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Analysis of DNA inversions in the shufflon of plasmid R64.

The shufflon, a multiple DNA inversion system in the plasmid R64, consists of four DNA segments flanked and separated by seven 19-bp repeat sequences. Site-specific recombinations mediated by the rci product occur between each inverted repeat sequence, resulting in inversions of the four segments independently or in groups. The seven 19-bp repeat sequences are classified into four types (repeat-a, -b, -c, and -d), according to their 3-bp variable sequences. We individually cloned A, B, and C segments of the R64 shufflon and determined the in vivo inversion frequency of each segment. The inversion frequencies of three segments differed greatly. The inversion frequency declined in the following order: segments A, B, and C. Synthetic 19-mer oligonucleotides corresponding to both strands of repeat-a, -b, -c, and -d sequences were inserted into appropriate sites of pBR322. The rci-mediated DNA inversion occurred between two synthetic inverted repeats, indicating that the 19-bp inverted repeat sequences are the sole elements required in cis for the shufflon system. The inversion frequencies of DNA segments flanked by various sequences indicate that the four types of repeat sequences determine the inversion frequency of the four DNA segments of the R64 shufflon. Deletion of a DNA segment flanked by direct repeat sequences could not be detected.

DNA Nucleotidyltransferases↗

Low-dose gadobenate dimeglumine versus standard-dose gadopentate dimeglumine for delayed contrast-enhanced cardiac magnetic resonance imaging.

RATIONALE AND OBJECTIVES: The purpose of our study was to compare gadopentate dimeglumine (Gd-DTPA) and gadobenate dimeglumine (Gd-BOPTA) for the evaluation of myocardial infarction (MI) and in the grading transmural extent on late-contrast enhanced cardiac magnetic resonance imaging. MATERIALS AND METHODS: Twenty-three patients with clinically proven MI were examined with the use of 0.2 mmol/kg Gd-DTPA and 0.1 mmol/kg Gd-BOPTA in 2 days interval. All patients were examined with the use of segmented two-dimensional inversion-recovery turbo fast-field echo pulse sequence with an inversion time 210-300 milliseconds. Fifteen minutes time delay was used on both examinations after the injection of contrast agent. Contrast-to-noise ratio between normal myocardium and infarcted myocardium and signal intensity ratio (SIR) of the enhanced myocardium to blood pool was derived and compared for each contrast agent. RESULTS: A total of 61 infarcted segments were analyzed. All of the infarcted segments were visualized on both Gd-BOPTA and Gd-DTPA enhanced images. There was statistically no significant difference between 0.2 mmol/kg Gd-DTPA and 0.1 mmol/kg Gd-BOPTA in the mean contrast-to-noise ratio (10.19 versus 10.22; P = .96), SNR (14.29 versus 14.25; P = .96), and SIR (4.34 versus 4.21; P = .38) of the infarcted segments. Intraobserver agreement (kappa) between Gd-DTPA and Gd-BOPTA were R1 = 91% and R2 = 86%. Interobserver agreements between the readers were Gd-DTPA = 85% and Gd-BOPTA = 88%. CONCLUSION: According to our data, the diagnostic efficacy of 0.1 mmol/kg dose Gd-BOPTA is equivalent to that of 0.2 mmol/kg Gd-DTPA for the assessment of MI on delayed enhanced magnetic resonance images.

Aged↗

Selective sweep at the Drosophila melanogaster Suppressor of Hairless locus and its association with the In(2L)t inversion polymorphism.

The hitchhiking model of population genetics predicts that an allele favored by Darwinian selection can replace haplotypes from the same locus previously established at a neutral mutation-drift equilibrium. This process, known as "selective sweep," was studied by comparing molecular variation between the polymorphic In(2L)t inversion and the standard chromosome. Sequence variation was recorded at the Suppressor of Hairless (Su[H]) gene in an African population of Drosophila melanogaster. We found 47 nucleotide polymorphisms among 20 sequences of 1.2 kb. Neutrality tests were nonsignificant at the nucleotide level. However, these sites were strongly associated, because 290 out of 741 observed pairwise combinations between them were in significant linkage disequilibrium. We found only seven haplotypes, two occurring in the 9 In(2L)t chromosomes, and five in the 11 standard chromosomes, with no shared haplotype. Two haplotypes, one in each chromosome arrangement, made up two-thirds of the sample. This low haplotype diversity departed from neutrality in a haplotype test. This pattern supports a selective sweep hypothesis for the Su(H) chromosome region.

