substantially increased (1-2 orders of magnitude) by the addition of E. coli ribosomal protein S1. We discuss the implications of this observation for the mechanism of action of the RegB ribonuclease in vitro and in vivo.
Specific, high-affinity RNA ligands to avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases were isolated from a combinatorial RNA library using the SELEX (systematic evolution of ligands by exponential enrichment) procedure. The selected RNA ligands bound their respective reverse transcriptases with approximately nanomolar dissociation constants. The ligands did not exhibit primary sequence conservation from selections against different target enzymes. Moreover, the selected ligands competed with the binding of template/primer complex and inhibited both the RNA-dependent DNA polymerase and the RNase H activities of the cognate reverse transcriptase. SELEX can yield both high-affinity and high-specificity oligonucleotide antagonists against specific members of a protein family.
185 clones with randomized ribosome binding sites, from position -11 to 0 preceding the coding region of beta-galactosidase, were selected and sequenced. The translational yield of each clone was determined; they varied by more than 3000-fold. Multiple linear regression analysis was used to determine the contribution to translation initiation activity of each base at each position. Features known to be important for translation initiation, such as the initiation codon, the Shine/Dalgarno sequence, the identity of the base at position -3 and the occurrence of alternative ATGs, are all found to be important quantitatively for activity. No other features are found to be of general significance, although the effects of secondary structure can be seen as outliers. A comparison to a large number of natural E.coli translation initiation sites shows the information profile to be qualitatively similar although differing quantitatively. This is probably due to the selection for good translation initiation sites in the natural set compared to the low average activity of the randomized set.
In Escherichia coli the unusual amino acid selenocysteine is incorporated cotranslationally at an in-frame UGA codon. Incorporation of selenocysteine relies, in part, on the interaction between a specialized elongation factor, the SELB protein, and a cis-acting element within the mRNA. Boundary and toeprint experiments illustrate that the SELB-GTP-Sec-tRNA(Sec) ternary complex binds to the selenoprotein encoding mRNAs fdhF and fdnG, serving to increase the concentration of SELB and Sec-tRNA(Sec) on these mRNAs in vivo. Moreover, toeprint experiments indicate that SELB recognizes the ribosome-bound message and that, upon binding, SELB may protrude out of the ribosomal-mRNA track so as to approach the large ribosomal subunit. The results place the mRNA-bound SELB-GTP-Sec-tRNA(Sec) ternary complex at the selenocysteine codon (as expected) and suggest a mechanism to explain the specificity of selenocysteine insertion. Cis-acting mRNA regulatory elements can tether protein factors to the translation complex during protein synthesis.
Our previous investigations of transforming growth factor types beta 1 and beta 2 (TGF beta s) showed negative or positive autocrine growth regulation of gliomas in vitro. Near-diploid gliomas were inhibited by the TGF beta s, whereas a stimulatory response correlated with progressive anaplasia and karyotypic divergence. We have tested the hypothesis that cytogenetic aberrations may be associated with conversion of TGF beta autoregulation from inhibitory to stimulatory among other cultured neuroectodermal tumors. Anchorage-independent growth and karyotypic aberrations supported the malignant nature in vitro of two medulloblastoma (MBL), two primitive neuroectodermal tumor (PNET), and two ependymoma (EPD) cultures. Transforming growth factor type beta 1 and/or TGF beta 2 RNA was evident by Northern blot analysis among these cell cultures. By radioreceptor assay active TGF beta was present in conditioned medium in concentrations of 0 to 14 ng/mL, whereas the total amount of active and latent TGF beta secreted was in the range of 3 to 118 ng/mL. Expression of the TGF beta radioreceptor (TGF beta-R) types I and II was shown by cross-linking assay. Responses to exogenous TGF beta were determined by [3H]-thymidine incorporation, cell counts, and anchorage-independent clonogenicity. Exogenous TGF beta was growth inhibitory for the near-diploid MBL, PNET, and EPD in vitro, as well as antagonistic to the mitogenic effect of epidermal growth factor (EGF) and insulin. In contrast, MBL, PNET, and EPD with a hyperdiploid subpopulation were stimulated to proliferate in monolayer culture or soft agar by TGF beta 1 and TGF beta 2. The growth response did not correlate with TGF beta-R type. Autocrine regulation was supported by antibody neutralization experiments performed with quiescent cells in the absence of exogenous TGF beta. Anti-TGF beta antisera enhanced the growth of TGF beta-inhibited cultures, whereas the TGF beta-stimulated cultures were inhibited by the antisera. Karyotypic divergence seemed to predict response as MBL, PNET, and EPD with hyperdiploid elements exhibited autocrine TGF beta-stimulation. In contrast, the near-diploid cultures were inhibited by the TGF beta s. By analogy with the gliomas, conversion of TGF beta autocrine regulation from inhibition to stimulation may be a late progression marker of anaplasia among MBL, PNET, and EPD. Secretion of this TGF, which serves both as a mitogen and immunosuppressive agent may contribute to the adverse prognosis of hyperdiploid neuroectodermal neoplasms of the central nervous system (CNS).
