Biomedical subjects

J A McCloskey

Publications and source records attributed to J A McCloskey.

At least 19 recordsLinked to original sources

PubMed1999

Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.

Animals↗

PubMed1999

Selective detection of ribose-methylated nucleotides in RNA by a mass spectrometry-based method.

Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification. Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are required for subunit assembly. Experimental difficulties in the mapping of ribose methylations increase with RNA molecular size and the complexity of mixtures resulting from nuclease digestion. A new and relatively rapid approach based on tandem mass spectrometry is described in which any of four ion reaction pathways occurring in the mass spectrometer can be monitored which are highly specific for the presence of 2'-O -methylribose residues. These pathways emanate from further dissociation of ribose-methylated mononucleotide (Nmp) ions formed in the electrospray ionization region of the mass spectrometer to then form the base, methylribose phosphate or PO(3)(-)anions. The mass spectrometer can be set for detection of generic ribose methylation (Nm) in oligonucleotides, selectively for each of the common methylated nucleo-sides Cm, Gm, Am or Um or for specific cases in which the base or sugar is further modified. By direct combination of mass spectrometry with liquid chromatography the method can be applied to analysis of complex mixtures of oligonucleotides, as for instance from synthetic or in vitro reaction mixtures or from nuclease digests of RNA. An example is given in which the single ribose-methylated nucleoside in Escherichia coli 16S rRNA (1542 nt), N(4),O-2'-dimethylcytidine, is detected in 25 pmol of a RNase T1 digest and localized to the fragment 1402-CCCGp-1405 in a single 45 min analysis.

Escherichia coli↗

PubMed1999

Determination of nearest neighbors in nucleic acids by mass spectrometry.

The identification of nearest-neighbor residues in nucleic acids provides useful constraints on establishment of base composition and sequence and is potentially applicable to a range of structural problems involving synthetic and natural polynucleotides. A new approach to this problem using electrospray ionization tandem mass spectrometry is based on measurement of precursor-product relationships derived from small fragment ions produced in the high-pressure ionization ("nozzle-skimmer") region of the instrument. Measured mass values of dinucleotide or other fragments, which give rise to mononucleotide ions N formed in the collision cell and transmitted by the second mass analyzer, establish the identities of residues adjacent to N. The technique is applicable to RNA and DNA, whether modified, or not, and is demonstrated using modified residues in nucleic acids up to the size of intact tRNA (76-mer). By monitoring of selected ion reaction channels, the method has been extended to LC/MS and to nearest-neighbor determinations directly in oligonucleotide mixtures.

DNA↗

PubMed1999

The RNA Modification Database: 1999 update.

The RNA Modification Database (http://medlib.med.utah.edu/RNAmods/) provides a comprehensive listing of naturally modified nucleosides in RNA. Each file includes: chemical structure; common name and symbol; type(s) of RNA in which found and corresponding phylogenetic distribution; Chemical s registry number and index name; and initial literature citations for structure characterization and chemical synthesis. New features include capability to search database files by name or substructural features, modifications in tmRNA, and links to related data and sites.

Archaea↗

PubMed1998

Reduced misreading of asparagine codons by Escherichia coli tRNALys with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position.

It has been suggested that modified nucleosides of the xm5(s2)U(m)34-type restrict the wobble capacity of the base, and that their function is to prevent misreading in the third position of the codon in mixed codon family boxes that encode two different amino acids. In this study in Escherichia coli, the misreading in vivo of asparagine codons in bacteriophage MS2 mRNA by different hypomodified derivatives of tRNALys, normally containing 5-methylaminomethyl-2-thiouridine (mnm5s2U34) in the wobble position, has been analysed. Contrary to what would be predicted from the general hypothesis for the function of mnm5s2U, it was found that the misreading of asparagine codons by tRNALys was greatly reduced in the mnmA (formerly asuE or trmU) and mnmE (formerly trmE) mutants which contain the hypomodified mnm5U34 and s2U34, respectively, instead of the fully modified mnm5s2U34. In addition, it was found that these hypomodified tRNAs were efficiently charged with lysine in vivo, under the growth conditions employed. The latter result is at variance with results obtained in vitro. The results are discussed in relation to the postulated function for modified nucleosides of the xm5s2U type.

Asparagine↗

PubMed1998

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Base Composition↗

PubMed1998

A novel wobble rule found in starfish mitochondria. Presence of 7-methylguanosine at the anticodon wobble position expands decoding capability of tRNA.

In the starfish mitochondrial (mt) genome, codons AGA and AGG (in addition to AGU and AGC) have been considered to be translated as serine. There is, however, only a single candidate mt tRNA gene responsible for translating these codons and it has a GCT anticodon sequence, but guanosine at the first position of the anticodon should base pair only with pyrimidines according to the conventional wobble rule. To solve this enigma, the mt tRNA GCUser was purified, and sequence determination in combination with electrospray liquid chromatography/mass spectrometry revealed that 7-methylguanosine is located at the first position of the anticodon. This is the first case in which a tRNA has been found to have 7-methylguanosine at the wobble position. It is suggested that methylation at N-7 of wobbling guanosine endows the tRNA with the capability of forming base pairs with all four nucleotides, A, U, G, and C, and expands the repertoire of codon-anticodon interaction. This finding indicates that a nonuniversal genetic code in starfish has been generated by base modification in the tRNA anticodon.

