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Intramolecular integration within Moloney murine leukemia virus DNA.

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features.

Bacteriophage lambda↗

Large inversion in Escherichia coli K-12 1485IN between inversely oriented IS3 elements near lac and cdd.

A companion study has shown that the inversion carried by strain 1485IN has one terminus between lac and proC and the other between his and cdd of the normal strain. Starting with this mapping data, we have done molecular work demonstrating that the inversion occurred by recombination between inversely oriented two IS3 elements, one present near lac and the other near the cdd locus; i.e., the inversion is IN(is3B-is3E). Evidence supporting this conclusion includes: (i) Normal and inversion strains share two short regions with identical restriction maps. One of these regions is near lac and the other near cdd. (ii) IS3 homology was detected in each of the terminus regions of both the normal and inversion strains. (iii) The sequence on one side of the original IS3 element near lac has been exchanged with the sequence on one side of the IS3 near cdd. Whether the inversion has occurred by one event of homologous recombination between the two IS3 elements or has been caused by involvement of IS3 elements on an F factor is discussed. Another rearrangement, probably related to inversion and deletion, was detected between the IS3 and cdd of the inversion strain.

Base Sequence↗

Localized conversion at the crossover sequences in the site-specific DNA inversion system of bacteriophage P1.

The crossover sites for site-specific C inversion consist of imperfect 12 bp inverted repeats with the dinucleotide TT at the center of symmetry. The phage P1 Cin recombinase acts not only at these cix sites but also less efficiently at cix-related sequences called quasi-cix sites, cixQ. When cixQ contains a central dinucleotide TT, crossover occurs in vivo at the 2 bp sequence TT in the normal and the quasi-cix sites. If cixQ carries only one T residue, inversion-associated localized conversion can occur at the mismatched position within the 2 bp sequence. The results indicate that Cin generates 2 bp staggered cuts in vivo and that reciprocal strand exchanges occur at these 2 bp crossover sequences.

Chromosome Inversion↗

[Value of turbo-spin-echo sequences in cerebral magnetic resonance tomography in children].

AIM: The value of turbo-spin-echo (TSE) sequences was compared with conventional spin-echo (SE) and inversion-recovery (IR) sequences for cerebral MRT in 70 children at 0.5 T and 1.5 T. In addition we evaluated whether proton weighted sequences (PD) were diagnostically important and in what proportion of cases. METHOD: Conventional T1 and T2 weighted SE and T2 weighted TSE sequences were used in all children. An IR sequence was performed in 39 patients. The various sequences were analysed semiquantitatively with regard to image quality, artifacts and the demonstration of normal and anatomical structures and pathological findings. RESULTS: By any criteria, TSE sequences were superior to conventional T2 weighted SE sequences at 0.5T and 1.5 T, requiring a shorter examination time (35-53%). In 8.6% the pathological finding was best seen on PD-SE sequences (5 glial scars, 1 tumor). CONCLUSION: Although TSE sequences are better than T2 SE sequences with regard to image quality and the demonstration of abnormalities, conventional double-echo SE sequences (with PD and T2 weighted images) cannot be entirely replaced by T2 weighted TSE sequences in children.

Adolescent↗

Intraorbital optic nerve signal hyperintensity on magnetic resonance imaging sequences in perioperative hypotensive ischemic optic neuropathy.

A 61-year-old man experienced severe bilateral posterior ischemic optic neuropathy after cardiac bypass surgery. Routine magnetic resonance imaging sequences were normal, but diffusion-weighted and fluid-attenuated inversion recovery (FLAIR) sequences showed abnormal hyperintensity within both intra-orbital optic nerves. This imaging abnormality has not been previously reported in this setting.

Basal Ganglia↗

Fast short-tau inversion-recovery MR imaging.

