Identical Insertion Research Articles

Abstract 4350: Clonal selection and parallel evolution during leptomeningeal dissemination of human and mouse medulloblastoma result in bicompartmental disease

Abstract Medulloblastoma metastasizes through the CSF in the leptomeningeal space to cover the brain and spinal cord. Mechanisms driving metastasis are currently unknown. It had been assumed that the primary tumor and its metastases share very similar biology. The Sleeping Beauty (SB) system is a novel functional genomics tool for cancer gene discovery. Ptch +/− mice develop localized medulloblastoma in 15% of cases, while ≈100% of Math1-SB11, SB transposon donor, Ptch +/− mice develop metastatic medulloblastoma. We sequenced over 158,000 insertion sites (Roche 454) from a series of >140 SB-induced primary medulloblastomas, as well as matched spinal and frontal lobe leptomeningeal metastases. Statistical analysis identified 359 commonly inserted genes (CIGs) in the primary tumors and 285 CIGs in the leptomeningeal metastases. Although primary tumors and their metastases always demonstrate a common transformed ancestor through sharing of identical clonal insertion sites, less than 10% of CIGs were found in both primary tumor and matched metastases. Matched spinal and frontal lobe metastases shared identical clonal insertions that were very highly subclonal in the primary tumor, suggesting that leptomeningeal dissemination arises only once, or from a single small subclonal population in the primary tumor. These data support the clonal selection model of metastasis, and suggest that MET-CIG insertions are acting as metastasis virulence genes. Similarity between metastases supports a model in which medulloblastoma is a bicompartmental disease (primary vs. metastases) that arises through parallel evolution in the two compartments. We validated our mouse model through copy number profiling of three matched trios of human medullolblastoma (primary and two metastases). In each case we demonstrate highly clonal regions of chromosomal gain/loss that are present in both metastatic samples, but are not apparent in the matched primary tumor. Similarly, profiling of promotor CpG island methylation in human primary medulloblastomas and matched metastases shows that within a given child the metastases are very similar to each other, but distinct from the primary tumor. PCR amplification was used to demonstrate a clonal 179 Kb deletion in a pair of human metastases, which was present in an extremely small subclone of the primary tumor, strongly supporting the clonal selection model during dissemination of human medulloblastoma. Our results support a model in which individual medulloblastomas metastases are similar to each other, but distinct from the primary tumor. This ‘bicompartmental model’ suggests a proximate reason for failure of current therapies, and that future clinical trials may need to address each compartment individually. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4350.

Investigation into the feasibility of using PRESAGE™/optical-CT dosimetry for the verification of gating treatments

This work presents an investigation into the use of PRESAGE™ dosimeters with an optical-CT scanner as a 3D dosimetry system for quantitative verification of respiratory-gated treatments. The CIRS dynamic thorax phantom was modified to incorporate a moving PRESAGE™ dosimeter-simulating respiration motion in the lungs. A simple AP/PA lung treatment plan was delivered three times to the phantom containing a different but geometrically identical PRESAGE™ insert each time. Each delivery represented a treatment scenario: static, motion (free-breathing) and gated. The dose distributions, in the three dosimeters, were digitized by the optical-CT scanner. Improved optical-CT readout yielded an increased signal-to-noise ratio by a factor of 3 and decreased reconstruction artifacts compared with prior work. Independent measurements of dose distributions were obtained in the central plane using EBT film. Dose distributions were normalized to a point corresponding to the 100% isodose region prior to the measurement of dose profiles and gamma maps. These measurements were used to quantify the agreement between measured and ECLIPSE® dose distributions. Average gamma pass rates between PRESAGE™ and EBT were >99% (criteria 3% dose difference and 1.2 mm distance-to-agreement) for all three treatments. Gamma pass rates between PRESAGE™ and ECLIPSE® 3D dose distributions showed excellent agreement for the gated treatment (100% pass rate), but poor for the motion scenario (85% pass rate). This work demonstrates the feasibility of using PRESAGE™/optical-CT 3D dosimetry to verify gating-enabled radiation treatments. The capability of the Varian gating system to compensate for motion in this treatment scenario was demonstrated.

Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3

BackgroundPlants engineered for abiotic stress tolerance may soon be commercialized. The engineering of these plants typically involves the manipulation of complex multigene networks and may therefore have a greater potential to introduce pleiotropic effects than the simple monogenic traits that currently dominate the plant biotechnology market. While research on unintended effects in transgenic plant systems has been instrumental in demonstrating the substantial equivalence of many transgenic plant systems, it is essential that such analyses be extended to transgenic plants engineered for stress tolerance. Drought-tolerant Arabidopsis thaliana were engineered through overexpression of the transcription factor ABF3 in order to investigate unintended pleiotropic effects. In order to eliminate position effects, the Cre/lox recombination system was used to create control plant lines that contain identical T-DNA insertion sites but with the ABF3 transgene excised. This additionally allowed us to determine if Cre recombinase can cause unintended effects that impact the transcriptome.ResultsMicroarray analysis of control plant lines that underwent Cre-mediated excision of the ABF3 transgene revealed only two genes that were differentially expressed in more than one plant line, suggesting that the impact of Cre recombinase on the transcriptome was minimal. In the absence of drought stress, overexpression of ABF3 had no effect on the transcriptome, but following drought stress, differences were observed in the gene expression patterns of plants overexpressing ABF3 relative to control plants. Examination of the functional distribution of the differentially expressed genes revealed strong similarity indicating that unintended pathways were not activated.ConclusionsThe action of ABF3 is tightly controlled in Arabidopsis. In the absence of drought stress, ectopic activation of drought response pathways does not occur. In response to drought stress, overexpression of ABF3 results in a reprogramming of the drought response, which is characterized by changes in the timing or strength of expression of some drought response genes, without activating any unexpected gene networks. These results illustrate that important gene networks are highly regulated in Arabidopsis and that engineering stress tolerance may not necessarily cause extensive changes to the transcriptome.

A family harboring a germ‐line N‐terminal C/EBPα mutation and development of acute myeloid leukemia with an additional somatic C‐terminal C/EBPα mutation

C/EBPalpha plays an essential role as a transcription factor in myeloid cell differentiation. Here, we describe a Japanese family in which two individuals with acute myeloid leukemia (AML) and one healthy individual had an identical 4-base pair insertion in the N-terminal region of CEBPA (350_351insCTAC), resulting in the termination at codon 107 (I68fsX107). The father and a son at diagnosis of AML had different in-frame insertion mutations in the C-terminal region of C/EBPalpha. These C-terminal mutations disappeared upon remission in both patients. Interestingly, the father showed different in-frame insertion mutations in the C-terminal CEBPA at the time of diagnosis and relapse. These data strongly suggest that the N-terminal C/EBPalpha mutation predisposes to the occurrence of a C-terminal C/EBPalpha mutation as a secondary genetic hit, causing AML.

A search for factors influencing etioplast–chloroplast transition

Chloroplast biogenesis in angiosperm plants requires the light-dependent transition from an etioplast stage. A key factor in this process is NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide. In a recent study the chloroplast outer envelope channel OEP16 was described to be involved in etioplast to chloroplast transition by forming the translocation pore for the precursor protein of PORA [Pollmann et al. (2007) Proc Natl Acad Sci USA 104:2019-2023]. This hypothesis was based on the finding that a single OEP16.1 knockout mutant in Arabidopsis thaliana was severely affected during seedling de-etiolation and PORA protein was absent in etioplasts. In contrast, in our study the identical T-DNA insertion line greened normally and showed normal etioplast to chloroplast transition, and mature PORA was present in etioplasts [Philippar et al. (2007) Proc Natl Acad Sci USA 104:678-683]. To address these conflicting results regarding the function of OEP16.1 for PORA import, we analyzed several lines segregating from the original OEP16.1 T-DNA insertion line. Thereby we can unequivocally show that the loss of OEP16.1 neither correlates with impaired PORA import nor causes the observed de-etiolation phenotype. Furthermore, we found that the mutant line contains at least 2 additional T-DNA insertions in the genes for the extracellular polygalacturonase converter AroGP1 and the plastid-localized chorismate mutase CM1. However, detailed examination of the de-etiolation phenotype and a genomewide transcriptional analysis revealed no direct influence of these genes on etioplast to chloroplast transition in Arabidopsis cotyledons.

