PubMed Health⌕ Search

Biomedical subjects

John F Atkins

Publications and source records attributed to John F Atkins.

At least 37 records · Page 2Linked to original sources
PubMed2005

Transcriptional slippage in bacteria: distribution in sequenced genomes and utilization in IS element gene expression.

BACKGROUND: Transcription slippage occurs on certain patterns of repeat mononucleotides, resulting in synthesis of a heterogeneous population of mRNAs. Individual mRNA molecules within this population differ in the number of nucleotides they contain that are not specified by the template. When transcriptional slippage occurs in a coding sequence, translation of the resulting mRNAs yields more than one protein product. Except where the products of the resulting mRNAs have distinct functions, transcription slippage occurring in a coding region is expected to be disadvantageous. This probably leads to selection against most slippage-prone sequences in coding regions. RESULTS: To find a length at which such selection is evident, we analyzed the distribution of repetitive runs of A and T of different lengths in 108 bacterial genomes. This length varies significantly among different bacteria, but in a large proportion of available genomes corresponds to nine nucleotides. Comparative sequence analysis of these genomes was used to identify occurrences of 9A and 9T transcriptional slippage-prone sequences used for gene expression. CONCLUSIONS: IS element genes are the largest group found to exploit this phenomenon. A number of genes with disrupted open reading frames (ORFs) have slippage-prone sequences at which transcriptional slippage would result in uninterrupted ORF restoration at the mRNA level. The ability of such genes to encode functional full-length protein products brings into question their annotation as pseudogenes and in these cases is pertinent to the significance of the term 'authentic frameshift' frequently assigned to such genes.

Adenosine↗
PubMed2005

P-site pairing subtleties revealed by the effects of different tRNAs on programmed translational bypassing where anticodon re-pairing to mRNA is separated from dissociation.

Programmed ribosomal bypassing occurs in decoding phage T4 gene 60 mRNA. Half the ribosomes bypass a 50 nucleotide gap between codons 46 and 47. Peptidyl-tRNA dissociates from the "take-off" GGA, codon 46, and re-pairs to mRNA at a matched GGA "landing site" codon directly 5' of codon 47 where translation resumes. The system described here allows the contribution of peptidyl-tRNA re-pairing to be measured independently of dissociation. The matched GGA codons have been replaced by 62 other matched codons, giving a wide range of bypassing efficiencies. Codons with G or C in either or both of the first two codon positions yielded high levels of bypassing. The results are compared with those from a complementary study of non-programmed bypassing, where the combined effects of peptidyl-tRNA dissociation and reassociation were measured. The wild-type, GGA, matched codons are the most efficient in their gene 60 context in contrast to the relatively low value in the non-programmed bypassing study.

Anticodon↗
PubMed2005

Expression levels influence ribosomal frameshifting at the tandem rare arginine codons AGG_AGG and AGA_AGA in Escherichia coli.

The rare codons AGG and AGA comprise 2% and 4%, respectively, of the arginine codons of Escherichia coli K-12, and their cognate tRNAs are sparse. At tandem occurrences of either rare codon, the paucity of cognate aminoacyl tRNAs for the second codon of the pair facilitates peptidyl-tRNA shifting to the +1 frame. However, AGG_AGG and AGA_AGA are not underrepresented and occur 4 and 42 times, respectively, in E. coli genes. Searches for corresponding occurrences in other bacteria provide no strong support for the functional utilization of frameshifting at these sequences. All sequences tested in their native context showed 1.5 to 11% frameshifting when expressed from multicopy plasmids. A cassette with one of these sequences singly integrated into the chromosome in stringent cells gave 0.9% frameshifting in contrast to two- to four-times-higher values obtained from multicopy plasmids in stringent cells and eight-times-higher values in relaxed cells. Thus, +1 frameshifting efficiency at AGG_AGG and AGA_AGA is influenced by the mRNA expression level. These tandem rare codons do not occur in highly expressed mRNAs.

Arginine↗
PubMed2004

Identification of a new antizyme mRNA +1 frameshifting stimulatory pseudoknot in a subset of diverse invertebrates and its apparent absence in intermediate species.

The expression of eukaryotic antizyme genes requires +1 translational frameshifting. The frameshift in decoding most vertebrate antizyme mRNAs is stimulated by an RNA pseudoknot 3' of the frameshift site. Although the frameshifting event itself is conserved in a wide variety of organisms from yeast to mammals, until recently no corresponding 3' RNA pseudoknot was known in invertebrate antizyme mRNAs. A pseudoknot, different in structure and origin from its vertebrate counterparts, is now shown to be encoded by the antizyme genes of distantly related invertebrates. Identification of the 3' frameshifting stimulator in intermediate species or other invertebrates remains unresolved.

