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This study investigated Pickering emulsion polymerization of styrene using self-assembled chitin nanofibers (CNFs) as stabilizers to produce CNF-based composite particles, which were further converted into hollow particles. Previously, we reported that regeneration from a chitin/1‑allyl‑3‑methylimidazolium bromide ion gel using methanol fabricated self-assembled CNFs. Prior to the emulsion polymerization, CNFs were maleylated by reaction with maleic anhydride in the presence of perchloric acid. After styrene was added to a dispersion of the produced anionic CNFs in ammonia aq., a mixture was ultorasonicated to give an emulsion, in which styrene droplets were stably surrounded by CNFs. Radical polymerization was then conducted in the presence of potassium persulfate as an initiator to produce the composite particles. Particle sizes became smaller as the amounts of CNFs increased. The hollow particles were prepared by solubilizing out the polystyrene cores with toluene.

We have already found that an amidinated chitin, which was prepared by the reaction of a partially deacetylated chitin with N,N-dimethylacetamide dimethyl acetal, was converted into an amidinium chitin bicarbonate with nanofiber morphology by CO2 gas bubbling and ultrasonic treatments in water. In this study, we performed the fabrication of composite materials of such cationic chitin nanofibers with an anionic polysaccharide, sodium alginate, by ion exchange. When the amidinium chitin bicarbonate nanofiber aqueous dispersion was added to an aqueous solution of sodium alginate, the composite material was agglomerated, which was isolated by centrifugation, filtration, and lyophilization, to form a manipulatable sheet. The morphology of the resulting sheet at nano-scale was evaluated by SEM measurement.

Ionizing radiation/DNA double-strand break repair/XRCC4. The mouse lymphoma cell line LX830 is an X-ray-hypersensitive mutant. Complementation tests between LX830 cells and radiation-sensitive mutants of M10 (Xrcc4 deficient cells) or SX10 (DNA ligase IV deficient cells) cells showed that M10 cells did not complement LX830 cells, but SX10 cells did, suggesting that LX830 cells would belong to the X-ray-cross complementation group (XRCC4). A sequence analysis of Xrcc4 cDNA in LX830 cells disclosed a transition of G to A at nucleotide position 129, which resulted in a change of tryptophan (43) to a termination codon. Transfection of the mouse Xrcc4 cDNA rescued the X-ray sensitivity of the mutant cells. LX830 is an Xrcc4-deficient cell line bearing a termination codon in exon 2 of the Xrcc4 gene and no wild-type Xrcc4 gene.

An ionizing radiation-sensitive mutant derivative of mouse lymphoma L5178Y cell, M10, is defective in rejoining DNA double-strand breaks (DSBs). The complementation test and the results of chromosome transfer suggested that M10 may belong to X-ray cross-complementation (XRCC) group 4. In the present study, sequence analysis of Xrcc4 cDNA in M10 cells disclosed a transversion of A (370) to T, which result in a change of arginine (124) to a termination codon. Interestingly, the mutation occurred in one allele and the transcripts of the Xrcc4 gene were expressed exclusively from the mutant allele. Transfection of M10 cells with the murine Xrcc4 cDNA completely rescued X-ray sensitivity of the mutant cells. M10 is a novel Xrcc-4-deficient cell line.

Norfloxacin (NFLX) caused induction of prophages VT1 and VT2 of enterohemorrhagic Escherichia coli O157 at subinhibitory concentrations. In time course experiments, we observed the following sequential events: upon induction, the phage genomes underwent multiplication; the amount of stx genes increased; and subsequently, large quantities of toxins VT1 and VT2 were produced. Further studies showed that the molecular mechanism of prophage induction is closely related to the RecA system since the prophage VT2 was not induced with NFLX in a recA mutant strain.

We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide “T” (9572) to “C” transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12.2% as compared with the normal cell line. To confirm the responsibility of thedna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild typedna-pkcs cDNA was observed. SX9 is a noveldna-pkcs-deficient cell line.