To enhance the versatility of the short-tau inversion-recovery (STIR) sequences, the authors determined a range of repetition time (TR) and inversion time (TI) combinations that suppress signal intensity from fat by study of both patient and phantom images. To make fast STIR images, variations in the following pulsing conditions were studied with use of an interactive computer program: decreasing the TR, limiting the number of excitations, and limiting the number of phase-encoding steps. The authors found that (a) STIR imaging need not be time consuming, (b) fat suppression can be accomplished at shorter TR by using shorter TI, and (c) short-TR fast STIR imaging is sensitive to enhancement with gadopentetate dimeglumine.

Humans↗

Magnetic resonance imaging and hydatidiform mole.

Five women with complete and partial hydatidiform mole were examined by magnetic resonance imaging (MRI). Spin-echo pulse sequences (T2 weighted) were found to provide excellent resolution, while inversion-recovery pulse sequences (T1 weighted) gave better pathological detail. The myometrium was clearly seen on each patient and no invasion by the tumour detected. This corresponded to the histological findings. The potential uses of magnetic resonance in the diagnosis of primary and recurrent trophoblastic disease and its role in management are discussed.

Female↗

Assessment of the extent of metastases of gastrointestinal carcinoid tumors using whole-body PET, CT, MRI, PET/CT and PET/MRI.

OBJECTIVE: To assess the diagnostic value of whole-body positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and the fusion of PET and CT (PET/CT) and PET and MRI (PET/MRI) in the detection of metastatic disease of gastrointestinal carcinoid tumors. MATERIALS AND METHODS: This prospective study included six patients with extensive nonresectable metastases of gastrointestinal carcinoid tumors which were consecutively examined from the base of the skull to the proximal thigh using a state-of-the-art PET/CT scanner and a 1.5 Tesla whole-body MRI scanner. PET was performed with a carbohydrated F-18-labeled somatostatin-receptor ligand ([ superset18 F]FP-Gluc-TOCA) using a Pico-3D PET scanner. CT was performed with a venous-dominant contrast-enhanced phase using a 16-slice CT scanner. MRI was performed with a coronal T2-weighted Half-Fourier Acquired Single-Shot Turbo Spin Echo (HASTE) sequence, a coronal T2-weighted Turbo-Short Tau Inversion-Recovery (STIR) sequence, a coronal T1-weighted Turbo Spin Echo (TSE) sequence and a high resolution axial T2-weighted TSE sequence. The data sets from PET and CT were fused automatically. The PET and MRI data sets were fused manually. Lesions were rated as metastases if they were not clearly identified as benign lesions according to standard radiological criteria. RESULTS: For PET, CT, MRI, PET/CT, and PET/MRI, the lesion-by-lesion based analysis showed an overall detection rate for liver metastases (n = 391) of 49.9% (P<.001), 37.1% (P<.001), 98.2%, 50.9% (P<.001) and 100%, for lymph node metastases (n = 37) of 91.9%, 83.8%, 64.9%, 100% and 97.3% and for osseous metastases (n = 12) of 100%, 8.3% (P<.005), 66.7%, 100% and 100%. CONCLUSIONS: PET as single modality revealed the most lymph node and osseous metastases. MRI as single modality revealed the most liver metastases. The combination of molecular/metabolic with anatomical/ morphological information improves the diagnostic accuracy for the detection of metastases in comparison to the single modalities. Whole-body PET/MRI is a very promising diagnostic modality for oncological imaging due to the missing radiation exposure and the high soft tissue resolution of MRI in contrast to CT.

Aged↗

A comparison of MR imaging with fast-FLAIR, HASTE-FLAIR, and EPI-FLAIR sequences in the assessment of patients with multiple sclerosis.