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The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.
Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet). The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet).
The ribosomal mRNA track was investigated by toeprinting 30S ribosomes, in the presence or absence of tRNA, using a variety of different ribosome-binding sites. We found that: (1) the ribosome, by itself, recognizes the mRNA translational initiation site; (2) the ribosomal mRNA track makes extensive contact with mRNA independent of tRNA and the start codon; (3) ribosome-mRNA complexes are less stable than complexes containing tRNA; and (4) toeprinting can be used to analyze the contour of the ribosomal mRNA track, yielding information on its "height" as well as its "length" dimension. Examination of several ribosome-binding sites, including those containing very stable secondary structure, indicated that the "height" of the mRNA track is quite roomy, while the nucleotide distance between the site of Shine-Dalgarno annealing, the P site, and the 3'-edge of the mRNA track is fixed. The data suggest a mechanism for tethering regulatory elements to the ribosome during translation.
The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct. Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (delta PR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.
The authors compared rates of physical and sexual abuse in women with eating disorders (N = 102) and general psychiatric disorders (N = 49). Relationships between sexual abuse and severity of eating disordered and psychiatric symptoms were also examined. While high rates of sexual abuse were found in the eating disordered sample, these rates were not significantly higher than those found in the general psychiatric population. No relationship between a history of sexual abuse and severity of eating disordered symptoms was found. However, within the eating disordered group, sexually abuse subjects reported more severe psychiatric disturbances of an obsessive and phobic nature than nonabused subjects. These findings suggest that while sexually abusive experiences may be related to increased psychological distress, they do not serve to increase eating disordered symptomatology.
We have applied the SELEX procedure (systematic evolution of ligands by exponential enrichment) to obtain RNA molecules that bind tightly to the Escherichia coli transcription termination factor rho. The starting pool was a population of RNA molecules 77 nucleotides (nt) long, in which was embedded a cassette of 30 nt of randomized sequence. The apparent dissociation constant of this RNA pool for hexameric rho factor was about 1 microM. After eight rounds of selection by filter binding, with RNA in either 10-fold or 40 to 100-fold excess at each step, the dissociation constant of the selected RNA had dropped by more than 500-fold to about 1 nM. Analysis of 29 clonal isolates from the population revealed that five had KDs substantially weaker than 10 nM (presumably background carryover), 40% were C-rich (as might have been predicted from rho's known substrate binding), and 40% had a strikingly preserved potential hairpin, in most cases of 6 base pairs with a 3 nt CAA loop and preceded by a CCCCA consensus. The rho-dependent trp t' terminator region includes a related potential hairpin structure; however, it is energetically unfavorable. The implications of the sequence findings for elucidating both static and dynamic aspects of rho factor recognition and response to its RNA target site are discussed.
DNA fragments which are intrinsically bent or curved migrate anomalously during electrophoresis through polyacrylamide gels. Starting with an initial population of approximately 10(12) unique DNA sequences, DNA which exhibited the kind of anomalous mobility associated with DNA bending was selected and enriched using a variation of the SELEX procedure. After seven rounds of selection and amplification, the vast majority of the remaining population of DNA fragments migrated as bent DNA. Cloning and sequencing of 30 individual sequences from this population has yielded information regarding the relationship between DNA sequence and bending. Some of the previous conclusions on DNA bending have been confirmed while others have been modified, by the results presented here. In addition, the dinucleotide base step CA/TG, which had not been thought to be a major factor in DNA bending, appears to be important.