The biological effects of active oxygen species were examined in a mutant mouse cell line (SL3-147) that is deficient in the repair of DNA double-strand breaks. This mutant cell line shows different sensitivities to X-rays, hydrogen peroxide, paraquat and menadione when compared to the wild-type cell line (LTA). SL3-147 was more sensitive to X-rays, hydrogen peroxide and paraquat, but was less sensitive to menadione in side by side comparisons to LTA cells. The greater number of DNA double-strand breaks in SL3-147 appears to account for the line's greater sensitivity to X-rays and paraquat. DNA damage other than double-strand breaks or injury to non-DNA targets, however, is responsible for the differences between LTA and SL3-147 in their sensitivities to hydrogen peroxide and menadione.

Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10.

DNA double-strand breaks induced by X- or neon beam-irradiation in a DNA double-strand break-repair-deficient mutant cell line (SL3-147) were examined. The increase in the number of DNA double-strand breaks was dose depend after irradiation with X-rays and neon beams and was enhanced by chromatin-proteolysis treatment before irradiation. These results suggest that the induction of DNA double-strand breaks by ionizing radiation, including heavy-ions, is influenced by the chromatin structure.

A method of chromosome painting on human sperm chromosomes using fluorescent in situ hybridization (FISH) is introduced. Sperm chromosome slides were prepared after in vitro fertilization of hamster eggs with human spermatozoa. The slides were treated by RNase A before FISH. Chromosome 4 was clearly and specifically painted in a majority of sperm-derived metaphase plates after an application of whole chromosome painting DNA probes of this chromosome. This is the first report of successful painting on human sperm chromosomes.

The mouse lymphoma (L5178Y) cell mutant M10 is defective in rejoining DNA double-strand breaks and is hypersensitive to ionizing radiation. The introduction of human chromosome 5 into M10 cells by microcell mediated chromosome transfer complemented the ionizing-radiation hypersensitivity defect of this cell line. The presence of chromosome 5 in the microcell hybrids was shown using PCR with chromosome-specific primers and fluorescence in situ hybridization. From this data we conclude that the gene that corrects the radiation hypersensitivity of M10 cells is located on chromosome 5 and tentatively assigned to the 5q14 to 5pter region. We designate this gene XRCC4L.

The mouse carcinoma cell line SX10 is a hypersensitive mutant to x-rays and bleomycin. An earlier complementation test suggests that SX10 would belong to x-ray-cross complementing group (XRCC) 4. However, in this study, a human XRCC4 expression vector failed to complement the SX10 phenotype. Consistent with the previous report, SX10 showed the same level of DNA-dependent protein kinase activity as the wild-type SR-1. We isolated and analyzed hybrids between SX10 and human diploid fibroblast cells and found that human chromosome 13 conferred the x-ray resistance to the hybrids, suggesting that a candidate gene would be located on this chromosome. Polymerase chain reaction analysis with these hybrids and x-ray-resistant transformants obtained by introducing human chromosomes into SX10 indicated that the mutant was likely to be defective in DNA ligase IV. Sequence analysis of the DNA ligase IV gene confirmed that a defect in SX10 was attributed to a transition of G to A at nucleotide position 1413 of the gene, leading to an amino acid substitution from Trp at residue 471 to a stop codon. Revertant clones (Rev1–3) derived from SX10 showed a restored x-ray resistance; Rev1 reverted to the original nucleotide G at position 1413, whereas Rev2 and Rev3 to C. Transfection of a mouse DNA ligase IV cDNA vector into SX10 restored the resistance to both x-rays and bleomycin. SX10 showed a reduced frequency of chromosomal integration of transfected DNA, but the revertants restored the frequency found in the wild-type cells. These results suggest a possible involvement of DNA ligase IV in the integration event of foreign DNA as well as a crucial role in DNA double-strand break repair.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460-kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex called Ku, which is comprised of 70 (Ku70) and 86 (Ku80) kDa subunits. Mutations that affect the expression of the catalytic or Ku80 subunits of DNA-PK disrupt both V(D)J recombination and DNA double-stranded break repair pathways. In this report, we show that two previously uncharacterized rodent cell lines that are defective in DNA double-stranded break repair express catalytically inactive DNA-PK. The DNA-PKcs from the DNA double-stranded break repair mutant cell lines IRS-20 and SX-9 assembles on double-stranded DNA but fails to function as a protein kinase. In addition to the kinase defect, the abundance of the DNA-PKcs from both of these cell lines is reduced relative to wild-type controls. These results suggest that the DNA-PKcs gene from each of these cell lines contains mutations that inactivate the enzymatic activity and the expression or stability of the gene product. These data further strengthen the hypothesis that DNA-PK-mediated protein phosphorylation is a necessary component of the DNA double-stranded break repair pathway.

