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PubMed1975

Site specific recA--independent recombination between bacterial plasmids: involvement of palindromes at the recombinational loci.

A recA-independent recombinational event is described which results in insertion of an entire plasmid genome at a unique site of another plasmid, and coincident excision of a precisely defined DNA segment originally present at the point of the insertion. The resulting recombinant molecules subsequently can undergo site-specific translocation of their component segments or inversion of their original DNA sequence orientation. The events observed entail nonreciprocal exchange of genetic material, and involve a discrete nucleotide sequences that is duplicated in rotationally symmetrical reverse orientation on plasmid DNA (i.e., inverted repeat; palindrome).

Base Sequence↗
PubMed1982

Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells.

We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescue of a latent AAV viral infection. In addition, the efficient rescue of viable AAV from the recombinant plasmid should facilitate the genetic analysis of AAV. Finally, the results of an analysis of the DNA from rescued virions indicate that an inversion of the AAV terminal sequences occurred during replication.

Cells, Cultured↗
PubMed2002

Coronary magnetic resonance angiography for assessment of the stent lumen: a phantom study.

PURPOSE: To evaluate the feasibility of visualizing the stent lumen using coronary magnetic resonance angiography in vitro. MATERIAL AND METHODS: Nineteen different coronary stents were implanted in plastic tubes with an inner diameter of 3 mm. The tubes were positioned in a plastic container filled with gel and included in a closed flow circuit (constant flow 18 cm/sec). The magnetic resonance images were obtained with a dual inversion fast spin-echo sequence. For intraluminal stent imaging, subtraction images were calculated from scans with and without flow. Subsequently, intraluminal signal properties were objectively assessed and compared. RESULTS: As a function of the stent type, various degrees of in-stent signal attenuation were observed. Tantalum stents demonstrated minimal intraluminal signal attenuation. For nitinol stents, the stent lumen could be identified, but the intraluminal signal was markedly reduced. Steel stents resulted in the most pronounced intraluminal signal voids. CONCLUSIONS: With the present technique, radiofrequency penetration into the stents is strongly influenced by the stent material. Thesefindings may have important implicationsforfuture stent design and stent imaging strategies.

Blood Vessel Prosthesis Implantation↗
PubMed2003

Optimal acquisition parameters for contrast enhanced magnetic resonance imaging after chronic myocardial infarction.

The aim of this study was to simplify the imaging of myocardial infarction based on theoretical aspects and patient variables and to define the optimal time for image acquisition. Thirteen patients with chronic myocardial infarction underwent magnetic resonance imaging. After injection of 0.2 mmol/kg body weight Gd-DTPA an inversion recovery turbo gradient echo sequence with different prepulse delays was applied every 3 to 5 minutes within an interval of 3 to 30 minutes. As parameters of investigation, the area of signal enhancement and the contrast between enhanced and nonenhanced myocardium were used. There was no influence of prepulse delay or time after contrast injection on the enhanced area. The contrast between enhanced and normal myocardium showed a peak at 6 minutes post Gd-DTPA injection and remained high. The contrast between blood and enhanced myocardium was best at 6 and 25 minutes with best intra- and interobserver variability. In conclusion, if a suitable contrast was achieved, the area of enhancement is independent of prepulse delay or imaging time. In most patients the highest contrast between blood, enhanced and normal myocardium is achieved 6 minutes and 25 minutes after contrast injection.

Aged↗
PubMed1996

Quantitative assessment of MRI lesion load in multiple sclerosis. A comparison of conventional spin-echo with fast fluid-attenuated inversion recovery.

In this study, we compared a fast fluid-attenuated inversion recovery (fast-FLAIR) sequence to conventional spin-echo (CSE) in the evaluation of brain MRI lesion loads of seven patients with clinically definite multiple sclerosis. Interleaved CSE (3000/20, 5 mm contiguous axial slices) and fast-FLAIR (9000/150/2200, 5 mm contiguous axial slices) sequences were performed on a 1.0 T machine. Lesions were counted consensually by two observers and then segmented independently by two other observers using a local thresholding technique, with subsequent manual editing in the case of poorly defined lesions. Four hundred and two lesions were detected in at least one of the two sequences: 128 were seen only on fast-FLAIR, 17 only on CSE. Forty-one lesions were larger on fast-FLAIR, while no lesion was considered larger on CSE. The numbers of periventricular (P = 0.05), cortical/subcortical (P = 0.02) and discrete (P = 0.05) lesions detected using fast-FLAIR were higher than those detected using CSE. The median lesion load was 7185 mm3 on CSE and 8418 mm3 on the fast-FLAIR, the average being 18% (range = 11.6-29%) higher on the fast-FLAIR images. Lesion contrast ratio was higher for lesions on the fast-FLAIR than on the CSE sequence (P < 0.0001). The percentages of poorly defined lesions which needed manual editing after the local thresholding technique was applied and the total time needed for the measurements were lower (P < 0.001) when fast-FLAIR images were used compared with CSE. This resulted in a reduced inter-rater coefficient of variation in measuring lesion volumes. Our data indicate that fast-FLAIR sequences are more sensitive than CSE in detecting multiple sclerosis lesion burden and that fast-FLAIR is a promising technique for natural history studies and clinical trials in multiple sclerosis.

