Automated system for purification of dye-terminator sequencing products eliminates up-stream purification of templates.

The quality of sequencing results is to a large extent determined by the purity of the template and the purification of the sequencing products. Fragments that can act as unspecific primers and templates are removed before gel analysis, and the background of unspecific signals is highly reduced. Purification of the sequencing products is needed to remove salts, nucleotides, proteins and template DNA that can interfere with the gel separation. We have developed a product, DYNAPURE Dye Terminator Removal, that specifically isolates and purifies the sequencing products in 10 min. The method is based on biotinylated sequencing primers and super-paramagnetic streptavidin beads. A PCR product is sequenced using a biotinylated sequencing primer, and the sequencing products are then bound to streptavidin beads in a 5-min reaction. The bead-DNA complexes are magnetically separated from the rest of the solution, and the remaining buffer constituents are washed away with TE buffer or with 70% ethanol. The whole procedure can be automated on liquid-handling robots fitted with a magnet station. The method eliminates purification of templates before cycle sequencing.

A novel mycobacterial antigen relevant to cellular immunity belongs to a family of secreted lipoproteins.

The gene sequence of a novel 24.1 kDa Mycobacterium tuberculosis protein was identified within the Sanger Centre (UK) M. tuberculosis genome database (cosmid MTCY24G1) by searching with a 126 bp DNA sequence isolated from a genomic M. leprae lambda gt11 library with M. leprae reactive human T cell clones as probes. The 24.1 kDa antigen is common to the vaccine strain Mycobacterium bovis BCG, as well as Mycobacterium leprae. The 699 bp open reading frame encodes a 233 amino acid long precursor protein with a signal peptide sequence for secretion and a consensus motif for lipid conjugation, which suggests that the mature protein is an exported lipoprotein antigen. The molecular mass of the mature protein antigen from M. leprae sonicate was shown to correspond to the deduced size of the M. tuberculosis protein by T cell Western analysis. Homology searches revealed two other similarly sized hypothetical secreted mycobacterial lipoproteins within the M. tuberculosis genome database.

Rapid, universal method to isolate PCR-ready DNA using magnetic beads.

A magnetic bead-based system for DNA isolation utilizing monodisperse beads was tested with the aim of producing a general approach for PCR-ready DNA. This commercially available system was originally designed for isolating PCR-ready DNA from human whole blood. We tested diverse organisms belonging to the major groups: bacteria, fungi, algae, vascular plants and vertebrates. Optimization of sample amounts and lysis conditions was done using several types of tissue (fish epithelium, plant leaves, mammalian liver and muscle tissues, fungal fruit-bodies and mycelium). The standard lysis conditions used for blood could be applied with good results for most bacteria, algae and vertebrates, while plant leaves and fungal fruit-bodies had to be mechanically broken to obtain proper lysis. For vascular plants and some cyanobacteria, lysis by heating to 65 degrees C gave better DNA yields than standard lysis at room temperature. In all cases, DNA suitable for PCR was prepared in less than 30 min. The PCR products yielded 350 to 500 bases of DNA sequence (99% accurate) by direct manual or automated sequencing.

Rapid isolation of PCR-ready DNA from blood, bone marrow and cultured cells, based on paramagnetic beads.

PCR-based methods for the analysis of genomic DNA are becoming increasingly common both in research and for routine purposes. A rapid, small-scale DNA isolation method is needed to take full advantage of the speed and automation potential of the PCR technology. We demonstrate the use of Dynabeads DNA DIRECT, a kit for the isolation of PCR-ready genomic DNA from whole blood, bone marrow or cultured cells in less than 10 min. The method is based on adsorption to magnetic beads prior to magnetic separation and involves no centrifugation steps or organic solvents. The yield and quality of the DNA is comparable to traditional large-scale methods. One isolation is enough for at least ten PCRs.

Semiquantitative polymerase chain reaction for t(14;18) in follicular lymphomas: a colorimetric approach.

BACKGROUND: Autologous bone marrow transplantation is increasingly being used in the management of several types of cancer, and to avoid reintroduction of malignant cells, bone marrow purging is often performed. In such cases, sensitive quantitation methods are needed both to assess the efficacy of the purging and for surveillance of patients in remission. Polymerase chain reaction (PCR) has the necessary sensitivity for this application, but it requires that the cancer cells can be recognized by a defined genetic abnormality. In addition, PCR is in principle a qualitative technique and must be modified for quantitative purposes. In follicular non-Hodgkin's lymphomas, the translocation t(14;18) (q32;q21) is common and is used here for model experiments. EXPERIMENTAL DESIGN: A PCR-based method for the quantitation of translocation-positive cells was developed on the basis of coamplification of cancer-specific target molecules with competitor molecules of known concentration. Gel electrophoresis was substituted by a colorimetric quantitation system to cope with patient PCR products of the same size as the competitor product and ease automation of the method at a later stage. Cell line Karpas 422, which contains the t(14;18) (q32;21) translocation, was used to validate the method. The method was used to assess the efficacy of a patient bone marrow purging where the initial infiltration levels were too low for traditional detection systems. RESULTS: A reproducible and near linear response was obtained between 70 pg and 200 ng K422 DNA, equivalent to 10 K422 cells and 30,000 K422 cells, respectively. Bone marrow infiltration in one patient was 0.6 to 0.7% before malignant cell removal and 3 to 7 x 10(-4) after removal. The corresponding figures for the other patient were 2% and 3 to 7 x 10(-4), respectively. CONCLUSIONS: The method presented has a sufficient dynamic range for applications like evaluation of bone marrow purging or monitoring of minimal residual disease. Adaptation of this method to other translocations is discussed.

