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A continuous supply of O(2) is important for itaconic acid production in Aspergillus terreus. Any interruption of aeration significantly reduces itaconic acid production. To overcome this effect, A. terreus M8 was transformed with the Vitreoscilla hemoglobin gene (vgb) which, as shown by Southern hybridization, was integrated into the recipient chromosome. The activity of the expressed hemoglobin was confirmed by a CO-difference spectrum. During itaconic acid production, the effect of a break in aeration during cultivation in the transformant with the vgb gene is alleviated. Additionally, the transformant shows improved itaconic acid production.

Objective. Increased sialylation has been reported in various kinds of cancers, but to date, sialylation of cervical carcinoma has never been evaluated. This study of the changes in messenger ribonucleic acid (mRNA) expression of the four sialyltransferases (ST3Gal I, ST3Gal III, ST3Gal IV, and ST6Gal I) in a normal cervix and that with FIGO stage IB1 squamous cell carcinoma was undertaken to assess the extent of sialylation associated with establishment of the carcinoma. Methods. Alterations in ST mRNA expression in FIGO IB1 cervical cancer ( n = 30) and normal cervixes ( n = 30) were examined by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). Results. ST6Gal I expression was enhanced in squamous cell carcinoma of the cervix ( P = 0.026, Mann–Whitney U test), but mRNA expression from the other three STs (ST3Gal I, ST3Gal III, and ST3Gal IV) was significantly down-expressed in squamous cell carcinoma of the cervix compared to the normal cervix ( P = 0.003, P P = 0.001, respectively). High ST6Gal I expression was associated with more invasive properties of cervical cancer, such as deep stromal invasion, lymph or vascular space involvement, and poor differentiation ( P = 0.010, P P Conclusions. A combination of enhanced ST6Gal I mRNA expression and decreased mRNA expression from ST3Gal I, ST3Gal III, and ST3Gal IV might be important in cervical cancer. Future studies will investigate whether RT-PCR detection of the expression of these enzymes can be helpful for prognostic purposes.

Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III. A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb. The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts. The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein. Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination. The pattern of expression of rec14 differs from that of previously analyzed S. pombe rec genes. Based upon mutant phenotypes and amino acid sequence similarities, we propose that S. pombe Rec14 is a functional homologue of S. cerevisiae Rec103.

Photoreactivating enzyme (DNA photolyase; deoxyribocyclobutadipyrimidine pyrimidine-lyase, EC 4.1.99.3) repairs UV damage to DNA by utilizing the energy of near-UV/visible light to split pyrimidine dimers into monomers. The enzyme is widespread in nature but is absent in certain species in a seemingly unpredictable manner. Its presence in humans has been a source of considerable controversy. To help resolve the issue we used a very specific and sensitive assay to compare photoreactivation activity in human, rattlesnake, yeast, and Escherichia coli cells. Photolyase was easily detectable in E. coli, yeast, and rattlesnake cell-free extracts but none was detected in cell-free extracts from HeLa cells or human white blood cells with an assay capable of detecting 10 molecules per cell. We conclude that humans most likely do not have DNA photolyase.

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.

Transforming growth factor beta-1 (TGF-beta1) is a multifunctional growth factor and known to inhibit the proliferation of epithelial cell. The relationship between serum TGF-beta1 level and the presence of cervical cancer was investigated in this study. The patients with cervical squamous cell carcinoma (SCC) had significantly lower level of serum TGF-beta1 (39.14 +/- 9.03 ng/mL; mean +/- SD) than those with myoma (49.17 +/- 9.38 ng/mL) and normal subjects (49.13 +/- 8.81 ng/mL), (p < 0.007 and p < 0.001, respectively). TGF-beta1 level was also lower in the patients with cervical intraepithelial neoplasia (CIN3) (42.07 +/- 9.38 ng/mL) than in normal controls (49.13 +/- 8.81 ng/mL) (p < 0.04). It suggested that diminution of the production of TGF-beta1 has close association with the neoplastic transformation of cervical epithelium.

Previously isolated meiotic recombination (rec) mutants of Schizosaccharomyces pombe define 16 complementation groups. The rec genes cloned and sequenced to date reveal little amino acid sequence identity to other reported proteins. We examined the rec mutants for alterations in meiotic events other than recombination to gain insight into the rec gene functions and to assess whether they affect recombination directly or indirectly. While mutations in the rec6–12, 14, 15 and 19 genes appeared to affect only meiotic recombination, a mutation in rec16 delayed meiotic DNA synthesis and, in some instances, reduced its amount; mitotic DNA synthesis was not detectably altered, indicating that the rec16 effect is limited to meiosis. In the rec16 mutant some meiotically induced transcripts (e.g., rec7 and 15) were significantly reduced in abundance, whereas others (e.g., rec10 and exo1) were induced and degraded with normal timing and extent during meiosis, indicating that the rec16 mutation leaves the basic meiotic program intact. These results indicate that the rec genes other than rec16 have their primary effect on meiotic recombination. In contrast, the rec16 gene product is essential for normal meiotic replication, recombination, and induction of some transcripts. These meiotic events may be coupled via a dependence of recombination and transcription on replication or via a cascade of gene expression.

DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.

Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction. The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA. No structural information on the enzyme is available at present. However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology. These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis. Trp277 (W277) in E. coli photolyase is conserved in all photolyases sequenced to date. We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis. Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis. Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.

The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca 2+ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The K cat / K m values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes.