Fusion Sequence Research Articles

TWO FUSION PREDICTORS FOR MULTISENSOR DISCRETETIME LINEAR SYSTEM

New multi-step fusion predictors for discrete-time linear dynamic systems with different types of observations are proposed. The multi-step fusion predictors are formed by a summation of the local Kalman filters/predictors with matrix weights depending only on time instants. According to fusion sequence, there could be two kinds of multi-step fusion predictors; the relationship between these fusion predictors is established. Then, the accuracy and computational efficiency of the fusion predictors are demonstrated on a first-order Markov process and a ground moving target indicator model with multisensor environment.

Direct Electrocatalytic mRNA Detection Using PNA-Nanowire Sensors

We report an electrochemical nucleic acids sensing system that exhibits high sensitivity and specificity when challenged with heterogeneous samples of RNA. The platform directly detects specific RNA sequences in cellular and clinical samples without any sample labeling or PCR amplification. The sensor features an electrode platform consisting of three-dimensional gold nanowires, and DNA or RNA hybridization is detected using an electrocatalytic reporter system. In this study, probes made of peptide nucleic acid (PNA) are used to detect a newly identified cancer biomarkera gene fusion recently associated with prostate cancer. The system is able to detect the fusion sequence with 100 fM sensitivity, and retains high sensitivity even in the presence of a large excess of non-complementary sequences. Moreover, the sensor is able to detect the fusion sequence in as little as 10 ng of mRNA isolated from cell lines or 100 ng total RNA from patient tissue samples. The PNA-nanowire nucleic acids sensor described is one of the first electrochemical sensors to directly detect specific mRNAs in unamplified patient samples.

Hormone-sensitive prostate cancer: a case of ETS gene fusion heterogeneity

Fusion of the hormone-regulated gene TMPRSS2 with ERG occurs in 50–70% of prostate cancers; fusions of ETV1 with one of several partners occur in approximately 10% of prostate cancers. These...

Variable Breakpoints Target PAX5 in Patients with Dicentric Chromosomes: A Model for the Basis of Unbalanced Translocations in Cancer

Although balanced translocations often result in the formation of chimaeric fusion genes, the analysis of unbalanced translocations has largely failed to identify target loci. This is due to the heterogeneity of the chromosomal breakpoints and the multiplicity of partner chromosomes. One such example is that of dicentric chromosomes found in patients with acute lymphoblastic leukaemia (ALL). We investigated patients with dic(7;9)(p11;p11~13) [n=13], dic(9;12)(p11~13;p13) [n=38] and dic(9;20)(p11~13;q11) [n=59] to determine the breakpoints of these translocations. FISH with probes for 9p showed that breakpoints were heterogeneous [n=54]. Apart from two cases with dic(9;20), all showed either deletion of the entire PAX5 gene [53.7%, n=29] or a breakpoint within the gene [42.6%, n=23]. For those cases with breakpoints within PAX5, FISH identified ETV6 as the partner in dic(9;12) cases (18/19 cases tested). Sequencing showed a novel breakpoint within intron 1 of ETV6 in a one case of dic(9;12). Molecular copy number counting (MCC) and long distance inverse PCR (LDI-PCR) identified five novel fusion sequences in which PAX5 partnered the LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21) and C20orf112 (20q11.1) loci. The PAX5-ASXL1 and PAX5-KIF3B fusion sequences were in opposing orientation, the remaining three fusions were in the same orientation but out of frame, implicating loss of PAX5 function. To confirm the significance of PAX5 disruption, we demonstrated that both the deletion and gene fusion events resulted in the same under-expression of PAX5 exons 1–2 (p<0.04), and 4–5 (p<0.04) in the patients with dicentric chromosomes, in association with differential expression of the PAX5 target genes, EBF1 (p<0.001), ALDH1A1 (p<0.001), ATP9A (p<0.01) and FLT3 (p<0.006). Genomic quantitative PCR (gqPCR) confirmed the heterozygous loss of PAX5 [n=16] and showed homozygous deletions involving the homologous allele in two patients. Mutation analysis [n=20] identified two cases with a single base pair deletion of exon 8 and a single base pair insertion into exon 2, both of which would translate into a truncated PAX5 protein. This is further support that the formation of the dicentric chromosome provided leukemic potential by abrogating normal PAX5 function in these cases. The same analysis of chromosome 20 in 26 dic(9;20) cases highlighted ASXL1 as the potential target gene in 62% [n=16] patients with the dic(9;20) tested, manifesting as recurrent fusion events and deletions as shown for PAX5. Three additional fusion sequences were identified; involving ASXL1, FRG1B (20q11.1) and LOC149950 (20q11.21) fused to sequence centromeric of PAX5. Here, we have shown for the first time, that specific gene loci may be the target of heterogeneous breakpoints in human cancer, acting through a variety of mechanisms. Although several investigations have failed to identify the key molecular events in patients with dicentric chromosomes, we report PAX5 and ASXL1 as key target genes, as a consequence of their involvement in multiple fusion genes and by deletion. This approach has considerable application to the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent while relatively few genes have been identified. In the absence of a dicentric chromosome, PAX5 is also targeted by interstitial deletions and copy number neutral LOH events, further supporting the importance of investigating the underlying molecular basis of unbalanced translocations. From the expansion of this approach into other tumour types, a large number of novel genes will surely emerge, expanding our understanding of carcinogenesis and ultimately leading to improved management of patients with cancer.

