Fusion Sequence Research Articles

Variant Philadelphia Translocation with BCR/ABL Fusion Gene Site In 10q24 In a Case of Chronic Myeloid Leukemia

AbstractAbstract 4839 Introduction.Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures:No relevant conflicts of interest to declare.

Metatarsal fusion pattern and developmental morphology of the Olduvai Hominid 8 foot: Evidence of adolescence

The morphology of the Olduvai Hominid (OH) 8 foot and the sequence of metatarsal epiphyseal fusion in modern humans and chimpanzees support the hypothesis that OH 8 belonged to an individual of approximately the same relative age as the OH 7 subadult, the holotype of Homo habilis. Modern humans and chimpanzees exhibit a variety of metatarsal epiphyseal fusion patterns, including one identical to that observed in OH 8 in which metatarsal 1 fuses before metatarsals 2–5. More than the metatarsal fusion sequence, however, the principal evidence of the youthful age of OH 8 lies in the morphology of metatarsals 1, 2, and 3. Because both OH 8 and OH 7 come from the same stratum at the FLK NN type site, the most parsimonious explanation of the OH 8 and OH 7 data is that this material belonged to the same individual, as originally proposed by Louis Leakey. The proposition that OH 8 belonged to an adult is unsupported by morphology, including radiographic evidence, and the fusion sequences in human and chimpanzee skeletal material reported here and in the literature.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Construction of an Expression Vector Containing a Novel Fusion Sequence from Middle Region of NS3 and Truncated Core Genes of Hepatitis C Virus

Construction of an Expression Vector Containing a Novel Fusion Sequence from Middle Region of NS3 and Truncated Core Genes of Hepatitis C Virus

Fusion of HMGA2 to COG5 in uterine leiomyoma

Uterine leiomyomas are smooth muscle tumors most commonly seen in middle-aged women. Approximately 10% of these tumors contain rearrangements of the chromatin-remodeling gene HMGA2 at the chromosome band 12q14.3. Herein, we report on a uterine leiomyoma with a novel HMGA2 fusion gene. A 44-year-old woman presented with a 20-cm mass uterine leiomyoma. From a histological standpoint, the tumor exhibited extensive hyalinization, very low mitotic activity (<1/10 HPH), and no cytologic atypia. Smooth muscle differentiation was confirmed by the expression of smooth muscle actin and desmin. Standard cytogenetic analysis showed the reciprocal translocation t(7;12)(q31.2;q14.3). Fluorescence in situ hybridization analysis confirmed a balanced rearrangement of the HMGA2 locus in 80% of the cells. 3'RACE reverse-transcription polymerase chain reaction identified the fusion of HMGA2 exon 4 to the COG5 locus on 7q31 (component of oligomeric golgi complex 5 isoform). The fusion sequence is predicted to encode a 96-amino acid chimeric protein that retains all three DNA-binding domains (AT hooks) of HMGA2, but that is shorter than the original HMGA2 protein. Since the general structure of the fusion gene is similar to other previously described HMGA2 fusions, its biologic activity is predicted to be likely similar.

Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

BackgroundThe chromosome of Streptomyces has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of Streptomyces avermitilis, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in Streptomyces.ResultsThirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable.ConclusionsVarious chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends.

Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer

Tyrosine kinase (TK) fusions are attractive drug targets in cancers. However, rapid identification of these lesions has been hampered by experimental limitations. Our in silico analysis of known cancer-derived TK fusions revealed that most breakpoints occur within a defined region upstream of a conserved GXGXXG kinase motif. We therefore designed a novel DNA-based targeted sequencing approach to screen systematically for fusions within the 90 human TKs; it should detect 92% of known TK fusions. We deliberately paired ‘in-solution’ DNA capture with 454 sequencing to minimize starting material requirements, take advantage of long sequence reads, and facilitate mapping of fusions. To validate this platform, we analyzed genomic DNA from thyroid cancer cells (TPC-1) and leukemia cells (KG-1) with fusions known only at the mRNA level. We readily identified for the first time the genomic fusion sequences of CCDC6-RET in TPC-1 cells and FGFR1OP2-FGFR1 in KG-1 cells. These data demonstrate the feasibility of this approach to identify TK fusions across multiple human cancers in a high-throughput, unbiased manner. This method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 µg of DNA, and circumvents the need for complex computational sequence analysis.