Nine mutants isolated from CHO · K1 cells with increased sensitivity to the lethal effect of plumbagin (PG), a powerful superoxide generator, were classified into five groups, A-E, according to their sensitivity to PG and methyl viologen (MV). Two mutants of group B (Pa13 and Pb4) were sensitive to both drugs, and two mutants of group C (Pa14 and Pa15) were moderately sensitive to PG and extremely sensitive to MV. To mitomycin C (MMC) these mutants showed cross-sensitivity; especially Pa13 and Pb4 (group B) were highly sensitive to MMC. Genetic complementation analyses of these four mutants were carried out using MV sensitivity. Sensitivity group B was divided into two complementation groups, I and II. Pa14 and Pa15 belonged to the same complementation group III. These four mutants were also classified into three complementation groups for MMC sensitivity. Because Pa13 and Pb4 were also sensitive to cis - diamminedichloroplatinum(II), they may have a defect in the repair of DNA crosslinks induced by these agents. A complementation group IV (Pa2 and Pa8) was also suggested based on the studies of MMC sensitivity.

The induction and repair of radiation-induced DNA damage was assessed in 3 mouse cell lines, including the parental L cell line, a radiation-sensitive, SL3-147 mutant cell line and the H5 revertant to radiation resistance. The yield of neither radiation-induced DNA single- nor double-strand breaks could explain the variable sensitivity of the 3 cell lines. Closure of DNA single-strand breaks proceeded at a similar rate in both the L and SL3-147 cell lines. Closure of DNA double-strand breaks however was significantly slower and less complete in the SL3-147 cell line than in either of the radiation-resistant cell lines. The results are consistent with the increased radiation sensitivity of the SL3-147 cell line resulting from a defect in their ability to repair radiation-induced DNA double-strand breaks.

The CHO UV-sensitive mutants UV24 and UV135 (complementation groups 3 and 5, respectively) are defective in nucleotide excision repair. After fusing each mutant with human lymphocytes, resistant hybrid clones showing genetic complementation were isolated by repeated exposure to UV radiation. Using a combination of isozyme markers, DNA probes, and cytogenetic methods to analyze the primary hybrids and their subclones, correction of the repair defect was shown to be correlated with the presence of a specific human chromosome in each case. Chromosome 2 corrected UV24, and the gene responsible was designated ERCC3. Line UV135 was corrected by human chromosome 13 and the gene designated ERCC5. The UV-sensitive mouse cell line, Q31, was shown not to complement UV135 and thus appears to be mutated in the same genetic locus (homologous to ERCC5) as UV135. Breakage of complementing chromosomes with retention of the genes correcting repair defects allowed the following provisional assignments: regional localization of ERCC5 to 13q14-q34, exclusion of ERCC3 from the region of chromosome 2 distal to p23, and relief of the ambiguity of ACP1 assignment (2p23 or 2p25) to 2p23 proximal to MDH1.

The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obatained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells.

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.