Animals↗
PubMed2004

Identification of regions in multiple sequence alignments thermodynamically suitable for targeting by consensus oligonucleotides: application to HIV genome.

BACKGROUND: Computer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants. However, even for regions that are equally conserved, their potential utility as hybridization targets varies. Mismatches in sequence variants are more disruptive in some duplexes than in others. Additionally, the propensity for self-interactions amongst oligonucleotides targeting conserved regions differs and the structure of target regions themselves can also influence hybridization efficiency. There is a need to develop software that will employ thermodynamic selection criteria for finding optimal hybridization targets in related sequences. RESULTS: A new scheme and new software for optimal detection of oligonucleotide hybridization targets common to families of aligned sequences is suggested and applied to aligned sequence variants of the complete HIV-1 genome. The scheme employs sequential filtering procedures with experimentally determined thermodynamic cut off points: 1) creation of a consensus sequence of RNA or DNA from aligned sequence variants with specification of the lengths of fragments to be used as oligonucleotide targets in the analyses; 2) selection of DNA oligonucleotides that have pairing potential, greater than a defined threshold, with all variants of aligned RNA sequences; 3) elimination of DNA oligonucleotides that have self-pairing potentials for intra- and inter-molecular interactions greater than defined thresholds. This scheme has been applied to the HIV-1 genome with experimentally determined thermodynamic cut off points. Theoretically optimal RNA target regions for consensus oligonucleotides were found. They can be further used for improvement of oligo-probe based HIV detection techniques. CONCLUSIONS: A selection scheme with thermodynamic thresholds and software is presented in this study. The package can be used for any purpose where there is a need to design optimal consensus oligonucleotides capable of interacting efficiently with hybridization targets common to families of aligned RNA or DNA sequences. Our thermodynamic approach can be helpful in designing consensus oligonucleotides with consistently high affinity to target variants in evolutionary related genes or genomes.

Base Sequence↗
PubMed2004

Factors that influence selection of coding resumption sites in translational bypassing: minimal conventional peptidyl-tRNA:mRNA pairing can suffice.

This study investigates bypassing initiated from codons immediately 5' of a stop codon. The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3' codon. This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized. These results have implications for re-pairing in ribosomal frameshifting. Shine-Dalgarno sequences in the mRNA can alter the distribution of landing sites observed. The bacteriophage T4 gene 60 nascent peptide, known to influence take-off in its native context, imposes stringent P-site pairing requirements, thereby limiting the number of suitable landing sites.

Base Sequence↗
PubMed2004

Readthrough of dystrophin stop codon mutations induced by aminoglycosides.

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (< 2% stop codon readthrough); and two appear refractory to aminoglycoside treatment. Readthrough levels do not substantially vary between different sources of gentamicin, and, for this set of mutations, the efficiency of termination at the premature stop codon mutation does not appear to correlate with disease severity.

Aminoglycosides↗
PubMed2004

A cryptic lysis gene near the start of the Qbeta replicase gene in the +1 frame.

The maturation/lysis (A2) protein encoded by the group B single-stranded RNA bacteriophage Qbeta mediates lysis of host Escherichia coli cells. We found a frameshift mutation in the replicase (beta-subunit) gene of Qbeta cDNA causes cell lysis. The mutant has a single base deletion 73 nucleotides (nt) 3' from the start of the replicase gene with consequent translation termination at a stop codon 129-131 nt further 3'. The 43-amino acid C-terminal part of the 67-amino acid product encoded by what in WT (wild-type) is the +1 frame, is rich in basic amino acids This 67-aa protein can mediate cell lysis whose characteristics indicate that the protein may cause lysis by a different mechanism and via a different target, than that caused by the A2 maturation/lysis protein. Synthesis of a counterpart of the newly discovered lysis product in wild-type phage infection would require a hypothetical ribosomal frameshifting event. The lysis gene of group A RNA phages is also short, 75 codons in MS2, and partially overlaps the first part of their equivalently located replicase gene, raising significant evolutionary implications for the present finding.

Allolevivirus↗
PubMed2004

P-site tRNA is a crucial initiator of ribosomal frameshifting.

The expression of some genes requires a high proportion of ribosomes to shift at a specific site into one of the two alternative frames. This utilized frameshifting provides a unique tool for studying reading frame control. Peptidyl-tRNA slippage has been invoked to explain many cases of programmed frameshifting. The present work extends this to other cases. When the A-site is unoccupied, the P-site tRNA can be repositioned forward with respect to mRNA (although repositioning in the minus direction is also possible). A kinetic model is presented for the influence of both, the cognate tRNAs competing for overlapping codons in A-site, and the stabilities of P-site tRNA:mRNA complexes in the initial and new frames. When the A-site is occupied, the P-site tRNA can be repositioned backward. Whether frameshifting will happen depends on the ability of the A-site tRNA to subsequently be repositioned to maintain physical proximity of the tRNAs. This model offers an alternative explanation to previously published mechanisms of programmed frameshifting, such as out-of-frame tRNA binding, and a different perspective on simultaneous tandem tRNA slippage.

