Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions: 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (αβ)₃ resulted first in monomerization (αβ), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, l-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA^Pyl). The PylRS-tRNA^Pyl pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N^?-(tert-butoxycarbonyl)-l-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the α-amino group of the substrate, whereas most aaRSs, including the structurally and genetically related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain α-groups: α-hydroxyacid, non-α-amino-carboxylic acid, N_α-methyl-amino acid, and d-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA^Pyl in E. coli in the presence of the α-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA^Pyl pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation.     

过表达周期蛋白Cyc28影响四膜虫的有性生殖进程

真核生物的细胞周期通过连续的激活和失活特定的周期蛋白/周期蛋白依赖性激酶复合物活性进行调控。嗜热四膜虫含有34种周期蛋白,有性生殖期特异表达的周期蛋白Cyc2和Cyc17在四膜虫小核减数分裂中发挥重要功能。本研究从嗜热四膜虫中鉴定出一种新的周期蛋白CYC28 (TTHERM_00082190)基因,预测编码266个氨基酸。实时荧光定量PCR表明,CYC28在有性生殖时期特异表达,且在4 h表达水平最高。通过同源重组构建获得MTT1启动子调控下的HA-CYC28突变体细胞。免疫荧光定位表明,HA-Cyc28定位在细胞质和凋亡的亲本大核中。分别构建CYC28敲除突变株和RNA干扰细胞株,对CYC28敲减突变体细胞的分析发现,营养生长和有性生殖期突变细胞发育正常。然而,过表达株Cyc28突变体引起原核染色体排列异常,原核不能完成有丝分裂形成配子核,有性生殖进程终止。结果表明,Cyc28参与细胞的有性生殖进程,它的正常表达和降解对原核有丝分裂的完成是必需的。     

周期蛋白Cyc2的过量表达及突变抑制嗜热四膜虫有性生殖发育

周期蛋白在细胞增殖过程中呈现周期性表达变化,不同的周期蛋白通过结合周期蛋白激酶(cyclin-dependent kinase,CDKs)及靶向特定蛋白质参与细胞有丝分裂和减数分裂过程的精确调控。嗜热四膜虫有性生殖期特异表达的B3型周期蛋白Cyc2(cyclin 2,Cyc2)对减数分裂的启始是必需的,但Cyc2蛋白的分子调控机制并不清楚。本研究通过0.1μg/mL和0.3μg/mL镉离子诱导突变细胞株OE-CYC2-B2086和OE-CYC2-CU428中CYC2基因在金属硫蛋白1基因(metallothionein gene 1,MTT1)启动子调控下上调表达。实时荧光定量PCR检测突变株OE-CYC2-B2086和OE-CYC2-CU428中CYC2的转录水平分别上调7.8倍和9.8倍。细胞有性生殖发育进程的荧光显微观察发现CYC2的表达上调并不影响有性生殖前期减数分裂的启始,但是干扰四膜虫有性生殖后期中新大核和新小核的正确形成。同时突变株OE-CYC2-B2086和OE-CYC2-CU428交配后,在镉离子诱导下不能产生有性生殖后代,但是该突变株分别和两种不同野生型细胞或CYC2敲除的突变细胞株交配能够恢复产生3%,15%或32%的有性生殖后代,有性生殖发育异常程度与CYC2的表达水平成正相关。进一步突变Cyc2第312位磷酸化位点丝氨酸为丙氨酸,获得CYC2单位点突变细胞株CYC2-S312A-B和CYC2-S312A-C。丝氨酸单位点突变阻止了四膜虫有性生殖期小核减数第1次分裂起始。结果表明周期蛋白2的表达水平和磷酸化修饰影响了不同阶段细胞核的功能,周期蛋白2对四膜虫有性生殖发育的正常进行是必需的。