New Insights to the MLL Recombinome Including 8 Novel Partner Genes ACTN4, C2CD3, DCP1A, FLNA, LAMC3, LOC100128568, NRIP3, and TNRC18.

Rearrangements of the MLL (mixed lineage leukemia) gene are associated with pediatric, adult and therapy-related acute leukemias of both the myeloid and lymphoid lineage. Today, about 60 MLL fusion partner genes have been characterized at the molecular level involving almost all chromosomes. Nearly one-third of these partner genes have been identified at the DCAL (Diagnostic Center of Acute Leukemia) where more than 700 MLL rearrangements, including 36 different partner genes, have been analyzed so far. 85% of the analyzed MLL rearrangements account for the 6 most frequent partner genes, and 15% for less frequent partner genes including the 8 novel partner genes ACTN4, C2CD3, DCP1A, FLNA, LAMC3, LOC100128568, NRIP3, and TNRC18. Here, we present an overview of all current known translocation partner genes. This overview indicates all the specific introns of the TPGs found to be involved in MLL translocations, their chromosomal locations and the type of genetic abnormality that is leading to the MLL fusion. Additionally, the current breakpoint cluster region (bcr) is also presented for the 10 most frequent partner genes, namely AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10, MLLT4 (AF6), ELL, EPS15, MLLT6 (AF17), MLLT11 (AF1Q) and partial tandem duplications (PTDs). These bcrs have been used as basis for the development of an long range multiplex PCR. This is an additional tool for the identification of genomic MLL breakpoints to enhance the diagnostic efficiency. Approximately 20% of all MLL rearrangements are complex ones. In this study, we have identified 109 (15%) complex rearrangements. Within these complex rearrangements, the 5' part of the MLL gene is generally fused in frame to one of the most frequent partner genes. But the 3' part of the MLL gene is fused in 45 cases (41%) to a novel so-called “reciprocal MLL partner gene” like ADSS or ATG16L2. This heterogeneous group of novel translocation partner genes had not been identified as fusion partners to the 5' part of the MLL gene before. Whether the pathobiology of complex rearrangements is different in comparison to their not complex counterparts has still to be proven. Also a novel “spliced MLL fusion” involving the AFF1 gene has been identified at the DCAL. In this case, the breakpoint is 90 kb up-stream of the AFF1 gene. Unfortunately, no cDNA/RNA was left to identify the chimeric fusion transcript. Nevertheless, the disease phenotype of a biphenotypic AL strengthens the involvement of the AFF1 gene. With these results, the number of genes involved in „ spliced MLL fusions“ has increased from seven to eight. For five patients, no partner gene could be identified at the molecular level. Also the “spliced MLL fusions” which is a mechanism to generate functional chimeric fusion transcripts with neighbouring genes described already for 8 partner genes of the MLL gene, could not be identified for these patients by RACE and RT-PCR. Furthermore, the determined patient-specific fusion sequences are succesfully used worldwide for minimal residual disease (MRD) monitoring to improve the treatment and outcome of acute leukemia patients.