BACKGROUND AND PURPOSE: Fast fluid-attenuated inversion-recovery (FLAIR) sequences are sensitive for detecting lesions in patients with multiple sclerosis (MS). More rapid fast-FLAIR imaging of the brain can be achieved by the concomitant use of half-Fourier acquisition single-shot turbo spin-echo (HASTE-FLAIR) and echo-planar imaging (EPI-FLAIR). The present study was performed in a large cohort of subjects to assess and compare the number and volume of brain lesions detected by the fast-FLAIR, HASTE-FLAIR, and EPI-FLAIR sequences in patients with MS. METHODS: Fast-FLAIR, HASTE-FLAIR, and EPI-FLAIR sequences were obtained from 46 consecutive MS patients. Lesions seen on each type of sequence were counted and classified by consensus by two observers. Lesion volumes were measured using a semiautomated segmentation technique based on local thresholding. RESULTS: The quality of the fast-FLAIR images was significantly better than that of HASTE-FLAIR and EPI-FLAIR images. Fast-FLAIR revealed significantly more lesions and higher lesion volumes than did HASTE-FLAIR and EPI-FLAIR. A similar number of large lesions was detected by the three sequences, but HASTE-FLAIR and EPI-FLAIR showed significantly fewer small and intermediate lesions than did fast-FLAIR. The number of lesions seen on HASTE-FLAIR and EPI-FLAIR images was similar. CONCLUSION: HASTE-FLAIR and EPI-FLAIR sequences revealed as many large MS lesions as fast-FLAIR. Because their acquisition times are only a fraction of that needed for fast-FLAIR sequences, they may be useful for making a rapid diagnosis of MS in uncooperative patients. Their reduced ability to detect smaller lesions indicates that they should not be used as a routine approach to imaging patients with MS.

Adult↗

Interleukin-1 alpha gene intron containing variable repeat region coding for the SP1 transcription factor recognition sequence is polymorphic.

Interleukin-1 alpha (IL-1 alpha) is a cytokine produced by a number of cell types including macrophages, fibroblasts, keratinocytes, and mesangial cells. We were interested in identifying a DNA restriction fragment length polymorphism (RFLP) for the IL-1 alpha gene for use in studies of genetic alteration in various human cancers. Human genomic DNA from 32 unrelated individuals was digested with various restriction enzymes, alone and in combination, and subjected to Southern blot analysis. Hybridization to 32P-labeled IL-1 alpha cDNA revealed an insertion-deletion-type polymorphic pattern. After digestion with RsaI, insertion-deletion-type polymorphic bands with sizes of 3.4 kb, 3.1 kb, and 2.8 kb and one invariant band of 0.8 kb were observed. These three alleles, designated A1, A2, and A3, had relative frequencies of 0.18, 0.06, and 0.78 with heterozygosity observed in 38% of the unrelated individuals studied. Evaluation of nine related individuals for this RsaI polymorphism was consistent with a Mendelian inheritance. Comparison of restriction patterns following Southern analysis of DNA digested with several different enzymes showed that the polymorphic region resides within the sixth intron. Furthermore, this RFLP results from a variable length region containing multiple copies of a recognition sequence for SP1, an imperfect copy of viral enhancer elements, and an inverse and complementary sequence of the glucocorticoid receptor binding site. The identified polymorphism may be of value in analyses of chromosome 2 and may help to elucidate mechanisms by which IL-1 alpha transcription is regulated.

Binding Sites↗

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae.

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than three-fold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.

Animals↗

Transposon 10 promoted deletions and inversions in the transfer genes of R100-1.

Spontaneous tetracycline-sensitive, transfer-deficient mutants of R100-1 were selected and analysed by genetic complementation tests and with the restriction endonuclease EcoR1. While some of the Tets Tra- mutants were caused by a single deletion event which removed the Tetr genes and extended into the neighbouring transfer genes, other mutants were the result of the deletion of the Tetr genes within Tn10 which was accompanied by an inversion of adjacent DNA sequences. A clustering of deletion and inversion endpoints occurred in the traA gene. Some of the transfer genes of R100-1 were assigned to EcoR1 fragments.

Chromosome Deletion↗

Nucleotide sequence of the thioredoxin gene from Escherichia coli.

