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[Molecular analysis of a double hyperunstable system y(2ns)sc(me)].

Molecular analysis of hyperunstable mutations in the adjacent yellow and scute loci was performed in a Drosophila melanogaster strain y2ns scme obtained earlier. The y2ns scme combination originated from the original y+ns mutation associated with the insertion into the regulatory region of the yellow gene of a chimeric sequence flanked by deletion copies of the P element. The new combination of mutations resulted from an inversion between P-element copies located in the adjacent yellow and scute loci. The inversion was flanked by two P-element copies at one side and one copy at the other side. All three P-element copies had various internal deletions and, therefore, could not code for active transposase. An introduction of the delta (2-3) gene for transposase induced mutagenesis in the y2ns scme strain. Most mutations observed were associated with either excision of a single P-element copy or reinversion resulting from the recombination between the ends of P elements located in the yellow and scute genes. Genetic analysis showed that the alleles resulting from the recombination at a high frequency reverted to the original state.

Alleles↗

RET rearrangements in radiation-induced papillary thyroid carcinomas: high prevalence of topoisomerase I sites at breakpoints and microhomology-mediated end joining in ELE1 and RET chimeric genes.

Children exposed to radioactive iodine after the Chernobyl reactor accident frequently developed papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase gene. Various types of RET rearrangements have been described. More than 90% of PTC with RET rearrangement exhibit a PTC1 or PTC3 type of rearrangement with an inversion of the H4 or ELE1 gene, respectively, on chromosome 10. To obtain closer insight into the mechanisms underlying PTC3 inversions, we analyzed the genomic breakpoints of 22 reciprocal and 4 nonreciprocal ELE1 and RET rearrangements in 26 post-Chernobyl tumor samples. In contrast to previous assumptions, an accumulation of breakpoints at the two Alu elements in the ELE1 sequence was not observed. Instead, breakpoints are distributed in the affected introns of both genes without significant clustering. When compared to the corresponding wildtype sequences, the majority of breakpoints (92%) do not contain larger deletions or insertions. Most remarkably, at least one topoisomerase I site was found exactly at or in close vicinity to all breakpoints, indicating a potential role for this enzyme in the formation of DNA strand breaks and/or ELE1 and RET inversions. The presence of short regions of sequence homology (microhomologies) and short direct and inverted repeats at the majority of breakpoints furthermore indicates a nonhomologous DNA end-joining mechanism in the formation of chimeric ELE1/Ret and Ret/ELE1 genes.

Base Sequence↗

Transposable genetic elements and plasmid evolution.

Transposable elements of DNA that are structurally defined and genetically discrete units seem to have an important role in the evolution of bacterial plasmids. Recombination occurring at the termini of such elements can result in the joining together of unrelated DNA segments that lack extensive nucleotide sequence homology. In addition, transposable elements serve as novel biological switches capable of turning on and off the expression of nearby genes as a consequence of their insertion into or excision from plasmid genomes.

Alleles↗

Techniques for manipulating chromosomal rearrangements and their application to Drosophila melanogaster. II. Translocations.

Translocations have long been valued for their segregational properties. This paper extends the utility of translocations by considering recombinational derivatives of pairs of simple reciprocal translocations. Three major derivative structures are noted. One of these derivatives is suitable for use in half-tetrad experiments. A second should find use in recombining markers with translocation breakpoints. The third is an insertional-tandem duplication: it has a section of one chromosome inserted into a heterologue with a section of the latter chromosome tandemly repeated about the breaks of the insert. All of these structures are contained in "constellations" of chromosomes that regularly segregate aneuploid-1 products (informationally equivalent to nonrecombinant adjacent-1 segregants) for one of the parental translocations but do not segregate euploid products. This is in contrast to the parental T1/T2 constellations which segregate euploid products but not aneuploid-1 products. Methods are described for selecting translocation recombinants on the basis of this dichotomy. Several examples of translocation recombinants have been recovered with these techniques, and the recombination frequencies seem to be consistent with those observed for crossovers between inversion breakpoints. Recombinant chromosomes tend to disjoin, but it is observed that the tendency may vary according to the region involved in the recombination, and it is suggested that this difference reflects a difference in chiasmata terminalization times. Special consideration is given to insertional-tandem duplications. Large insertional-tandem duplications are useful in gene dosage studies and other experiments that require an insert from one chromosome to another. Large duplications can be deleted to form small duplications. To generate a small insert for a specified region, it is only necessary to have one translocation with a breakpoint flanking the region of interest. The second translocation can have a breakpoint quite far from the region: an insertional-tandem duplication containing the region that has one closely flanking breakpoint can be deleted to create a smaller duplication that has two closely flanking breakpoints.