Discordance of MLL-Rearranged (MLL-R) Infant ALL in Monozygotic Twins with Spontaneous Clearance of Preleukemic Clone in Unaffected Twin

The concordance rate for acute leukemia in infant monozygotic twins is estimated to be between 50 and 100%. The leukemic clone is thought to develop in one twin and transfer to the other twin via placental anastamoses. This hypothesis is supported by studies documenting identical, clonal, non-constitutional fusion sequences in concordant leukemias of monozygotic twins. For the ~80% of infant leukemias that are MLL-R+, the twin concordance rate is thought to be 100%, with a very short latency period between the diagnosis of the two twins. This suggests that either the MLL fusion is sufficient to cause leukemia, or it promotes the rapid acquisition of additional mutations required for the development of overt disease.We report a case of discordance in an infant monozygotic twin pair in which twin A presented with MLL-ENL+ ALL and twin B, who was transiently RT-PCR+ for MLL-ENL in bone marrow (BM) and blood, remains healthy 2.5 years later. Twin A was diagnosed at age 9 months (T=0) with CD10(−) pre-B ALL, cytogenetics t(11;19)(q23;p13.3), FISH+ for MLL-R, RT-PCR+ for MLL-ENL fusion transcript. Twin B was healthy with normal CBC and physical exam (PE). At T+7wks, twin B's CBC and PE remained normal, but RT-PCR on peripheral blood mononuclear cells (PBMNC) was MLL-ENL+. Three weeks later (T+10wks), BM aspirate was also RT-PCR+ for MLL-ENL. Morphology, FISH and cytogenetics were normal, as were CBC and PE. Two weeks later (T+12wks), twin B developed neutropenia (WBC= 2,880/ml; ANC=461) and thrombocytopenia (29,000/ml) one week after a viral URI (fever, rhinorrhea, cough, diffuse erythematous rash). He had no lymphadenopathy or hepatosplenomegaly on PE. Review of his PB smear confirmed the cytopenias and revealed atypical lymphocytosis and activated monocytes compatible with a viral process, but no lymphoblasts. Large platelets were noted, suggesting a diagnosis of acute ITP. No treatment was given. Repeat CBC 2 days later showed recovery of counts with WBC of 6,390/ml (ANC=1150) and platelets of 117,000/ml. RT-PCR of PBMNC was negative for MLL-ENL; BM aspirate was not performed. One week later, CBC had normalized. Over the next 4 months, monthly CBC and PE were normal, and monthly RT-PCR of PBMNC were negative. Twin B remains healthy with no clinical or laboratory evidence of leukemia at T+2.5yrs.Twin A was treated according to COG protocol P9407. End-induction BM showed morphologic remission, normal cytogenetics and negative FISH, although RT-PCR remained MLL-ENL+. End-consolidation BM showed continued remission, and was negative for MLL-ENL by cytogenetics, FISH and RT-PCR. At the end of P9407 therapy, an additional 1 year of maintenance therapy was given (total duration of therapy = 2 years). Two months after completion of therapy (26 months from diagnosis), twin A experienced an isolated testicular relapse of CD10(−) pre-B ALL, MLL-ENL+. He received reinduction chemotherapy and testicular radiation and is currently receiving post-induction chemotherapy.To our knowledge, this is the first report of discordant MLL-R leukemia in infant monozygotic twins, and the first report of documented spontaneous clearance of a detectable preleukemic clone in a healthy “twin B”. There are reports of twins discordant for MLL-R leukemia, but placental status was either unknown, dichorionic, or the leukemia developed in the first twin at 5 years of age and was likely postnatally acquired. The temporal association of the disappearance of the preleukemic clone in twin B with an episode of viral-induced, self-limited thrombocytopenia and neutropenia suggests the possibility that the immune process that mediated the cytopenias may also have been responsible for clearance of the preleukemic clone.This report confirms prior reports that clonal MLL-R cells are shared between monozygotic twins. However, this case presents evidence against the hypothesis that MLL rearrangements (specifically the MLL-ENL fusion) are sufficient for the development of overt leukemia. It is possible that a viral-induced autoimmune reaction may have aborted the progression of twin B's MLL-ENL+ preleukemic clone to overt leukemia by eradicating the clone prior to the acquisition of additional mutations. It is also possible that twin B's preleukemic clone persists at a level beneath the sensitivity of our RT-PCR assay. A less speculative conclusion is that short latency concordance of infant MLL-R leukemia in monozygotic twins is not universal.