Several spontaneous revertants able to grow in restrictive temperature (39°C) were isolated from a temperature-sensitive mutant (ts3) of murine leukemic cells L5178Y. The revertants showed reduced plating effi-ciencies and prolonged doubling times at 39°C when compared with wild type cells. The alanyl-tRNA synthetase activity was thermolabile in both ts3 cells and the revertants, indicating suppression as the mechanism of reversion. It was also demonstrated that glycerol protected the enzyme from thermal inac-tivation. The specific activities of alanyl-tRNA synthetase and alanine amino-transferase did not differ much in the wild type, mutant, and revertant cells. This indicates the unlikelihood of thermolabile enzyme overproduction or of increased intracellular concentration of L-alanine causing phenotypic suppres-sion in these revertants. The mechanism of sunnressinn is discussed.

The mutant mouse lymphoma cell M10, which is sensitive to methyl methanesulfonate and ionizing radiation, was compared with the parental L5178Y cells for mutation induction after γ-irradiation. The rate of induced mutations to 6-thioganine resistance in L5178Y cells was 2−3 x 10 −7 per R, as determined after exposure ranging from 25 to 500 R. The induced mutation frequency per unit dose per locus in M10 cells was about 4 times higher than that in L5178Y cels at the lower doses of exposure (25–75 R), but it declined sharply at the higher doses of γ-rays (100–150 R). The rate of induced mutation per unit cell killing in M10 cells was nearly the same as that in L5178Y cells when they were compared at the levels of lower cell killing.

Three mutagen-sensitive mutants, MS-1, M10 and Q31, have been isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline 1-oxide (4NQO), and Q31 cells are cross-sensitive to UV and 4NQO. Lines resistant to 6-thioguanine (TGr) and 5-bromo-2′-deoxyuridine (BUr) were isolated from L5178Y and these three mutagen-sensitive mutants. All the TGr lines were sensitive to 5-bromo-2′-deoxyuridine and HAT medium and all the BUr lines were sensitive to 6-thioguanine and HAT medium. The hybrids homozygous for the mutagen-sensitive markers showed nearly the same sensitivity to UV, 4NQO, X-rays and MMS as their parental TGr and BUr lines. The hybrids constructed by fusing L5178Y BUr and TGr lines from each of MS-1, M10 and Q31 displayed the normal UV, X-ray and MMS resistancy of L5178Y cells. Thus the UV-, X-ray- and MMS-sensitive markers in MS-1, M10 and Q31 were recessive in somatic cell hybrids. The 4NQO-sensitive phenotype, however, behaved codominantly in somatic cell hybrids.

Three X-ray-sensitive mutants (LX821, LX827 and LX830) have been isolated from mouse-lymphoma L5178Y cells. These mutants are much more sensitive to the lethal effects of ionizing radiation than the parental L5178Y cells but are as resistant to ultraviolet radiation as L5178Y cells. We have previously isolated a mutant M10 that is sensitive to methyl methanesulfonate (MMS) and cross sensitive to ionizing radiation and 4-nitroquinoline 1-oxide (4NQO). Unlike M10 cells, newly isolated mutants were not sensitive to MMS and were less sensitive to 4NQO. These results indicate that new mutants may be deficient in the repair of DNA damage specific to ionizing radiation. LX821 and LX827 cells were concomitantly resistant to 5-bromodeoxyuridine, whereas LX830 cells were not.

A temperature-sensitive mutant (ts2) of murine leukemic L5178Y cells accumulates aberrant mitotic cells and subsequently, multinucleate cells upon exposure to a restrictive temperature. Temperature-shift experiments using synchronized cell populations demonstrate that the execution point of ts2 cells is located between the early and mid S phase in the cell cycle. Mutant cells can achieve cell division when they are shifted down by the time of karyokinetic stage. However, multinucleate cells, once formed, are unable to undergo cytokinesis even though transferred to a permissive temperature. Cytofluorometric analysis indicates that unequal chromosome distribution among daughter nuclei takes place during multinucleation. The mechanism of defective karyo-and cyto-kinesis in ts2 cells is discussed.