Rapid development of PCR-based genome-specific repetitive DNA junction markers in wheat

In hexaploid wheat (Triticum aestivum L.) (AABBDD, C=17 000 Mb), repeat DNA accounts for approximately 90% of the genome, of which transposable elements (TEs) constitute 60%-80%. Despite the dynamic evolution of TEs, our previous study indicated that the majority of TEs are conserved and collinear between the homologous wheat genomes, based on identical insertion patterns. In this study, we exploited the unique and abundant TE insertion junction regions identified from diploid Aegilops tauschii to develop genome-specific repeat DNA junction markers (RJM) for use in hexaploid wheat. In this study, both BAC end and random shotgun sequences were used to search for RJM. Of the 300 RJM primer pairs tested, 269 (90%) amplified single bands from diploid Ae. tauschii. Of these 269 primer pairs, 260 (97%) amplified hexaploid wheat and 9 (3%) amplified Ae. tauschii only. Among the RJM primers that amplified hexaploid wheat, 88% were successfully assigned to individual chromosomes of the hexaploid D genome. Among the 38 RJM primers mapped on chromosome 6D, 31 (82%) were unambiguously mapped to delineated bins of the chromosome using various wheat deletion lines. Our results suggest that the unique RJM derived from the diploid D genome could facilitate genetic, physical, and radiation mapping of the hexaploid wheat D genome.

Cost-effective HRMA pre-sequence typing of clone libraries; application to phage display selection

BackgroundMethodologies like phage display selection, in vitro mutagenesis and the determination of allelic expression differences include steps where large numbers of clones need to be compared and characterised. In the current study we show that high-resolution melt curve analysis (HRMA) is a simple, cost-saving tool to quickly study clonal variation without prior nucleotide sequence knowledge.ResultsHRMA results nicely matched those obtained with ELISA and compared favourably to DNA fingerprinting of restriction digested clone insert-PCR. DNA sequence analysis confirmed that HRMA-clustered clones contained identical inserts.ConclusionUsing HRMA, analysis of up to 384 samples can be done simultaneously and will take approximately 30 minutes. Clustering of clones can be largely automated using the system's software within 2 hours. Applied to the analysis of clones obtained after phage display antibody selection, HRMA facilitated a quick overview of the overall success as well as the identification of identical clones. Our approach can be used to characterize any clone set prior to sequencing, thereby reducing sequencing costs significantly.

In vivo attenuation of recombinant murine gammaherpesvirus 68 (MHV-68) is due to the expression and immunogenicity but not to the insertion of foreign sequences

Recombinant herpesviruses are increasingly utilized to study herpesvirus biology. For recombinant viruses carrying insertions of foreign sequences, attenuated phenotypes in vivo have been frequently observed. In most cases, the underlying mechanisms were not clear or have not been investigated. In this study, we used a recombinant murine gammaherpesvirus 68 (MHV-68), carrying a cassette for the expression of the non-structural protein NS3 of Hepatitis C virus (MHV-68-NS3), to systematically address the question whether the insertion of a defined foreign sequence (NS3) interferes with the biological properties of the recombinant virus in vivo, and to analyze the underlying mechanism. We show that while MHV-68-NS3 is attenuated in vivo, recombinant MHV-68 carrying identical genomic inserts but unable to express the NS3 protein, are not attenuated. Moreover, we provide evidence that the attenuated phenotype of MHV-68-NS3 is caused by the immune response. Our findings are important for the in vivo use of recombinant MHV-68 carrying insertions of marker genes, reporter genes or genes of model antigens. They are also relevant for the potential application of MHV-68 as gene delivery vector.

Transgene-Induced Gene Silencing Is Not Affected by a Change in Ploidy Level

BackgroundWhole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids) of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species) are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana.Methodology/Principal FindingsDiploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS) inverted repeat (hairpin) constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA.Conclusions/SignificanceOur results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Chronic Myeloid Leukemia Patients Undergoing Kinase Inhibitor Therapy

Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia (CML), a minority of patients acquire mutations in the BCR-ABL kinase domain, resulting in imatinib resistance. The spectrum of kinase domain mutations discovered to date is quite heterogeneous, consisting almost exclusively of single nucleotide substitutions affecting key amino acids that regulate drug binding or BCR-ABL function. Here, we describe an alternative kinase domain insertion/truncation mutation in three CML patients undergoing kinase inhibitor therapy. In each of these patients, direct DNA sequencing of BCR-ABL RT-PCR products revealed that the same 35 nucleotides from ABL intron 8 had been inserted at the normal exon 8 to 9 splice junction. This 35-bp intronic sequence was flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion created a premature translational stop codon after 10 intron-encoded amino acids (amino acid 484). This resulted in truncation of 653 C-terminal amino acids, which included part of the kinase domain and the entire "last exon" region. These findings demonstrate that kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy.