Adult↗
PubMed1994

A Drosophila third chromosome Minute locus encodes a ribosomal protein.

Minutes (M) are a group of over 50 phenotypically similar Drosophila mutations widely believed to affect ribosomal protein genes. This report describes the characterization of the P element-induced M(3)95A(Plac92) mutation [allelic to M(3)95A]. This mutation can be reversed by the mobilization of the P element, demonstrating that the mutation is caused by insertion of this transposable element. The gene interrupted by insertion of the P element was cloned by use of inverse polymerase chain reaction. Nucleotide sequence analysis revealed a 70-75% identity to the human and rat ribosomal protein S3 genes, and to the Xenopus ribosomal protein S1a gene. At the amino acid level, the overall identity is approximately 78% for all three species. This is only the second time that a Minute has been demonstrated to encode a ribosomal protein.

Amino Acid Sequence↗
PubMed2002

Efficient recovery of centric heterochromatin P-element insertions in Drosophila melanogaster.

Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.

Animals↗
PubMed1986

Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.

Chromosome Deletion↗
PubMedDate not supplied

Comparison of multishot echo-planar fluid-attenuated inversion-recovery imaging with fast spin-echo fluid-attenuated inversion-recovery and T2-weighted imaging in depiction of white matter lesions.

PURPOSE: To compare a multishot echo-planar fluid-attenuated inversion-recovery (EPI-FLAIR) sequence with fast spin-echo FLAIR (F-FLAIR) and fast spin-echo T2-weighted (FSE-T2W) sequences in depiction of white matter lesions. METHODS: Thirty-five patients with various white matter lesions were included in this prospective study. Two independent readers for lesion detection (lesion size, >2 mm) compared sequences quantitatively. In 22 patients, contrast was calculated between periventricular hyperintensity (PVH) and the cerebrospinal fluid (CSF). RESULTS: EPI-FLAIR revealed more lesions than FSE-T2W (p < 0.01). However, F-FLAIR revealed more lesions than EPI-FLAIR (p < 0.01). For PVH-to-CSF contrast, EPI-FLAIR demonstrated significantly higher contrast than FSE-T2W. There were no differences in PVH-to-CSF contrast between EPI-FLAIR and F-FLAIR. CONCLUSIONS: This study shows that EPI-FLAIR has distinct advantages over FSE-T2W in the depiction of white matter lesions. Although EPI-FLAIR reduces imaging time by more than 60% relative to F-FLAIR, it cannot replace F-FLAIR for the detection of lesions in the cerebral white matter.

Aged↗
PubMed1996

An aroA mutant of Yersinia pestis is attenuated in guinea-pigs, but virulent in mice.

This study describes a PCR-based approach for the production of a rationally attenuated mutant of Yersinia pestis. Degenerate primers were used to amplify a fragment encoding 91.45% of the aroA gene of Y. pestis MP6 which was cloned into pUC18. The remainder of the gene was isolated by inverse PCR. The gene was sequenced and a restriction map was generated. The Y. pestis aroA gene had 75.9% identity with the aroA gene of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain GB using the suicide vector pGP704. A stable aro-defective mutant. Y. pestis GB aroA, was isolated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response against challenge with the virulent Y. pestis strain GB. Unusually for an aro-defective mutant, the Y. pestis aroA mutant was virulent in mice, with a median dose which induced morbidity of death similar to that of the wild-type, although time to death was significantly prolonged.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2004

Thouless-Anderson-Palmer approach for lossy compression.

We study an ill-posed linear inverse problem, where a binary sequence will be reproduced using a sparse matrix. According to the previous study, this model can theoretically provide an optimal compression scheme for an arbitrary distortion level, though the encoding procedure remains an NP-complete problem. In this paper, we focus on the consistency condition for a dynamics model of Markov-type to derive an iterative algorithm, following the steps of Thouless-Anderson-Palmer approach. Numerical results show that the algorithm can empirically saturate the theoretical limit for the sparse construction of our codes, which is also very close to the rate-distortion function.

Journal Article↗
PubMed2003

Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO(2) atmosphere.

This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the BleR (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO2 atmosphere for optimal growth. The DNA flanking the BleR insert of one of the high-CO2-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO2 atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO2-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO2-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.

Animals↗
PubMed1995

A rapid PCR-based test for the endogenous viral element ev3 of chickens.

A short fragment of chicken genomic DNA encompassing the insertion site of the endogenous avian leucosis viral element ev3 was isolated using the inverse polymerase chain reaction (inverse PCR) technique. The nucleotide sequence of the unoccupied site was used to design PCR primers that can be used to unambiguously determine the genetic status of any chicken, with respect to ev3. Screening of a small number of individuals from exotic breeds of chickens suggested that the frequency of ev3 is highly variable. The ev3 integration site shows a high degree of sequence homology with the macrophage-specific tyrosine kinase gene, bmk, in mice.