Animals↗

PubMed1998

The RNA modification database--1998.

The RNA modification database provides a comprehensive listing of posttranscriptionally modified nucleosides from RNA, and is maintained as an updated version of the initial printed report [Limbach,P.A., Crain,P.F. and McCloskey,J.A. (1994) Nucleic Acids Res. , 22, 2183-2196]. Information provided for each nucleoside includes: the type of RNA in which it occurs and phylogenetic distribution; common chemical name and symbol; Chemical Abstracts registry number and index name; chemical structure; initial literature citations for structural characterization or occurrence, and for chemical synthesis. The data are available through the World Wide Web at: http://www-medlib.med.utah/RNAmods/RNAmods .html

Computer Communication Networks↗

PubMed1998

Applications of mass spectrometry to the characterization of oligonucleotides and nucleic acids.

Mass spectrometry-based techniques continue to undergo active development for applications to nucleic acids, fueled by methods based on electrospray and matrix-assisted laser desorption ionization. In the past two years, notable advances have occurred in multiple interrelated areas, including sequencing techniques for oligonucleotides, approaches to mixture analysis, microscale sample handling and targeted DNA assays, and improvements in instrumentation for greater sensitivity and mass resolution.

Chromatography, Liquid↗

PubMed1998

Posttranscriptional modifications in 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfataricus.

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfactaricus by using combined chromatography-mass spectrometry-based methods. Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75 degrees C, compared with 11 and 23 sites, respectively, in Escherichia coli. We structurally characterized 10 different modified nucleosides in 16S rRNA, 64% (24 residues) of which are methylated at O-2' of ribose, and 8 modified species in 23S rRNA, 86% (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain. From cultures grown at progressively higher temperatures, 60, 75, and 83 degrees C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23 degrees C range shown for 2'-O-methyladenosine in 16S rRNA (21% increase) and for 2'-O-methylcytidine (24%) and 2'-O-methylguanosine (22%) in 23S rRNA. These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment.

Alkylation↗

PubMed1997

Biosynthesis of archaeosine, a novel derivative of 7-deazaguanosine specific to archaeal tRNA, proceeds via a pathway involving base replacement on the tRNA polynucleotide chain.

Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15. We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity. The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis. The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H. volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone. Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H. volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H. volcanii cells. Thus, this novel TGT in H. volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.

Amino Acid Sequence↗

PubMed1997

The RNA modification database.

The RNA modification database provides a comprehensive listing of posttranscriptionally modified nucleosides from all RNAs, and is maintained as an updated version of the initial printed report [Limbach,P.A., Crain,P.F. and McCloskey,J.A. (1994)Nucleic Acids Res. , 22, 2183-2196]. Information provided for each nucleoside includes: the RNA in which it occurs and phylogenetic distribution; common chemical name and symbol; Chemical Abstracts registry number and index name; chemical structure; initial literature citations for structural characterization or occurrence, and for chemical synthesis. The data are available through the WWW and via anonymous ftp.

Databases, Factual↗

PubMed1997

Collision-induced dissociation of polyprotonated oligonucleotides produced by electrospray ionization.

The efficient production of polyprotonated oligonucleotides, studied at n < or = 19, occurs from water/propan-2-ol solutions over an ammonium acetate concentration range between 2.5 and 40 mm and a pH range from 5 to 11. Average charge-state levels observed were approximately half of those found in mass spectra of polyanionic oligonucleotides, reflecting differences in sites of ionization: heterocyclic bases for protonation and phosphodiester backbone for deprotonation. Collision-induced dissociation mass spectra show three principal reaction paths: (1) release of protonated bases, with abundances dictated largely by base proton affinity; (2) phosphodiester chain cleavage at C3'-O3' indicative of sequence in the 3'-->5' direction; and (3) chain cleavage concomitant with base loss giving furan-type ions indicative of sequence in the 5'-->3' direction, analogous to reactions of polyanionic oligonucleotides. Thymine residues undergo very little protonation, resulting in characteristic absence of phosphodiester cleavage on the 3' side of T sites, producing mass-ladder gaps representing dinucleotides.

Acetates↗

PubMed1997

Posttranscriptional modification of tRNA in psychrophilic bacteria.