Self‐Assembly of Thermally Responsive Amphiphilic Diblock Copolypeptides into Spherical Micellar Nanoparticles

As structure–property relationships for protein self-assembly have been elucidated, advances in chemistry and structural biology have facilitated the development of biologically inspired polypeptides through chemical and biosynthetic schemes that have afforded novel protein-based films, fibers, micelles, and gels. In a number of instances, reversible protein self-assembly has been driven by welldefined conformational changes of peptide units induced in response to an external stimulus. Indeed, designed molecular assembly of stimuli-responsive peptides has emerged as a “bottom-up” approach for creating complex, but ordered, hierarchical structures from simple amino acid building blocks. As illustrated by the design of diand triblock polypeptides, microand nanoscale features can be tuned by control of the amino acid sequence, molecular weight, and secondary structure of the peptide. In particular, amphiphilic block copolypeptides can self-assemble into a variety of diverse structures, including rods, cylinders, spheres, and vesicles. Although diblock copolymers consisting of chemically and conformationally distinctive individual polypeptide blocks have been produced by chemical and biosynthetic schemes, to date, relatively few recombinant amphiphilic diblock polypeptides have been synthesized. Given the capacity to incorporate targeting ligands, cell membrane fusion sequences, receptor activating peptides, fluorescent or chelating groups, as well as the ability to tailor pharmacokinetics, biodistribution, and peptide stability, significant opportunities exist for micelles or vesicles produced from recombinant protein block copolymers. Elastin-mimetic polypeptides based on the pentameric repeat sequence (Val-Pro-Gly-Xaa-Gly) undergo thermal and pH-responsive self-assembly in aqueous solution. Spontaneous phase separation of the polypeptide coincides with a conformational rearrangement of local secondary structure above a unique transition temperature (Tt) determined by the chemical identity of the fourth amino acid (Xaa) in the pentapeptide repeat. Recent studies have demonstrated the potential of engineered materials derived from elastin in a broad range of biomedical and biotechnological applications and, in particular, drug delivery. 8] Characteristically, elastin-mimetic blocks that contain hydrophobic amino acids in the fourth amino acid position, such as tyrosine, display a conformational transition from random coil to repetitive type II b turns at temperatures well below 37 8C, whereas blocks that contain a charged amino acid in this position, such as glutamic acid, persist as a random coil throughout the physiologic temperature range. Thus, we postulated that amphiphilic diblock copolymers bearing glutamic acid and tyrosine residues in Nand C-terminal blocks, respectively, would promote micelle formation by temperature-induced self-assembly with a core–shell structure. Moreover, we speculated that at a sufficiently high density of glutamic acid units, charge repulsion would limit the association of the hydrophilic blocks andminimize micelle aggregation. Micelles stabilized by self-assembly alone are typically unstable in a complex environment containing naturally occurring amphiphiles, such as plasma proteins, glycolipids, and lipopeptides. Therefore, by positioning cysteine residues between blocks, we hypothesized that highmolecular-weight protein aggregation or uncontrolled micelle–micelle association would be avoided by nanoparticle stabilization through disulfide cross-linking. These studies represent the first report of thermally responsive and crosslink stabilized protein micelles produced through the tailored design of recombinant amphiphilic diblock copolymers. Two amphiphilic diblock polypeptides (ADP1 and ADP2) were synthesized and self-assembled into micellar structures with consecutive cysteine residues incorporated at the core– shell interface (Scheme 1). Expression of the diblock synthetic genes in E. coli expression strain, BL21(DE3), afforded recombinant protein polymers in high yield after immobilized-metal-affinity chromatography (IMAC) purification from the cell lysate. Mass spectrometry confirmed a correspondence between the observed and expected masses of the respective diblocks with consistent sequence composition by amino acid analysis. The presence of cysteine residues within the polypeptide chain was characterized by the use of a thiolreactive fluorescent dye (see the Supporting Information). Differential scanning calorimetry (DSC) demonstrated an endothermic transition at around 10 8C for both diblock copolymers, which conforms to the established relationship between the position of the transition temperature and the mole fraction of tyrosine in elastin-mimetic protein poly[*] Dr. W. Kim, Dr. E. L. Chaikof Emory University Departments of Biomedical Engineering and Surgery Georgia Institute of Technology, School of Chemical Engineering 101 Woodruff Circle, Rm 5105, Atlanta, GA 30322 (USA) Fax: (+1)404-727-3667 E-mail: echaiko@emory.edu