Novel Group I Introns Encoding a Putative Homing Endonuclease in the Mitochondrial cox1 Gene of Scleractinian Corals

Analyses of mitochondrial sequences revealed the existence of a group I intron in the cytochrome oxidase subunit 1 (cox1) gene in 13 of 41 genera (20 out of 73 species) of corals conventionally assigned to the suborder Faviina. With one exception, phylogenies of the coral cox1 gene and its intron were concordant, suggesting at most two insertions and many subsequent losses. The coral introns were inferred to encode a putative homing endonuclease with a LAGLI-DADG motif as reported for the cox1 group I intron in the sea anemone Metridium senile. However, the coral and sea anemone cox1 group I introns differed in several aspects, such as the intron insertion site and sequence length. The coral cox1 introns most closely resemble the mitochondrial cox1 group I introns of a sponge species, which also has the same insertion site. The coral introns are also more similar to the introns of several fungal species than to that of the sea anemone (although the insertion site differs in the fungi). This suggests either a horizontal transfer between a sponge and a coral or independent transfers from a similar fungal donor (perhaps one with an identical insertion site that has not yet been discovered). The common occurrence of this intron in corals strengthens the evidence for an elevated abundance of group I introns in the mitochondria of anthozoans.

AtPTR3, a wound-induced peptide transporter needed for defence against virulent bacterial pathogens in Arabidopsis

Mutation in the wound-induced peptide transporter gene AtPTR3 (At5g46050) of Arabidopsis thaliana has been shown to affect germination on media containing a high salt concentration. The heterologous expression in yeast was utilized to verify that the AtPTR3 protein transports di-and tripeptides. The T-DNA insert in the Atptr3-1 mutant in the Arabidopsis ecotype C24 revealed two T-DNA copies, the whole vector sequence, and the gus marker gene inserted in the second intron of the AtPTR3 gene. An almost identical insertion site was found in the Atptr3-2 mutant of the Col-0 ecotype. The AtPTR3 expression was shown to be regulated by several signalling compounds, most clearly by salicylic acid (SA), but also methyl jasmonate (MeJA) and abscisic acid. Real-time PCR experiments suggested that the wound-induction of the AtPTR3 gene was abolished in the SA and JA signalling mutants. The Atptr3 mutant plants had increased susceptibility to virulent pathogenic bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tomato, and produced more reactive oxygen species when grown on media containing paraquat or rose bengal. Public microarray data suggest that the AtPTR3 expression was induced by Pseudomonas elicitors and by avirulent P. syringae pathovars and type III secretion mutants. This was verified experimentally for the hrpA mutant with real-time PCR. These results suggest that AtPTR3 is one of the defence-related genes whose expression is reduced by virulent bacterium by type III dependent fashion. Our results suggest that AtPTR3 protects the plant against biotic and abiotic stresses.

High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin phoe.

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 microg/ml) while the other (KP-1) was susceptible (MIC of 0.5 microg/ml); both contained the bla(ACT-1), bla(SHV-1), and bla(TEM-1) beta-lactamases. bla(ACT-1) in both strains was encoded on a large approximately 150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo under antibiotic pressure, generating the carbapenem-resistant clone. This is the first study in the Enterobacteriaceae where expression of the phosphate-regulated PhoE porin has been associated with resistance to antimicrobials. Our results with this pair of Klebsiella clinical isolates highlight the complex and evolving nature of multiple drug resistance in this species.

Polyclonal Long-Term MFGS-gp91phox Marking in Rhesus Macaques after Nonmyeloablative Transplantation with Transduced Autologous Peripheral Blood Progenitor Cells

We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34+ cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34+ progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking.

The auxiliary field method in quantum mechanical four-fermi models: a study towards chiral condensation in QED

A study for checking the validity of the auxiliary field method (AFM) is made in quantum mechanical four-fermi models which act as prototypes of models for chiral symmetry breaking in quantum electrodynamics. It has been shown that AFM, defined by an insertion of Gaussian identity to path integral formulae and by the loop expansion, becomes more accurate when taking higher order terms into account under the bosonic model with a quartic coupling in 0- and 1-dimensions as well as the model with a four-fermi interaction in 0-dimension. The case is also confirmed in terms of two models with the four-fermi interaction among N species in 1-dimension (the quantum mechanical four-fermi models): higher order corrections lead us towards the exact energy of the ground state for a whole region including the weak as well as the strong coupling. It is found that the second model belongs to a ‘WKB exact class’ that has no higher order corrections other than the lowest correction. Discussions are also made for unreliability on the continuous time representation of path integration and for a new model of QED as a suitable probe for chiral symmetry breaking.

