Abstract
To identify the specificity of base substitutions, a novel experimental system was established based on rifampicin-resistant (Rif r) mutant screening and sequencing of the defined region of the rpoB gene in E. coli. We focused on comparing mutational spectra of base substitutions induced by either low energy nitrogen ion beam implantation or 60Co-gamma rays. The most significant difference in the frequency of specific kinds of mutations induced by low energy nitrogen ion beam was that CG <FONT FACE=Symbol>®</font>TA transitions were significantly increased from 32 to 46, AT <FONT FACE=Symbol>®</font>TA transversions were doubled from 7 to 15 in 50 mutants, respectively. The preferential base substitutions induced by nitrogen ion beam implantation were CG <FONT FACE=Symbol>®</font>TA transitions, AT <FONT FACE=Symbol>®</font>GC transitions, AT <FONT FACE=Symbol>®</font>TA transversions, which account for 92.13% (82/89) of the total. The mutations induced by 60Co-gamma rays were preferentially GC <FONT FACE=Symbol>®</font>AT and AT <FONT FACE=Symbol>®</font>GC transitions, which totaled 84.31% (43/51).
base substitution; low-energy nitrogen ion beam implantation; 60Co-gamma rays; Escherichia coli
MUTAGENESIS
RESEARCH ARTICLE
Comparison of base substitutions in response to nitrogen ion implantation and 60Co-gamma ray irradiation in Escherichia coli
Chuan-Xiao XieI; An XuI; Li-Jun WuI; Jian-Min YaoI; Jian-Bo YangII; Zeng-Liang YuI*
IKey Laboratory of Ion Beam Bioengineering Chinese Academy of Sciences, Institute of Plasma Physics, Anhui, China
IIInstitute of Atomic Energy, Anhui Academy of Agricultural Science, Hefei, Anhui Province, China
Correspondence Correspondence to Zeng-Liang Yu Key Laboratory of Ion Beam Bioengineering Chinese Academy of Sciences, Institute of Plasma Physics Hefei P.O.Box 1126 Anhui, Chin Email: cxxie@ipp.ac.cn
ABSTRACT
To identify the specificity of base substitutions, a novel experimental system was established based on rifampicin-resistant (Rifr) mutant screening and sequencing of the defined region of the rpoB gene in E. coli. We focused on comparing mutational spectra of base substitutions induced by either low energy nitrogen ion beam implantation or 60Co-gamma rays. The most significant difference in the frequency of specific kinds of mutations induced by low energy nitrogen ion beam was that CG ® TA transitions were significantly increased from 32 to 46, AT ® TA transversions were doubled from 7 to 15 in 50 mutants, respectively. The preferential base substitutions induced by nitrogen ion beam implantation were CG ® TA transitions, AT ® GC transitions, AT ® TA transversions, which account for 92.13% (82/89) of the total. The mutations induced by 60Co-gamma rays were preferentially GC ® AT and AT ® GC transitions, which totaled 84.31% (43/51).
Key words: base substitution, low-energy nitrogen ion beam implantation, 60Co-gamma rays, Escherichia coli.
Introduction
The mutational spectrum induced by ionizing radiation has been an issue of long-standing interest in radiation biology (Grosowsky et al., 1988). Southern blotting analysis permits the partitioning of ionizing radiation-induced mutagenesis into detectable deletions and major genomic rearrangements and into point mutations (Grosowsky et al., 1988; (Grosowski et al., 1986). Methods based on specific locus PCR were established to determine the mutational spectrum of fairly large fragments (Hei et al., 1997; Wu et al., 1999). The molecular nature of the point mutations, however, has been left unresolved (Grosowsky et al., 1988). Point mutations comprise base substitutions (transitions and transversions), frameshifts, small deletions and insertions. Among them, base substitutions represent approximately 2/3 of the point mutations analyzed induced by ionizing radiation (Grosowsky et al., 1988). The mechanism by which ionizing radiation produces mutagenicity is not entirely understood at present, which is partially due to little evidence from the mutational spectrum. Assays for transitions, transversions, frameshifts at specific sites and small deletions are based upon Lac+ reversion of a specific mutation located within the lacZ gene in the F' plasmid. A number of lacZ constructs and strains were developed in this experimental system (Cupples et al., 1990; Cupples and Miller 1989; Ohta et al., 1999). In particular, the lacZ system has been widely used in mutation spectrum evidence for specific mutations from defined constructs. Yang et al. (1997) reported some in vitro mutational spectra of low-energy nitrogen ion beam implantation by using the lacZ constructs. However, it should be noted that the DNA molecules interact in vivo with many molecules and chemicals such as proteins and lipids, indicating that the structure of DNA molecules is more complex than naked DNA. The interaction between an ion beam and DNA is different in vitro and in vivo. Therefore, it is necessary to establish an experimental system to study how the low-energy nitrogen ion beam induces a mutation in vivo.
Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase, which is encoded by the rpoB gene, can confer resistance to rifampicin (Jin et al., 1988; Jin and Gross 1988). Increased mutagenesis to rifampicin resistance reveals that base substitutions in rpoB confer E. coli cells this capacity (Jin et al., 1988; Matic et al., 1997). Here, we designed two pairs of primers for specific regions of the rpoB gene. The corresponding regions were PCR-amplified and sequenced for analyzing the base substitutions. We compared the base substitution mutations induced by nitrogen ion beam implantation with those induced by gamma radiation, in order to analyze the mutagenicity of this procedure.
Materials and Methods
Media and strain
Culture media LB (1% tryptone, 0.5% yeast extract, 1% NaCl) or MM (minimal medium: 0.05% L-asparagine, 0.02% MgSO4, 0.001% FeSO4, 1% glucose) were used, solidified when required with 1.5% agar, and supplemented with rifampicin (150 mg/mL) and streptomycin (30mg/mL), respectively, as appropriate when needed. The E. coli K-12 strain AB1157 was kindly provided by M.G. Marinus from the University of Massachusetts Medical School. Its genotype is F-thr-1 ara-14 leuB6 (gpt-proA)62 lacY1 tsx-33 supE44(AS) galK2(Oc) hisG4(Oc) rfbD1 mgl-51 rpoS396(Am) rpsL31(StrR) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1.
Nitrogen ion beam implantation, 60Co-gamma ray irradiation
Overnight cultures were diluted ~100-fold and grown in LB medium until early log phase (optical density at 600 nm [OD600] = ~0.3). Aliquots of 10 mL were pelleted and resuspended in 10 mL of MM salt solution with 10% glycerol for nitrogen ion treament. The liquid was spread onto the surface of plates. The ion implanted was nitrogen, with an energy of 10 keV, and the dosages (fluence) used were 1.3×1014, 2.6×1014, 3.9×1014, 5.2×1014, 6.5×1014, 7.8×1014, 9.1×1014, 10.4×1014, 11.7×1014 ions/cm2. During implantation, the pressure of the target chamber was ~10-3 Pa, and the temperature of the implantation environment was estimated to be approximately 0 °C. The cell-containing liquid was irradiated with 60Co-gamma rays at doses of 0.5 Gy, 1 Gy, 2 Gy, 5 Gy, 10 Gy, 20 Gy, 50 Gy, 80 Gy, 100 Gy, and 120 Gy, respectively.
Mutant screening, mutant frequency, and determination of survivors
The treated and control cells were washed down with 10 mL MM salt solution, and centrifuged for 1 min at 9,000 rpm. The precipitates were resuspended with 5 mL LB medium and incubated at 37 °C for 30 min at 220 rpm. After resuspension in MM salt solution, 50 mL of appropriate dilutions (104 ~ 106 fold) were spread onto MM plates to determine the total count, and 100 mL of them, with an appropriate cell density, were spread onto LB-rif plates to screen the mutant. The mutant frequencies were calculated by dividing the mean number of mutants by the average number of total cells.
Primer design, oligomer synthesis, PCR amplification and sequencing
The primer pairs for amplification of the regions were designed by the program WEBPRIMER (Stanford University) and reevaluated by the program PRIMER DESIGN. All primers were synthesized in the ExpediteTM Nucleic Acid Synthesis System (workstation) in TaKaRa (Dalian Corporation).
After confirmation by streak culture on LB-rif plates, the mutant clones were toothpicked from the plates and suspended in 200 mL of 1Pfu PCR buffer, boiled for 10 min in the PCR apparatus (Perkin-Elmer 9600) for lysing the bacteria. Then, the content of each tube was divided into two tubes for two different rpoB region amplifications. The PCR mixture was then completed, and 40 cycles (30 s at 94 °C, 30 s at 58 °C, and 45 s at 72 °C) were performed. The final composition of the PCR mixture was 1Pfu PCR buffer with Mg2+, 75 pmol of both primers, 200 mM each dNTP (TakaRa, Dalian), and 3 U of cloned Pfu polymerase (TakaRa, Dalian). The PCR products were purified with a DNA purification Kit (Wizard® SV Gel and PCR Clean-Up System, Promega) with low-melting agarose gel and sent to sequencing (TaKaRa, Dalian). DNA sequencings were all performed in both directions, using a Perkin-Elmer Applied Biosystems Model 377 DNA Sequencer in TaKaRa (Dalian) Corporation.