Animals↗

A glutamate-dependent acid resistance gene in Escherichia coli.

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.

Acids↗

Application of multiple inversion recovery for suppression of macromolecule resonances in short echo time (1)H NMR spectroscopy of human brain.

Macromolecules contribute broad "background" resonances to the (1)H NMR brain spectra at short echo times. The application of long echo times is the most widely used method for removing these resonances. Here, it is demonstrated that these background resonances may be suppressed at short echo times using multiple inversion recovery (MIR). In the technique presented, the MIR sequence consists of four adiabatic inversion pulses, applied preparatory to a 20-ms echo time stimulated echo localization sequence. The inversion times (359, 157, 69, and 20 ms) were selected to preferentially suppress macromolecules with longitudinal relaxation times between 38 and 300 ms. While the resulting spectra have lower overall signal-to-noise, baseline contributions from macromolecules are greatly reduced. Unlike the typical long TE acquisitions, the short TE MIR acquisition preserves the myo-inositol resonance.

Adult↗

Molecular analysis of T-cell receptor transcripts in a human T-cell leukemia bearing a t(1;14) and an inv(7); cell surface expression of a TCR-beta chain in the absence of alpha chain.

The polymerase chain reaction (PCR) was used to amplify cDNA in order to characterize normal and hybrid T-cell receptor (TCR) gene rearrangements derived from a T-cell acute lymphoblastic leukemia (T-ALL) bearing a chromosome 7 inversion. The nucleotide sequence analysis of the amplified product showed the presence of an out-of-frame V beta/J gamma/C gamma transcript and an in-frame V gamma/J gamma/C beta transcript which result from an interlocus recombination between the TCR-beta and gamma loci and the transcription of the reciprocal hybrid TCR gene. The sequence analysis of the reciprocal DNA segment directly involved in the breakpoint of the inversion showed a recombination between a J gamma-sequence heptamer signal and a coding J beta gene segment. The exonuclease nibbling of each DNA extremity and the non-templated nucleotide insertion observed at both coding and reciprocal joints demonstrate that the inversion is mediated by the lymphocyte recombinase complex. During T-cell differentiation, TCR-beta genes are rearranged prior to TCR-alpha genes. In the present case of T-ALL, we have shown that TCR-delta genes are rearranged at both loci, excluding productive rearrangements of TCR-alpha genes. The molecular analysis of the normal TCR genes derived from the leukemic cells revealed the presence of productively rearranged TCR-beta, gamma, and delta genes. Cell surface staining of these cells showed the presence of CD3 molecules and of a TCR-beta chain corresponding to the normal beta allele expressed in a disulfide-linked complex with a protein which is not TCR-alpha, gamma, or delta. This could represent the cell surface expression of the hybrid TCR-V gamma/C beta protein or the expression of a TCR-beta homodimer.

Adult↗

Sequential gel electrophoretic analysis of esterase-2 in two populations of Drosophila buzzatii.

Sequential electrophoresis, using three different buffer systems on cellulose acetate gels, was used to characterize the allelic variation for esterase-2 in two populations of D. buzzatii in Australia that are separated by 550 km. Twenty-five alleles were detected, of which nine were unique to one population, eight unique to the other, and only eight were common to both populations. Allele frequencies within each population were significantly different between the two major chromosome sequences (standard and j inversion), and for each chromosome sequence allele frequencies were significantly different between populations. Observed allelic frequency distributions were not significantly different from those predicted for selective neutrality using the homozygosity test statistic. However, estimates of the effective sizes of the populations derived from their observed differentiation, together with the history of the species in Australia, provide support for some form of balancing selection affecting at least some of the alleles.

