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Products and implied mechanism of H chain switch recombination.

The Ig H chain switch is a DNA recombination event. The recombination occurs between two or more switch regions, areas of tandem sequence duplication that lie upstream of the corresponding H chain C region genes. We have determined the DNA sequence at four recombination sites in three molecularly cloned, rearranged switch regions. All eight donor and recipient recombination sites are at the common pentamers GGGGT, GAGCT, and GGTGG. One of the switch recombination events is an inversion of S gamma 3 sequences. Another of the recombinational events is an internal S gamma 1 deletion, which may be switch enzyme mediated. These results, together with other switch recombination site sequences, suggest that switch recombination is mediated by cutting enzymes with modest specificity and religation enzymes with no specificity.

Base Sequence↗

[Magnetic resonance of the brain: comparative assessment of conventional sequences versus fast sequences].

The conventional Spin-Echo sequences have been the most used acquisition techniques on Magnetic Resonance Imaging (MRI) of the brain, since the beginning of 1990s. Recently, however, technological developments proposed such new fast acquisition techniques, with a dramatic reduction in acquisition time as Gradient Echo, Turbo Spin Echo, Gradient Spin Echo ed Echo Planar sequences. We investigated the comparative adequacy of the new fast sequences, in brain MR studies versus conventional sequences, on medium field strength MR equipment (.5 T). On in vitro exams S/N ratio was evaluated and on in vivo study C/N ratio between white substance grey substance and fluid were evaluated, in 15 healthy volunteers. All the exams were carried out on Philips NT5 Gyroscan MR unit operating at .5 T, with 15 mT/m power gradients. Both conventional and fast sequences Spin-Echo (SE), Inversion Recovery (IR), Gradient-Echo (GE), Turbo Spin-Echo (TSE), Gradient Spin-Echo (GRASE) and Echo Planar (EPI), all optimized to obtain T1, T2 and T1-IR weighted images, were performed. The sequences were evaluated separately comparing different acquisition techniques relative to the same type of contrast (T1, T2, and T1-IR). The results indicate the superiority of the fast acquisition techniques, both in terms of quality and acquisition times, except for T1-weighted images, where the conventional Spin-Echo sequences confirmed to be superior. In conclusion, the present study suggests that fast acquisition techniques can improve the conventional protocol of sequences, in the anatomical representation of the normal brain even with medium field strength equipment only on T2 and T1-IR weighted images. On T1-weighted imaging conventional SE acquisition technique is still the best choice. The time saving obtained with fast sequences can therefore be used for an overall improvement in the quality of images, and to apply accessory acquisition plans on routine exams.

Brain↗

Short tau inversion recovery and three-point Dixon water-fat separation sequences in acute traumatic bone fractures at open 0.35 tesla MRI.

OBJECTIVE: Fat suppression can be used to improve the diagnostic confidence in traumatic bone fractures of the extremities. We compared a three-point Dixon "sandwich" water-fat separation (WFS) sequence, resulting in a water-only and a fat-only image set after one excitation, with the STIR sequence on an open 0.35 T superconductive MR system. DESIGN AND PATIENTS: T1-weighted, STIR (2000/40 [TR/TE]), and WFS (2000/36 [TR/TE]) MR images were prospectively obtained in 27 patients with 40 radiologically diagnosed fractures immediately after first-line treatment. Signal-to-noise (S/N) ratio, contrast-to-noise (C/N) ratio, and bone marrow edema volumes were measured together with qualitative parameters (four-point scale). RESULTS: WFS was significantly superior to STIR in all quantitative parameters (better S/N ratio, P<0.001; better C/N ratio, P<0.001; larger marrow edema, P <0.023; Wilcoxon signed rank test). Visibility of bone marrow edema, visibility of fracture line, and preservation of anatomical details were better with the WFS sequence ( P<0.001, P<0.001, P<0.001, respectively; ANOVA). Fat saturation was rated more homogeneous, however, with the STIR sequence (not significant; P<0.101). CONCLUSION: On the basis of qualitative and quantitative assessments, the three-point Dixon "sandwich" water-fat separation sequence was consistently superior to the STIR sequence in the delineation of traumatic fractures.

Adipose Tissue↗

Experimental acute intracerebral hemorrhage. Value of MR sequences for a safe diagnosis at 1.5 and 0.5 T.