Determination of base substitutions
The base substitutions were determined by a run of blastn (blast 2 sequences) between the sense strand sequence of the wild type and mutants on line on the website (http://www.ncbi.nih.gov/blast/). The subject sequence is the sequence of the same region as in E. coli K-12 strain MG1655 (GenBank ACCESSION: U00096).
Results
Survival fraction and mutant frequency determination
Figure 1 shows the surviving fraction and mutant frequencies of E. coli cells irradiated by either nitrogen ion beam with various dosages or 60Co-gamma rays. As compared to 60Co-gamma rays (Figure 1 B), the 10 keV low-energy nitrogen ion beam induced a different pattern of cytoxicity (Figure 1 A). The moderate decreased pattern of the survival curve showed that low-energy nitrogen ion beam implantation may have a moderate cytotoxic effect on E. coli cells. At the dosage of 50% cell killing, the mutant frequency of nitrogen ion beam implantation (Figure 1 C: 6.5×1014 ions/cm2) reached 9.5×10-7, which is ~10-fold higher than that of 60Co-gamma rays (Figure 1 D: 10 Gy mutant frequency = 9.3×10-8). This suggests that low-energy nitrogen ion beam implantation might generate higher frequency of mutagenicity, specially base substitutions, as they were identified by Rifr mutant screening.
Primers
To analyze the Rifr selection mutation and non-selective mutation spectrum, we here chose two regions for determination of the mutation (Figure 2). Region A (492 bp in size) is the highly conserved region, which confers the E. coli cell the capacity of Rifr, when an appropriate base substitution is induced. Region B (530 bp in size) covers a non-conserved region of the rpoB coding sequence (419 bp, from 3611 to 4029), 77 bp of non-coding sequence between rpoB and rpoC genes, 34 bp coding sequence of rpoC (from 1 to 34). The primer sequences are listed in Table 1.
Optimization of PCR amplification
Since there is no single set of working conditions fulfilling the requirements of all PCR amplifications, the factors related to these reactions, including reaction component concentrations and procedures (time, temperature parameters and cycles), need to be adjusted within theoretically suggested ranges for efficient amplification of specific targets. In the present study, the bacterial lysate was used directly as PCR template. Briefly, mutant bacteriolysis was achieved in 1pfu PCR buffer at 95 °C for 10 min in the PCR apparatus. The content of each tube was divided into two tubes for two different rpoB region amplifications in the same mutant. 50 Rifr mutant clones induced by the 10 keV nitrogen ion beam (three of them shown in Figure 3 A) and by 10 Gy 60Co-gamma rays (three of them shown in Figure 3 B) were picked respectively to amplify the two defined regions. The optimized PCR components and procedures are described in Materials and Methods.
rpoB region A is a sensitive region for determination of base substitutions
After amplification and purification, the PCR products were sequenced in both directions. The sequences were compared with the corresponding wild-type sequence by the blastn program (blast the two sequences http://www. nibi.nlm.nih.gov/BLAST.html) to find the mutations. Each mutation site was also verified by comparing the sense strand with its complementary sequence. The distribution and types of mutations are summarized in Table 2. Ninety-seven out of a hundred of the mutants had at least one base substitution mutation in region A. Ninety-nine percent of the mutants had no base changes in region B. This finding suggests that most of the mutations (99%) are derived from the selection of rifampicin resistance, and that region A of the rpoB gene is the sensitive region for determining the specificity of base substitutions.
In addition, our observation is consistent with the sequence studies of a number of other laboratories. Severinov et al. (1993) analyzed mutations in rpoB leading to Rifr. They found that most of the base substitutions responsible for the Rifr phenotype lied in two clusters. Even though the full length of this gene is 4029 bp, the Rifr responsible region was only 177 bp. Previous studies (Jin and Gross 1988; Miller et al., 2002; Phanchaisri et al., 2002) identified 47 single-base substitutions at 29 sites and distributed among 21 coding positions. Most of them (46/47) lie in the region A defined in this research, whereas the mutations of the base substitution type leading to Rifr in rpoB covered all kinds of transitions and transversions.
