Mutations Ivs4nt1, 47delCT, and G148S identified in the phenylalanine hydroxylase gene by RT-PCR of illegitimate transcripts and chemical cleavage of mismatch.
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Base Sequence↗
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Determinants of RNA hairpin loop-loop complex stability.
Complexes formed by RNA hairpin loops with complementary loop sequences derived from Escherichia coli RNA I and RNA II, which are involved in the control of DNA replication of plasmid ColE1, have been analyzed to determine the sequence and structural elements required to achieve full affinity. Of particular interest is the origin of the enhanced stability of the complex formed by hairpin loops whose loop sequences have been inverted 5' to 3' with respect to wild-type sequences. Full complementarity of the two interacting loops is required to achieve full or enhanced affinity, while the stems of the two hairpins can differ. The major determinant of enhanced affinity lies in the base-pairs formed at positions 1 and 7 of the loops, together with the two base-pairs of each stem which are closest to the loop. Sequence variation in the middle of the loops, or further down the stem away from the loops, exerts only a modest influence on complex stability. We incorporate these results into a model for the loop-loop interaction which accounts for the importance of positions one and seven and the first two nucleotides of the stem, while providing potentially unique structures for recognition by the RNA one modulator protein.
Base Sequence↗
In vitro resolution of the dimer bridge of the minute virus of mice (MVM) genome supports the modified rolling hairpin model for MVM replication.
Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.
Animals↗
Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.
The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.
Amino Acid Sequence↗
Tn3 transposition immunity is conferred by the transposase-binding domain in the terminal inverted-repeat sequence of Tn3.
A series of mutant terminal inverted repeats (IRs), having 2 bp substitutions at various sites within the 38-bp IR sequence of the ampicillin-resistance transposon Tn3, were tested for transposition immunity to Tn3. Mutations within region 1-10 in the IR did not affect transposition immunity, while mutations within region 13-38 inactivated the immunity function. These two regions corresponded to domain A which was not bound specifically by Tn3 transposase and to domain B which was bound by the transposase, respectively. This indicates that specific binding of transposase to domain B within the IR sequence is responsible for transposition immunity.
Ampicillin Resistance↗
T2 mapping of human femorotibial cartilage with turbo mixed MR imaging at 1.5 T: feasibility.
The feasibility of a high-spatial-resolution technique for mapping T1 and T2 in articular cartilage in the human knee was evaluated. The technique, turbo mixed magnetic resonance (MR) imaging, is based on a pulse sequence in which inversion-recovery and spin-echo measurements are interleaved. The sequence was first validated in a phantom experiment in which T1 and T2 values obtained with an accepted spectroscopic technique were correlated with those obtained by using a clinical magnetic resonance imager with the turbo mixed technique. T2 maps were obtained with turbo mixed imaging in 25 volunteers (17 men, eight women; mean age, 30.8 years; range, 23-45 years). A high correlation (r = 0.99) was found between T1 and T2 values obtained at spectroscopy and those obtained at turbo mixed imaging. Relative differences in the range of cartilage relaxation times between the two techniques were less than 20%. Turbo mixed imaging in human volunteers showed T2 cartilage relaxation times that corresponded with previously published data. Turbo mixed imaging, thus, is feasible for T2 mapping of cartilage.
Adult↗
Simultaneous assessment of flow and BOLD signals in resting-state functional connectivity maps.
We have recently demonstrated using functional magnetic resonance imaging the presence of synchronous low-frequency fluctuations of signal intensities from the resting human brain that have a high degree of temporal correlation (p < 0.0001) both within and across the sensorimotor cortex. A statistically significant overlap between the resting-state functional connectivity map and the task-activation map due to bilateral finger tapping was obtained. Similar results have been obtained in the auditory and visual cortex. Because the pulse sequence used for collecting data was sensitive to blood flow and blood oxygenation, these low-frequency fluctuations of signal intensity may have arisen from variations of both. The objective of this study was simultaneously to determine the contribution of the blood oxygenation level signal and the flow signal to physiological fluctuations in the resting brain using the flow-sensitive alternating inversion recovery pulse sequence. In all subjects, the functional connectivity maps obtained from BOLD had a greater coincidence with task-activation maps than the corresponding functional connectivity maps obtained from blood-flow signals at the same level of statistical significance. Results of this study suggest that while variations in blood flow might contribute to functional connectivity maps, BOLD signals play a dominant role in the mechanism that gives rise to functional connectivity in the resting human brain.
Adolescent↗
Recombination within the upstream gene of duplicated myelin basic protein genes of myelin deficient shimld mouse results in the production of antisense RNA.
The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5' boundary and the nucleotide sequence surrounding the 3' boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.
Animals↗
Molecular characterisation of the enolase gene from the human malaria parasite Plasmodium falciparum. Evidence for ancestry within a photosynthetic lineage.