2 mutant mouse cells M10 and Q31 were examined for chromosomal aberrations induced by ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO), as compared with mouse lymphoma L5178Y cells. Q31 cells are UV- and 4NQO-sensitive cells isolated from L5178Y cells. M10 cells are similar but are sensitive to ionizing radiation and 4NQO. After treatment with UV or 4NQO, chromatid-type aberrations in these cell strains were induced more frequently in the first mitotic cells, at late fixation times. After UV exposure (2.4 J./m2), the maximal frequencies of chromatid-type breaks in Q31 cells were about 5 times higher than in L5178Y cells. In M10 cells such breaks were only as frequent as in L5178Y cells. After 4NQO treatment (50 ng/ml) the frequencies of chromatid-type breaks in M10 and Q31 cells were significantly higher than in L5178Y cells. From these results and those of previous studies (Takahashi et al., 1982), M10 cells may be considered hypersensitive to γ-rays and 4NQO, but not to UV, and thus react similarly to L5178Y cells. The hypersensitivity of M10 cells to 4NQO may result from a defect in the ionizing-radiation repair mechanism as has been suggested to occur in ataxia telangiectasia (AT) cells. Q31 cells are hypersensitive to UV and 4NQO, but not to γ-rays. Q31 cells may be considered to be deficient in a UV-like repair pathway. In conclusion, characteristics of murine M10 and Q31 cells are compared with those of human AT and xeroderma pigmentosum (XP) cells.

Mouse cells in culture are inefficient in excision of pyrimidine dimers induced by ultraviolet irradiation (UV) (Lehmann and Kirk-Bell, 1972). Despite this inefficiency, mouse cells are as resistant to the lethal effects of UV as are excisionproficient human cells (Rauth et al., 1974; Sato and Setlow, 1981). The mechanism of tolerance by which mouse cells survive UV irradiation is not clear. UV-sensitive mouse cell mutants, if isolated, would be useful for the study of this mechanism. We have isolated a UV-sensitive mutant, Q31, from mouse lymphoma L5178Y cells (Sato and Hieda, 1979), and this mutant has been examined for excision and postreplication repairs of UV damage (Sato and Setlow, 1981). The results show that the parental mouse cells remove 10-20% of induced dimers during the 24-h incubation period and that Q31 cells cannot remove detectable amounts of dimers. Hence the lack of excision repair may be to some extent responsible for the elevated UV sensitivity in mutant cells. Post-replication repair, which will represent another tolerance mechanism operative in mouse cells, was examined to find out whether this mechanism is missing from mutant Q31 cells; the increase in molecular weights of newly synthesized DNA after irradiation was compared between mutant and parental cells, The size of DNA synthesized immediately after irradiation was smaller in mutant than in parental cells, but the rate of DNA-chain elongation was not much different between mutant and parental cells (Sato and Setlow, 1981). However, the changes in the rates of DNA synthesis during the post-irradiation incubation period have not been determined yet. For this report we measured the rates of DNA synthesis after irradiation and observed an affected recovery of UV-inhibited DNA synthesis in mutant cells.

A high-voltage generating machine which could generate semi-rectangular pulses in PBS solution was constructed, and the effects of field strength and duration of the pulse on electric pulse-mediated transformation of mouse mammary carcinoma FM3A cells by a linear form of plasmid pSV2neo DNAs were examined. In parallel, cell survival and growth after pulsing were analyzed. When the field strength and duration of the pulse were increased, the transformation frequency increased, although the cell survival rate decreased. Under the best conditions, the transformation frequency was 2 X 10(-4), which was 80 times higher than that obtained by the calcium phosphate coprecipitation method.

The radiation-sensitive mutant M10 of mouse lymphoma L5178Y cells was examined for its ability to rejoin DNA single-strand breaks induced by gamma-rays. The alkaline sucrose gradient sedimentation analysis revealed that M10 cells repaired single-strand breaks but simultaneously produced increasing amounts of small DNA fragments with time of postirradiation incubation, something which was not observed in L5178Y cells. Since small fragments did not appear in M10 cells irradiated at room temperature, DNA fragmentation may result from cold treatment during irradiation followed by incubation at 37 degrees C. This indicates that the cold susceptibility is characteristic of M10 cells and is not related to radiation sensitivity of this mutant. This conclusion is supported by the finding that no DNA degradation takes place after cold treatment with a subsequent incubation in the other radiosensitive mutant LX830 that belongs to the same complementation group as M10.