Comparison of the specificity of base substitutions
To compare the mutagenicity of the two mutagens, a total of 100 independent mutants (50 each) in rpoB genes were sequenced. The frequency of total detected nitrogen ion-induced mutation reaches 89, while the 60Co-gamma ray-induced mutation frequency is only 51 (Table 2). The transitions prominently account for 75.28% and 84.31% of base substitutions induced by nitrogen ion beam and 60Co-gamma rays, respectively. On the other hand, the types of base substitutions are different between the two mutagens. Two types of base substitutions (GC ® CG transversions, AT ® GC transitions) were induced by low-energy nitrogen ion beam, but were not found in cells treated with 60Co-gamma rays, whereas AT ® CG transversions were not found in low-energy nitrogen ion beam implantation, but were found in cells treated with 60Co-gamma rays (Table 2).
The treatment was done with double-stranded DNA, and, thus, it was impossible to discriminate from which strand the position of the damage was derived. The types of base substitutions were grouped as CG ® TA transitions, AT ® GC transitions, AT ® TA transversions, and GC ® CG transversions. For instance, a CG ® TA transition can also be derived from a GC ® AT transition on the complementary strand. The specificity of base substitutions derived from low-energy nitrogen ion beam implantation and 60Co-gamma ray-irradiated cells is also summarized in Table 2. The most significant difference in the frequency of specific kinds of mutations induced by low-energy nitrogen ion beam was that CG ® TA transitions were significantly increased from 32 to 46, and AT ® TA transversions were doubled from 7 to 15, as compared to the frequencies of gamma ray-induced mutations. In summary, the frequencies of nitrogen ion beam implantation-induced mutations showed that the preference of base substitutions induced by nitrogen ion beam were GC ® AT transitions, AT ® GC transitions, and AT ® TA transversions which account for 92.13% (82/89) of the total, while the mutations induced by 60Co-gamma ray were mainly GC ® AT transitions, and AT ® GC transitions, 84.31% (43/51).
Discussion
Nitrogen ion beam implantation and mutation specificity
During the treatment of E. coli cells with 10 keV nitrogen ion beam implantation, the air pressure was 10-3 Pa and the temperature was as low as 0 °C in the target chamber. The working conditions of the ion implantation were not suitable for living cells. Even though they were protected with 10% glycerol, 15% of them died during treatment, as compared to a complete control (data not shown). Phanchaisri et al. (2002) reported a similar cell-killing effect on E. coli cells, when treated with Ar+ at a working condition of 10-4 Pa and approximately 0 °C. We may estimate that the E. coli cell has a side surface of about 0.5 to 1 mm2. Therefore, around 105 ~ 106 ions per cell were bombarded on the cell surface when the cells were spread flat. Yu et al. (2002) had found evidence that most of Ar+ with energy of 30 keV can only penetrate the cell wall into ~100 to 200 nm, at a fluence of 1.5×1015 ions/cm2. Accidentally, rare ions could reach a depth of 10 mm in the plant cell wall (Yu et al., 2002). In the present study, 10 keV nitrogen ions at the fluence rank of 1014 to 1015 were used to implant the E. coli cells. We presumed that the cytoplasmic effect might play the most important role in the induction of the mutation, as in the study of Hei et al. (1997) in a mammalian system, using exact numbers of alpha particles for the irradiation of cytoplasm.
Since mutational spectra convey only the end point of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult to assess the mutational specificity of mutagens directly from them. Exposure to ionizing radiation can damage DNA directly, but the predominant pathway arises from radiolysis of H2O, which results in the formation of reactive species such as OH.. There is evidence indicating that ROS can react with DNA (Wang et al., 1998). The gamma radiation-induced mutations were derived from these direct and/or indirect DNA damages. 8-OH-dG and 5-OH-dC might offer some explanations for the preference of GC ® AT transition, according to the summary of potential correlation between mutations observed in oxidant-induced mutational spectra (Hirano et al., 2001; Wang et al., 1998). The greater number of types and higher frequencies of nitrogen ion beam-induced mutations suggest that the mechanisms of nitrogen ion mutagenesis are more complicated than those of gamma-ray radiation. This complexity might result from the complex interactions between nitrogen ions and the target molecules. Low-energy nitrogen ion beams could not only generate ionizing radiation effects similar to gamma-ray radiation, but also the ions themselves could play a role in the formation of adducts. Some in vitro studies performed in our laboratory seem to support this point. New amino acids were synthesized in component solutions, and the nitrogen ion itself might also provide a nitrogen group in this reaction (Shi et al., 2001b). As for nucleotides, nitrogen ion implantation might produce an effect of damage and form some adducts (Shi et al., 2001a). As a heavy ion, the high LET irradiation effect (Hendry 1999) might be considered, even though the mutant screening system was aimed at determining base substitution. The cascade effects of high LET irradiation might also play a role in forming the base substitution mutations. We also compared the results with our former naked DNA irradiation studies, in which the transitions were mainly from CG to TA and from AT to GC, and the transversions were mainly from CG to AT and from CG to GC (Yang et al., 1997). This simple mutational spectrum indicated that the interaction between naked DNA in vitro and nitrogen ions was also likely to be simple. Therefore, more types and species of reactive adducts could be generated in the process of implantation than in that of gamma ray irradiation and naked DNAs. Apparently, a reactive accelerated nitrogen ion group and its series products could act as component of adducts. Studies about the roles of heavy ions in the formation of the reactive DNA adducts are underway, to explain the detailed mechanisms of the specificity of base substitutions.