Characterization of a promoter region supporting transcription of a novel human beta-galactoside alpha-2,6-sialyltransferase transcript in HepG2 cells.

In humans, two transcripts encoding beta-galactoside alpha-2,6-sialytransferase (EC 2.4.99.1.) have previously been described. One of the transcripts is widely expressed, whereas the other is restricted to mature B-cells. In this study we demonstrate the existence of a third transcript in the hepatoma cell-line HepG2. The expression of this transcript is controlled by a promoter region which efficiently supports transcription in HepG2 cells, and which harbours putative binding sites for liver-enriched and acute phase inducible transcription factors.

An HLA-DRw53-restricted T-cell epitope from a novel Mycobacterium leprae protein antigen important to the human memory T-cell repertoire against M. leprae.

Cellular immunity mediated by T cells plays a major role in protection against intracellular infections, including leprosy, a chronic disease caused by Mycobacterium leprae. In this work, we describe CD4+ T-cell clones, isolated from healthy humans immunized with M. leprae, which recognize a novel M. leprae protein antigen previously isolated from a lambda gt11 DNA expression library. On the basis of the deduced primary structure of the carboxyl-terminal part of the antigen, we have used a synthetic-peptide approach to exactly define the T-cell epitope recognized. Importantly, major histocompatibility complex restriction studies showed that the epitope is presented by an HLA-DRw53 molecule which is frequently expressed in many populations. In addition, we have demonstrated that a long-term cell-mediated immunity response against the peptide epitope is present after immunization with M. leprae. In conclusion, the M. leprae T-cell epitope described here fulfills the primary criteria for subunit vaccine candidates against leprosy.

A simple subtraction method for the isolation of cell-specific genes using magnetic monodisperse polymer particles.

In this study we present a simple subtraction method for the isolation of cell-type-specific genes using magnetic beads. Biotinylated first-strand cDNA is generated from one cell type and immobilized onto magnetic streptavidin beads. Poly A+ RNA, isolated from a different cell type by use of oligo-dT beads, is then hybridized to the immobilized cDNA. Beads with hybridized mRNA are subsequently removed from the solution by attraction to a magnet. The cell-specific mRNA, left in solution, is finally converted to a radiolabeled cDNA probe in order to screen cDNA libraries. In this study, we present an example of a successful subtraction strategy involving three cell types: the pre-B-cell line Reh; 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated Burkitt's lymphoma line Daudi; and TPA-stimulated T lymphocytes. This strategy was chosen due to our interest in gene products known to be expressed in TPA-stimulated B and T lymphocytes, but not in Reh cells. Several previously unknown genes were identified.

Tissue specific expression and cDNA structure of a human transcript encoding a nucleic acid binding [oligo(dC)] protein related to the pre-mRNA binding protein K.

In human cells at least 20 different proteins or groups of proteins have been identified that are associated with hnRNAs. These proteins (designated A1-U) are highly abundant in the nucleus. In this study, we present the sequence of a novel cDNA clone, sub2.3, isolated from a human lymphocyte cDNA library. The predicted amino acid sequence shows homology to repeated domains in the human hnRNA binding protein K (hnRNP K), which are believed to be of functional importance. hnRNP K is among the major oligo(rC/dC) binding proteins in vertebrate cells and we show here that the protein product of sub2.3 also binds to oligo(dC). This is shown by a novel approach where we demonstrated specific binding of in vitro translated sub2.3 protein to biotinylated oligo(dC) which was immobilized on magnetic streptavidin-coated Dynabeads. Moreover we found that the sub2.3 transcript is expressed in a tissue dependent manner with the highest expression observed in several lymphoid tissues and skeletal muscle. The gene was also abundantly expressed in several lymphoid cell lines and the hepatoma cell line HepG2 while a low expression was observed in the HL60 myeloid cell line and in the HeLa cervical carcinoma cell line. In conclusion, this study presents the cDNA sequence of a novel transcript which shows tissue specific expression and encodes a protein with oligo(dC) binding specificity in vitro.

