BiochemistryCloning of the DNA-binding subunit of human nuclear factor cB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor a (transcription factor/protein purifilcation/DNA sequencing/regulation of nuclear factor ucB expression/multigene family) RALF MEYER*, EUNICE N. HATADA*, HANS-PETER HOHMANNt, MONIKA HAIKERt, CORNELIA BARTSCH*, URS R6THLISBERGERt, HANS-WERNER LAHMt, ERNST J. SCHLAEGERt, ADOLPHUS P. G. M. VAN ABSTRACTThe DNA binding subunit of nuclear factor KB (NF-acB), a B-cell protein that interacts with the inmunoglobulin K light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor a (TNFa), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-cB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-KB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFa or phorbol ester. Thus, both factors not only activate NF-KB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-KB.

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.

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