Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes.

BACKGROUND: Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels. RESULTS: HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10-fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre-treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5-fold). In addition, the constructs with the 3' deletion were more responsive to stimulation by polyamine addition than those with the 5' deletion. CONCLUSIONS: The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell-based assay for the analysis of polyamines or their analogues on translational frameshifting.

Sequence specificity of aminoglycoside-induced stop condon readthrough: potential implications for treatment of Duchenne muscular dystrophy.

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside-induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside-induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside-induced readthrough in the order C > U > A > or = G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33-base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low-level aminoglycoside-induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed.

The traY gene product and integration host factor stimulate Escherichia coli DNA helicase I-catalyzed nicking at the F plasmid oriT.

F plasmid conjugative transfer is initiated by the introduction of a site- and strand-specific nick within the plasmid origin of transfer (oriT). Genetic studies have shown nick formation to be dependent on both the traI and traY genes. However, highly purified TraIp, the traI gene product, nicks oriT in a site- and strand-specific manner in the absence of the traY gene product (TraYp) in vitro (Matson, S.W., and Morton, B.S. (1991) J. Biol. Chem. 266, 16232-16237). Analysis of the oriT region has revealed binding sites for TraYp and the host protein integration host factor (IHF). To explore possible interactions occurring at oriT, highly purified TraIp, TraYp, and IHF were incubated with a supercoiled oriT-containing DNA substrate. A marked enhancement of the nicking reaction catalyzed by TraIp was observed in a reaction that required both TraYp and IHF. In addition, TraIp was able to nick a linear oriT-containing double-stranded DNA substrate when IHF and TraYp were present in the reaction; such a substrate is not nicked by TraIp alone. Individual protein concentration requirements for the supercoiled and linear nicking reactions were similar, and the reactions occurred at equal velocity, suggesting that they are biochemically identical. Concentrations of TraYp and IHF that yield half-maximal activity in the nicking assays compare well with the reported KD values for the IHF and TraYp binding sites in oriT. These data, coupled with data presented in the accompanying report, suggest that TraYp and IHF bind independent of one another, forming a nucleo-protein complex with oriT that can be recognized and nicked by TraIp.

Stepwise assembly of a relaxosome at the F plasmid origin of transfer.

A central step in the transfer of genetic information during bacterial conjugation of the Escherichia coli F plasmid involves the formation of a protein-DNA complex, called the relaxosome, at the origin of transfer. During conjugation, the relaxosome introduces a site- and strand-specific nick from which the physical transfer of a single strand of DNA is initiated. At least two F-encoded proteins, TraIp (traI gene product) and TraYp (traY gene product), and one host-encoded protein, integration host factor, are involved in this process. In this report, we use DNase I protection and electron microscopic techniques to investigate the mechanism of relaxosome formation. Our results show that TraYp and integration host factor form a protein-DNA complex that facilitates the binding of TraIp to assemble a relaxosome capable of introducing a site- and strand-specific nick at the origin of transfer. This nick is identical to that observed during conjugation.

Disruption of a topoisomerase-DNA cleavage complex by a DNA helicase.

The type II DNA topoisomerases are targets for a variety of chemotherapeutic agents, including the antibacterial quinolones and several families of antitumor drugs. These agents stabilize an enzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drug-stabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons.

A comparison of four PTSD measures' convergent validities in Vietnam veterans.

We compared the convergent validities of four commonly used post-traumatic stress disorder (PTSD) measures in 80 help-seeking Vietnam veterans by contrasting their intercorrelations. When scored as continuous severity or frequency measures, the Mississippi Scale for Combat-related PTSD's and the Post-Traumatic Stress Disorder Interview's (PTSD-I's) concordances with other measures were similar to one anothers' and generally larger than those of either the Diagnostic Interview Schedule (DIS) PTSD module or the MMPI PTSD scale. However, when used only to identify stress disorder's presence or absence, the four techniques' concordances were nearly identical. This suggested that the four measures have similar convergent validities when used simply to identify PTSD, but that the PTSD-I and Mississippi scale offer better convergent validity than the MMPI or DIS instruments when used as severity measures.

A cluster of strong topoisomerase II cleavage sites is located near an integrated human immunodeficiency virus.

The Human Immunodeficiency Virus (HIV) integrates into host cellular DNA as a double strand DNA molecule. Here a previously studied HIV isolate was examined for binding and cleavage by topoisomerase II in vitro within the 5' LTR region and human flanking DNA. A cluster of strong binding and cleavage sites in the human sequences was located approximately 850 bp upstream from the integration site. This region maps to a locus consisting of a complex repeating element, and alternating purine/pyrimidine sequences. Topoisomerase II binding and cleavage sites were also located within the HIV 5' LTR, in particular a site overlying the DNA sequence coding for TAR, another inverted repeat element in the DNA.

HMf, a histone-related protein from the hyperthermophilic archaeon Methanothermus fervidus, binds preferentially to DNA containing phased tracts of adenines.