Effect of treatment with two intravaginal inserts on the uterine and vaginal microflora of early postpartum beef cows

To describe the bacterial species found within the uterus and vagina of early postpartum (10 to 20 d) beef cows treated for 14 d with (i) two intravaginal (CIDR-B) progesterone releasing inserts (C-P4; n=31); (ii) two identical but blank inserts (C-BL; n=15); (iii) untreated controls (CON; n=15). It was hypothesised that due to the locally immunosuppressive effects of progesterone on the uterus, the bacterial microflora of C-P4 would be altered by this treatment in contrast to CON and C-BL. Cows were enrolled at two intervals 7 d apart. Blood samples were collected at 0, 7 and 14 d after beginning treatments for subsequent progesterone assay. A triple guarded swabbing technique was used to collect bacteriological samples from the uterus of every cow on days 0, 7 and 14 following CIDR insertion. Swabs were also collected from the inserts and vagina of every cow on day 14. Due to the small sample sizes, only descriptive statistics were generated. Plasma progesterone levels were maintained at mid-luteal phase concentrations by the intravaginal progesterone releasing inserts (C-P4: 4.2 +/- 0.4 ng/mL at 7 d; 3.6 +/- 0.2 ng/mL at 14 d), although increased progesterone concentrations were found in 4/15 CON and 9/15 C-BL cows on day 14. Bacteria were isolated from 32/61 (52%) of all uterine samples collected at the time of insertion. Uterine and vaginal swabs from CON cows showed a marked reduction in isolates over time such that 14 d after insertion only 1/15 uterine swabs grew bacteria. In contrast, C-BL and C-P4 treated cows failed to show reductions in the number of uterine or vaginal isolates at 14 d after device insertion. Heavy growths of Pseudomonas aeruginosa and Actinomyces pyogenes were found on the intravaginal inserts from C-BL and C-P4 cows. Cows enrolled in the second week of the study that received intravaginal inserts (C-P4 + C-BL) were more likely to have Pseudomonas isolated from the uterus than those enrolled in week 1 (1/18 versus 14/28). The presence of two intravaginal inserts, regardless of hormone content, substantially altered the profile of uterine and vaginal bacteria in early postpartum beef cows. It was suspected that because of the early stage at insert application, the cervix had not involuted sufficiently to provide an adequate microbial barrier to the uterus. Pseudomonas aeruginosa was the predominant species isolated from both uterine and insert cultures after 14 d of treatment but may have been a contaminant, due to the greater proportion of cows infected with it that had been enrolled in the second week of the study.

The Chloroplast Genome Sequence of the Green Alga Pseudendoclonium akinetum (Ulvophyceae) Reveals Unusual Structural Features and New Insights into the Branching Order of Chlorophyte Lineages

One major lineage of green plants, the Chlorophyta, is represented by the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The Prasinophyceae occupies the most basal position in the Chlorophyta, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae remains unresolved. The chloroplast genome sequences currently available for representatives of three chlorophyte classes have revealed that this genome is highly plastic, with Chlamydomonas (Chlorophyceae) and Chlorella (Trebouxiophyceae) showing fewer ancestral features than Nephroselmis (Prasinophyceae). We report the 195,867-bp chloroplast DNA (cpDNA) sequence of Pseudendoclonium akinetum (Ulvophyceae), a member of the class that has not been previously examined for detailed cpDNA analysis. This genome shares common evolutionary trends with its Chlorella and Chlamydomonas homologs. The gene content, number of ancestral gene clusters, and abundance of short dispersed repeats in Pseudendoclonium cpDNA are intermediate between those observed for Chlorella and Chlamydomonas cpDNAs. Although Pseudendoclonium cpDNA features a large inverted repeat, its quadripartite structure is unusual in displaying an rRNA operon transcribed toward the large single-copy (LSC) region and a small single-copy region containing 14 genes that are normally found in the LSC region. Twenty-seven group I introns lie in nine genes and fall within four subgroups (IA1, IA2, IA3, and IB); 19 encode putative homing endonucleases, and 7 have homologs at identical insertion sites in other chlorophyte or streptophyte organelle genomes. The high similarity observed among the 14 IA1 and 7 IA2 introns and their encoded endonucleases suggests that many introns arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. Interestingly, one intron (in atpA) and some of the dispersed repeats also reside in Pseudendoclonium mitochondria, providing strong evidence for interorganellar lateral transfer of these genetic elements. Phylogenetic analyses of 58 cpDNA-encoded proteins and genes support the hypothesis that the Ulvophyceae is sister to the Trebouxiophyceae but cannot eliminate the hypothesis that the Ulvophyceae is sister to the Chlorophyceae. We favor the latter hypothesis because it is strongly supported by phylogenetic analyses of gene order data and by independent structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters.