Aneuploidy↗

Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP.

Among recombinant phages carrying integrated hepatitis B virus (HBV) DNA sequences cloned from the human hepatoma-derived cell line huSP, one clone, lambda hu-489, revealed some unusual features. The 2.25-kilobase Eco D fragment from the insert of this clone hybridized to the HBV DNA probe only and its nucleotide sequence was determined. The viral sequence, as well as a cellular flanking sequence, showed extensive rearrangement accompanied by inverted repetition. The Eco D fragment contained HBV DNA from the 5'-end region of gene S to the middle of gene X, followed by a long cellular flanking sequence. Moreover, a part of gene X was found inversely repeated at the head of the same gene S in a head-to-head configuration truncated by the same cellular sequence. Therefore, the same junction sequence of viral DNA and the cellular sequence was found at two different sites in the Eco D fragment in opposite polarities.

Base Sequence↗

Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of Drosophila melanogaster.

R2 retrotransposons insert into the rRNA-encoding units (rDNA units) that form the nucleoli of insects. We have utilized an R2 integration system in Drosophila melanogaster to study transcription of foreign sequences integrated into the R2 target site of the 28S rRNA genes. The exogenous sequences were cotranscribed at dramatically different levels which closely paralleled the level of transcription of the endogenous R1 and R2 elements. Transcription levels were inversely correlated with the number of uninserted rDNA units, variation in this number having been brought about by the R2 integration system itself. Females with as few as 20 uninserted rDNA units per X chromosome had expression levels of endogenous and exogenous insertion sequences that were 2 orders of magnitude higher than lines that contained over 80 uninserted rDNA units per chromosome. R2 insertions only 167 bp in length exhibited this range of transcriptional regulation. Analysis of transcript levels in males suggested R2 insertions on the Y chromosome are not down-regulated to the same extent as insertions on the X chromosome. These results suggest that transcription of the rDNA units can be tightly regulated, but this regulation gradually breaks down as the cell approaches the minimum number of uninserted genes needed for survival.

Animals↗

Thrombopoietin-stimulated ex vivo expansion of human bone marrow megakaryocytes.

The effect of thrombopoietin (TPO) on megakaryocytopoiesis (MKP) has been mainly studied using clonogenic assays in murine systems. In this study, we evaluated MKP in liquid culture using human bone marrow cells. While interleukin 3 (IL-3) and stem cell factor (SCF) are potent activators of TPO-stimulated MKP in the murine system, only IL-3 exhibited synergistic activity with TPO in cultures of human bone marrow. The IL-3 effect on TPO-stimulated megakaryocyte (MK) proliferation, expressed as the absolute number of MKs per seeded CD34+ cell, was more pronounced with purified CD34+ cell (8 +/- 1.6 SE versus 2.8 +/- 0.7 SE in the presence and absence of IL-3, respectively) than with mononuclear cells (MNC) (16 +/- 2.8 SE versus 11 +/- 2.0 SE). This effect of IL-3 on TPO-stimulated MK proliferation was due to a general proliferation of all cell types since the relative frequency of MKs (32.1 +/- 3 SE and 55.8 +/- 3 SE in MNC and CD34+ cells, respectively) was not affected by IL-3. The effect of TPO alone, TPO + IL-3, TPO + SCF, and TPO + IL-3 + SCF on MK proliferation was examined in MNC and CD34+ cultures. Greater numbers of MK per seeded CD34+ were observed in MNC compared to CD34+ cultures under all conditions except when TPO was added with both IL-3 and SCF. The enhancing effect of MNC was also observed on MK ploidy in the presence of TPO and IL-3. While proliferation and ploidy increase with TPO concentration in the murine system, they are inversely related in the human system. A significant 2.5-fold enhancement of TPO-induced MK proliferation was observed when purified CD34+ cells were cultured in inserts separated from human bone marrow stroma, indicating that soluble stimulatory factors are released from the stroma. These observations will be useful for ex vivo expansion of MKs to treat post-transplant or chemotherapy-associated thrombocytopenia.

