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Biomedical subjects

L Gold

Publications and source records attributed to L Gold.

At least 37 records · Page 2Linked to original sources
PubMed1997

In vitro selection of self-cleaving RNAs with a low pH optimum.

RNAs that undergo a rapid site-specific cleavage at low pH have been selected by in vitro selection (the SELEX process). The cleavage does not require the addition of any divalent metal ions, and is in fact inhibited by divalent metal ions, spermine, or high concentrations of monovalent metal ions. This low pH catalyzed cleavage results in a 2',3'-cyclic phosphate at the 3' end and a free hydroxyl at the 5' end. The reaction proceeds with a calculated rate of 1.1 min-1 at room temperature in cacodylate buffer at pH 5.0. The rate of cleavage is dependent on the pH and shows an optimum around pH 4.0. The rate constant is independent of RNA concentration, indicating to an intramolecular reaction. Autocatalytic cleavage at low pH, in the absence of a metal ion requirement, adds to the reaction possibilities that may have existed on the prebiotic earth.

Catalysis↗
PubMed1997

Single-stranded RNA recognition by the bacteriophage T4 translational repressor, regA.

The T4 protein, RegA, is a translational repressor that blocks ribosome binding to multiple T4 messages by interacting with the mRNAs near their respective AUG start codons. Other than the AUG, there are no obvious similarities between the affected mRNAs. High affinity RNA ligands to RegA were isolated using SELEX (systematic evolution of ligands by exponential enrichment). The selected RNAs exhibited the consensus sequence 5'-AAAAUUGUUAUGUAA-3'. The AUG was invariant, suggesting that it is the primary effector of binding specificity. The UU immediately 5' to the AUG and the upstream poly(A) tract were highly conserved among the selected RNAs. Boundary and footprinting experiments are consistent with the consensus sequence defining the RegA-binding site. Interestingly, chemical modification and nuclease digestion data indicate that the RNA-binding site is single-stranded, as if RegA discriminates between targets based on their primary sequence, not their secondary structure. Minor variations from the consensus at positions other than the universally conserved AUG have little effect on RegA binding, but accumulation of mutations has a profound effect on the interaction. Comparison of the in vivo targets for RegA to the SELEX-generated consensus suggests a repression pattern whereby the translation of individual messages is sequentially halted until the least similarly affected message, the regA gene itself, is repressed.

Bacteriophage T4↗
PubMed1997

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.

Adenosine↗
PubMed1997

Libraries for genomic SELEX.

An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX.

Binding Sites↗
PubMed1997

From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops.

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?

Base Sequence↗
PubMed1997

Enhanced urokinase plasminogen activation in chronic pancreatitis suggests a role in its pathogenesis.

BACKGROUND & AIMS: Urokinase plasminogen activator (uPA) regulates plasmin generation from plasminogen. The aim of this study was to analyze the role of the plasminogen activator/plasmin system in chronic pancreatitis (CP). METHODS: Using Northern blot analysis, in situ hybridization, and immunohistochemistry, the expression of uPA, its receptor (uPAR), plasminogen activator inhibitor 1 (PAI-1), and transforming growth factor beta 1 (TGF-beta 1) was studied in 14 patients undergoing pancreatic resection for CP. Normal control pancreatic tissue was obtained through an organ donor program. RESULTS: Eight of 14 CP samples showed concomitant increased expression (P < 0.001) of uPA (5.2-fold), uPAR (5.9-fold), and TGF-beta 1 (8.8-fold) messenger RNA (mRNA) compared with normal controls. PAI-1 mRNA expression was increased (6.5-fold; P < 0.001) in all CP samples. By in situ hybridization, moderate to strong mRNA staining of all four factors was present in acinar cells, some ductal cells, and areas with ductal metaplasia in CP samples. A similar staining pattern was found by immunohistochemistry. Intense mRNA and immunostaining for all of these factors in CP samples was associated with a higher degree of pancreatic damage. CONCLUSIONS: uPA and its receptor may contribute to the lytic damage observed in CP by plasmin generation. Similarly, increased amounts of plasmin may activate latent TGF-beta, thereby leading to the accumulation of fibrotic tissue.

Adolescent↗
PubMed1997

Calcifying odontogenic cyst: a clinicopathologic study of 57 cases with immunohistochemical evaluation for cytokeratin.

PURPOSE: A clinicopathologic study of all cases accessioned as calcifying odontogenic cyst (COC) from 1971 to 1996 from the files of the Oral Pathology Laboratory at Temple University School of Medicine was undertaken. MATERIALS AND METHODS: Microscopic slides and clinical histories of cases diagnosed as calcifying odontogenic cyst were reviewed and analyzed. Ten cases were processed for cytokeratin immunohistochemical staining. RESULTS: Fifty-seven cases were reviewed, 28 males and 29 females. Patients' ages ranged from 7 to 83 years, with a mean age of 49.8 years. Thirty-four cases involved the mandible and 23 were from the maxilla. Seventeen were reported in peripheral locations, and 38 occurred centrally within the jaws. Two were found both centrally and peripherally. The most common clinical sign for central lesions was a radiolucency sometimes associated with a jaw expansion. The most common clinical complaint for peripheral lesions was a nodular growth on the gingiva. Although lining epithelial cells were strongly positive for cytokeratin, full-brown ghost cells and disintegrating ghost cells were nonreactive. CONCLUSION: Calcifying odontogenic cyst can occur in any age-group, intraosseously or extraosseously, and as a solid lesion. No recurrences were found after surgical removal in the current series.

