Replicative Bypass of Abasic Site in Escherichia coli and Human Cells: Similarities and Differences

Abasic [apurinic/apyrimidinic (AP)] sites are the most common DNA damages, opposite which dAMP is frequently inserted (‘A-rule’) in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a ‘C-rule’, ‘A-rule’, or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z), a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the ‘A-rule’ was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z→T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36–67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.     

DNA Polymerase II of Escherichia Coli in the Bypass of Abasic Sites in Vivo

The function of DNA polymerase II of Escherichia coli is an old question. Any phenotypic character that Pol II may confer upon the cell has escaped detection since the polymerase was discovered 24 yr ago. Although it has been shown that Pol II enables DNA synthesis to proceed past abasic sites in vitro, no role is known for it in the bypass of those lesions in vivo. From a study of phage S13 single-stranded DNA, we now report SOS conditions under which Pol II is needed for DNA synthesis to proceed past abasic sites with 100% efficiency in vivo. Overproduction of the GroES(+)L(+) heat shock proteins, which are members of a ubiquitous family of molecular chaperones, eliminated this requirement for Pol II, which may explain why the role of Pol II in SOS repair had eluded discovery. Mutagenesis accompanied SOS bypass of abasic sites when the original occupant had been cytosine but not when it had been thymine; the quantitative difference is shown to imply that adenine was inserted opposite the abasic sites at least 99.7% of the time, which is an especially strict application of the A-rule. Most, but not all, spontaneous mutations from Rif(s) to Rif(r), whether in a recA(+) or a recA(Prt(c)) cell, require Pol II; while this suggests that cryptic abasic lesions are a likely source of spontaneous mutations, it also shows that such lesions cannot be the exclusive source.     

Human Trophoblast Cells Are Permissive to the Complete Replicative Cycle of Human Cytomegalovirus

Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.     

Giant Cells of Escherichia coli

A mutant strain of Escherichia coli K-12 produced amorphous cells when grown in a variety of media. The lon(-) allele, known to increase the radiation sensitivity of the cytokinesis mechanism, was introduced into the mutant by means of conjugation. Cells of this recombinant strain grew, after exposure to radiation, into giant amorphous cells, approximately 500 to 1,000 times the volume of a normal E. coli cell. These giant cells are analogous to the filaments formed after the irradiation of lon(-) rod-shaped cells.     

重组人碱性成纤维细胞生长因子(bFGF)融合蛋白的高效表达及鉴定

碱性成纤维细胞生长因子（ｂＦＧＦ）参与了许多细胞生长和分化的调控过程。本文采用重组ＤＮＡ技术在大肠杆菌中高效表达了人ｂＦＧＦ。首先将编码人ｂＦＧＦ基因克隆到ｐＸＴ表达载体中与其上游的一短Ｓ导肽共一阅读框架,ｂＦＧＦ基因的表达受强的Ｔ７启动子调控。采用ＢＬ２１（ＤＥ３）大肠杆菌作为宿主菌,用ＩＰＴＧ诱导ＢＬ２１（ＤＥ３）细菌合成的Ｔ７ＲＮＡ聚合酶,后者可催化高水平的ｂＦＧＦ基因表达,其ｂＦＧＦ产量可占总菌体蛋白的４２.５％。采用肝素Ｓｅｐｈａｒｏｓｅ一步亲和层析法直接从诱导后的细菌裂解产物中得到纯化的重组人ｂＦＧＦ蛋白。经Ｗｅｓｔｅｒｎ印迹分析证明该蛋白可被人ｂＦＧＦ特异性单克隆抗体所识别。进一步研究证明该蛋白具有刺激ＮＲ６Ｒ－３Ｔ３成纤维细胞增殖的生物学活性,并且这一活性可被人ｂＦＧＦ特异性中和抗体所中和。     

Similarities in control of mini-F plasmid and chromosomal replication in Escherichia coli.

In Escherichia coli, concentrations of a mini-F plasmid with two origins of replication (oriV and oriS) were 50% lower in fast-growing cells than in slow-growing cells. By contrast, a mini-F plasmid deleted for oriV maintained a uniform concentration in both fast- and slow-growing cells, and in this behavior the plasmid mimicked the control by the host of chromosomal origin (oriC) concentration.     

Tracer Studies to Locate the Site of Platinum Ions Within Filamentous and Inhibited Cells of Escherichia coli

The distribution of platinum ions within Escherichia coli after the induction of filaments with cis-Pt(NH(3))(2)Cl(4), and after growth inhibition by PtCl(6) (2-), has been determined with radioactive metal compounds ((191)Pt, with a half-life of approximately 3 days) by the simple chemical procedure of Roberts et al. In the filamentous cells, the platinum metal is associated with metabolic intermediates, nucleic acids, and cytoplasmic proteins; whereas, in inhibited cells, the platinum is combined only with the cytoplasmic protein. Similar experiments with gram-positive cells of Bacillus cereus and Staphyloccus aureus, which show no filamentous growth in the presence of cis-Pt(NH(3))(2)Cl(4), reveal that the metal complex does penetrate the cell wall and subsequently becomes bound predominantly by metabolic intermediates.     