PURPOSE: To determine the detectability of intracerebral hematomas with MR imaging at 1.5 T and 0.5 T with fluid attenuated inversion recovery turbo spinecho (FLAIR) and gradient-echo sequences. MATERIAL AND METHODS: Twenty-seven intracerebral hematomas were created in 25 piglets by injection of venous blood into the brain through a burr hole. All were imaged with T2*-weighted gradient echo sequences (fast field echo, FFE), T2-weighted fluid attenuated inversion recovery turbo spin-echo sequences (FLAIR), T2-weighted turbo spin-echo (TSE) and T1-weighted spinecho sequences. Follow-up was performed on the 2nd, 4th and 10th postoperative days. Ten animals were additionally investigated with similar sequences at 0.5 T. Histologic correlation was obtained in all cases. RESULTS: T2* FFE sequences detected all acute intracerebral hematomas and demonstrated the size correctly at 1.5 T and 0.5 T. The conspicuity was better at 1.5 T. FLAIR sequences were unreliable in the hyperacute phase at 1.5 T: However. subarachnoid and intraventricular extension was best appreciated with FLAIR images. T2 TSE images were incapable of detecting paraventricular and subarachnoid hemorrhages, but clearly demonstrated intracerebral blood in other locations. TI-weighted images were insensitive to hemorrhage in the acute state but very useful in subacute and chronic hematomas. CONCLUSION: The safe and reliable diagnosis of intracerebral hemorrhage is probably possible with MR imaging at 1.5 T and 0.5 T even of hematomas less than 90 min old, but requires the application of at least FLAIR, T2* FFE and T1 sequences and is therefore time consuming.

Acute Disease↗

Imaging of water and fat fractions in high-field MRI with multiple slice chemical shift-selective inversion recovery.

Fat and water fractions were quantified at high field using a chemical shift-selective inversion recovery (CSS-IR) sequence to address the major difficulties encountered at high field by phase-sensitive techniques used for fat/water discrimination. Water- and fat-suppressed images were perfectly registered, which is a prerequisite for quantification. Immunity of the inversion pulse to B(1) field modulations and off-resonance effects was tested for two adiabatic inversion pulses, with hyperbolic secant and asymmetric hyper-pulse waveforms. Taking into account adiabaticity, immunity to off-resonance, radiofrequency power requirements, and specific absorption rate, the former was chosen. A close correlation was found between fat content measured by CSS-IR at 4.7 T (R(2)= 0.97, P < 0.001) and 9.4 T (R(2)= 0.99, P < 0.05) and that measured by Soxhlet extraction. Different sources of bias (low signal-to-noise ratio, magnetization transfer effect) are discussed. J. Magn. Reson. Imaging 2000;12:488-496.

Adipose Tissue↗

Novel HeLa topoisomerase II is the II beta isoform: complete coding sequence and homology with other type II topoisomerases.

DNA topoisomerase (topo) II mediates DNA strand passage in an ATP-dependent reaction. Human cell lines express at least two genetically distinct forms of the enzyme, topo II alpha (p170) and II beta (p180). Previously, we isolated a novel HeLa cDNA clone (CAA5) that partially encodes a protein homologous to topo II alpha (Austin, C.A. and Fisher, L.M. (1990) FEBS Lett. 266, 115-117). In this paper we show that CAA5 encodes a C-terminal segment of human topo II beta. We report here for the first time cDNA clones spanning the entire coding sequence. Overlapping clones specifying the 3' end of the cDNA have been isolated, mapped and sequenced. The missing 5' coding sequence was obtained by an inverse PCR protocol and from a specifically primed cDNA library. Human topo II beta is a 1621 amino acid protein which is closely homologous to topo II alpha in the N-terminal three quarters of its sequence. In contrast, the C-terminal segments of the alpha and beta sequences show considerable divergence suggesting these regions may mediate different cellular functions of the two isoforms. Southern blot analysis of yeast and Drosophila DNA using human alpha and beta specific probes detected a single topo II homologue in these lower eukaryotes. Comparison of the protein sequence for human topo II beta with other type II topoisomerases revealed several conserved motifs and has allowed identification of the likely ATPase- and DNA breakage-reunion domains.

Base Sequence↗

DNA triple-helix formation at pyrimidine-purine inversion sites.

A systematic investigation of a series of triplex forming oligonucleotides (TFOs) containing alpha-, and beta-thymidine, alpha- and beta-N7-hypoxanthine, and alpha- and beta-N7 and N9 aminopurine nucleosides, designed to bind to T-A inversion sites in DNA target sequences was performed. Data obtained from gel mobility assays indicate that T-A recognition in the antiparallel triple-helical binding motif is possible if the nucleoside alpha N9-aminopurine is used opposite to the inversion site in the TFO.