Adolescent↗
PubMed1997

SELEX and the evolution of genomes.

The interrupted genome structures of complex multicellular organisms have most likely changed the evolution of the regulation of metabolism and development. Wasted intron sequences make regulation of gene expression in (for example) mammals appear to be unnecessarily complicated. The recent discoveries that globular RNA molecules are very much like the antigen-combining sites of antibodies suggest that intronic RNA may be used to help solve the problems raised by this complexity.

Evolution, Molecular↗
PubMed1997

Post-SELEX combinatorial optimization of aptamers.

In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.

Base Sequence↗
PubMed1997

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.

Base Sequence↗
PubMed1996

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.

Anti-HIV Agents↗
PubMed1996

A specific RNA structural motif mediates high affinity binding by the HIV-1 nucleocapsid protein (NCp7).

Current research indicates that the nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) interacts with a variety of RNA substrates during the progression of the viral life cycle. The RNA features specifically recognized by the protein, however, have yet to be identified. SELEX was used to generate a set of RNAs whose affinities for nucleocapsid were on the order of 2 x 10(-9) M. Comparative analysis revealed that each RNA contains a highly conserved fourteen nucleotide sequence-block. Computer modeling and structure probing experiments indicate that the RNA ligands use the consensus sequence to fold into hairpins with an identical asymmetric bulge. The presence of the nucleocapsid protein protects the asymmetric bulge from ribonuclease attack, suggesting that it is the key element in protein recognition. A search for similar structural motifs within the HIV genome reveals several potential interaction sites for the nucleocapsid protein.

Base Sequence↗
PubMed1996

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.

Base Sequence↗
PubMed1996

Viral persistence in neurons alters synaptic plasticity and cognitive functions without destruction of brain cells.

Neurons have a restricted expression of MHC heavy chain molecules which prevents presentation of antigens of infecting viruses. As a result, such infected cells escape immune surveillance and allow the establishment of noncytolytic persistent infection. Here we show that a chronic noncytolytic viral infection both in vitro and in vivo selectively perturbed the expression of GAP-43, a protein that plays a central role in neuronal plasticity processes accompanying learning and memory. GAP-43 expression was greatly decreased in the hippocampus, an area of heightened viral replication, while synaptic density was preserved. Concurrently, the ability to learn tasks was significantly impaired in these persistently infected mice. Yet, infected neurons remained free from structural injury.

Animals↗
PubMed1996

Inhibitory RNA ligand to reverse transcriptase from feline immunodeficiency virus.

High-affinity, high-specificity RNA ligands for reverse transcriptase from feline immunodeficiency virus (FIV) were isolated from an RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. The selected RNA ligands bound to FIV reverse transcriptase with dissociation constants in the nanomolar range. One of the ligands was a potent inhibitor of the RNA-dependent DNA polymerase activity of both the recombinant and the virion-derived FIV reverse transcriptase. It also inhibited the reverse transcriptase from an FIV mutant that is resistant to 3'-azido-3'-deoxythymidine (AZT). The inhibition of FIV reverse transcriptase was competitive with respect to template-primer and noncompetitive with respect to deoxyribonucleoside 5'-triphosphates. This ligand was specific for the FIV enzyme and did not inhibit other reverse transcriptases tested (avian myeloblastosis virus, Moloney murine leukemia virus, and human immunodeficiency virus type 1).

Animals↗
PubMed1996

In vitro selection of RNA specifically cleaved by bacteriophage T4 RegB endonuclease.

T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4 messages, rendering them nonfunctional. Not all -GGAG- sites are processed equally by RegB; those found at the Shine-Dalgarno sequences and in intercistronic regions are processed with higher efficiency than the -GGAG- sites located in coding regions. The low activity of RegB observed in vitro is enhanced by 1-2 orders of magnitude by the Escherichia coli ribosomal protein S1. We have used SELEX (systematic evolution of ligands by exponential enrichment) on a combinatorial RNA library to obtain molecules that are specifically cleaved by T4 RegB endonuclease in the presence of S1. The sequences obtained contain the required -GGAG- tetranucleotide and were unusually enriched in adenosine and cytosine nucleotides. No consensus structure or sequence motif other than -GGAG- was conserved among the selected molecules. The majority of the RNAs are entirely dependent on S1 for RegB-catalyzed cleavage; however, a few RNAs are found to be S1 independent but are cleaved by RegB with much lower rates.

Bacteriophage T4↗
PubMed1996

In vitro evolution of the DNA binding sites of Escherichia coli methionine repressor, MetJ.

The SELEX procedure was used to study the recognition between the E. coli methionine repressor (MetJ) and its DNA binding sites. DNA ligands with high affinity for either the holo-repressor or apo-repressor were isolated from a pool of molecules randomized over 20 base-pairs. Among 90 DNA ligands selected by holo-repressor binding, roughly 90% contain variations of two tandem, perfect eight base-pair Met-boxes, which are the consensus deduced from natural met operators. Base-pairs that are important, for specific interactions with the protein are highly conserved. The data also reveal the importance of the non-contacted operator base-pairs in facilitating the conformational changes in the operator which must occur for repressor binding. There are also effects due to the sequences of the base-pairs immediately flanking the operator site. DNA ligands selected by apo-repressor share a very similar, but not identical, consensus with that selected by holo-repressor, suggesting that the corepressor does not greatly alter the specificity of repressor binding.

Bacterial Proteins↗