In order to establish a simple and efficient method of electroporation applicable to gene transfer in mammalian cells, the effects of various pulsing conditions on viability and transformation efficiency were studied. By investigating the effects of temperature during and after the pulse, field strength, pulse length, and pulsing media, we have established a standard method: a single decaying pulse with a duration of around 1 ms at room temperature in a medium containing physiological concentrations of salts, including Ca2+ and Mg2+. Application of the standard method to various types of cells, both floating and adherent, resulted in an efficient transformation by pSV2neo DNA. Furthermore, the electroporation-mediated transformation of tsBN2 cells by a cosmid clone (∼ 45 kb) carrying the human gene RCC1 was successful.

A new method (viral suicide method) for the isolation of UV-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with UV-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their UV sensitivity. Nineteen of 238 clones examined were sensitive to UV irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their UV sensitivity for 4 months in culture. UV sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (D0) are 2.9, 3.7, and 7.8 J/m2 for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to X- irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability.

We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers (22). We divided the mutants into two groups, "highly sensitive" and "moderately sensitive" mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG.

About fifty suppressor sensitive (sus) mutants of phage ∅80 were isolated after hydroxylamine treatment, and these were classified into fourteen cistrons. In vitro and in vivo complementation experiments revealed that at least five cistrons were concerned with head formation and that at least six cistrons were concerned with tail formation in ∅80. Some presumed “early” mutants were also found. Defective lysogens were isolated from a ∅80-lysogenic nonpermissive strain using the colicin plate method (Gratia, 1966). In these strains deletions which affected the colicin B receptor gene extended for varying distances into prophage ∅80. Marker rescue experiments were carried out with these deletion lysogens by infecting various sus mutants, and the gene order in prophage ∅80 was determined. Clustering of all head genes and also of tail genes, as in phage λ, were demonstrated by the results of the prophage deletion mapping as well as the two-factor crosses performed among the sus mutants. Moreover, the gross gene arrangement of ∅80 was also similar to that of phage λ: namely, the cluster of head genes was found to be located at one end of the vegetative map of ∅80, being followed by that of tail genes, and presumed “early” genes are located at the other end of the map.

Transducing variants of coliphage λ have been isolated from a donor strain in which the normal prophage-host relationship is altered by a deletion that extends from the galE gene of the host into the Q gene of the prophage. The 40 λ gal strains isolated fall into 8 classes. Two classes do not carry the deletion of the donor strain and must have arisen in the primary recipient. The most common class contains phage genes SR A-J and part of gene Q, and might arise by cutting at the att site. One class of λ gal contain all known λ genes except for a large section of gene Q. Lysogens of this class lyse late after induction, liberating a small amount of phage. A further mutation ( qin ) allows plaque formation by these Q-deletion phages, apparently by shortening the latent period. The qin mutation entails a physical loss of DNA, genetic loss of the gal genes and a loss of ability to complement genes S and R in trans .

Mutations in the anthranilate synthetase eistron can alter the capacity to synthesize other enzymes of the tryptophan pathway in Escherichia coli. On the other hand, a deletion over the anthranilate synthetase eistron leads to non-repressible transcription of tryptophan genes. From this evidence it is concluded that the operator should be localized at the anthranilate synthetase eistron extremity of the operon.