Animals↗
PubMed2004

Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides.

Evidence is presented that morpholino, 2'-O-methyl, phosphorothioate, and RNA antisense oligonucleotides can direct site-specific -1 translational frameshifting when annealed to mRNA downstream from sequences where the P- and A-site tRNAs are both capable of repairing with -1 frame codons. The efficiency of ribosomes shifting into the new frame can be as high as 40%, determined by the sequence of the frameshift site, as well as the location, sequence composition, and modification of the antisense oligonucleotide. These results demonstrate that a perfect duplex formed by complementary oligonucleotides is sufficient to induce high level -1 frameshifting. The implications for the mechanism of action of natural programmed translational frameshift stimulators are discussed.

Animals↗
PubMed2003

Sequences that direct significant levels of frameshifting are frequent in coding regions of Escherichia coli.

It is generally believed that significant ribosomal frameshifting during translation does not occur without a functional purpose. The distribution of two frameshift-prone sequences, A_AAA_AAG and CCC_TGA, in coding regions of Escherichia coli has been analyzed. Although a moderate level of selection against the first sequence is evident, 68 genes contain A_AAA_AAG and 19 contain CCC_TGA. The majority of those tested in their genomic context showed >1% frameshifting. Comparative sequence analysis was employed to assess a potential biological role for frameshifting in decoding these genes. Two new candidates, in pheL and ydaY, for utilized frameshifting have been identified in addition to those previously known in dnaX and nine insertion sequence elements. For the majority of the shift-prone sequences no functional role can be attributed to them, and the frameshifting is likely erroneous. However, none of frameshift sequences is in the 306 most highly expressed genes. The unexpected conclusion is that moderate frameshifting during expression of at least some other genes is not sufficiently harmful for cells to trigger strong negative evolutionary pressure.

Base Sequence↗
PubMed2003

Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs.

Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.

Base Sequence↗
PubMed2003

Deletion of selenoprotein P alters distribution of selenium in the mouse.

Selenoprotein P (Se-P) contains most of the selenium in plasma. Its function is not known. Mice with the Se-P gene deleted (Sepp(-/-)) were generated. Two phenotypes were observed: 1) Sepp(-/-) mice lost weight and developed poor motor coordination when fed diets with selenium below 0.1 mg/kg, and 2) male Sepp(-/-) mice had sharply reduced fertility. Weanling male Sepp(+/+), Sepp(+/-), and Sepp(-/-) mice were fed diets for 8 weeks containing <0.02-2 mg selenium/kg. Sepp(+/+) and Sepp(+/-) mice had similar selenium concentrations in all tissues except plasma where a gene-dose effect on Se-P was observed. Liver selenium was unaffected by Se-P deletion except that it increased when dietary selenium was below 0.1 mg/kg. Selenium in other tissues exhibited a continuum of responses to Se-P deletion. Testis selenium was depressed to 19% in mice fed an 0.1 mg selenium/kg diet and did not rise to Sepp(+/+) levels even with a dietary selenium of 2 mg/kg. Brain selenium was depressed to 43%, but feeding 2 mg selenium/kg diet raised it to Sepp(+/+) levels. Kidney was depressed to 76% and reached Sepp(+/+) levels on an 0.25 mg selenium/kg diet. Heart selenium was not affected. These results suggest that the Sepp(-/-) phenotypes were caused by low selenium in testis and brain. They strongly suggest that Se-P from liver provides selenium to several tissues, especially testis and brain. Further, they indicate that transport forms of selenium other than Se-P exist because selenium levels of all tissues except testis responded to increases of dietary selenium in Sepp(-/-) mice.

Animals↗
PubMed2003

RECODE 2003.

The RECODE database is a compilation of translational recoding events (programmed ribosomal frameshifting, codon redefinition and translational bypass). The database provides information about the genes utilizing these events for their expression, recoding sites, stimulatory sequences and other relevant information. The Database is freely available at http://recode.genetics.utah.edu/.

Animals↗
PubMed2003

Maintenance of the correct open reading frame by the ribosome.

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon-anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.

Anticodon↗
PubMed2003

Translational recoding signals between gag and pol in diverse LTR retrotransposons.

Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol. To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana. In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame. This was particularly true for the Ty1/copia group retroelements. Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting. Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame. None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame. Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames. These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism.

Base Sequence↗