Constitutive expression of c-myc does not relieve cAMP-mediated growth arrest in human lymphoid Reh cells.

The Reh cell system is suitable for evaluating events important for control of proliferation independently of mechanisms involved in differentiation, as Reh cells are unable to differentiate. In the human pre-B cell line Reh, activation of adenylate cyclase by forskolin induces a five to tenfold rapid, transient down-regulation of steady-state c-myc RNA within 4 hours. Concurrently, the cells are strongly growth arrested in the G1 phase of the cell cycle. To clarify if the observed growth arrest could be relieved by constitutive expression of c-myc, an exogenous c-myc gene under constitutive promoter control was introduced into Reh cells by electroporation. The c-myc-expressing construct pDMmycHyg contained human c-myc exons 2 and 3 driven by the Mo-MLV LTR and conferred hygromycin resistance. Exogenous c-myc RNA transcripts and protein were constitutively expressed in the transfected clones at levels roughly twice as high as the level in nontransfected cells. Total c-myc protein levels were unchanged upon treatment of transfected clones with forskolin. Yet, the transfected cells were not released from growth arrest. Furthermore, the transfected Reh cells did not differentiate upon forskolin treatment. Constitutive overexpression of c-myc is therefore not sufficient for relieving forskolin-mediated effects on growth arrest in Reh cells.

Cell-specific expression of human beta-galactoside alpha 2,6-sialyltransferase transcripts differing in the 5' untranslated region.

In humans, two cDNAs have been isolated encoding beta-galactoside alpha 2,6-sialyltransferase, differing only in part of the 5' untranslated region. Primer extension data show that the two cDNAs are near full-length clones. RNase protection analysis of different cell types showed that the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a B-cell library resided only in mature B cells. In contrast, the transcript corresponding to the alpha 2,6-sialyltransferase cDNA isolated from a placenta library was found in all cells tested. Our results also indicate the existence of a third alpha 2,6-sialyltransferase transcript in the hepatoma cell line HepG2. Mature B cells were found to express high amounts of alpha 2,6-sialyltransferase mRNA, compared to other cell types tested, as shown by Northern blot analysis. Moreover there was an increased expression of beta-galactoside alpha 2,6-sialyltransferase mRNA in activated B cells compared to resting B cells. In vitro transcription and translation of the cDNAs resulted in a protein of 45 kDa, but the transcripts were translated with different efficiency, suggesting a role for the 5' untranslated region in regulation of translation. We have also made an alpha 2,6-sialyltransferase construct lacking the specific 5' regions of the two cDNAs. A transcript generated from this construct was translated more efficiently in vitro than the two alpha 2,6-sialyltransferase cDNAs.

T-cell receptor tau delta +/CD3+4-8-T- cell acute lymphoblastic leukemias: a distinct subgroup of leukemias in children. A report of five cases.

In a 10-year study of T-cell acute lymphoblastic leukemias (T-ALL) in children, we have identified five cases expressing the T-cell receptor tau delta (TCR tau delta). The incidence (26%) of TCR tau delta+T-cell leukemias in our material was high. Clinically, the TCR tau delta+ leukemias represented a distinct subgroup of T-cell leukemias. Mean age at onset of disease, 1.8 years, was remarkably low for mature T-cell leukemias. White blood cell counts were high, lymph node enlargements were discrete, and no mediastinal tumors were seen. Four of five TCR tau delta+ leukemias carried rearrangements of the C tau 2 gene, and transcribed the T-early alpha genetic element.

The B cell antigen CD75 is a cell surface sialytransferase.

In this work we have isolated a cDNA clone encoding the B cell antigen CD75. The amino acid sequence of CD75 is shown to be identical to that of human alpha 2,6 sialyltransferase, believed to be primarily associated with the Golgi complex. This is the first demonstration of cell surface expression of sialytransferase which, in B cells, may play an important role in intercellular adhesion and antigen presentation events.

Gene isolation with human T lymphocyte probes. Isolation of a gene that expresses an epitope recognized by T cells specific for Mycobacterium bovis BCG and pathogenic mycobacteria.

We have used human CD4+ T lymphocyte clones as primary probes to identify and isolate lambda gt11 rDNA clones that express epitopes recognized by T cells. The method that we describe here permits a direct survey of T cell epitope coding sequences in genomic DNA or cDNA libraries. A lambda gt11 library of Mycobacterium leprae DNA was screened with M. leprae-reactive human T cell clones as probes, allowing the isolation of a M. leprae DNA clone encoding the unidentified Ag. This DNA clone differs in restriction maps from those previously identified by antibody probes and encodes an epitope that is unique to vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin and pathogenic mycobacteria. This method is generally applicable and should expedite the study of Ag and epitopes important to the T cell response in infections and in autoimmune diseases.