Alleles↗

Transfer of Bifidobacterium inopinatum and Bifidobacterium denticolens to Scardovia inopinata gen. nov., comb. nov., and Parascardovia denticolens gen. nov., comb. nov., respectively.

Bifidobacterium inopinatum Crociani et al. 1996 and Bifidobacterium denticolens Crociani et al. 1996 have distinct phenotypic characteristics and low G+C contents compared with other bifidobacteria. In the 16S rRNA phylogenetic tree, these two species grouped in an independent subcluster. In our previous work, partial heat-shock protein 60 (HSP60) gene-sequence analysis also indicated that these two species had distinct taxonomic positions. In this work, the complete HSP60 genes of five representative bacterial strains were sequenced by using an inverse PCR method. The complete sequence similarities turned out to be at the same level as those of the partial genes, thus confirming the result based on partial sequence analysis. On the basis of all the evidence mentioned above, it is proposed that these two species should be transferred to two new genera as Scardovia inopinata gen. nov., comb. nov., and Parascardovia denticolens gen. nov., comb. nov.

Bacterial Typing Techniques↗

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs.

5' Untranslated Regions↗

MR of the kidneys, liver, and spleen in paroxysmal nocturnal hemoglobinuria.

The magnetic resonance (MR) findings in the liver, kidneys, and spleen in eight patients with paroxysmal nocturnal hemoglobinuria (PNH) were retrospectively reviewed to determine whether characteristic features could be demonstrated. Eight patients underwent abdominal MR examinations by gradient echo sequences (seven patients), spin-echo sequences (seven patients), and inversion recovery (one patient). Signal intensities of the kidneys, liver, and spleen were visually evaluated. Autopsy and liver biopsy correlation were available in one case each. Renal signal intensity was decreased in all eight patients by either gradient-echo or T2-weighted sequences and in the single inversion recovery sequence. Hepatic signal intensity was decreased in three of eight patients on spin- and gradient-echo images. Splenic signal intensity was decreased in three of eight patients on spin- and gradient-echo images, and in two of these was manifest as focal low signal spots (Gamna-Gandy bodies). While the signal intensity in the renal cortex is typically decreased in patients with PNH, signal intensities in the liver and spleen are variable. Low signal intensity in the kidneys is due to hemosiderin deposition resulting from intravascular hemolysis, whereas low signal intensity in the liver or spleen may be due to either transfusion siderosis, or as a consequence of hepatic or portal venous thrombosis.

Adolescent↗

Comparative analysis of invertebrate Tc6 sequences that resemble the vertebrate V(D)J recombination signal sequences (RSS).

Invertebrate cells lack the p53 recombination checkpoint but contain mobile DNA sequences that transpose by a mechanism in part shared with excision of the V(D)J recombination signal sequences (RSS). In this work, inversion, deletion, and duplication of sequences associated with an invertebrate C. elegans Tc6 element is described. The structure of this C. elegans sequence and other dispersed Tc6 elements suggests that covalently closed 'hairpin' structures are not unique to excision of the V(D)J RSS by the RAG proteins, but rather can be generated by transposases at transposon termini leading to characteristic inversion and duplication events. Comparative analysis of recombination events at invertebrate sequences resembling the vertebrate V(D)J RSS may be useful in understanding V(D)J recombination-mediated recombination events in malignant vertebrate cells or genetic diseases such as ataxia telangectasia, in which the p53 recombination checkpoint is defective.

Animals↗

How to search for RNA structures. Theoretical concepts in evolutionary biotechnology.