Adenine↗

Myocardial viability: assessment with three-dimensional MR imaging in pigs and patients.

PURPOSE: To prospectively evaluate the correlation between a three-dimensional (3D) delayed enhancement magnetic resonance (MR) imaging sequence and a two-dimensional (2D) delayed enhancement MR imaging sequence for noninvasive assessment of myocardial viability in pigs and patients. MATERIALS AND METHODS: The pig and patient studies were approved by the responsible authorities, and patients gave written informed consent. MR imaging was performed by using a rapid 3D inversion-recovery balanced steady-state free precession sequence and a 2D segmented inversion-recovery fast low-angle shot sequence as the reference standard. Fourteen pigs with reperfused (n=7) or nonreperfused (n=7) myocardial infarction and 17 patients (13 men, four women; mean age, 64.9 years+/-8.6 [standard deviation]) suspected of having myocardial infarction were included. Linear regression analysis and Bland-Altman analysis were used to compare the infarction volumes. RESULTS: In 10 of the 14 pigs the induction of myocardial infarction was successful. In these pigs, altogether 81 segments with myocardial infarction were demonstrated by both MR sequences, and agreement between the two sequences for classification of transmural extent of myocardial infarction was 99.7%. The infarction volume determined by using 3D MR imaging (4.64 cm3+/-2.48) in the pigs highly correlated with that of 2D MR imaging (4.65 cm3+/-2.39, r=0.989, P<.001) and that of staining by using triphenyltetrazolium chloride (4.67 cm3+/-2.44, r=0.996, P<.001). Thirteen of the 17 patients examined showed myocardial infarction in 34 myocardial segments with both sequences, and agreement between the two sequences for classification of transmural extent of myocardial infarction was 98.6%. In the patients, the infarction volume determined with both sequences highly correlated (9.71 cm3+/-7.47 for the 3D sequence vs 10.01 cm3+/-8.04 for the 2D sequence, r=0.982, P<.001). The breath-hold time necessary for the 3D MR imaging (21.0+/-2.3 seconds) was significantly shorter than that for 2D MR imaging (188.3+/-20.2 seconds, P<.001). CONCLUSION: Myocardial infarction volumes obtained with the 3D MR imaging sequence are highly correlated and in good agreement with volumes obtained with the 2D MR imaging standard approach and reduced the acquisition time by a factor of nine.

Animals↗

Fast in vivo water quantification in rat brain oedema based on T(1) measurement at high magnetic field.

BACKGROUND: In vivo water content determination based on magnetic resonance (MR) method is of importance in clinical practice as well as in animal studies to follow up the treatment given in order to reduce brain oedema. The methods proposed in the literature so far are largely time consuming. The aim of this study was to find a fast in vivo water quantification method having real advantage for patients suffering from critical conditions. METHOD: Cold injury was applied to provoke brain oedema in fourteen rats. T(1) values of both the oedematous area and the contralateral normal cortex were determined by two independent methods 24 hours after the cold impact. First, from a series of images recorded by inversion recovery spin echo (IRSE) sequence and then by progressive saturation experiment performed by localised MR spectroscopy using stimulated echo acquisition mode (STEAM). To reduce the acquisition time, a two-element repetition time array was optimised for the STEAM experiment, whereas four inversion times were used for T(1) mapping. Both methods were validated against gel phantoms with known T(1) values. After the MR measurements the animals were sacrificed and the water contents of the regions of interest were determined by gravimetric wet-dry method. FINDINGS: The reciprocals of the in vivo measured T(1) values were correlated with the reciprocals of the brain water contents. STEAM experiment showed stronger correlation (r=0.96) than IRSE (r=0.93). In addition, STEAM provided more accurate T(1) values in the phantom study. Determination of brain water content based on T1 measurement does work also at high magnetic field. Determination of brain water content by Magnetic Resonance Spectroscopy is feasible within 2 minutes. INTERPRETATION: Using the presented fast method, water content can be determined within a couple of minutes in animal experiments as well as in the daily clinical practice.

Animals↗

Contrast manipulation in NMR imaging.

The past few years have shown rapid growth of NMR imaging in both image quality and diagnostic usefulness. It has become apparent, as the images have been published, that both inter- and intra-group imaging of the same underlying pathology produces images which can have vastly differing appearance. This effect is mainly due to imaging techniques which use different pulse sequence types and timings thus varying the relative contribution of the protpn density, T1, and T2 properties of the tissues. In this paper we investigate the contrast manipulation effects and methods for SNR optimization for the saturation recovery, inversion recovery, spin echo, and inversion recovery spin echo pulse sequences when applied to three clinically relevant imaging tasks.