Posttranscriptional modification in tRNA is known to play a multiplicity of functional roles, including maintenance of tertiary structure and cellular adaptation to environmental factors such as temperature. Nucleoside modification has been studied in unfractionated tRNA from three psychrophilic bacteria (ANT-300 and Vibrio sp. strains 5710 and 29-6) and one psychrotrophic bacterium (Lactobacillus bavaricus). Based on analysis of total enzymatic hydrolysates by liquid chromatography-mass spectrometry, unprecedented low amounts of modification were found in the psychrophiles, particularly from the standpoint of structural diversity of modifications observed. Thirteen to 15 different forms of posttranscriptional modification were found in the psychrophiles, and 10 were found in L. bavaricus, compared with approximately 29 known to occur in bacterial mesophiles and 24 to 31 known to occur in the archaeal hyperthermophiles. The four most abundant modified nucleosides in tRNA from each organism were dihydrouridine, pseudouridine, 7-methylguanosine, and 5-methyluridine. The molar abundances of the latter three nucleosides were comparable to those found in tRNA from Escherichia coli. By contrast, the high levels of dihydrouridine observed in all three psychrophiles are unprecedented for any organism in any of the three phylogenetic domains. tRNA from these organisms contains 40 to 70% more dihydrouridine, on average, than that of the mesophile E. coli or the psychrotroph L. bavaricus. This finding supports the concept that a functional role for dihydrouridine is in maintenance of conformational flexibility of RNA, especially important to organisms growing under conditions where the dynamics of thermal motion are severely compromised. This is in contrast to the role of modifications contained in RNA from thermophiles, which is to reduce regional RNA flexibility and provide structural stability to RNA for adaptation to high temperature.

Base Composition↗

PubMed1996

Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.

Chromatography, High Pressure Liquid↗

PubMed1996

Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry.

Procedures are described for interpretation of mass spectra from collision-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing of oligonucleotides of completely unknown structure at approximately the 15-mer level and below, from DNA or RNA. Identification of sequence-relevant ions that are produced from extensive fragmentation in the quadrupole collision cell are based primarily on (1) recognition of 3'- and 5'- terminal residues as initial steps in mass ladder propagation, (2) alignment of overlapping nucleotide chains that have been constructed independently from each terminus, and (3) use of experimentally measured molecular mass in rejection of incorrect sequence candidates. Algorithms for sequence derivation are embodied in a computer program that requires < 2s for execution. The interpretation procedures are demonstrated for sequence location of simple forms of modification in the base and sugar. The potential for direct sequencing of components of mixtures is shown using an unresolved fraction of unknown oligonucleotides from ribosomal RNA.

Algorithms↗

PubMed1996

Conformational flexibility in RNA: the role of dihydrouridine.

In order to further understand the structural role of the modified nucleoside dihydrouridine in RNA the solution conformations of Dp and ApDpA were analyzed by one- and two-dimensional proton NRM spectroscopy and compared with those of the related uridine-containing compounds. The analyses indicate that dihydrouridine significantly destabilizes the C3'-endo sugar conformation associated with base stacked, ordered, A-type helical RNA. Equilibrium constants (Keq = [C2'-endo]/[C3'-endo]) for C2'-endo-C3'-endo interconversion at 25 degrees C for Dp, the 5'-terminal A of ApDpA and D in ApDpA are 2.08, 1.35 and 10.8 respectively. Stabilization of the C2'-endo form was shown to be enhanced at low temperature, indicating that C2'-endo is the thermodynamically favored conformation for dihydrouridine. DeltaH values show that for Dp the C2'-endo sugar conformation is stabilized by 1.5 kcal/mol compared with Up. This effect is amplified for D in the oligonucleotide ApDpA and propagated to the 5'-neighboring A, with stabilization of the C2'-endo form by 5.3 kcal/mol for D and 3.6 kcal/mol for the 5'-terminal A. Post-transcriptional formation of dihydrouridine therefore represents a biological strategy opposite in effect to ribose methylation, 2-thiolation or pseudouridylation, all of which enhance regional stability through stabilization of the C3'-endo conformer. Dihydrouridine effectively promotes the C2'-endo sugar conformation, allowing for greater conformational flexibility and dynamic motion in regions of RNA where tertiary interactions and loop formation must be simultaneously accommodated.

Drug Stability↗

PubMed1996

Structural characterization of U*-1915 in domain IV from Escherichia coli 23S ribosomal RNA as 3-methylpseudouridine.

Mass spectrometry-based methods have been used to study post-transcriptional modification in the 1900-1974 nt segment of domain IV in 23S rRNA of Escherichia coli, a region which interacts with domain V in forming the three- dimensional structure of the peptidyl transferase center within the ribosome. A nucleoside constituent of M r 258 (U*)which occurs at position 1915, within the highly modified oligonucleotide sequence 1911-psiAACU*Apsi-1917, was characterized as 3-methylpseudouridine (m3psi). The assignment was confirmed by chemical synthesis of m3psi and comparison with the natural nucleoside by liquid chromatography-mass spectrometry. 3-Methylpseudouridine is previously unknown in nature and is the only known derivative of the common modified nucleoside pseudouridine thus far found in bacterial rRNA.

Base Sequence↗