The relation between RNA sequences and minimum free energy secondary structures is viewed as a mapping from sequence space into shape space. The properties of such mappings depend strongly on the ratios of the numbers of sequences and structures and, hence, substantial differences are observed between samples of structures derived from AUGC, pure AU or pure GC sequences. Statistical analysis of large samples is used to demonstrate that structures from AUGC sequences are much less sensitive to point mutations than those from sequences containing exclusively AU or GC. The frequency with which a structure is realized in sequence space is inversely proportional to some power c > 1 of the structure's frequency rank, thus following a (generalized) Zipf law. For long sequences the exponent approaches c = 1. An inverse folding algorithm is used to compute samples of sequences folding into the same secondary structure. These sequences are distributed randomly in sequence space. Common structures form extended neutral networks along which populations can migrate through the entire sequence space without changing structure. In this migration, moves of Hamming distance d = 1 and d = 2 are accepted in order to allow for base and base pair exchanges, respectively. Around any arbitrarily chosen sequence a ball that contains sequences folding into all common structures can be drawn. This ball has a diameter that is much smaller than the diameter of sequence space. Hence, only a small fraction of sequence space needs to be searched in order to find a given structure. The results derived from the mapping of sequences into structures are used to suggest a rationale for evolutionary searches on RNA structures: selection cycles with high and low mutation rates applied in alternation. Generalizations of the results to RNA 3-D structures and protein structures are discussed.

Algorithms↗

Anxiolytic-like action of neurokinin substance P administered systemically or into the nucleus basalis magnocellularis region.

There is evidence that the neurokinin substance P plays a role in neural mechanisms governing learning and reinforcement. Reinforcing and memory-promoting effects of substance P were found after it was injected into several parts of the brain and intraperitoneally. With regard to the close link between anxiety and memory processes for negative reinforcement learning, the aim of the present study was to gauge the effect of substance P on anxiety-related behaviors in the rat elevated plus-maze and social interaction test. Substance P was tested at injection sites where the neurokinin has been shown to promote learning and to serve as a reinforcer, namely in the periphery (after i.p. administration) and after injection into the nucleus basalis magnocellularis region. When administered i.p., substance P had a biphasic dose-response effect on behavior in the plus-maze with an anxiolytic-like action at 50 microg/kg and an anxiogenic-like one at 500 microg/kg. After unilateral microinjection into the nucleus basalis magnocellularis region, substance P (1 ng) was found to exert anxiolytic-like effects, because substance P-treated rats spent more time on the open arms of the plus-maze and showed an increase in time spent in social interaction. Furthermore, the anxiolytic effects of intrabasalis substance P were sequence-specific since injection of a compound with the inverse amino acid sequence of substance P (0.1 to 100 ng) did not influence anxiety parameters. These results show that substance P has anxiolytic-like properties in addition to its known promnestic and reinforcing effects, supporting the hypothesis of a close relationship between anxiety, memory and reinforcement processes.

Animals↗

Shoulder MRI refinements: differentiation of rotator cuff tear from artifacts and tendonosis, and reassessment of normal findings.

One of the difficulties with rotator cuff imaging lies in the normal variability of the tendon's signal. There may be intermediate signal present within the tendon because of magic-angle phenomenon, muscle and tendon fiber interdigitation, or tendinopathy related to degenerative changes or overuse injury. Partial and complete rotator cuff tears should be distinguishable from these causes of intermediate signal if water signal is reliably identified. This article reviews the important issue of distinguishing between rotator cuff tear and other causes of high signal in the rotator cuff, including artifacts and tendonosis. We include a review of the literature and a brief report of a study we conducted on 20 shoulders of 14 asymptomatic, young volunteers. In this study, the rotator cuff tendons were evaluated for abnormal signal at different TE values to determine at what TE the interpreters were able to confidently distinguish the high-signal intensity of a tear (water) from the intermediate signal intensity associated with artifact and tendinopathy. Readers were able to distinguish water and tendon signal in 70% to 100% of fast-spin echo (FSE) fat-saturated images with TE of 66, but there was interobserver variability at this TE, suggesting that it is less reliable than 88 ms in the identification of rotator cuff tears. By using FSE fat-saturated sequences with TE of 88 and fast spin echo inversion recovery (FSEIR) sequences, readers at all levels of experience were able to differentiate water signal intensity from tendon signal intensity in 100% of cases. Therefore, we suggest that either FSEIR images or FSE fat-saturated images with TE greater than 66 be used to facilitate the differentiation of fluid signal from intermediate increased signal intensity in rotator cuff imaging. Additionally, this article reviews the normal findings of shoulder magnetic resonance imaging (MRI) as revealed by the asymptomatic subjects included in our study, and assesses these findings in respect to previous publications. The normal features reviewed include the subacromion-subdeltoid (SA/SD) bursa, the biceps tendon sheath, the acromioclavicular (AC) joint, and the greater tuberosity of the humerus. A small amount of fluid was commonly seen in the SA/SD bursa, as well as the biceps tendon sheath. Subjective down-sloping of the acromion in the coronal plane, mild degenerative change of the AC joint, and undersurface spurring of the AC joint were uncommon in our normal subjects. Cystic change limited to the posterior aspect of the greater tuberosity was identified in 15% to 45% of shoulders.

