Automated film reader for DNA sequencing based on homomorphic deconvolution

Journal ArticleAn automated reader for electrophoresis based DNA sequencing methods is described that provides fast and accurate sequence determination. Digitized sequencing lanes are processed with homomorphic blind deconvolution in preparation for peak detection, interlane alignment, peak refinement and base calling. Initial reads from direct blot sequencing films have error rates of about 1% at the rate of 5 nucleotides/s. Typical read lengths are 500-600 nucleotides. The described reader is a significant improvement over existing readers and could be an essential component in the sequencing efforts of the Human Genome Project

Drosophila gene for antizyme requires ribosomal frameshifting for expression and contains an intronic gene for snRNP Sm D3 on the opposite strand

Journal ArticlePreviously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5* open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the 11 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5* ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype

Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides

Journal ArticleEvidence is presented that morpholino, 2_x0001_-O-methyl, phosphorothioate, and RNA antisense oligonucleotides can direct sitespecific âˆ'1 translational frameshifting when annealed to mRNA downstream from sequences where the P- and A-site tRNAs are both capable of re-pairing with âˆ'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 âˆ'1 frameshifting. The implications for the mechanism of action of natural programmed translational frameshift stimulators are discussed

Dual-luciferase reporter system for studying recoding signals

Journal ArticleA new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme

Effects of 3'-terminal phosphates in RNA produced by ribozyme cleavage

Journal ArticleDuring the course of a structural investigation of a selenocysteine insertion element from rat Type 1 iodothyronine 59-deiodinase (D1 SECIS), we wished to prepare milligram quantities of an RNA that comprised the upper stem/loop of this RNA structure (41 nt; Fig+ 1A)+ This stem/loop is a stimulator required for the insertion of selenocysteins at a specific internal UGA stop codon (Berry et al+, 1991)+ Construction of the upper stem/ loop RNA required that the base of the stem end with four noncanonical base pairs (Walczak et al+, 1996), with no additional base pairing below this terminal quartet, as determined from in vitro assays (Martin et al+, 1998)+ This RNA has been produced synthetically for crystallization trials, so we sought a thorough comparison between this molecule and the RNA produced by run-off transcription+ Because the RNA to be studied begins with an adenosine and ends in a uridine, a ribozyme was used at each end to produce homogeneous RNA+ The system used to accomplish this has been described previously (Ferre-D™Amare & Doudna, 1996) and the resulting T7-produced RNA would differ from the synthetic RNA only by the 39- cyclic phosphate+ In making preliminary imino proton NMR assignments for both of these RNAs, we concluded that the base pairing for both RNAs is nearly identical, as evident from the one-dimensional NMR spectra collected in 90% H2O (Fig+ 2)+ Close inspection shows that there are subtle differences near the RNA helix en

P-site tRNA is a crucial initiator of ribosomal frameshifting

Journal ArticleThe 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

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

Journal ArticleABSTRACT: 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

Upstream stimulators for recoding

Journal ArticleRecent progress in elucidation of 5' stimulatory elements for translational recoding is reviewed. A 5' Shine-Dalgarno sequence increases both +l and ? I frameshift efficiency in several genes; examples cited include the E. coli prfB gene encoding release factor 2 and the thuiX gene encoding the y and t subunits of DNA polymerase III holoenzyme. The spacing between the Shine-Dalgarno sequence and the shift site is critical in both the +l and ? 1 frameshift cassettes; however, the optimal spacing is quite different in the two cases. A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5' sequences have been shown to be vital for this frameshift event. Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase. The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation. A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur

Diverse bacterial genomes encode an operon of two genes, one of which is an unusual class-I release factor that potentially recognizes atypical mRNA signals other than normal stop codons

Journal ArticleABSTRACT: Background: While all codons that specify amino acids are universally recognized by tRNA molecules, codons signaling termination of translation are recognized by proteins known as class-I release factors (RF). In most eukaryotes and archaea a single RF accomplishes termination at all three stop codons. In most bacteria, there are two RFs with overlapping specificity, RF1 recognizes UA(A/G) and RF2 recognizes U(A/G)A. The hypothesis: First, we hypothesize that orthologues of the E. coli K12 pseudogene prfH encode a third class-I RF that we designate RFH. Second, it is likely that RFH responds to signals other than conventional stop codons. Supporting evidence comes from the following facts: (i) A number of bacterial genomes contain prfH orthologues with no discernable interruptions in their ORFs. (ii) RFH shares strong sequence similarity with other class-I bacterial RFs. (iii) RFH contains a highly conserved GGQ motif associated with peptidyl hydrolysis activity (iv) residues located in the areas supposedly interacting with mRNA and the ribosomal decoding center are highly conserved in RFH, but different from other RFs. RFH lacks the functional, but non-essential domain 1. Yet, RFH-encoding genes are invariably accompanied by a highly conserved gene of unknown function, which is absent in genomes that lack a gene for RFH. The accompanying gene is always located upstream of the RFH gene and with the same orientation. The proximity of the 3' end of the former with the 5' end of the RFH gene makes it likely that their expression is co-regulated via translational coupling. In summary, RFH has the characteristics expected for a class-I RF, but likely with different specificity than RF1 and RF2. Testing the hypothesis: The most puzzling question is which signals RFH recognizes to trigger its release function. Genetic swapping of RFH mRNA recognition components with its RF1 or RF2 counterparts may reveal the nature of RFH signals. Implications of the hypothesis: The hypothesis implies a greater versatility of release-factor like activity in the ribosomal Asite than previously appreciated. A closer study of RFH may provide insight into the evolution of the genetic code and of the translational machinery responsible for termination of translation

Uninterrupted translation through putative 12-nucleotide coding gap in sequence of carA: business as usual

Journal ArticlePrevious work of others reported an untranslated stretch of 12 nucleotides in the 5' coding sequence of carA from Pseudomonas aeruginosa. However, N-terminal protein sequencing of carA-lacZ translational fusions shows that these 12 nucleotides are normally translated in a continuous triplet manner, both in P. aeruginosa and in Escherichia coli