A Synthesis of New Rigid Fluorescent Bichromophoric Probes for Studying Mechanisms of Donor-Donor Energy Migration

Three new fluorescent probes were synthesized for improving the method of studying donor-donor energy migration (DDEM). Each probe has two identical fluorescent 7-diethylaminocoumarin-3-carbonyl groups attached to a rigid bisteroid dodecacyclic spacer through additional inserts. In two probes, the inserts are beta-Ala and L-Ser residues, which provide for a different nearest environment of the fluorophores. The third probe has identical beta-Ala inserts. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

A site‐specific insertion sequence in flax genotrophs induced by environment

A single-copy 5.7 kilobase (kb) DNA fragment, termed Linum Insertion Sequence 1 (LIS-1), has been identified and characterized. This is one of the DNA changes associated with the environmentally induced heritable changes resulting in stable lines termed genotrophs in flax (Linum usitatissimum). The insertion sequence and its insertion site have been cloned from genomic libraries and sequenced. PCR products across the insertion and surrounding regions have also been cloned and sequenced. The 5.7 kb DNA fragment is inserted into a 3.7 kb EcoRI fragment in the plastic line (Pl) with the generation of a 3 base pair duplication at the insertion site, as well as additional sequence changes. The identical insertion was also found in other genotrophs and flax varieties. The intact element was not present in Pl but appeared to be generated by a reproducible series of complex rearrangements or insertion events. LIS-1 is the result of a targeted, highly specific, complex insertion event that occurs during the formation of some of the genotrophs, and occurs naturally in many flax and linseed varieties.

Evidence for Watson−Crick and Not Hoogsteen or Wobble Base Pairing in the Selection of Nucleotides for Insertion Opposite Pyrimidines and a Thymine Dimer by Yeast DNA Pol η

We have recently reported that pyrene nucleotide is preferentially inserted opposite an abasic site, the 3'-T of a thymine dimer, and most undamaged bases by yeast DNA polymerase eta (pol eta). Because pyrene is a nonpolar molecule with no H-bonding ability, the unusually high efficiencies of dPMP insertion are ascribed to its superior base stacking ability, and underscore the importance of base stacking in the selection of nucleotides by pol eta. To investigate the role of H-bonding and base pair geometry in the selection of nucleotides by pol eta, we determined the insertion efficiencies of the base-modified nucleotides 2,6-diaminopurine, 2-aminopurine, 6-chloropurine, and inosine which would make a different number of H-bonds with the template base depending on base pair geometry. Watson-Crick base pairing appears to play an important role in the selection of nucleotide analogues for insertion opposite C and T as evidenced by the decrease in the relative insertion efficiencies with a decrease in the number of Watson-Crick H-bonds and an increase in the number of donor-donor and acceptor-acceptor interactions. The selectivity of nucleotide insertion is greater opposite the 5'-T than the 3'-T of the thymine dimer, in accord with previous work suggesting that the 5'-T is held more rigidly than the 3'-T. Furthermore, insertion of A opposite both Ts of the dimer appears to be mediated by Watson-Crick base pairing and not by Hoogsteen base pairing based on the almost identical insertion efficiencies of A and 7-deaza-A, the latter of which lacks H-bonding capability at N7. The relative efficiencies for insertion of nucleotides that can form Watson-Crick base pairs parallel those for the Klenow fragment, whereas the Klenow fragment more strongly discriminates against mismatches, in accord with its greater shape selectivity. These results underscore the importance of H-bonding and Watson-Crick base pair geometry in the selection of nucleotides by both pol eta and the Klenow fragment, and the lesser role of shape selection in insertion by pol eta due to its more open and less constrained active site.