We have isolated and characterised the gene encoding the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase) from the human malaria parasite Plasmodium falciparum. This was achieved using a combination of cDNA sequencing and inverse-PCR techniques. The gene maps to chromosome 10 of the parasite. We have also mapped two further glycolytic enzyme genes, glyceraldehyde-3-phosphate dehydrogenase and triose-phosphate isomerase, to chromosome 14. The enolase gene encodes a protein of 446 amino acids (48.7 kDa), and all amino acid residues implicated in substrate/cofactor binding and catalysis are conserved in the malarial enolase molecule. The predicted protein sequence displays approximately 60-70% identity to enolase molecules of other eukaryotes, the closest relationship with its homologues seen amongst the seven fully described glycolytic pathway enzymes of P. falciparum. Of particular significance in this well conserved molecule is a characteristic 5-amino-acid insertion sequence that is identical in position and virtually identical in primary structure to that which is otherwise found uniquely in plant enolase proteins. This pentapeptide, together with other features of the plasmodial sequence, points to a common ancestry with photosynthetic organisms at the level of a protein-encoding nuclear gene, thus extending earlier analyses of nuclear small-subunit ribosomal RNA genes, and of an extrachromosomal circular 35-kb DNA element found in P. falciparum, which have also indicated such a relationship.
Amino Acid Sequence↗
Correlation of some MR findings in ankylosing spondylitis.
Results of the spinal magnetic resonance (MR) in patients with ankylosing spondylitis (AS) were evaluated prospectively in correlation with age, symptoms duration and some inflammatory tests. 20 patients with AS were prospectively examined by complex clinical and laboratory tests. All were examined with X-ray and 1,5T magnetic resonance. The visibility, margination and extent of acute and chronic pathological vertebral lesions were qualitatively and quantitatively compared statistically. The axial form of AS strongly prevailed (70%). If acute AS vertebral changes are evaluated CRP did not correlated with local vertebral changes, erythrocyte sedimentation rate and functional tests BASDAI and BASFI correlated with vertebral changes on the various significance level due to the MR sequence used. Chronic spinal changes correlated with age of the patients and duration of the symptoms. BASFI test with chronic changes did not correlate. MR is the best method for evaluating changes in vertebrae and in the adjacent soft tissues. The excellent evaluation of acute and chronic local MR changes of vertebral marrow is provided by Braun's scoring system. In acute AS changes short T1 inversion recovery (STIR) sequence showed most vertebral marrow changes, while in the chronic AS changes T1 weighted images were the statistically significantly best sequence in depicting the marrow and ligamentous pathology.
Adult↗
Two chloroplast DNA inversions originated simultaneously during the early evolution of the sunflower family (Asteraceae).
The chloroplast DNA (cpDNA) inversion in the Asteraceae has been cited as a classic example of using genomic rearrangements for defining major lineages of plants. We further characterize cpDNA inversions in the Asteraceae using extensive sequence comparisons among 56 species, including representatives of all major clades of the family and related families. We determine the boundaries of the 22-kb (now known as 22.8 kb) inversion that defines a major split within the Asteraceae, and in the process, we characterize the second and a new, smaller 3.3-kb inversion that occurs at one end of the larger inversion. One end point of the smaller inversion is upstream of the trnE-UUC gene, and the other end point is located between the trnC-GCA and rpoB genes. Although a diverse sampling of Asteraceae experienced substantial length variation and base substitution during the long evolutionary history subsequent to the inversion events, the precise locations of the inversion end points are identified using comparative sequence alignments in the inversion regions. The phylogenetic distribution of two inversions is identical among the members of Asteraceae, suggesting that the inversion events likely occurred simultaneously or within a short time period shortly after the origin of the family. Estimates of divergence times based on ndhF and rbcL sequences suggest that two inversions originated during the late Eocene (38-42 MYA). The divergence time estimates also suggest that the Asteraceae originated in the mid Eocene (42-47 MYA).
Amino Acid Sequence↗
Large T-antigen and sequences within the regulatory region of JC virus both contribute to the features of JC virus DNA replication.
The requirements for the DNA replication of the human papovavirus JC were analyzed using JC T-antigen as well as the T-antigens of the related viruses SV40 and BK. With all three T-antigens, the boundary of the core origin mapped on the early side to position 5093 of the viral genome. In conjunction with earlier studies, the core origin of DNA replication was therefore defined as a 68-bp region which, similar to the SV40 core origin, contains three major structural elements, early palindrome, T-antigen binding site II, and A/T-rich tract. Replication was stimulated by sequences flanking the core origin on the early side. Specifically, the stimulating sequences on the early side were identified as T-antigen binding site I. The degree to which flanking sequences were able to stimulate viral DNA replication was dependent on the T-antigen used in the experiment, with JC T-antigen relying most and BK T-antigen relying least on the flanking sequences. SV40 T-antigen showed an intermediate dependence. The same hierarchy was observed when replication activities were compared. BK T-antigen was more active in replicating DNA than SV40 T-antigen, which in turn was more effective than JC T-antigen. Dependence on flanking sequences is, thus, inversely correlated to the replicating activity of the respective T-antigen, showing that, in addition to the origin, the T-antigen contributes to the characteristics of JC virus DNA replication.
Antigens, Viral, Tumor↗
Cloning and nucleotide sequence of the pullulanase gene of Thermus thermophilus HB8 and production of the enzyme in Escherichia coli.