Bessel fusion multipliers

In this paper we study properties of a Bessel multiplier when the symbol involved belongs to l p . Furthermore, we introduce the concept of Bessel fusion multiplier which generalizes a Bessel multiplier for Bessel fusion sequences. We study their behavior when the symbol belongs to l p and some continuity properties.

Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein

The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

The Fetal Cleft Palate: V. Elucidation of the Mechanism of Palatal Clefting in the Congenital Caprine Model

Failure of palatal shelf fusion at the precise gestational time point when shelf elevation and migration normally occur results in clefting. The present study defined the mechanism of clefting in the congenital caprine model by evaluating the temporal sequence of palatal shelf fusion. Six Spanish-type goats pregnant with twins were gavaged for 10 days (gestational days 32 to 41) with anabasine. Goats were examined with ultrasound throughout this period to assess fetal movement and evaluated histologically. Twelve untreated, unclefted fetuses served as controls. After anabasine induction, real-time ultrasound confirmed a dramatic decrease in fetal movement. Hyperflexion of the neck occurred throughout the experimental period compared with control fetuses, in which spontaneous neck movements occurred. Histologic evaluation of the induced fetuses demonstrated the tongue wedged between the palatal shelves throughout the period of fusion. The shelves remained vertically oriented without elevation, ultimately resulting in clefting. The unclefted, control group demonstrated progressive elevation and migration of the palatal shelves between gestational days 38 and 40, with contact and fusion of the medial edge epithelia in the midline. Palatal clefting in the congenital caprine model occurred concomitant with reduced fetal movement. Resultant neck hyperflexion causes the tongue to obstruct medial migration of the palatal shelves and fusion, whereas the shelves themselves are either directly prevented from elevation secondary to anabasine impairment or indirectly secondary to the obstructing tongue. Although the molecular mechanism for the teratogenic effects of anabasine remains elusive, the authors have demonstrated an association between reduced fetal movement and palatal clefting in their congenital caprine model.

Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter

To excise a selectable marker gene from transgenic plants, a new binary expression vector based on the 'genetically modified (GM)-gene-deletor' system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Histochemical staining for GUS activity showed that, upon induction by heat shock, all exogenous DNA, including the selectable marker gene ipt, beta-glucuronidase (gusA) gene and the FLP recombinase gene, between two loxP/FRT sites was eliminated efficiently from primary transgenic tobacco plants. Molecular analysis further confirmed that excision of the marker gene (ipt) was heritable and stable. Our approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants.

Isolation of a Recent Korean Epizootic Strain of Newcastle Disease Virus from Eurasian Scops Owls Affected with Severe Diarrhea

Velogenic Newcastle disease virus (NDV) was recovered from two dead Eurasian Scops Owls (Otus scops) from a wildlife rescue center in Korea during 2005. Phylogenetic analysis based on the sequence of the partial fusion (F) protein revealed that the isolates had the highest level of homology to recent Korean NDV strains from poultry.

Thermal Formation of Polycyclic Aromatic Hydrocarbons from Cyclopentadiene (CPD)

Polycyclic aromatic hydrocarbon growth from cyclopentadiene (CPD) pyrolysis was investigated using a laminar flow reactor operating in a temperature range of 600 to 950℃. Major products from CPD pyrolysis are benzene, indene and naphthalene. Formation of observed products from CPD is explained as follows. Addition of the cyclopentadienyl radical to a CPD π-bond produces a resonance-stabilized radical, which further reacts by one of three unimolecular channels: intramolecular addition, C-H bond β-scission, or C-C bond β-scission. The intramolecular addition pathway produces a 7-norbornenyl radical, which then decomposes to indene. Decomposition by C-H bond β-scission produces a biaryl intermediate, which then undergoes a ring fusion sequence that has been proposed for dihydrofulvalene-to-naphthalene conversion. In this study, we propose C-C bond β-scission pathway as an alternative reaction channel to naphthalene from CPD. As preliminary computational analysis, Parametric Method 3 (PM3) molecular calculation suggests that intramolecular addition to form indene is favored at low temperatures and C-C bond β-scission leading to naphthalene is predominant at high temperatures.

