Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Foreign protein expression from S phase specific promoters in continuous cultures of recombinant CHO cells

Foreign protein expression from the commonly used SV40 promoter has been found to be primarily during the S-phase of the cell cycle. Simple mathematical models with this cell cycle phase dependent expression of foreign protein suggest that the specific production rate will be proportional to the cell growth rate, which is particularly disadvantageous in high cell density fed-batch or perfusion bioreactors. In this study we investigate this predicted relationship between the production rate and growth rate by culturing recombinant CHO cells in a continuous suspension bioreactor. One CHO cell line, GS-26, has been stably transfected with the plasmid pSVgal, which contains the E. coli lac Z gene under the control of the SV40 promoter. This GS-26 cell line was grown in suspension cultures over a range of specific growth rates in batch and continuous modes. The intracellular -galactosidase activity was assayed using a standard spectrophotometric method after breaking the cells open and releasing the enzyme. A strong growth associated relationship is found between the intracellular -galactosidase content and the specific growth rate in batch and continuous cultures, as predicted.     

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry

Summary The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90°) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40–60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained as control cells progress through the cell cycle. At later times postinfection (>42 h), total protein decreased due to cellular changes resulting from viral replication and cell death.     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene

As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.Abbreviations ble bleomycin resistance gene - Hm hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene     

Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

血清SAA、YKL-40及SP-A的联合检测对小儿难治性肺炎支原体肺炎的预测价值分析

摘要 目的：探究血清淀粉样蛋白A（SAA）、几丁质酶样蛋白YKL-40和肺表面活性物质相关蛋白-A（SP-A）表达水平对的联合检测对小儿难治性肺炎支原体肺炎（RMPP）的预测价值。方法：纳入2019年11月至2021年12月期间我院收治的60例MPP患儿作为研究对象,依据病情最终转归将其分为RMPP组和普通肺炎支原体肺炎（GMPP）组,另纳入同期于我院行体格检查的60例健康儿童作为对照组。收集所有受试儿童的临床资料、实验室指标及影像学结果,采用酶联免疫吸附法（ELISA）检测血清SAA、YKL-40和SP-A的表达水平,应用受试者工作特征曲线（ROC）判定各指标单项检测和联合检测的预测效能。结果：MPP组患儿和对照组儿童一般资料相比无统计学差异（P＞0.05）。依据病情最终转归,60例MPP患儿中共有23例（38.33 %）进展为RMPP,RMPP组与GMPP组患儿在白介素-6（IL-6）、C-反应蛋白（CRP）、降钙素原（PCT）、D-二聚体（D-D）等实验室指标以及肺不张、胸腔积液等影像学特征方面相比有统计学差异（P＜0.05）。RMPP组和GMPP组血清SAA、YKL-40和SP-A的表达水平显著高于对照组（P＜0.05）,同时RMPP组血清SAA、YKL-40和SP-A的表达水平显著高于GMPP组（P＜0.05）。血清SAA的最佳截断值为39.75 mg/L,预测RMPP发生的ROC曲线下面积为0.894（95%CI：0.861-0.925）,敏感度为78.26 %,特异度为86.67 %;血清YKL-40的最佳截断值为31.85 ng/mL,预测RMPP发生的ROC曲线下面积为0.754（95%CI：0.634-0.873）,敏感度为73.91 %,特异度为67.21 %;血清SP-A的最佳截断值为35.59 ng/mL,预测RMPP发生的ROC曲线下面积为0.761（95%CI：0.640-0.891）,敏感度为73.91 %,特异度为75.00 %;SAA+YKL-40-1+SP-A三者联合检测预测RMPP的AUC为0.914（95%CI：0.871-0.957）,敏感度为91.30 %,特异度为91.66 %。结论：血清SAA、YKL-40和SP-A表达水平的检测可作为预测RMPP发生的重要生物学指标,且三者联合检测的预测效能较高,可为临床尽早诊断RMPP、尽早干预、改善患儿预后提供一定的帮助。     

真核生物DNA复制起始调控

复制起始调控是真核生物复制调控机制的重要环节,也是细胞生长调控的核心问题．对SV40病毒和酵母体系的研究为阐明真核生物的复制起始机制及其与细胞周期的关系提供了线索．目前,与DNA复制起始有关的多种蛋白质因子（如核蛋白P₁,DNA单链结合蛋白,DNA聚合酶α,增殖细胞核抗原等）的作用机理逐渐明朗,周期依赖的调控特点得到了证实文章着重介绍了DNA复制起始在细胞周期中的两个调控点及各种周期蛋白在该点的作用,文中还涉及复制起始异常与肿瘤发生的关系.