A 3.4-kb SphI fragment carrying the pullulanase gene of Thermus thermophilus HB8 was cloned. Based on the nucleotide sequence of it and the flanking region analyzed by direct sequencing of the inverse PCR product, an expression vector was constructed. The E. coli cells harboring the plasmid produced an about 80-kDa protein having pullulanase activity, the optimum temperature of which was 70 degrees C.
Amino Acid Sequence↗
Serial imaging of canine myocardial infarction by in vivo nuclear magnetic resonance.
Anterior infarction was produced in eight dogs to characterize serial changes in nuclear magnetic resonance signal intensity within the infarct zone. Magnetic resonance imaging was done on the day of infarction, on day 4, 5 or 6, on day 13 and day 20 using a 0.15 tesla (6.25 MHz) resistive imager. Electrocardiographically triggered spin echo (30, 45 and 60 ms echo times) and inversion recovery (400 to 500 ms inversion time) pulse sequences were employed to obtain single slice images. On day 20, the excised hearts were sectioned and examined to determine infarct location and extent. In the spin echo images, signal intensity within the ischemic zone was visibly increased in seven of the eight dogs on the day of infarction, and in all dogs by days 4 to 6. Signal intensity remained elevated in all but two dogs at day 20. With inversion recovery imaging, changes in the infarct zone were highly variable; both ill defined increases and decreases in signal intensity were noted. With a 30 ms echo time, signal intensity in the infarct zone was increased on average 29.8 +/- 24.1% above that in normal myocardium on the day of infarction. The relative signal intensity increased to 62.4 +/- 23.5% during the first 2 weeks after infarction (p less than 0.05), then decreased to 12.0 +/- 18.5% by day 20 (p less than 0.05). Similar changes were detected in the images using the 45 and 60 ms echo times. Nuclear magnetic resonance imaging therefore is able to detect regions of myocardial infarction and follow evolutionary changes in signal intensity within the infarct zone with healing.
Animals↗
The role of macronuclear DNA sequences in the permanent rescue of a non-mendelian mutation in Paramecium tetraurelia.
The paramecium tetraurelia mutant called d48 has a complete copy of the A surface protein gene in its micronuclei, but lacks the A gene in the macronucleus. Previous experiments have shown that microinjection of a plasmid containing the entire A gene or a large portion of the gene into the macronucleus of d48 rescued the cell line after formation of a new macronucleus (autogamy). Here we show that several different regions of the A gene can rescue d48, but 100% of the activity cannot be localized to a single, defined region. Inversion of a sequence contained within an A gene plasmid had no measurable effect on rescue efficiency and co-injection of two different plasmids results in enhancement of rescue activity despite the non-contiguous form of the DNA sequences. Both these results suggest that no specific product (RNA or protein) with defined end points is made from the rescuing fragment. A unique restriction site was created in the A gene and used to demonstrate that the injected DNA does not serve as a direct template for the synthesis of the new macronuclear DNA. Models to explain the action of the injected DNA are discussed.
Animals↗
Depth pulse sequences for surface coils: spatial localization and T1 measurements.
The depth pulse sequences theta;[2 theta(+/- x, +/- y)]2 and 2 theta;theta(+/- x);[2 theta(+/- x, +/- y)]2 have been implemented with a 20 mm diameter two-turn surface coil operating at 31P resonance (89.96 MHz). In these sequences theta refers to the pi/2 rf pulse at the center of the sensitive region of the coil, +/- x and +/- y denote the four orthogonal phases of the rf pulses, and ";" represents an optional brief delay (e.g., 4 microseconds) between pulses to facilitate switching between different phases. Localization of the sensitive region was demonstrated with phantom samples by in vivo monitoring of rat livers and detection of necrotic regions of subcutaneously implanted tumors. The inversion-recovery pulse sequence, 2 theta-tau-theta(+/- x);[2 theta(+/- x, +/- y)]2, where tau is a variable delay, was employed to measure the spin-lattice relaxation time of a selected region, which could be varied by changing the pulse width and the size of the surface coil.
Animals↗
Background suppression with multiple inversion recovery nulling: applications to projective angiography.
We have developed a technique to accurately null the longitudinal magnetization (Mz) of background material. This suppression involves first saturating the longitudinal magnetization (Mz) of a region, and then applying several nonselective inversions. The inversions are timed relative to the saturation such that Mz is nulled across a broad range of T1 at a predetermined time after the initial saturation. B1 and B0 inhomogeneity, which could lead to inaccurate suppression, are dealt with by the combination of a multiple tip saturation sequence and four adiabatic inversion pulses. The suppression sequence can be used to form projective angiograms by selectively tagging the imaging region with the saturation pulse. After the inversions are played out, a projection taken through the tag region when Mz is nulled will only contain signal from blood that has flown into the region after the saturation. Since only two dimensions are acquired, the technique can acquire gated projection angiograms in reasonable scan times. Representative inflow MIR angiograms of the carotid arteries and renal arteries show excellent background suppression.
Carotid Arteries↗
Large-scale identification of virulence genes from Streptococcus pneumoniae.
Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.
Animals↗