Monoclonal Antibodies That Bind to Common Epitopes on the Dengue Virus Type 2 Nonstructural-1 and Envelope Glycoproteins Display Weak Neutralizing Activity and Differentiated Responses to Virulent Strains: Implications for Pathogenesis and Vaccines

The abilities of monoclonal antibodies (MAbs) that bind to defined sequential epitopes on the dengue virus (DENV) nonstructural-1 (NS1) glycoproteins to cross-react with epitopes on the DENV envelope (E) glycoproteins were investigated. In this study, some of these MAbs cross-reacted with the DENV type 2 (DENV-2) E glycoprotein and with synthetic peptides representing X-ray crystallographically confirmed surface-exposed regions on this glycoprotein. MAb 1G5.3 cross-reacted with the flavivirus-conserved 101-WGNGCGLFG-109 fusion sequence, the 273-SSGNL-277 DENV-2 hinge region sequence, and the 156-GKHGKEIKIT-165 sequence of virulent DENV-2 strains. MAb 1G5.4-A1-C3 cross-reacted with the 67-NTTTESRCPT-76 and 156-GKHGKEIKIT-165 sequences of virulent DENV-2 strains, the 338-EIMDLDNRHV-347 sequence from a highly virulent DENV-2 (M2) strain, and two epitopes on a virulent DENV-3 strain (288-KMDKLELKG-296 and 323-RVEYRGEDAP-332), which all contained target ELK/KLE-type motifs (underlined). These MAbs showed reduced cross-reactions with the corresponding sequences from weakly pathogenic strains of all four DENV serotypes and had either no (MAb 1G5.4-A1-C3) or weak (MAb 1G5.3) neutralizing activity against them. MAb 1G5.3 more strongly neutralized DENV-2 strains with higher pathogenic capacities, while MAb 1G5.4-A1-C3 showed increasing neutralizing titers against the virulent DENV-3 strain and the moderately virulent and highly virulent (M2) DENV-2 strains. These cross-reactions with the E glycoprotein accord with the observation that MAb 1G5.3 caused dramatic and lethal antibody-enhanced replication (AER) of a DENV-2 strain in vivo. Together with in vivo AER studies of these DENV strains using MAb 1G5.4-A1-C3, these results may account for the increased pathogenic capacities of such strains, which is likely to have important implications for pathogenesis and vaccines.

Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection

Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS ( E. coli OmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Patients with chronic myeloid leukemia harbor the chromosomal translocation t(9;22), which corresponds to fusion of the BCR and ABL genes at the DNA level. The translated fusion product is an oncogenic protein with increased ABL tyrosine kinase activity causing cell transformation. To date, reverse transcriptase-polymerase chain reaction is considered the most sensitive method available for detecting low copy numbers of the BCR-ABL gene fusion. Recently, Cepheid introduced its GeneXpert-based assay for the identification of the BCR-ABL gene fusion in cells from blood samples. This system comprises a walk-away self-contained instrument that combines cartridge-based microfluidic sample preparation with reverse transcriptase-polymerase chain reaction-based fluorescent signal detection and BCR-ABL and ABL Ct (threshold cycle) determination. The difference between the BCR-ABL Ct and ABL Ct (DeltaCt) is expected to represent the ratio of the two populations of mRNAs and ultimately the percentage of neoplastic cells present. We tested whether this BCR-ABL fusion detection system could be used as a clinical diagnostic tool for monitoring patients with minimal residual disease of chronic myelogenous leukemia. We report similar performance characteristics, including limit of detection, specificity, sensitivity, and precision, of this automated BCR-ABL fusion detection system to those of a manual TaqMan reverse transcriptase-polymerase chain reaction-based test.

‘GM‐gene‐deletor’: fused loxP‐FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants

Pollen- and seed-mediated transgene flow is a concern in plant biotechnology. We report here a highly efficient 'genetically modified (GM)-gene-deletor' system to remove all functional transgenes from pollen, seed or both. With the three pollen- and/or seed-specific gene promoters tested, the phage CRE/loxP or yeast FLP/FRT system alone was inefficient in excising transgenes from tobacco pollen and/or seed, with no transgenic event having 100% efficiency. When loxP-FRT fusion sequences were used as recognition sites, simultaneous expression of both FLP and CRE reduced the average excision efficiency, but the expression of FLP or CRE alone increased the average excision efficiency, with many transgenic events being 100% efficient based on more than 25,000 T(1) progeny examined per event. The 'GM-gene-deletor' reported here may be used to produce 'non-transgenic' pollen and/or seed from transgenic plants and to provide a bioconfinement tool for transgenic crops and perennials, with special applicability towards vegetatively propagated plants and trees.