The nucleotide sequence of the thioredoxin gene from Escherichia coli was determined. The structural gene was identified on a cloned 3-kb PvuII fragment by hybridization with a synthetic oligodeoxyribonucleotide corresponding to a part of the amino acid sequence of thioredoxin. Restriction-enzyme fragments were used as templates in the dideoxy sequence method, directly and after subcloning into M13mp8. A segment of 450 nucleotides was determined using both strands, alternatively, without extensive overlaps. The sequence contains the thioredoxin coding region, a potential ribosome-binding site, and a putative promotor region. The predicted amino acid sequence differs by two inversions from the previously given thioredoxin sequence. The revised sequence is presented and the results further show that thioredoxins from E. coli B and K12 are identical.

Amino Acid Sequence↗

Soft tissue twisting injuries of the knee.

OBJECTIVE: Twisting injuries occur as a result of differential motion of different tissue types in injuries with some rotational force. These injuries are well described in brain injuries but, to our knowledge, have not been described in the musculoskeletal literature. We correlated the clinical examination and MR findings of 20 patients with twisting injuries of the soft tissues around the knee. DESIGN AND PATIENTS: We prospectively followed the clinical courses of 20 patients with knee injuries who had clinical histories and MR findings to suggest twisting injuries of the subcutaneous tissues. Patients with associated internal derangement of the knee (i.e., meniscal tears, ligamentous or bone injuries) were excluded from this study. MR findings to suggest twisting injuries included linear areas of abnormal dark signal on T1-weighted sequences and abnormal bright signal on T2-weighted or short tau inversion recovery (STIR) sequences and/or signal to suggest hemorrhage within the subcutaneous tissues. These MR criteria were adapted from those established for indirect musculotendinous junction injuries. RESULTS: All 20 patients presented with considerable pain that suggested internal derangement on physical examination by the referring orthopedic surgeons. All presented with injuries associated with rotational force. The patients were placed on a course of protected weight-bearing of the affected extremity for 4 weeks. All patients had pain relief by clinical examination after this period of protected weight-bearing. CONCLUSIONS: Twisting injuries of the soft tissues can result in considerable pain that can be confused with internal derangement of the knee on physical examination. Soft tissue twisting injuries need to be recognized on MR examinations as they may be the cause of the patient's pain despite no MR evidence of internal derangement of the knee. The demonstration of soft tissue twisting injuries in a patient with severe knee pain but no documented internal derangement on MR examination may allow the orthopedic surgeon to elect for a trial of conservative nonsurgical management.

Adult↗

The transient processes in multi-pulse nitrogen-14 NQR.

It has been demonstrated that transient processes, observed in a single crystal of NaNO2 acted upon by pulse sequences MW-2 and MW-4 and their modifications with 180 degrees flip angle of the pulses (Solid State Nucl. Magn. Resonance 10 (1997) 63; Sov. Phys.-JETP 88(5) (1999) 1580), which manifest themselves in the oscillating form of the NQR signals envelope, can be explained in the frames of a two-particle model. It has been proved that the nature of echo signals in the effective field of multi-pulse sequences received by the inversion of the phase of the sequence pulses or by introducing an additional 180 degrees pulse is connected with re-focusing of accumulated digressions of the flip angle from the ideal 180 degrees pulse. Experimental results of observing single and multiple echoes in a number of powdered nitrogenated substances in the effective field of various sequences at room temperature have been presented.

Journal Article↗

Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions.

Three random translational beta-glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.

Amino Acid Sequence↗

Alpha beta T-cell development is not affected by inversion of TCR beta gene enhancer sequences: polar enhancement of gene expression regardless of enhancer orientation.

V(D)J recombination and expression of the T-cell receptor beta (TCRbeta) gene are required for the development of the alphabeta T lymphocyte lineage. These processes depend on a transcriptional enhancer (Ebeta) which acts preferentially on adjacent upstream sequences, and has little impact on the 5' distal and 3' proximal regions of the TCRbeta locus. Using knock-in mice, we show that alphabeta T-cell differentiation and TCRbeta gene recombination and expression are not sensitive to the orientation of Ebeta sequences. We discuss the implication of these results regarding the mode of enhancer function at this locus during T lymphocyte development.

Animals↗

Germline transformation of the butterfly Bicyclus anynana.

Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.

Animals↗