Cerebral Infarction↗

From sequences to shapes and back: a case study in RNA secondary structures.

RNA folding is viewed here as a map assigning secondary structures to sequences. At fixed chain length the number of sequences far exceeds the number of structures. Frequencies of structures are highly non-uniform and follow a generalized form of Zipf's law: we find relatively few common and many rare ones. By using an algorithm for inverse folding, we show that sequences sharing the same structure are distributed randomly over sequence space. All common structures can be accessed from an arbitrary sequence by a number of mutations much smaller than the chain length. The sequence space is percolated by extensive neutral networks connecting nearest neighbours folding into identical structures. Implications for evolutionary adaptation and for applied molecular evolution are evident: finding a particular structure by mutation and selection is much simpler than expected and, even if catalytic activity should turn out to be sparse of RNA structures, it can hardly be missed by evolutionary processes.

Base Composition↗

A large AZFc deletion removes DAZ3/DAZ4 and nearby genes from men in Y haplogroup N.

Deletion of the entire AZFc locus on the human Y chromosome leads to male infertility. The functional roles of the individual gene families mapped to AZFc are, however, still poorly understood, since the analysis of the region is complicated by its repeated structure. We have therefore used single-nucleotide variants (SNVs) across approximately 3 Mb of the AZFc sequence to identify 17 AZFc haplotypes and have examined them for deletion of individual AZFc gene copies. We found five individuals who lacked SNVs from a large segment of DNA containing the DAZ3/DAZ4 and BPY2.2/BPY2.3 gene doublets in distal AZFc. Southern blot analyses showed that the lack of these SNVs was due to deletion of the underlying DNA segment. Typing 118 binary Y markers showed that all five individuals belonged to Y haplogroup N, and 15 of 15 independently ascertained men in haplogroup N carried a similar deletion. Haplogroup N is known to be common and widespread in Europe and Asia, and there is no indication of reduced fertility in men with this Y chromosome. We therefore conclude that a common variant of the human Y chromosome lacks the DAZ3/DAZ4 and BPY2.2/BPY2.3 doublets in distal AZFc and thus that these genes cannot be required for male fertility; the gene content of the AZFc locus is likely to be genetically redundant. Furthermore, the observed deletions cannot be derived from the GenBank reference sequence by a single recombination event; an origin by homologous recombination from such a sequence organization must be preceded by an inversion event. These data confirm the expectation that the human Y chromosome sequence and gene complement may differ substantially between individuals and more variations are to be expected in different Y chromosomal haplogroups.

Chromosome Mapping↗

Complete Dipolar Decoupling of 13C and Its Use in Two-Dimensional Double-Quantum Solid-State NMR for Determining Polymer Conformations

A multiple-pulse technique for complete dipolar decoupling of directly bonded 13C-labeled sites is described. It achieves significant spectral simplifications in a recently introduced two-dimensional double-quantum solid-state NMR experiment for determining torsion angles. Both homonuclear and heteronuclear dipolar couplings are removed by combining a 13C multiple-pulse sequence with continuous-wave irradiation on the protons. The 13C sequence has a fundamental 10-pulse cycle which is a significantly modified magic-sandwich-echo sequence. The crucial heteronuclear decoupling is achieved by breaking the 360 degrees "inner" pulses in the magic sandwich into 90 degrees pulses and spacing them by 1H 360 degrees pulse lengths. Spectral artifacts typical of multiple-pulse sequences are eliminated by phase shifts between cycles. In contrast to many other multiple-pulse decoupling sequences, the long window in the cycle is the dwell time and can be longer than the inverse dipolar coupling, which makes the sequence practical for direct detection even with long pulse ring-down times. A modification of the sequence to scale the chemical shift and increase the effective spectral width is also presented. The 1D and double-quantum 2D experiments are demonstrated on polyethylene with 4% 13C-13C spin pairs. The potential of this approach for distinguishing segmental conformations is illustrated by spectral simulations of the two-dimensional ridge patterns that correlate double-quantum and single-quantum chemical-shift anisotropies. Copyright 1998 Academic Press.

Journal Article↗

A 189-bp repeat region within the human cytomegalovirus replication origin contains a sequence dispensable but irreplaceable with other sequences.