Identification of Leukemia-Specific Fusion Gene Transcripts with a Novel Oligonucleotide Array

Identification of specific chromosomal translocations is essential for the diagnosis and prognosis of leukemia. In this study, we employ DNA microarray technology to detect chromosomal aberrations in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), as well as in leukemic cell lines. Reverse transcription using a random 9-mer primer was performed with total RNA from patients and leukemic cells lines. Multiplex PCR reactions using four groups of primer sets were then performed for amplification of cDNA from reverse-transcribed total RNA samples. Normal and fusion sequences were distinguished by hybridization of the amplified cDNA to a selective oligonucleotide array (SOA) containing 20-30mer synthetic probes. A total of 23 sets of oligomers were fabricated on glass slides for the detection of normal and fusion genes, as follows: BCR/ABL, AML/EAP, AML/ETO, AML/MDS, PML/RARA, NUMA1/RARA, PLZF/RARA, and CBFB/MYH. Gene translocation in leukemia was effectively identified with the SOA containing various leukemia-specific fusion and normal control sequences. Leukemic fusion sequences from patients and cell lines hybridized specifically to their complementary probes. The probe sets differing by approximately 50% at their 5' or 3' ends could distinguish between normal and fusion sequences. The entire process of detection was completed within 8 hours using the SOA method. Probe sets on SOA can effectively discriminate between leukemia-specific fusion and normal sequences with a chip hybridization procedure. The oligonucleotide array presents several advantages in identifying leukemic gene translocations, such as multiplex screening, relatively low cost, and speed.

Inv(11)(q21q23) Fuses MLL to the NOTCH Co-Activator Mastermind-Like 2 in Secondary T Cell Acute Lymphoblastic Leukemia.

Development of a secondary leukemia after chemotherapeutic treatment for childhood acute leukemia is associated with rearrangements of the MLL gene on chromosome 11q23 and characteristically results in acute myeloid leukemia (AML). We identified the NOTCH co-activator Mastermind-like 2 as MLL fusion partner in two cases of pediatric secondary leukemia. The MLL-MAML2 fusion results from a cryptic inv(11)(q21q23) only detectable by interphase fluorescence in situ hybridization with an MLL split signal probe. With a latency of twenty months and five years after initial diagnosis for MLL negative AML and precursor B acute lymphoblastic leukemia (ALL), respectively, both patients developed a therapy-related T-cell acute lymphoblastic leukemia.MAML2 is the first MLL fusion partner involved in human Notch-signaling and was only recently identified as recurrent translocation fusion partner in a subset of salivary gland tumors. The genomic MLL breakpoint shows similar localization and sequence features described for etoposide induced treatment-related AML. MLL-MAML2 positive cells were detectable up to two years prior to clinical apparent secondary leukemia in one case. The discrepant dynamics of clone expansion quantified by either the genomic fusion sequence or Ig/TCR gene rearrangements as clone specific markers suggests a selection process within the inversion positive population and the need for additional mutation events to promote overt leukemic disease. Whole genome expression profiles demonstrated differential expression of both typical MLL and NOTCH downstream genes, which suggests a modulatory role of the MAML2 transcriptional activation domain in MLL leukemogenesis and lineage assignment induced by the MLL-MAML2 fusion protein.MSM21620813WSF3401600.

Multi-agent decision fusion for motor fault diagnosis

Improvement of recognition rate is ultimate aim for fault diagnosis researchers using pattern recognition techniques. However, the unique recognition method can only recognise a limited classification capability which is insufficient for real-life application. An ongoing strategy is the decision fusion techniques. In order to avoid the shortage of single information source coupled with unique decision method, a new approach is required to obtain better results. This paper proposes a decision fusion system for fault diagnosis, which integrates data sources from different types of sensors and decisions of multiple classifiers. First, non-commensurate sensor data sets are combined using an improved sensor fusion method at a decision level by using relativity theory. The generated decision vectors are then selected based on correlation measure of classifiers in order to find an optimal sequence of classifiers fusion, which can lead to the best fusion performance. Finally, multi-agent classifiers fusion algorithm is employed as the core of the whole fault diagnosis system. The efficiency of the proposed system was demonstrated through fault diagnosis of induction motors. The experimental results show that this system can lead to super performance when compared with the best individual classifier with single-source data.

Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli

In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.