Animals↗

The scars of newborn intensive care.

Many techniques used in neonatal intensive care are invasive and the risk of producing skin damage is high. To investigate this, 100 consecutive survivors of neonatal intensive care (gestation 26-42 weeks, median 32) were examined in detail by a single observer at 16-29 months of age, and the scar severity, site and likely cause noted. Scarring was present in all infants although it was usually trivial. The total number of scars was inversely related to gestational age and directly related to the duration of intensive care. Eleven children had cosmetically or functionally significant lesions caused by chest drain insertion, extravasation of intravenous fluid or skin stripping by adhesive tape. To reduce the frequency and severity of skin damage, neonatal staff need to be aware that many routine procedures may lead to long term scarring. In particular, more careful wound closure after chest drain removal is needed.

Cicatrix↗

Molecular structure of canine LINE-1 elements in canine transmissible venereal tumor.

We determined the 4251-bp sequence of open reading frame 2 (ORF2) of canine LINE-1 retroposon that encodes 1275 amino acids. The truncated LINE-1 inserts associated with transmissible venereal tumor (TVT) of dogs contained the 1378-bp LINE-1 insert (TVT-LINE) flanked by 10-bp direct repeats upstream to c-myc gene. The TVT-LINE elements were composed of 416 bp inverse sequences homologous to the complementary strand of the LINE-1, a 5-bp deletion and 962-bp sequences homologous to the 3' region of the LINE-1.

Animals↗

Ancient haplotypes resulting from extensive molecular rearrangements in the wheat A genome have been maintained in species of three different ploidy levels.

Plant genomes, in particular grass genomes, evolve very rapidly. The closely related A genomes of diploid, tetraploid, and hexaploid wheat are derived from a common ancestor that lived <3 million years ago and represent a good model to study molecular mechanisms involved in such rapid evolution. We have sequenced and compared physical contigs at the Lr10 locus on chromosome 1AS from diploid (211 kb), tetraploid (187 kb), and hexaploid wheat (154 kb). A maximum of 33% of the sequences were conserved between two species. The sequences from diploid and tetraploid wheat shared all of the genes, including Lr10 and RGA2 and define a first haplotype (H1). The 130-kb intergenic region between Lr10 and RGA2 was conserved in size despite its activity as a hot spot for transposon insertion, which resulted in >70% of sequence divergence. The hexaploid wheat sequence lacks both Lr10 and RGA2 genes and defines a second haplotype, H2, which originated from ancient and extensive rearrangements. These rearrangements included insertions of retroelements and transposons deletions, as well as unequal recombination within elements. Gene disruption in haplotype H2 was caused by a deletion and subsequent large inversion. Gene conservation between H1 haplotypes, as well as conservation of rearrangements at the origin of the H2 haplotype at three different ploidy levels indicate that the two haplotypes are ancient and had a stable gene content during evolution, whereas the intergenic regions evolved rapidly. Polyploidization during wheat evolution had no detectable consequences on the structure and evolution of the two haplotypes.

Chromosome Deletion↗

Complete inversion of the uterus during caesarean section: a case report.

A case of complete inversion of the uterus through the uterine incision at the time of Caesarean section is presented. The possible aetiological factors were a short cord and fundal insertion of the placenta. Disappearance of the lower segment incision and the raw appearance of the endometrial surface of a completely inverted uterus posed a diagnostic dilemma. Prompt understanding and immediate repositioning by a simple technique prevented any further complications. This rare complication of Caesarean section and its easy principles of management are described for the benefit of an unwary obstetrician.

