Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3' fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3'-untranslated region of the gene.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

Cell cycle-dependent changes in tissue transglutaminase mRNA levels in bovine endothelial cells.

The pattern of transglutaminase gene expression through the cell cycle was examined by Northern blot analysis using cultured bovine endothelial cells and a cDNA probe. When the cells reached confluency or were arrested in G0/G1 phase by nutrition deprivation, transglutaminase mRNA rose to a very high level; S- and M-phase extracts showed high and low levels, respectively. Subcellular localization studies by sucrose gradient centrifugation and immunostaining demonstrated that the majority of transglutaminase is present in cytosols throughout the cycle. The cell cycle-dependent changes in the transglutaminase mRNA levels strongly support the implicated involvement of the enzyme in cell growth, differentiation, and senescence.     

The mechanism of placental alkaline phosphatase induction in vitro

The mechanism of placental alkaline phosphatase (PLAP) induction by prednisolone in a uterine cervical epidermoid cancer cell line SKG-IIIa was investigated in vitro by enzyme-cytochemistry, enzyme immunoassay, Northern and Southern blot analysis, and in situ hybridization. Enzyme-cytochemical alkaline phosphatase (ALP) staining and immunoassay revealed increased levels of PLAP (heat-stable ALP) in prednisolone-treated cells. Northern blot analysis and in situ hybridization showed increased amounts of PLAP mRNA. Southern blot analysis indicated that PLAP was not a product of an amplified or rearranged gene. These findings suggest that the induction of PLAP mRNA in SKG-IIIa cells by prednisolone in turn increased the levels of PLAP.     

CENP—B的基因表达与细胞周期关系的研究

本文以HeLa细胞为材料研究一种着丝粒蛋白CENP-B的基因表达与细胞周期及细胞核骨架的关系。将HeLa细胞同步在不同周期时相,以流式细胞光度术、同位素掺入和ACA着丝粒染色等方法检测细胞同步化效果。我们分别提取了各周期时相细胞的总RNA和Poly(A)~ RNA,用Dot blot和Northern blot杂交方法研究CENP-B在细胞周期中的表达。结果表明,CENP-B基因在细胞周期中的各个时相均有表达,但表达的强度差别很大:G2期表达最强,S期最弱,G1期中的表达介于二者之间;有意义的是CENP-B基因在M期仍然有较强的表达,表现出其在细胞周期中表达的持续性;这种表达的持续性反映了一种可能性:着丝粒、动粒蛋白不断合成,但直到S期后进入G2期时着丝粒、动粒蛋白到一定临界浓度时才开始组装新的动粒。另外,着丝粒、动粒蛋白的持续合成对着丝粒、动粒功能的发挥可能是必需的。用Bam H I限制性内切酶消化处于不同细胞周期时相的HeLa细胞核骨架,提取与核骨架紧密结合的DNA,用~(32)P标记的cDNA为探针研究CENP-B基因与细胞核骨架的结合与其表达的关系。结果证明,在G2期细胞中CENP-B基因表达最强,与细胞核骨架结合最为紧密,G1期细胞中次之,S期中CENP-B基因与核骨架结合最弱,说明CENP-B基因与细胞核骨架结合的紧密度影响其表达强度。     

Application and limitations of differential hybridization in the isolation of T cell-specific cDNA clones

We investigated the limitations and effectiveness of differential hybridization in the cloning of T cell-specific cDNA (complementary DNA) molecular clones. By using the technique with T cell and B cell cDNA probes, together with Northern blot analysis, we successfully isolated cDNA clones exclusively expressed in T cells from 1 X 10(4) plaque-forming units of a T cell hybridoma. These clones represent 0.068% of the mass of the cytoplasmic mRNA. Our result shows that differential hybridization is an effective procedure when used in combination with Northern blot analysis for screening of genes selectively expressed in T cells.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Evidence for increased translational efficiency in the induction of P450IIE1 by solvents: analysis of P450IIE1 mRNA polyribosomal distribution

The potential for enhanced translational processing of P450IIE1 mRNA during the early phase of P450IIE1 induction by pyridine or acetone was assessed by hybridization analysis of polyribosomal P450IIE1 mRNA distribution in rat hepatic tissue. Optical absorbance profiles of polyribosomal fractions exhibited an apparent shift at 5 h following pyridine administration relative to control. Slot and Northern blot analyses for P450IIE1 mRNA in the cytoplasmic extracts isolated from 5 h pyridine-treated rats demonstrated a shift in distribution of P450IIE1 message toward heavier polyribosomal fractions and Northern blot analysis suggested the presence of different populations of P450IIE1 mRNA. Slot blot analyses also demonstrated a shift in the polyribosomal distribution of P450IIE1 mRNA at 12 h following pyridine treatment; in contrast, hybridization analysis for P450IA1 revealed no shift in polyribosomal distribution of P450IA1 mRNA. Acute acetone administration to animals also resulted in a similar shift in polyribosomal distribution of P450IIE1 mRNA as compared to control. These data suggest that P450IIE1 mRNA shifts toward larger polyribosomes following acute exposure of animals to pyridine or acetone and provide evidence that induction of P450IIE1 at early times following acute pyridine or acetone administration involves enhanced translational efficiency through increased loading of ribosomes on P450IIE1 mRNA.     

Identification and characterization of an ovary-selective isoform of epoxide hydrolase

A novel ovary-selective gene was identified by suppression subtractive hybridization (SSH) that is expressed only during the mouse periovulatory phase of a stimulated estrous cycle. Analysis of the protein encoded by the full-length cDNA revealed that the majority of it, with the exception of the first 44 amino acids, matched soluble epoxide hydrolase (Ephx2, referred to as Ephx2A). By comparing the cDNA sequence of this newly identified variant of soluble epoxide hydrolase (referred to as Ephx2B) with the mouse genome database, an exon was identified that corresponds to its unique 5' cDNA sequence. Through the use of an Ephx2A-specific probe, Northern blot analysis revealed that this mRNA was also expressed in the ovary, with the highest level of expression occurring during the luteal phase of a stimulated estrous cycle. In situ hybridization revealed that Ephx2B mRNA expression was restricted to granulosa cells of preovulatory follicles. Ephx2A mRNA expression, however, was detectable in follicles at different stages of development, as well as in the corpus luteum. Total ovarian epoxide hydrolase activity increased following the induction of follicular development, and remained elevated through the periovulatory and postovulatory stages of a stimulated estrous cycle. The change in enzyme activity paralleled the combined mRNA expression profiles for both Ephx2A and Ephx2B, thus supporting a role for epoxide metabolism in ovarian function.