The human cytomegalovirus (HCMV) replication origin exhibits a strain-dependent difference in the number of copies of a 189-bp region: the AD169 and Towne strains contain one and three copies of the region, respectively. A nearly complete deletion of the 189-bp repeat region of the Towne strain does not eliminate the origin's ability to initiate DNA synthesis. Here we report that the replication ability of the HCMV replication origin in infected cells disappeared after replacements of an internal sequence (152 bp) of the 189-bp repeat region with lambda DNA of identical and different lengths as well as after introduction of multiple nucleotide substitutions within the 152-bp internal sequence of the 189-bp repeat. In contrast, a variation in the copy number of 189-bp region (either one or two copies) or an inversion of the 152-bp internal sequence of the 189-bp repeat maintained replication abilities similar to those of the wild-type origin of the Towne strain. These results indicate that the 189-bp repeat region within the HCMV replication origin is not just a dispensable spacer sequence but instead contains an irreplaceable sequence that may play a supporting role in HCMV DNA replication.

Amino Acid Substitution↗

Kinetic studies of the modulation of ada promoter activity by upstream elements.

We have determined the kinetics of initiation of transcription of the wild-type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M-1 and 1.7 x 10(-2) s-1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild-type sequence with an up-mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (-0.05), leading to a 12-fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

MR imaging of the ankle at 3 Tesla and 1.5 Tesla: protocol optimization and application to cartilage, ligament and tendon pathology in cadaver specimens.

The objective of this study was to optimize ankle joint MR imaging in volunteers at 1.5 Tesla (T) and 3.0 T, and to compare these optimized sequences concerning image quality and performance in assessing cartilage, ligament and tendon pathology in fresh human cadaver specimens. Initially our clinical ankle protocol consisting of T1-weighted (-w), fat-saturated (fs) T2-w, and short tau inversion-recovery fast spinecho (FSE) sequences was optimized at 1.5 T and 3.0 T by two radiologists. For dedicated cartilage imaging, fs-intermediate (IM)-w FSE, fs spoiled gradient echo, and balanced free-precession steady-state sequences were optimized. Using the optimized sequences, thirteen cadaver ankle joints were imaged. Four radiologists independently assessed these images concerning image quality and pathology. All radiologists consistently rated image quality higher at 3.0 T (all sequences p<0.05). For detecting cartilage pathology, diagnostic performance was significantly higher at 3.0 T (ROC-values up to 0.93 vs. 0.77; p<0.05); the fs-IM FSE sequence showed highest values among the different sequences. Average sensitivity for detecting tendon pathology was 63% at 3.0 T vs. 41% at 1.5 T and was significantly higher at 3.0 T for 2 out of 4 radiologists (p<0.05). Compared to 1.5 T, imaging of the ankle joint at 3.0 T significantly improved image quality and diagnostic performance in assessing cartilage pathology.

Aged↗

Molecular cloning and localization of an abundant novel protein of Plasmodium berghei.

Screening of Plasmodium berghei genomic libraries using DNA insert corresponding to the 3' half of P. falciparum 70-kDa heat shock protein gene identified several abundant clones which represent a novel gene in the parasite. The complete sequence was obtained using an approach based on inverse polymerase chain reaction. Analysis of the deduced amino acid sequence revealed the presence of 19 imperfect repeats of the sequence Gly-Gly-Met-Pro toward the carboxy terminus. Except for the similar sequence repeated seven times in the malarial 70-kDa heat shock protein, the sequence of the cloned gene product is very different. Moreover, the sequence also revealed acidic and basic domains in the protein which are more than 60% similar in sequence to functional domains present in numerous DNA binding transcription factors. A 56-kDa protein was identified by immunoprecipitation from labeled P. berghei extract using antisera raised in mice against gene products expressed in Escherichia coli. The protein is present in all the different life cycle stages of the parasites as revealed by immunoelectron microscopy.

Amino Acid Sequence↗

GS-Aligner: a novel tool for aligning genomic sequences using bit-level operations.

A novel algorithm, GS-Aligner, that uses bit-level operations was developed for aligning genomic sequences. GS-Aligner is efficient in terms of both time and space for aligning two very long genomic sequences and for identifying genomic rearrangements such as translocations and inversions. It is suitable for aligning fairly divergent sequences such as human and mouse genomic sequences. It consists of several efficient components: bit-level coding, search for matching segments between the two sequences as alignment anchors, longest increasing subsequence (LIS), and optimal local alignment. Efforts have been made to reduce the execution time of the program to make it truly practical for aligning very long sequences. Empirical tests suggest that for relatively divergent sequences such as sequences from different mammalian orders or from a mammal and a nonmammalian vertebrate GS-Aligner performs better than existing methods. The program and data can be downloaded from http://pondside.uchicago.edu/~lilab/ and http://webcollab.iis.sinica.edu.tw/~biocom.

Algorithms↗