Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli

The bifunctional enzyme chorismate mutase (CM)-prephenate dehydratase (PD), which is encoded by the pheA gene of Escherichia coli, catalyses the two consecutive key steps in phenylalanine biosynthesis. To utilize the enzyme for metabolic engineering of phenylalanine-producing Corynebacterium glutamicum KY10694, the intact gene was cloned on a multicopy vector to yield pEA11. C. glutamicum cells transformed with pEA11 exhibited a more than tenfold increase in CM and PD activities relative to the host cells. Moreover, the level of pheA expression was further elevated a fewfold when cells were starved of phenylalanine, suggesting that the attenuation regulation of pheA expression functions in heterogeneous C. glutanicum. Plasmid pEA11 encoding the wild-type enzyme was mutated to yield pEA22, which specified CM-PD exhibiting almost complete resistance to end-product inhibition. When pEA22 was introduced into KY10694, both the activities of CM and PD were highly maintained throughout the cultivation, thus leading to a 35% increased production (23 g/l) of phenylalanine.     

Molecular analysis of the dhp1 + gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST 2 and RAT 1 genes of Saccharomyces cerevisiae

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.     

The 3′ → 5′ exonucleases of both DNA polymerases δ and ε participate in correcting errors of DNA replication in Saccharomyces cerevisiae

DNA polymerases II (ε) and III(δ) are the only nuclear DNA polymerases known to possess an intrinsic 3′ → 5′ exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase δ and ε 3′ → 5′ exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10² and 10¹, respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 × 10³-fold. Analysis of mutation rates of double mutants indicated that the 3′ → 5′ exonucleases of DNA polymerases δ and ε can act competitively and that, like the 3′ → 5′ exonuclease of DNA polymerase δ the 3′ → 5′ exonuclease of DNA polymerase ε acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3′ → 5′ exonucleases of DNA polymerases δ and ε act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.     

Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining

In northwest European countries maternal age is increasing. This will lead to an increase of the prevalence of Down syndrome conceptuses. Meanwhile, the increased use of prenatal cytogenetic diagnosis (PCD) will lead to a decrease in the prevalence of Down syndrome among livebirths. We were interested to know what the result of these two opposite developments would be in the near future, and we describe here a model to quantify these processes and the resulting livebirth prevalence of Down syndrome. The model is demonstrated for The Netherlands from 1992 to 2001. The predicted livebirth prevalence for The Netherlands in 1992 is 1.36 per 1000. Demographic factors will cause an increase to 1.76 per 1000 in 2001 with present indications for PCD and a utilization ratio of 50%. An increase of the utilization ratio to 90% in 2001 will lead to a prevalence of 1.22 per 1000, a little less than the present prevalence. Alternative screening programs, including maternal serum screening, could lead to a further decrease of the livebirth prevalence. The model described here can be used for evaluation of the consequences of alternative forms of Down syndrome screening.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

Gas chromatographic—mass spectrometric determination of tiopronin in human blood using acrylic acid esters as a derivatization reagent for the thiol group

A gas chromatographic—mass spectrometric method for determining tiopronin, which has a thiol group, in human blood has been described. To prevent the oxidative degradation of tiopronin in the blood, its thiol group was immediately protected by treatment with isobutyl acrylate, which reacted readily with tiopronin in a 0.1 M Na₂HPO₄ solution (pH 9.1). The reaction was quantitative within 30 min. The blood sample was deproteinized and purified by a combination of liquid—liquid extraction and solid-phase extraction. Finally, the carboxyl moiety of the ester adduct was derivatized to the pentafluorobenzyl ester. The derivatives of tiopronin and the internal standard were analysed with gas chromatography—mass spectrometry. The precision of the method was satisfactory, and the calibration curve had good linearity in the concentration range investigated. The limit of determination of tiopronin in blood was estimated to be ca. 1 ng/ml.     

Canopy photosynthetic production in a Japanese larch forest. II. Estimation of the canopy photosynthetic production

Seasonal changes and yearly gross canopy photosynthetic production were estimated for an 18 year old Japanese larch (Larix leptolepis) forest between 1982 and 1984. A canopy photosynthesis model was applied for the estimation, which took into account the effect of light interception by the non-photosynthetic organs. Seasonal changes in photosynthetic ability, amount of canopy leaf area and light environment within the canopy were also taken into account. Amount of leaf area was estimated by the leaf area growth of a single leaf. The change of light environment within the canopy during the growing season was estimated with a light penetration model and the leaf increment within the canopy. Canopy respiration and surplus production were calculated as seasonal and yearly values for the three years studied. Mean yearly estimates of canopy photosynthesis, canopy respiration and surplus production were 37, 13 and 23 tCO₂ ha⁻¹ year⁻¹, respectively. Vertical trend, seasonal changes and yearly values of the estimates were analyzed in relation to environmental and stand factors.