MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.

The genetic basis of early T-cell precursor acute lymphoblastic leukaemia.

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300).

A novel retinoblastoma therapy from genomic and epigenetic analyses.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated.

CREST maps somatic structural variation in cancer genomes with base-pair resolution.

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.

Analyzing and visualizing expression data with Spotfire.

This unit assumes the reader is familiar with the Spotfire environment, has successfully installed Spotfire, and has uploaded and prepared data for analysis. It presents numerous methods for analyzing microarray data. Specifically, the first two protocols describe methods for identifying differentially expressed genes via the t-test/ANOVA and the distinction calculation respectively.

Loading and preparing data for analysis in spotfire.

This unit strictly focuses on data preparation within Spotfire. Microarray data exist in a variety of formats, which often depend on the particular array technology and detection instruments used. The first protocols in this unit describe loading Affymetrix and GenePix data into Spotfire.

An overview of Spotfire for gene-expression studies.

Spotfire DecisionSite for Functional Genomics (referred to here as Spotfire) is a powerful data mining and visualization application with use in many disciplines. This unit provides an overview of Spotfire's utility in analyzing gene expression data obtained from DNA microarray experiments. Analysis of microarray data requires software-based solutions able to handle and manipulate the enormous amount of data generated.

An overview of Spotfire for gene-expression studies.

Spotfire DecisionSite for Functional Genomics (referred to here as Spotfire) is a powerful data mining and visualization application with use in many disciplines. This unit provides an overview of Spotfire's utility in analyzing gene expression data obtained from DNA microarray experiments. Analysis of microarray data requires software-based solutions able to handle and manipulate the enormous amount of data generated.

Influenza in migratory birds and evidence of limited intercontinental virus exchange.

Migratory waterfowl of the world are the natural reservoirs of influenza viruses of all known subtypes. However, it is unknown whether these waterfowl perpetuate highly pathogenic (HP) H5 and H7 avian influenza viruses. Here we report influenza virus surveillance from 2001 to 2006 in wild ducks in Alberta, Canada, and in shorebirds and gulls at Delaware Bay (New Jersey), United States, and examine the frequency of exchange of influenza viruses between the Eurasian and American virus clades, or superfamilies.

Persistent host markers in pandemic and H5N1 influenza viruses.

Avian influenza viruses have adapted to human hosts, causing pandemics in humans. The key host-specific amino acid mutations required for an avian influenza virus to function in humans are unknown. Through multiple-sequence alignment and statistical testing of each aligned amino acid, we identified markers that discriminate human influenza viruses from avian influenza viruses.

A novel approach for the analysis of T-cell reconstitution by using a T-cell receptor beta-based oligonucleotide microarray in hematopoietic stem cell transplantation.

Objective: Analysis of T-cell population diversity is important to hematopoietic stem cell transplantation (HSCT). The millions of specificities in T-cell receptor (TCR) hypervariable complementarity- determining region 3 (CDR3) precludes detection of all T-cell populations by antibody-based flow cytometry. An alternative method, the TCR CDR3 spectratyping assay, involves multiple polymerase chain reaction (PCR) analyses and is interpreted only qualitatively.

Large-scale sequence analysis of avian influenza isolates.

The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes.

Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells.

To elucidate the genomics of cellular responses to cancer treatment, we analyzed the expression of over 9,600 human genes in acute lymphoblastic leukemia cells before and after in vivo treatment with methotrexate and mercaptopurine given alone or in combination. Based on changes in gene expression, we identified 124 genes that accurately discriminated among the four treatments. Discriminating genes included those involved in apoptosis, mismatch repair, cell cycle control and stress response.

Characterization of the MLL partner gene AF15q14 involved in t(11;15)(q23;q14).

Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4.

Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling.

Treatment of pediatric acute lymphoblastic leukemia (ALL) is based on the concept of tailoring the intensity of therapy to a patient's risk of relapse. To determine whether gene expression profiling could enhance risk assignment, we used oligonucleotide microarrays to analyze the pattern of genes expressed in leukemic blasts from 360 pediatric ALL patients. Distinct expression profiles identified each of the prognostically important leukemia subtypes, including T-ALL, E2A-PBX1, BCR-ABL, TEL-AML1, MLL rearrangement, and hyperdiploid >50 chromosomes.

Cloning, genomic organization and chromosomal localization of the gene encoding the murine sodium-dependent, purine-selective, concentrative nucleoside transporter (CNT2).

A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides.

Biotechnology core laboratories: An overview.

An assessment of the capabilities of biotechnology core facilities requires access to current data on state-of-the-art technologies, personnel, space, services, financial issues, and the demand for such facilities. Data on these topics should be useful to researchers, facility personnel, administrators, and granting agencies.To obtain such data, the Association of Biomolecular Resource Facilities (ABRF) conducted a general survey on the operation and technical capabilities of core facilities.

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp.

cDNA cloning, expression pattern, and chromosomal localization of Mlf1, murine homologue of a gene involved in myelodysplasia and acute myeloid leukemia.

The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARalpha). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown.

Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32).

Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10, a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD), in MALT lymphomas due to the recurrent t(1;14)(p22;q32). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD.

Comparison of activation of CPT-11 by rabbit and human carboxylesterases for use in enzyme/prodrug therapy.

Several recent studies have examined the possibility of producing tumor-specific cytotoxicity with various enzyme/ prodrug combinations. The enzymes are targeted to tumor cells either with antibodies (ADEPT, antibody directed enzyme prodrug therapy) or with viruses (VDEPT). The goal of the present study was to identify an appropriate enzyme for use in activating the prodrug 7-ethyl-10-[4-(1-piper-idino)-1-piperidino]carbonyloxycamptothe cin (CPT-11).

HERF1, a novel hematopoiesis-specific RING finger protein, is required for terminal differentiation of erythroid cells.

The AML1/core binding factor beta (CBFbeta) transcription factor is essential for definitive hematopoiesis; however, the downstream pathways through which it functions remain incompletely defined. Using a differential cloning approach to define components of this pathway, we have identified a novel gene designated HERF1 (for hematopoietic RING finger 1), whose expression during development is dependent on the presence of functional AML1/CBFbeta. HERF1 contains a tripartite RING finger-B box-alpha-helical coiled-coil domain and a C-terminal region homologous to the ret proto-oncogene-encoded finger protein.

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region.

Two candidate downstream target genes for E2A-HLF.

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation, is thought to drive the leukemic transformation of early B-cell precursors by repressing an evolutionarily conserved apoptotic pathway. To test this hypothesis, we sought to identify downstream targets of E2A-HLF in t(17;19)+ pro-B leukemia cells (UOC-B1) that had been transfected with a zinc-inducible vector encoding a dominant-negative suppressor (E2A-HLF[dn]) of the oncoprotein. Representational difference analysis of mRNAs from E2A-HLF(dn)+ UOC-B1 cells grown with (E2A-HLF inactive) or without (E2A-HLF active) the addition of zinc yielded several differentially expressed cDNA fragments that were individually subcloned.