Artifacts↗

Molecular mechanisms for transposition of drug-resistance genes and other movable genetic elements.

Transposition is proposed to be responsible for the rapid evolution of multiply drug-resistant bacterial strains. Transposons, which carry the genes encoding drug resistance, are linear pieces of DNA that range in size from 2.5 to 23 kilobase pairs and always contain at their ends nucleotide sequences repeated in inverse order. In some transposons the terminal inverted repeat sequences are capable of independent movement and are called insertion sequences. Transposons carry a gene that encodes transposase(s), the enzyme(s) responsible for recombination of the transposon into another DNA molecule. Studies on transposable genetic elements in bacteria have not only given insight into the spread of antibiotic resistance but also into the process of DNA movement.

Anti-Bacterial Agents↗

Spin-echo and STIR MR imaging of sports-related muscle injuries at 1.5 T.

Seventy patients with clinically diagnosed athletic muscle injuries of varying severity were studied with MR imaging at 1.5 T. Twenty underwent follow-up MR studies. In all cases, SE T1-weighted and double-echo T2-weighted pulse sequences were used. These were supplemented by short T1 inversion recovery (STIR) sequence in 36 cases. Muscle injuries were more readily seen with STIR images than with SE T2-weighted images. In both initial assessment and follow-up of tears, the use of the STIR technique allowed the greatest lesion/muscle contrast. Short TR, short TE SE images provided anatomic detail and were an adjunct to T2-dependent SE images in the evaluation of organized hematomas (11 cases). Follow-up MR studies in 20 patients at variable time intervals allowed demonstration of regression of the tear in 11 cases, fibrous scar formation in 5 cases, and recurrence of the tear in 4 cases. Evolution of hematomas into scar and into cyst was demonstrated in three and two cases, respectively. Owing to the additive effect of T1 and T2 mechanisms, the STIR sequence is well suited for initial evaluation and can replace T2-weighted images in the follow-up of muscle trauma.

Adolescent↗

Cloning and porin activity of the major outer membrane protein P1 from Coxiella burnetii.

Coxiella burnetii, the etiological agent of Q fever, is a gram-negative obligate intracellular bacterium. Two striking characteristics of this microorganism are its ability to thrive within a phagolysosome and its ability to persist in the environment outside a host cell. These abilities have been attributed to the existence of C. burnetii developmental cycle variants: large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC). Variants differ in protein profiles, including differential expression of a major outer membrane protein (MOMP) of C. burnetii, designated P1. The approximately 29-kDa MOMP is highly expressed in LCV, down-regulated in SCV, and not apparent in SDC. We sought to characterize P1 through purification of native protein for N-terminal analysis, cloning, and functional studies. Highly purified P1, extracted from C. burnetii membranes by using the zwitterionic detergent Empigen, allowed the determination of N-terminal and internal peptide sequences. The entire P1 coding locus was cloned by PCR amplification based upon these peptide sequences, followed by inverse PCR. Comparison of the predicted P1 amino acid sequences among the C. burnetii isolates Nine Mile, Koka, Scurry, and Kerns indicated a high degree of conservation. Structural prediction suggests that the peptide has a predominantly beta-sheet conformation, consistent with bacterial porins. Typical porin characteristics were observed for native P1, including detergent solubilization properties, heat modification of purified protein, and channel formation in a planar lipid bilayer. Characterization of differentially expressed P1 as a porin increases our understanding of the function of morphological variants and their role in pathogenesis.

Amino Acid Sequence↗