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.

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.

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 Agtll 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

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In humans, two cDNAs have been isolated encoding P-galactoside a2,6-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 a2,6-sialyltransferase cDNA isolated from a B-cell library resided only in mature B cells. In contrast, the transcript corresponding to the a2,6-sialyltransferase cDNA isolated from a placenta library was found in all cells tested. Our results also indicate the existence of a third a2,6-sialyltransferase transcript in the hepatoma cell line HepG2. Mature B cells were found to express high amounts of a2,6-sialyltransferase mRNA, compared to other cell types tested, as shown by Northern blot analysis. Moreover there was an increased expression of P-galactoside a2,6-sialyltransferase mRNA in activated B cells compared to resting B cells. la 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 a2,6-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 a2,6-sialyltransferase cDNAs.The expression of cell-surface carbohydrate groups is subjected to changes during development, differentiation and oncogenic transformation. Moreover, there is strong evidence that specific terminal sugar structures play important roles in processes like cell motility and cellular interactions [l -61. Termination of sugar structures in a cell is dependent on several parameters; among these are the differential expression of sugar transferases in cells, the ratio between enzymes competing for the same substrate [7, 81 and sugar branch specificity of the enzymes [9]. P-Galactoside a2,6-sialyltransferase (a2,6-sialyltransferase) is expressed in many tissues, including mature B cells, which express high levels of its transcripts [lo, 111 (and this study). This enzyme is known to catalyse the attachment of sialic acid to the N-linked oligosaccharide structure