Abstract A18: A novel targeted capture and massively parallel sequencing platform for systematic discovery of tyrosine kinase fusions in cancer

Abstract Purpose: To identify systematically unknown kinase fusions in an unbiased manner from minimal amounts of tumor-derived genomic DNA. Background: Tyrosine kinase (TK) fusion proteins drive tumorigenesis of many different cancers through deregulated kinase activity. Since cancer cells are dependent on such aberrant signaling, TK fusions are attractive drug targets, and effective therapies targeting these lesions have translated rapidly into the clinic. Unfortunately, only a limited number of TK fusions have been found, because current methods for their identification lack sufficient throughput to allow for systematic analyses of hundreds of tumors. We hypothesized that tumors contain as yet unidentified TK fusions whose discovery has been hindered by these experimental limitations. Experimental Procedures: To overcome these challenges, we performed an in silico analysis of the protein and genomic breakpoint sequences of known cancer-derived TK fusions (n=59). All TK fusions identified to date contain an intact GXGXXG kinase motif. Fusion points at the genomic level occurred within a defined region upstream of this motif, evoking a high throughput screening strategy. We therefore applied DNA capture (Agilent SureSelect) technology to target specifically these regions where breakpoints were likely to occur. Our custom DNA capture platform included all 90 human TKs (and AKT-1, -2, -3 and BRAF), and should capture ∼92% of known TK fusions. The captured DNA was then subjected to 454 massively parallel sequencing. We chose 454 sequencing because the long read length (∼200 nts) would allow for identification of breakpoints that occurred far upstream of the GXGXXG motif. To validate this platform, we used DNA (1.5 g per sample) from thyroid cancer cells (TPC-1) and acute myeloid leukemia cells (KG-1) with known fusions at the mRNA level but unknown fusion points at the genomic level. The recovered sequences were analyzed using two independently derived novel computational algorithms designed specifically for this application. Results: Approximately 60,000 and 100,000 captured 454 sequences were recovered from TPC-1 and KG-1 cells, respectively. TK-containing sequences were enriched ∼776 fold across both samples, indicating the efficiency of our capture method. Candidate fusion sequences were validated by PCR with breakpoint-spanning primers. Among the 15 candidate fusions identified from computational analyses of the TPC-1 sequences, only the CCDC6-RET fusion was validated by direct sequencing of the PCR products. Whereas the CCDC6-RET sequence juxtaposed intronic elements from the fusion partners, the FGFR1OP2-FGFR1 fusion sequence in KG-1 cells proved more complex. The only validated fusion sequence among the 30 candidates contained two non-contiguous portions of FGFR1. Further analysis with long-range PCR revealed that this sequence was actually part of the FGFR1OP2-FGFR1 fusion sequence, which contained inverted exonic regions between the sequences of the two fusion partners. No fusion sequences were recovered from an additional 3 control cell lines without known fusions. Conclusions: We have developed a unique high throughput platform to map genomic fusion points involving TKs in an unbiased manner. Using this technology, we were able to identify readily the genomic sequences of 2 TK fusions mapped previously at the mRNA level. This novel method is distinct from other similar efforts, because it focuses specifically on targets with therapeutic potential, uses only 1.5 g of DNA per sample, and circumvents the need for complex computational sequence analysis. We now plan to use this screening method to search for novel TK fusions in highly annotated tumor samples. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A18

Atypical mRNA fusions in PML‐RARA positive, RARA‐PML negative acute promyelocytic leukemia

