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J N Vournakis

Publications and source records attributed to J N Vournakis.

At least 37 records · Page 2Linked to original sources
PubMed1983

Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.

A direct approach to determining the sequence specificities of equilibrium binding drugs by using the DNase protection technique is described. The method utilizes singly end-labeled restriction fragments and partial digestion of the drug fragment complex with DNase I. Microdensitometry of autoradiograms produced after electrophoretic separation of digestion products allows determination of sequences that are affected by drug binding. The feasibility of the technique for locating small ligands bound to DNA and its eventual use as a quantitative thermodynamic approach to studying ligand binding to heterogeneous DNA as a function of sequence is illustrated by using actinomycin D and Netropsin.

Animals↗
PubMed1982

Secondary structure of Bombyx mori and Dictyostelium discoideum 5S rRNA from S1 nuclease and cobra venom ribonuclease susceptibility, and computer assisted analysis.

The 5S rRNAs from Bombyx mori and Dictyostelium discoideum were end-labeled with [32-P] at either the 5' or 3' end and sequenced using enzymatic digestion. The secondary structure of these molecules was studied using the single-strand specific S1 nuclease and the base-pair specific cobra venom ribonuclease. Computer analysis of these results was performed and was used to generate a consensus secondary structure for each molecule. A comparison of these results with those of other workers is presented.

Animals↗
PubMed1982

Computer-aided prediction of RNA secondary structures.

A brief survey of computer algorithms that have been developed to generate predictions of the secondary structures of RNA molecules is presented. Two particular methods are described in some detail. The first utilizes a thermodynamic energy minimization algorithm that takes into account the likelihood that short-range folding tends to be favored over long-range interactions. The second utilizes an interactive computer graphic modelling algorithm that enables the user to consider thermodynamic criteria as well as structural data obtained by nuclease susceptibility, chemical reactivity and phylogenetic studies. Examples of structures for prokaryotic 16S and 23S ribosomal RNAs, several eukaryotic 5S ribosomal RNAs and rabbit beta-globin messenger RNA are presented as case studies in order to describe the two techniques. Anm argument is made for integrating the two approaches presented in this paper, enabling the user to generate proposed structures using thermodynamic criteria, allowing interactive refinement of these structures through the application of experimentally derived data.

Animals↗
PubMed1981

Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures.

In recent years, there has been a growing appreciation of the potential applications of 5'-32P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity [gamma-32P]ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure.

Adenosine Triphosphate↗
PubMed1980

Secondary structure of mouse and rabbit alpha- and beta-globin mRNAs: differential accessibility of alpha and beta initiator AUG codons towards nucleases.

The nucleotide sequence from the 5' terminus inward of one third of mouse alpha- and beta maj-globin messenger RNAs has been established. In addition, using 5' 32P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit alpha- and beta-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit beta-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the alpha-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with beta-globin mRNA in rabbit reticulocyte is 30--40% faster than for alpha-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.

Animals↗
PubMed1979

Sequence and secondary structure of Drosophila melanogaster 5.8S and 2S rRNAs and of the processing site between them.

Drosophila melanogaster 5.8S and 2S rRNAs were end-labeled with 32p at either the 5' or 3' end and were sequenced. 5.8S rRNA is 123 nucleotides long and homologous to the 5' part of sequenced 5.8S molecules from other species. 2S rRNA is 30 nucleotides long and homologous to the 3' part of other 5.8S molecules. The 3' end of the 5.8S molecule is able to base-pair with the 5' end of the 2S rRNA to generate a helical region equivalent in position to the "GC-rich hairpin" found in all previously sequenced 5.8S molecules. Probing the structure of the labeled Drosophila 5.8S molecule with S1 nuclease in solution verifies its similarity to other 5.8S rRNAs. The 2S rRNA is shown to form a stable complex with both 5.8S and 26S rRNAs separately and together. 5.8S rRNA can also form either binary or ternary complexes with 2S and 26S rRNA. It is concluded that the 5.8S rRNA in Drosophila melanogaster is very similar both in sequence and structure to other 5.8 rRNAs but is split into two pieces, the 2S rRNA being the 3' part. 2S anchors the 5.8S and 26S rRNA. The order of the rRNA coding regions in the ribosomal DNA repeating unit is shown to be 18S - 5.8S - 2S - 26S. Direct sequencing of ribosomal DNA shows that the 5.8S and 2S regions are separated by a 28 nucleotide spacer which is A-T rich and is presumably removed by a specific processing event. A secondary structure model is proposed for the 26S-5.8S ternary complex and for the presumptive precursor molecule.

