The nucleotide sequence of the portion of a Bacillus subtilis (strain PAP115) 3 kb Pst I fragment which contains an endo-beta-1, 4-glucanase gene has been determined. This gene encodes a protein of 499 amino acid residues (Mr = 55,234) with a typical B. subtilis signal peptide. Escherichia coli which has been transformed with this gene produces an extracellular endoglucanase with an amino-terminus corresponding to the thirtieth encoded amino acid residue. The gene is preceded by a cryptic reading frame with a rho-independent terminator structure, and itself has such a structure in the immediate 3'-flanking region. We have also identified, in the 5'-flanking region, nucleotide sequences which resemble promoter elements recognized by Bacillus RNA polymerase E sigma 43. Comparison of the encoded amino acid sequence to other known beta-glucanases reveals a small region of similarity to the encoded protein of the Clostridium thermocellum celB gene. These similar regions may contain substrate-binding and/or catalytic sites.

The extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS-PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0-7.0. Under the conditions tested, the activity is maximal between 45 and 50 degrees C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

The induction of alpha-amylase by starch has been studied in the filamentous fungus Aspergillus oryzae. Low levels of alpha-amylase activity were found in both intracellular and extracellular samples from glucose-grown cultures. However, alpha-amylase activity increased when starch was the sole carbon source. The intracellular enzyme activity was induced by a factor of approximately 6.5, while the extracellular activity increased 20-fold over that found in the glucose-grown cultures. Regardless of the carbon source or cellular location, the molecular weight of the active protein was 52 500 +/- 1800 and only this protein reacted with antibodies specific for alpha-amylase. A parallel study of the in vitro translated proteins directed by poly(A)+ RNA fractions indicated a presumptive alpha-amylase with a similar isoelectric point but with a molecular weight of approximately 54 000. This protein was most prevalent using RNA from early, exponentially growing cultures in starch medium. Immunoprecipitation data indicate that the abundance of alpha-amylase transcripts decreases dramatically after the first 12 h, reflecting an initial transcription control for the expression of this enzyme.

Using a filter binding assay, we have detected and partially purified a protein from human placenta that has a high affinity for N-acetoxy-2-acetylaminofluorene-modified double-stranded DNA (AAF-[3HJDNA) of bacteriophage 17. This protein has been partially purified from a 1 M NaC extract of a crude nuclear fraction by a combination of ion-exchange and nucleic acid affinity chromatography. With Although the excision repair of chemical damage clearly shares many features with the repair of UV-induced damage (1), recent experiments have suggested that there may be differences in the ways in which lesions produced by these two types of agents are recognized and removed (2, 3). If differences do exist, a class of proteins may exist that selectively recognizes DNA modified by chemical carcinogens. This logic has led us to search for DNA-binding proteins that recognize N-acetoxy-2-acetylaminofluorene (NA-AAF)modified DNA (AAF-DNA).To assay for such proteins, we have used filter binding techniques (4) similar to those used to demonstrate DNA-binding proteins that specifically recognize UV-damaged DNA (5) and partially depurinated DNA (6). In this paper we report the partial purification and characterization of a DNA-binding protein from human placenta that shows strong preferential binding to AAF-DNA and DNA modified with methyl methanesulfonate or methylnitrosourea (MMS-DNA or MNU-DNA, respectively) as compared to single-and double-stranded DNA, supercoiled bacteriophage OX174 DNA, partially depurinated DNA, glucosylated bacteriophage T4 DNA, or UV-irradiated DNA.MATERIALS AND METHODS Preparation of Binding Substrates. Bacteriophage T7[3H]DNA (1.21 X 105 cpm/,ug, unless otherwise indicated) was prepared from phage grown and isolated as described in the literature (7,8), and the DNA was purified from NaDoDSO4-lysed, proteinase K-digested (Merck) phage (9).AAF-[3H]DNA was prepared as described (10).

The yeast Schwanniom?ces alluvius ferments soluble starch to ethanol at a conversion efficlen~---c~-of greater than 95%. Only trace amounts of side products are detectable.