Reciprocal RARA-PML transcripts are not detected in approximately 25% of patients with PML-RARA positive acute promyelocytic leukemia (APL), but the reasons for this are poorly understood. We studied 21 PML-RARA positive/RARA-PML negative cases by bubble PCR and multiplex long template PCR to identify the genomic breakpoints. Additional RT-PCR analysis was performed based on the DNA findings. Three cases were found to have complex rearrangements involving a third locus: the first had a PML-CDC6-RARA forward DNA fusion and expressed a chimeric PML-CDC6-RARA mRNA in addition to a PML-RARA. The other two had HERC1-PML and NT_009714.17-PML genomic fusion sequences at their respective reciprocal breakpoints. Six patients were falsely classified as RARA-PML negative due to deletions on chromosome 15 and/or 17, or alternative splicing leading to atypical RARA-PML fusion transcripts, which were not identified by conventional RT-PCR assays. This study demonstrates that the frequency of RARA-PML expression has been underestimated and highlights remarkable complexity at chromosomal breakpoint regions in APL even in cases with an apparently simple balanced t(15;17)(q24;q12).

Evolutionary Changes Affecting Rapid Identification of 2008 Newcastle Disease Viruses Isolated from Double-Crested Cormorants

A morbidity-mortality event involving virulent Newcastle disease virus (NDV) in wild double-crested cormorants (Phalacrocorax auritus) occurred in North America in the summer of 2008. All 22 viruses isolated from cormorants were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene. However, the USDA-validated reverse transcription-PCR assay targeting the fusion gene that is specific for virulent isolates identified only 1 of these 22 isolates. Additionally, several of these isolates have been sequenced, and this information was used to identify genomic changes that caused the failure of the test and to revisit the evolution of NDV in cormorants. The forward primer and fusion probe were redesigned from the 2008 cormorant isolate sequence, and the revised fusion gene test successfully identified all 22 isolates. Phylogenetic analyses using both the full fusion sequence and the partial 374-nucleotide sequence identified these isolates as genotype V, with their nearest ancestor being an earlier isolate collected from Nevada in 2005. Histopathological analysis of this ancestral strain revealed morphological changes in the brain consistent with that of the traditional mesogenic pathotypes in cormorants. Intracerebral pathogenicity assays indicated that each of these isolates is virulent with values of >0.7 but not more virulent than earlier isolates reported from Canada.

Immunologically silent cancer clone transmission from mother to offspring

Rare cases of possible materno-fetal transmission of cancer have been recorded over the past 100 years but evidence for a shared cancer clone has been very limited. We provide genetic evidence for mother to offspring transmission, in utero, of a leukemic cell clone. Maternal and infant cancer clones shared the same unique BCR-ABL1 genomic fusion sequence, indicating a shared, single-cell origin. Microsatellite markers in the infant cancer were all of maternal origin. Additionally, the infant, maternally-derived cancer cells had a major deletion on one copy of chromosome 6p that included deletion of HLA alleles that were not inherited by the infant (i.e., foreign to the infant), suggesting a possible mechanism for immune evasion.

Double stranded RNA tricks melanoma cells into committing suicide

Double stranded RNA tricks melanoma cells into committing suicide

Bcr-Abl fusion sequences do not induce immune responses in mice when administered in mouse polyomavirus based virus-like particles

Mouse polyomavirus-like particles (MPyV-VLPs) carrying inside a fragment of the Bcr-Abl hybrid protein containing the epitope of chronic myeloid leukemia fusion region were prepared. A sequence encoding 171 amino acids covering Bcr-Abl breakpoint was fused to the C-terminal part of VP3 minor protein connecting it to the VP1 capsomeres. Chimeric particles, the Bcr-Abl VLPs, were tested for their ability to induce Bcr-Abl specific immune response in mice after their intranasal (i.n.) or intraperitoneal (i.p.) administration without any other adjuvants. Bcr-Abl VLPs induced strong anti-VP1 immune response in both i.n. and i.p. immunized mice. As expected, neither IgG nor IgM anti-Bcr-Abl specific antibodies were detected in the sera of immunized animals. Surprisingly, no specific CTL (cytotoxic T-lymphocyte) activity was proved using two different methods (in vitro cytotoxicity assay with CFSE-labeled target cells and highly sensitive cytotoxicity assay using MHC class I Bcr-Abl specific pentamers). In addition, no proliferative response of T-cells of i.n. immunized mice after in vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells was observed. Taken together, Bcr-Abl breakpoint epitopes appeared to be weak immunogens and even MPyV-VLPs did not provide sufficient adjuvant ability to support induction of immune responses specific to Bcr-Abl fusion zone epitope.