Experimental system
It has been deduced that mismatch DNA damages were the most important source of mutations induced by nitrogen ion beam implantation in naked DNA (see review by Yu, 2000). We here constructed a novel experimental system through which the specificity of base substitutions in living cells can be detected and analyzed. The E. coli chromosome rpoB gene region A contains the two clusters responsible for Rifr. Previous studies (Jin and Gross 1988; Miller et al., 2002; Severinov et al., 1993) have identified 47 single-base substitutions which cover all kinds of transitions and transversions in region A. Our experiment was time and cost-saving because it was based on a single E. coli strain and did not require preparation of chromosome or plasmid DNA. Moreover, it was sensitive and efficient for determining the specificity of base substitutions. We also sequenced the region B of the rpoB gene, in order to obtain the nonselective mutation. However, only one T insertion mutation induced by low energy nitrogen ion beam implantation was identified through this screening system, suggesting that it is very hard to identify nonselective mutations based on this system.
Generally, E. coli strains have an average mutation rate to rifampicin resistance of about 1×10-8, also called spontaneous mutation rate. Some strains have a spontaneous mutation rate of about 2.0×10-9 (Matic et al., 1997). We here found a lower spontaneous mutation rate of about 1×10-9 (Figure 1 C and Figure 1 D, dosage = 0). The difference might be due to our protocols, since the cell concentration for mutant screening was around ~109 fold higher than the survival-determining one. A different loss in the process of manipulation might lead to this fairly lower spontaneous mutation rate. Theoretically, after irradiated or implanted, the mutant cells have a physiological delay to stabilize the mutant phenotype of up to 4 ~ 5 generations. Routinely, the treated cells are grown overnight (Cupples et al., 1990; Cupples and Miller 1989). Here, the treated cells were grown in an enriched culture medium for only 30 min, and a little longer to wait for spreading. This time period allowed the cells to replicate and divide just one round. Though the mutant phenotype was not quite stable, the homologous mutants could be avoided in cultures, due to cell division. The short culture time might also result in lower mutant frequencies determined in mutagens, because it may have been too short for some mutants to express their mutant phenotype. It would be necessary to have information about how long and how well the culture time was after treatment with mutagens. This work is in progress.
New sites leading to Rifr
Previous studies (Jin and Gross 1988; Miller et al., 2002; Severinov et al., 1993) have identified 47 single-base substitutions at 29 sites and distributed among 21 coding positions leading to Rifr in E. coli. Here, we identified two new Rifr-determining sites (Table 2). The complete new site determined in this study is located at nucleotide site 1551 and amino acid site 517. GC ® CG transversion at 1551 caused a histidine substitute, Gln517, and led to a fairly high Rifr capacity. The other new site determined is located at 1692. When dC1692 was replaced by a dT, it resulted in a change of Pro564 into leucine. The synonymous mutation of the second site had been reported previously (Miller et al., 2002)[11], but the nucleotide substitution, dC1692 ® dT1692, had not yet been identified. This finding might supplement the Rifr analysis in E. coli and some other pathogenic bacteria.
Acknowledgements
This work was partly supported by grants from the National Natural Science Foundation of China (General Program n. 10375066 & n. 30170234). We thank research assistants Yu, L.X. and Liu, X.H. for their help in nitrogen ion beam implantation.
EditorAssociado: Carlos F.M. Menck
Received: July 25, 2003;
Accepted: November 14, 2003.
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Publication Dates
-
Publication in this collection
20 July 2004 -
Date of issue
2004
History
-
Received
25 July 2003 -
Accepted
14 Nov 2003