Specific tryptophan messenger RNA molecules synthesized at an early stage of de-repression in Escherichia coli were analysed by sucrose density-gradient sedimentation. In these experiments, the following results were obtained: (a) on the tryptophan operon, macromessenger RNA molecules of sedimentation coefficient about 33 s were synthesized; (b) the messenger RNA molecules made from mutants in which only anthranilie acid synthetase (anth) cistron or nearly half the tryptophan operon of the A cistron side is preserved were smaller in size than those from the intact tryptophan operon; (c) the synthesis of the messenger RNA on the tryptophan operon was initiated at the beginning of the anth cistron (operator) and proceeded to the end of the A cistron. Following the progress of synthesis, the messenger RNA grew in molecular size. In the wild type strain W3102, anth messenger RNA only existed as an initial and incomplete product among the tryptophan messenger RNA; (d) upon repression, the tryptophan repressor blocked the initiation of specific messenger RNA synthesis by associating with the operator site. The synthesis of the messenger, once initiated from the operator, is not brought to a halt immediately by tryptophan repression, but probably proceeds to the other end of the operon. In short, it is concluded that tryptophan messenger RNA molecules are multicistronic; the so-called operator to which the tryptophan repressor has an affinity, is located on the anth cistron side, and is the initiation site for the synthesis of the tryptophan messenger RNA molecules.

Messenger RNA was detected which formed a specific hybrid with DNA of the tryptophan region carried by bacteriophage φ80. Since the synthesis of specific messenger RNA corresponds to the genetic properties and physiological activities of the tryptophan genes in Escherichia coli cells, messenger RNA is considered to be specifically related to the genetic information of the tryptophan synthesizing system. Through the hybridization technique with DNA's from φ80 dt 0 which carries the entire region of the tryptophan system, and φ80 pt 1 which carries the tryptophan region with a deletion of the anthranilic acid synthetase cistron, a specific messenger RNA corresponding to the anthranilic acid synthetase cistron was detected. Because the rate of synthesis of specific tryptophan messenger RNA was reduced by the addition of tryptophan, it is concluded that regulation in the tryptophan system operates at the gene level.

The density of bacteriophage φ80 was measured by density gradient equilibrium centrifugation in CsCI solutions. This density was the same in phages derived from a lysogenic or a lytic growth cycle. The distribution of low-frequency transducing (LFT) particles of φ80 in a CsCl density gradient was shown to be much broader than that of plaque-forming particles. The transducing phages in the LFT lysates appear to transmit their characteristic densities, as well as definite try markers, to the φ80 dt particles in the high-frequency transducing lysates derived from them. The φ80 dt particles can be divided into two classes according to their requirement for helper phages in transduction. One class transduces more efficiently in the presence of active helper phage, while with the other class the active phage does not enhance or even reduces the transduction frequency. A new transducing phage type, which is not defective since it multiplies without help from the active phage, has been isolated. This phage, called φ80 pt (plaque-forming tryptophan), forms a small turbid plaque. A tryptophan auxotroph lysogenized by φ80 pt becomes try+, although it always segregates out some try− nonlysogenic cells. The buoyant density of φ80 pt in CsCI solution is greater than that of φ80, suggesting that a larger genetic fragment is carried in this phage.

IT may be of great help in the elucidation of regulatory systems in mammalian cells if we could isolate temperature-conditional mutants and identify their mutated functions. I report here that a temperature-sensitive mutant of L5178Y murine leukaemic cells has a thermolabile L-alanyl-tRNA synthetase.

A UV-sensitive mutant, Q31, isolated from mouse-lymphoma L5178Y cells, was studied for excision and post-replication repairs. A nearly equal number of UV endonuclease-sensitive sites was induced by UV in L5178Y, Q31, and human Raji cells. L5178Y cells irradiated with 10 J/m2 removed 18% of sensitive sites from DNA of detection, whereas Raji cells eliminated about 60% of the sites. These results during incubation for 24 h, and Q31 cells removed 3% of the sites, a fraction less than the limit indicate that mouse-lymphoma cells are capable of excision repair to a limited extend as compared with human cells and that mutant Q31 cells are essentially devoid of dimer excision. The newly synthesized DNA was of smaller size in UV-irradiated and unirradiated Q31 cells than that in the corresponding L5178Y cells, but the DNAs in both cell strains increased to comparable sizes after a 2-h chase.