大肠杆菌yijP侵袭蛋白诱导人脑微血管内皮细胞凋亡

为研究大肠杆菌的脑微血管内皮细胞侵袭基因yijP的功能,将yijP基因（1．04kb）克隆到pQE30表达载体,构建表达产物为N末端带有6个组氨酸（His）序列的yijP汇合蛋白,以M15（pREP4）为受体菌,大量表达（His）6-yijP汇合蛋白,利用Ni—NTA亲和层析纯化汇合蛋白,将经透析法复性的一定浓度的（His）6-yijP蛋白加入到体外培养的人脑微血管内皮细胞中,结果显示yijP蛋白对人脑微血管内皮细胞有较强的细胞毒作用：在相差显微镜下可观察到细胞皱缩、胞膜呈泡状膨出,随着时间延长细胞逐渐脱落;荧光显微镜下可见细胞核呈现为致密团块状或圆形浓染颗粒状,呈凋亡样改变：DNA琼脂糖凝胶电泳可见DNA阶梯状条带;流式细胞仪显示在正常二倍体峰之前出现一个亚二倍体峰;Western印迹可检测到caspase-3的活性片段。这些现象均出现在yijP蛋白作用于人脑微血管内皮细胞的16h之后,提示在大肠杆菌侵袭人脑微血管内皮细胞过程中,yijP蛋白可能起到诱导脑微血管内皮细胞迟发性凋亡的毒素作用。     

Differences and Similarities: Comparative Methods in Mastication

Comparison is the key to biological study and both similaritiesand differences aid in sharpening our understanding of structuresand processes. The initial comparison represents an observationallevel; followed by decisions aboutpossible homology, analogyand homoplasy which represent levels that have less certainty.Analogous homoplasies are presumably the most useful conditionsfor characterizing functional constraints. Operational criteriaare discussed, as are minimal technical standards for procedurespermitting characterization of functions and roles in the masticatorysystem.     

Gap-Directed Translesion DNA Synthesis of an Abasic Site on Circular DNA Templates by a Human Replication Complex

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged “minicircle” DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.     

Giant Cells of Escherichia coli: a Morphological Study

Bacterial growth without division was observed in a giant cell-producing strain of Escherichia coli K-12. Giant cell production is controlled by the lon(-) (failure of cell division after irradiation) and mon(-) (formation of irregularly shaped cells) genes. Irradiation of a lon(-)mon(-) strain (P678-A(4)) with low doses of ultraviolet or ionizing radiation results in the production of large, amorphous giant cells with 500 to 1,000 times the volume of the nonirradiated parents. The concentration of NaCl in the growth medium was found to influence irradiated-cell morphology. Low concentrations (0.2% NaCl) resulted in elongated cells, whereas spherical giant cells were produced in the presence of high salt (1% NaCl) concentrations.Thin-section electron microscopy revealed an extensive network of intracellular membranes forming vacuoles, vesicles, and cisternae. These structures bear a striking resemblance to the rough and smooth membranes (endoplasmic reticulum, Golgi complex, vacuoles, etc.) found in eucaryotic cells.     

Acridine Binding by Escherichia coli: pH Dependency and Strain Differences

Acridine dye binding by cells of Escherichia coli has been characterized in terms of a number of parameters. There is a temperature-dependent, readily reversible binding of acriflavine which occurs to a greater extent with acridine-sensitive mutants of E. coli K-12 than with wild-type E. coli B or K-12. There is an essentially irreversible internal binding of acriflavine which occurs when the cellular permeability barriers are destroyed or altered by heat-treatment, elevated pH, treatment with toluene or phenethyl alcohol, or infection with bacteriophage T2 or T4. Both the reversible and the irreversible binding of acridines occurs more effectively with the acridine dye acriflavine than with the related dye proflavine, and still less effectively with 9-aminoacridine and quinacrine. These properties of acridine binding can be correlated with various inhibitory effects of the dyes on the cells.     

Localization of Anionic Phospholipids in Escherichia coli Cells

Cardiolipin (CL) is an anionic phospholipid with a characteristically large curvature and is of growing interest for two primary reasons: (i) it binds to and regulates many peripheral membrane proteins in bacteria and mitochondria, and (ii) it is distributed asymmetrically in rod-shaped cells and is concentrated at the poles and division septum. Despite the growing number of studies of CL, its function in bacteria remains unknown. 10-N-Nonyl acridine orange (NAO) is widely used to image CL in bacteria and mitochondria, as its interaction with CL is reported to produce a characteristic red-shifted fluorescence emission. Using a suite of biophysical techniques, we quantitatively studied the interaction of NAO with anionic phospholipids under physiologically relevant conditions. We found that NAO is promiscuous in its binding and has photophysical properties that are largely insensitive to the structure of diverse anionic phospholipids to which it binds. Being unable to rely solely on NAO to characterize the localization of CL in Escherichia coli cells, we instead used quantitative fluorescence microscopy, mass spectrometry, and mutants deficient in specific classes of anionic phospholipids. We found CL and phosphatidylglycerol (PG) concentrated in the polar regions of E. coli cell membranes; depletion of CL by genetic approaches increased the concentration of PG at the poles. Previous studies suggested that some CL-binding proteins also have a high affinity for PG and display a pattern of cellular localization that is not influenced by depletion of CL. Framed within the context of these previous experiments, our results suggest that PG may play an essential role in bacterial physiology by maintaining the anionic character of polar membranes.