Identification of novel HMGA2 fusion sequences in lipoma: Evidence that deletion of let‐7 miRNA consensus binding site 1 in the HMGA2 3′ UTR is not critical for HMGA2 transcriptional upregulation

Lipoma is a benign tumor composed of mature adipocytes and one of the most common mesenchymal tumors seen in adults. Rearrangement of HMGA2 in chromosome band 12q15 has been found in approximately 60-70% of ordinary lipomas with cytogenetic abnormalities. Herein, we report two novel HMGA2 fusion sequences in lipomas with chromosome 12 rearrangements. Cytogenetic studies showed 12q abnormalities in both cases, and fluorescence in situ hybridization (FISH) confirmed the involvement of HMGA2 in each instance. Rapid amplification of cDNA ends (RACE) PCR experiments revealed that one lipoma contained a fusion of the HMGA2 3' untranslated region (UTR) to a genomic area downstream of the DYRK2 locus on 12q15; the second lipoma showed a fusion of the HMGA2 3' UTR to a genomic sequence upstream of the DCN locus on 12q21. In both instances the breakpoint on HMGA2 occurred downstream to let-7 miRNA (microRNA) consensus binding site (CBS) 1. These two and several other previously reported tumors containing HMGA2 3' UTR rearrangements show breakpoints after let-7 miRNA CBS 1, which suggests that the elimination of this miRNA binding site is not critical for driving HMGA2 transcriptional upregulation.

Predicting protein subcellular location: exploiting amino acid based sequence of feature spaces and fusion of diverse classifiers

A novel approach CE-Ploc is proposed for predicting protein subcellular locations by exploiting diversity both in feature and decision spaces. The diversity in a sequence of feature spaces is exploited using hydrophobicity and hydrophilicity of amphiphilic pseudo amino acid composition and a specific learning mechanism. Diversity in learning mechanisms is exploited by fusion of classifiers that are based on different learning mechanisms. Significant improvement in prediction performance is observed using jackknife and independent dataset tests.

A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design

We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.

Gender-specific pattern differences of the ossification centers in the pediatric elbow

Only a limited number of studies have investigated the age ranges in which the secondary centers of the elbow appear and ossify. Knowledge of sequence, gender differences and age ranges can aid in accurate assessment of radiographs, especially in cases of injury. To determine the sequence and general age ranges in which each ossification center both appears and fuses, and also to identify differences between genders. This study included 412 sets of radiographs of children's elbows that were analyzed prospectively by a single experienced pediatric radiologist. The presence as well as state of fusion of each ossification center was noted. The ages of the children ranged from 2 months to 17 years. In girls, the radial head and medial epicondyle appeared at the same age. In boys, there was a trend towards the radial head appearing earlier than the medial epicondyle. There was no statistically significant difference between the age at which the trochlea and olecranon appeared. Our results demonstrate a statistically significant difference between genders in both appearance and fusion. All centers both appeared and fused earlier in girls, with the exception of the appearance of the capitellum. The sequence of appearance and fusion was similar between genders. Ossification centers at the elbow both appear and fuse earlier in females but the normal range in age for the times of appearance and fusion of these centers is quite wide for both sexes.

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.

Applications of Transposon-Based Gene Delivery System in Bacteria

Mobile genetic segments, or transposons, are also referred to as "jumping genes" as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved ATrich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as fmgenetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis(STM),DNA or cDNA sequencing, transposon site hybridization(TraSH), and scanning linker mutagenesis(SLM). Therefore, transposon-mediated genetic engineering is a baluable discipiline for the study of bacterial physiology and pathogenesis in living hosts.