A micromachined chemical amplifier was successfully used to perform the polymerase chain reaction (PCR) in continuous flow at high speed. The device is analogous to an electronic amplifier and relies on the movement of sample through thermostated temperature zones on a glass microchip. Input and output of material (DNA) is continuous, and amplification is independent of input concentration. A 20-cycle PCR amplification of a 176-base pair fragment from the DNA gyrase gene of Neisseria gonorrhoeae was performed at various flow rates, resulting in total reaction times of 90 seconds to 18.7 minutes.

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The expression of collagen VI, an adhesive glycoprotein of the extracellular matrix, is completely inhibited in virally transformed fibroblasts and in many cell lines derived from spontaneous mesenchymal tumors. Here we present evidence that DNA methylation plays an important role in this inhibition: (a) The mRNA level for DNA methyltransferase is highly increased in simian virus 40 (SV40)-transformed fibroblasts compared with normal cells and this increase correlates with the decrease of the mRNA level for collagen VI. Keywords: collagen; DNA methylation; extracellular matrix ; oncogenic transformation ; promoter.Tumor cells display dramatic differences in their phenotype when compared with normal cells. They often exhibit a roundish shape, show irregularities on their cell surface and contain a disrupted cytoskeleton. It is likely that these changes contribute to the invasive and metastatic properties of malignant cells. Today, it is generally accepted that the alterations of the phenotype are caused by subtle changes in the synthesis rate of a distinct set of proteins (termed transformation-sensitive proteins) that are directly or indirectly involved in cell adhesion and attachment. Fibroblasts transformed by tumor viruses are frequently used in the laboratory as a model system to investigate transformation-associated alterations. Such fibroblasts display the characteristic phenotype of a tumor cell and have the advantage that the normal counterpart, the original cell, is available for comparative studies.We have recently used this model system to characterize the molecular events that lead to the transformed phenotype. A subtractive cDNA library was prepared from mRNA of normal fibroblasts and their simian virus 40 (SV4O)-transformed counterparts with the objective of obtaining a comprehensive set of cDNA clones for proteins that are specifically lost upon oncogenic transformation [ 11. As expected, this library contained many clones that coded for proteins of the extracellular matrix or the cytoskeleton, including collagen VI, fibronectin and vinculin [ 1 -31.Collagen VI had already been under investigation in our laboratory for several years as a typical example of a transformation-sensitive protein [4] (Mr 600 000) composed of three different polypeptide chains, al(VI), a2(VI) and a3(VI), and forms a fibrillar network independent from the interstitial collagen fibers (for review see [5, 61). The major part of the molecule consists of globular domains which share significant sequence similarity with the A domains of von Willebrand factor and the I domain of some integrin receptors [6, 71. The collagenous triple helix contributes less than 25 % to the entire molecular mass, yet it is this domain that has given the molecule its name. Collagen VI promotes attachment and spreading of fibroblasts in vitro similar to fibronectin. Consistent with this function, the protein is found in intimate contact with the plasma membrane where it interacts with integrins of the pl family [6].We and others found that the synt...

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