HMf, a histone-related protein from Methanothermus fervidus, was found to bind preferentially to a DNA that is intrinsically bent as a result of the presence of phased oligo(dA) tracts. The intergenic regions in M. fervidus DNA are A+T rich and frequently contain oligo(dA) tracts, some of which may have the size and phasing required to create a net bending in one direction. The binding of HMf to bent DNA could play a direct role in gene expression and stabilization of the genome of this organism.

Phased adenine tracts in double-stranded RNA do not induce sequence-directed bending.

Tracts of four to six adenines phased with the DNA helix produce a sequence-directed bending of the helix axis. Here, using gel electrophoresis and electron microscopy (EM), we have asked whether a similar motif will induce bending in a duplex RNA helix. Single-stranded RNAs were transcribed either from short synthetic DNA templates or from Crithidia fasciculata kinetoplast bent DNA, and the complementary single-stranded RNAs were annealed to produce duplex RNA molecules containing blocks of four to six adenines. Electrophoresis on polyacrylamide gels revealed no retardation of the RNAs containing phased blocks of adenines relative to duplex RNAs lacking such blocks. Examination by EM showed most of the molecules to be straight or only slightly bent. Thus, in contrast to DNA duplexes, phased adenine tracts do not induce sequence-directed bending in double-stranded RNA. Analysis of the distribution of molecule shapes for the highly bent C. fasciculata DNA showed that the adenine blocks do not act cooperatively to induce DNA bending and that the molecules must equilibrate between a spectrum of bent shapes.

Drosophila topoisomerase II-DNA interactions are affected by DNA structure.

The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.

Spondee recognition threshold as a function of set size.

Recognition thresholds for spondaic words were obtained in normal listeners under conditions of varying set size. Six sets of spondees were derived from the 36-word corpus of a Northwestern University recording of CID W-1 spondaic words. One of these sets represented the full list of 36 spondees, whereas the remaining five were comprised of 27, 18, 9, 6, and 3 spondees selected to be homogeneous in level and slope characteristics. Results revealed a systematic and reliable effect wherein mean threshold decreased from 19.1 dB SPL to 12.2 dB SPL as set size was reduced from 36 to 3 spondees. The data are consistent with past research on human information processing in that spondee recognition threshold increased linearly with the number of bits per stimulus.

Multidimensional scaling of quality judgments of speech signals processed by hearing aids.

Perceptual dimensions underlying similarity ratings and preference judgments of the quality of hearing-aid-processed speech were derived via multidimensional-scaling procedures, and were correlated with 15 indices of electroacustic performance to determine those electroacoustic characteristics contributing maximally to quality judgements. Connected discourse was tape recorded through 20 conventional hearing aids and presented in a paired-comparison paradigm to ten normal listeners for similarity ratings and quality preference judgments. Analysis of the group similarity matrices revealed only one definitive perceptual dimension, low-cutoff frequency, that was common to the listeners. In a three-dimensional INDSCAL solution, this electroacoustic factor correlated--0.87 with dimension 1. Listeners strongly preferred hearing aids with relatively low low-cutoff frequencies; dimension 1 correlated--0.76 with their preferences. The quality judgments of some listeners were found via KYST analysis to exhibit a multidimensional structure, with low-cutoff frequency remaining the most salient dimension. Secondary dimensions varied across these several listeners, and included response irregularity, equivalent input noise level, high-cutoff frequency, and intermodulation distortion. Thus, the data provided evidence that, while low-cutoff frequency dominates listener judgments a multidimensional model may underlie the perceptual strategies used by some listeners to form quality judgments of hearing-aid-processed speech.

The influence of hearing aid nonlinear distortion on two auditory tasks.

A study is reported which examines the influence of hearing aid nonlinear distortion on multitone pitch perception. Normal-hearing subjects produced results indicating that hearing aid-generated harmonic and intermodulation components did significantly affect pitch judgments. Substantial differences in the amount of distortion between the two test aids was not, however, a critical factor. A follow-up study using closed-set intelligibility materials in noise further confirmed that relatively large measurable differences in nonlinear distortion between hearing aids do not seem to have the clinical significance which is often presumed.

Listener-assessed intelligibility of hearing aid-processed speech.

The capacity of listeners to yield reliable and valid preferences for hearing aid-processed speech was evaluated by the method of paired comparisons. Discourse by a male talker with General American dialect was processed by five hearing aids under conditions of quiet and a background of multitalker babble. Hearing aid-processed Revised Central Institute for the Deaf Sentences spoken by the same male talker and embedded in the same multitalker background were used as stimuli in establishing criterion intelligibility performance. All stimuli were tape-recorded and delivered via monaural earphone to 90 normal listeners. Test-retest results revealed reliability coefficients of 0.94 and 0.65 on the paired-comparison condition in quiet and in babble, respectively. A low-positive relationship was observed between responses on the two subjective tasks. Essentially negligible correlations were found between subjective preferences on these individual conditions and performance on the sentence task. General conditions are hypothesized under which paired-comparison judgments of intelligibility might be expected to correspond favorably with objective performance.