Animals↗
PubMed1994

Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis.

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.

Amino Acid Sequence↗
PubMedDate not supplied

A general method for optimizing tissue discrimination in magnetic resonance imaging.

The basic partial differential formulation for small differences has been applied to the equations for the inversion recovery and saturation recovery sequences with spin-echo rephase pulses and the spin-echo sequence itself. The result is a generalized expression which is proportional to tissue discrimination expressed as the signal-difference-to-noise ratio for a pair of tissues. The expression is a function of the fractional differences in the tissue relaxation times and proton densities, T1, T2, and n, respectively, and the sequence parameters TR and TI, or TE. From it can be derived the conditions which lead to optimum tissue discrimination for a given pulse sequence. It is also possible to predict the optimum pulse sequence to use for any pair of tissues of interest. The method requires a knowledge of the fractional differences in T1, T2, and n for both tissues.

Biophysical Phenomena↗
PubMed1984

Chronic subdural hematoma: demonstration by magnetic resonance.

The ability of magnetic resonance (MR) to identify intracranial hematomas was tested in five patients with clinical and computed tomographic signs of chronic subdural hematoma. The extracerebral collections were displayed as a zone of bright intensity using the T1-weighted inversion recovery (IR 1,500/400) sequence, reflecting the lesions' short T1 relaxation times. The collections also showed high intensity using the spin echo (SE) sequence, with a longer delay of 100 ms and 160 ms, reflecting the long T2 relaxation time. The spin echo sequence with a repetition time of 500 ms and an echo delay of 160 ms (SE 500/160) almost effaced other structures in the image, thus increasing the specificity of this pulse scheme for detection of chronic blood collections. Although in two of the five patients the subdural hematomas were in the isodense CT phase, all were easily visualized with MR.

Adult↗
PubMed1995

Nontraumatic pediatric musculoskeletal MR imaging: comparison of conventional and fast-spin-echo short inversion time inversion-recovery technique.

PURPOSE: To compare conventional short inversion time inversion-recovery (STIR) with fast spin-echo (FSE) STIR techniques to evaluate suspected nontraumatic musculoskeletal abnormalities. MATERIALS AND METHODS: Thirty STIR and FSE-STIR examinations in 26 pediatric patients with suspected nontraumatic musculoskeletal abnormalities were prospectively evaluated. Qualitative (subjective) and quantitative (five-point rank score) analyses of the images were performed. RESULTS: FSE-STIR was faster than STIR (mean, 2 minutes 25 seconds and 6 minutes 35 seconds, respectively). Fat suppression was slightly better with STIR. Image degradation due to motion was judged similar. Lesion contrast to muscle was slightly better with STIR than FSE-STIR, and lesion contrast to fat was equivalent. Qualitatively, lesion conspicuity was similar: All lesions were seen with both techniques. CONCLUSION: FSE-STIR can replace STIR when an inversion-recovery fat-suppression sequence is desired. Considerable imaging time is saved.

Adolescent↗
PubMed2005

Androgen mediation of thrombocytosis in epithelial ovarian cancer biology.

PURPOSE: Preoperative thrombocytosis (platelet count > 400 x 10(9)/L) at initial exploration for epithelial ovarian carcinoma is associated with decreased surgical cytoreducibility and poor survival. Platelets express androgen receptor (AR), which contains a polymorphic CAG trinucleotide repeat sequence of which the length inversely correlates with AR transactivation function. We hypothesized that androgen-mediated thrombocytosis promotes aggressive ovarian cancer biology. EXPERIMENTAL DESIGN: Sixty-three patients with epithelial ovarian carcinoma underwent genotype analysis of the CAG repeat polymorphism in AR. Medical records were reviewed to assess preoperative thrombocytosis, surgical findings, and survival. Data were examined using the Fisher's exact, logistic regression, and Kaplan-Meier analyses. RESULTS: AR CAG repeat lengths ranged from 8 to 27, with a median of 23. Fifteen of 63 patients (23.8%) showed preoperative thrombocytosis. Short AR allelotype (< or = 20 CAG repeats) was associated with a higher incidence of thrombocytosis (P = 0.04). The combination of short AR allelotype and thrombocytosis was the only significant factor that predicted inability to achieve optimal surgical cytoreduction (P = 0.02). Women with short AR allelotype and thrombocytosis showed statistically decreased progression-free survival (13 versus 37 months, P = 0.01) and overall survival (37 versus 65 months, P = 0.02) when compared with women with long AR allelotype and normal platelet counts. On multivariate analyses, suboptimal cytoreduction was the only significant factor predictive of disease-specific overall survival (P = 0.0002) but the combination of short AR allelotype and thrombocytosis approached statistical significance (P = 0.08). CONCLUSIONS: Androgen modulation of thrombocytosis may promote aggressive epithelial ovarian cancer biology.

Alleles↗