Whether Zn2+ for the polymerase system was selected inevitably or by historical accident

Zinc ion was found in various kinds of polymerases and involved in the activation of the 3'-hydroxyl group of primer chain's growing end. In order to investigate whether zinc ion was selected for nucleotide synthesizing system inevitably or by historical accident, ab initio molecular orbital calculations were carried out and the results suggest that zinc ion may have been selected inevitably.     

Gene expression and immunohistochemical localization of basic fibroblast growth factor in renal cell carcinoma.

Renal cell carcinoma is known as a neoplastic condition of renal tubular cells and usually shows a hypervascular tumor in angiographic examination. We examined the presence of basic fibroblast growth factor (bFGF) in human renal cell carcinoma. To determine if alterations in bFGF gene expression are present in human renal cell carcinoma, paired samples of normal and neoplastic renal tissue from 6 patients were analyzed for bFGF mRNA content by Northern blot hybridization. In 4 out of 6 patients, tumor tissue expressed bFGF mRNA 2 to 4 times greater than corresponding normal tissue. Two patients showed minimal elevation of tumor bFGF mRNA. The localization of bFGF in the renal cell carcinoma tissue was also examined using immunohistochemical staining, and it was found that bFGF was positively stained at the nuclei of tumor cells and the cell surface. These results suggest that increased expression of bFGF may be associated with neoplastic growth in renal tubular epithelial cells and neovascularization.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Effects of Far-Red Light on K+ Fluxes in a Colorless Mutant of Chlorella

In a colorless mutant of Chlorella kessleri, far-red light significantlyenhanced the K⁺ efflux. This effect was abolished by the K⁺channel-blocker tetraethylammonium acetate. Using cyanine dyeto monitor membrane potential, we deduced that the K⁺ effluxunder far-red light was probably accompanied by hyperpolarizationof the plasmalemma. (Received August 30, 1993; Accepted November 16, 1993)     

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.