Adult↗

Low-field magnetic resonance imaging of early subchondral cyst-like lesions in induced cranial cruciate ligament deficient dogs.

Six healthy adult male mongrel dogs underwent cranial cruciate ligament transection in the left stifle. Survey radiography of both stifles and low-field (0.064 T) MRI of the left stifle were performed preoperatively and at 2, 6, and 12 weeks postoperatively. Focal changes in signal intensity were seen with MRI in the subchondral bone of the medial tibial condyle at 2 and 6 weeks postoperatively. At 12 weeks postoperative, a cyst-like lesion was detected using MRI in the subchondral bone of the medial tibial condyle in 4 of 6 dogs and a less defined lesion at this site in the remaining 2 dogs. The cyst-like lesion was spherical in shape and showed typical characteristics of fluid with low signal intensity on T1-weighted images, high signal intensity on T2-weighted images and high signal intensity on inversion recovery images. The lesion was seen in the subchondral bone of the caudal medial and/or middle region of the tibial plateau slightly cranial to the insertion of the caudal cruciate ligament. No subchondral cysts were seen in the tibia on radiographs. Histopathologically, the tibia was characterized by a loose myxomatous phase of early subchondral cyst formation.

Animals↗

A multiresolution restoration method for cardiac SPECT imaging.

In this study we present a multiresolution based method for restoring cardiac SPECT projections. Original projections were decomposed into a set of sub-band frequency images by using analyzing functions localized in both the space and frequency domain. This representation allows a simple denoising and restoration procedure by discarding high-frequency channels and performing inversion only in low frequencies. The method was evaluated in bull's eye reconstructions of a realistic cardiac chest phantom with a custom-made liver insert and 99mTc liver-to-heart activity ratios (LHAR) of 0:1, 1.5:1, 2.5:1, and 3.5:1. The cardiac phantom in free air was used as the reference standard. Reconstructions were performed by filtered backprojection using (1) no correction; (2) restoration without attenuation correction; (3) attenuation correction without restoration; and (4) restoration and attenuation correction. The attenuation correction was carried out with the Chang's method for one iteration. Results were compared with those obtained using an optimized prereconstruction Metz filter. Quantitative analysis was performed by calculating the normalized chi-square measure and mean +/- s.d. of bull's eye counts. In reconstructions with high liver activity (LHAR > 2), attenuation correction without restoration severely distorted the polar maps due to the spill-over of liver activity into the inferior myocardial wall. Both restoration methods when combined with an attenuation correction compensated this artifact and yielded uniform polar maps similar to that of the standard reference. There was no visual or quantitative difference between the performance of Metz filtering and multiresolution restoration. However, the main advantage of the multiresolution method is that it states a more concise and straightforward approach to the restoration problem. Multiresolution based methods does not require information about the object image or optimization processes, such as in conventional nuclear medicine restoration filters. In addition, the method is easy to implement using DFT techniques and potentially can be extended to noniterative spatially shift-invariant restorations in SPECT.

Biophysical Phenomena↗

Site-specific inversion sequence of the herpes simplex virus genome: domain and structural features.

The genome of herpes simplex virus-1 consists of two covalently linked components, L and S, that invert relative to each other. The L and S components consist of unique DNA sequences bracketed by inverted repeats. The inverted repeats of the L component are designated ab and b' a' and those of the S component are designated a' c' and ca. The number of a sequences at the termini and at the L-S component junction varies from one to several copies. Insertion into the middle of the L component of a DNA fragment consisting of 156 base pairs (bp) of the b sequence, an entire a sequence of 501 bp, and 618 bp of the c sequence created a new site through which additional inversions in the genome occurred. Comparison of the nucleotide sequences of DNA fragments containing one and two a sequences defined the domain of the a sequence. The single a sequence consists of two 20-bp direct repeats (designated as DR1) bracketing a region that contains 19 tandem direct repeats of a 12-bp sequence (DR2) adjacent to three direct repeats of a 37-bp sequence (DR4), in addition to short stretches of unique sequences. The fragment with two tandem a sequences contained three copies of DR1-i.e., the intervening DR1 was shared by the two a sequences. Furthermore, one a sequence contained 22 copies of DR2 and two copies of DR4 whereas the second a sequence contained 19 copies of DR2 and two copies of DR4. These observations suggest that (i) amplification of the number of terminal and internal a sequences is the consequence of intramolecular or intermolecular recombination through DR1, (ii) the number of copies of DR2 and DR4 within the a sequence is not fixed and may vary as a consequence of unequal crossing over or slippage, and (iii) inversion results from intramolecular recombination between terminal and inverted a sequences.

Animals↗

Inverse planning for interstitial gynecologic template brachytherapy: truly anatomy-based planning.

PURPOSE: Commercially available optimization schemes generally result in an undesirable dose distribution, because of the particular shapes of tumors extending laterally from the tandem. Dose distribution is therefore manually obtained by adjusting relative dwell time values until an acceptable solution is found. The objective of this work is to present the clinical application of an inverse planning dose optimization tool for the automatic determination of source dwell time values in the treatment of interstitial gynecologic templates. METHODS AND MATERIALS: In cases where the tumor extends beyond the range of the tandem-ovoid applicator, catheters as well as the tandem are inserted into the paravaginal and parametrial region in an attempt to cover the tumor volume. CT scans of these patients are then used for CT-based dose planning. Dose distribution is obtained manually by varying the relative dwell times until adequate dose coverage is achieved. This manual planning is performed by an experienced physician. In parallel, our in-house inverse planning based on simulated annealing is used to automatically determine which of all possible dwell positions will become active and to calculate the dwell time values needed to fulfill dose constraints applied to the tumor volume and to each organ at risk. To compare the results of these planning methods, dose-volume histograms and isodose distributions were generated for the target and each organ at risk. RESULTS: This procedure has been applied for the dose planning of 12 consecutive interstitial gynecologic templates cases. For all cases, once the anatomy was contoured, the routine of inverse planning based on simulated annealing found the solution to the dose constraints within 1 min of CPU time. In comparison, manual planning took more than 45 min. The inverse planning-generated plans showed improved protection to organs at risk for the same coverage compared to manual planning. CONCLUSION: This inverse planning tool reduced the planning time significantly and produced improved plans with reduced dose to the organs at risk. Furthermore, the inverse planning approach improves the physician's control over treatment. The focus becomes the physician's prescription to the target and his or her compromise due to dose to normal structures.

Algorithms↗

Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition.

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.

Chromosome Inversion↗

The evolving genome of Salmonella enterica serovar Pullorum.

Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas. However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.

Chromosome Mapping↗

Mapping of transposable element Dissociation inserts in Brassica oleracea following plant regeneration from streptomycin selection of callus.

To investigate the potential of heterologous transposons as a gene-tagging system in broccoli (Brassica oleracea var. italica), we have introduced a Dissociation ( Ds)-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct. In three successive selfed generations of plants, there was no evidence of germinal-excision events. In a previous study, we overcame this apparent inability to produce B. oleracea plants with germinal excisions by performing a novel tissue-culture technique to select for fully green shoots from seed with somatic excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%), and molecular analysis showed that the plants contained the equivalent of a germinal-excision event. In this study, we followed the previous work by using inverse and nested PCR to generate probes of flanking genomic DNA adjacent to independently reinserted Ds elements, and these were hybridised to DNA from a double-haploid mapping population of B. oleracea. Seventeen Ds insertions and the original Ds T-DNA site have been localised, and these are spread over six (out of nine) linkage groups. Distribution of inserts show that 15 were found on a different linkage group to the original 'launch' site, and of these 11 were found to be clustered on two separate groups. Previous studies in other plant species have found that germinal excision of Ds predominantly moves to sites linked close to the donor site. However, this study shows a potential to produce plants with Ds insertion scattered over many unlinked sites.

Blotting, Southern↗