Animals↗
PubMed1977

The effect of magnesium and manganese ions on the structure and template activity for reverse transcriptase of polyribocytidylate and its 2'-0-methyl derivative.

The secondary structure of the hydrogen bonded hybrids polycytidylate-oligodeoxguanylate (poly(rC)-(dG)12-18 and poly (2'-oMe) cytidylate-oligodeoxyguanylate (poly (rCm)-(dG)12-18 was studied at several magnesium and manganese ion concentrations. These hybrids are effective template-primer complexes for the synthesis of poly(dG) by avian myeloblastosis virus (AMV) DNA polymerase under disparate ionic conditions. Circular dichroism spectra and thermal melting data were obtained as a function of ion concentration, including conditions that allow optimum rates of poly (dG) synthesis by each complex. These studies demonstrate that both hybrids can change conformation and stability depending on their ionic environment. Comparison of enzyme activity and physical data suggest that the polymerase recognizes particular secondary structure features. Changes in the activity of the AMV polymerase can be induced by varying the Mg++ and Mn++ concentrations alone and in combination. These variations in enzyme activity are correlated with observed changes in the base-stacking alignment of the synthetic template primers. The ions, therefore, seem to affect enzyme activity by altering the conformation of the polnucleotide complexes.

Avian Myeloblastosis Virus↗
PubMed1977

End labeling of enzymatically decapped mRNA.

A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.

Adenosine Triphosphatases↗
PubMed1977

Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease.

S1 nuclease isolated from Aspergillus oryzae has been used to investigate the secondary structure of rabbit globin messenger RNA (mRNA). The enzyme, which is specific for single stranded nucleotides, digests globin mRNA to a limited extent, with 65-75% of the mRNA nucleotides resistant to digestion under mild conditions. This limited digestion is not due to enzyme inactivation, but rather to the normal activity of the single-strand nuclease. The reaction was studied as a function of temperature, salt and enzyme concentration. Analysis of the products of digestion on 20% acrylamide- 7M urea slab gels reveals a stable pattern of unique fragments ranging in size from 9 to 71 nucleotides. Separated alpha and beta globin mRNAs show similar, but not identical gel patterns, indicating strong structural similarities between the two species. The high degree of nuclease resistance, along with the fragment patterns seen on polyacrylamide gels, gives evidence to support a model of rabbit globin mRNA which contain specific, rather than random, helical structure.

Amylases↗
PubMed1975

Full length and discrete partial reverse transcripts of globin and chorion mRNAs.

Rabbit globin mRNA was copied by AMV reverse transcriptase in the presence of various concentrations of deoxyribonucleotides (dNTPs). The cDNAs were analyzed by electrophoresis under denaturing conditions in formamide-polyacrylamide gels. Discrete size products were detected, ranging from 65 to 650 nucleotides-that is, up to the full length of the mRNA template. Increasing the concentrations of all four dNTPs stimulated formation of full-length transcripts and made the incomplete copies less abundant. Hybridization and nuclease digestion experiments indicated that the full-size product is indeed a complete transcript of globin mRNA. Similar results were obtained with chorion mRNAs. The possible usefulness of the discrete partial transcripts is discussed.

Animals↗
PubMed1975

Electrophoretic patterns of deadenylylated chorion and globin mRNAs.

Treatment of silkmoth chorion mRNAs with calf thymus RNase H (EC 3.1.4.34; RNA-DNA-hybrid ribonucleotidohydrolase) in the presence of oligo(dT) specifically and effectively removes the 3'-terminal poly(A) sequences. Excision of non-poly(A) fragments cannot be detected. Under these conditions, RNase H leads to increased electrophoretic homogeneity of rabbit globin mRNA, presumably as a result of removal of poly(A) sequences that are inherently variable in length. Treatment with RNase H converts the three diffuse zones of messages for the several chorion proteins into multiple sharp bands.

Adenosine Monophosphate↗