Publisher Summary This chapter presents the electric pulse-mediated method of gene transfer in mammalian cells. A mouse lymphoma cell line L5178Y is used to select various mutagen-sensitive mutants. This cell line and its mutants grow in suspension and are easy to handle. Electric pulse-mediated gene transfer, also designated as electroporation or electropermeabilization, utilizes the increase in permeability because of local breakdown of plasma membranes. During this period, extracellular materials such as DNA can enter the cell. This breakdown, if not too large, is rapidly repaired resuming cell viability. The DNA introduced by this method is not exposed to lysosomes and is not degraded, making carrier DNA unnecessary. Calcium phosphate-unresponsive lymphoid cells can be transformed with DNA by using this method. The effects of electric pulses on cell viability is examined by using the intermediate-voltage decaying pulse generator.

Fourteen mutants exhibiting conditional induction of sister chromatid exchanges (SCEs) and/or chromosomal aberrations (CAs) were selected out of 65 temperature-sensitive (for growth) mutants from mouse FM3A cells treated with N-methyl-N′-nitro-N-nitrosoguanidine. The mutants with chromosomal abnormality at the nonpermissive temperature were classified into three groups. The group 1 mutant manifested mainly SCEs at the nonpermissive temperature. The group 2 mutants manifested both SCEs and CAs. The group 3 mutants, including ts-131b, a mutant defective in DNA replication and previously described, manifested only CAs. A substantial number of SCEs accompanied by CAs at the site of SCEs were observed in some of the group 2 mutants at the nonpermissive temperature. The results suggest that there are at least three processes involved in the formation of SCEs and CAs, one is common to both phenomena while the others are independent. Some other mutants belonging to the groups 1 and 2 displayed a high incidence of interchromosomal chromatid exchanges at the nonpermissive temperature, suggesting that the same genetic defects leading to high induction of SCEs correlate with induction of chromosomal rearrangements. From these results, the group 2 mutants were subdivided further into five classes, and the mutants in each class had different cytogenetic properties from one another. This indicates defects in at least several genes that participate in the production of SCEs or CAs. The mutants described should be useful for analyzing the mechanisms of SCE or CA formation, and also the mechanism of chromosomal rearrangements.

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30–37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6–8 fold more sensitive to 4NQO than L5178Y cells in terms of D 10 values (dose required to reduced survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.

A multiple mutagen-sensitive mutant (XUM1) of mouse T-cell lymphoma line, L5178Y, is hypersensitive to ionizing radiation, ultraviolet (UV) light, and cross-linking agents (such as mitomycin C). The frequency of transfection for XUM1 cells after exposure to calcium phosphate-coprecipitated pSV2neo DNA was more than 10(4)-fold less effective than that for Ltk-aprt- (LTA) cells. Other transfection methods (DEAE-dextran and polybrene-DMSO) were not effective for L5178Y and XUM1 cells. The transfection-proficient trait of LTA cells was demonstrated to be genetically dominant by examining the the transfection frequency in hybrid clones constructed between XUM1 and LTA cells. To circumvent the problem with XUM1, the LTA genes necessary for transformation processes were introduced into XUM1 cells by constructing hybrids between XUM1 and LTA cells irradiated with X-rays which causes directional chromosome elimination for hybrid cells. Four of 194 hybrid clones tested were transfection-proficient and hypersensitive to UV (XL102, XL107, XL215, and XL216). All four clones were not hypersensitive to X-rays or mitomycin C. The frequencies of transfection for XL102 and XL216 were nearly the same level as that for LTA cells. The efficiency of transfection for XL107 and XL215 was 10 to 100-fold lower than that for LTA cells.

Two independently isolated radiation-sensitive mutants, M10 and LX830, of mouse L5178Y cells were compared for their sensitivity to various DNA-damaging agents. These two mutants share hypersensitivity to gamma rays, bleomycin, arabinofuranosyl cytosine (Ara-C) and aphidicolin. But they differ in that M10 is more sensitive to killing by methyl methanesulfonate (MMS) than LX830 or L5178Y, whereas LX830 is more sensitive to inactivation by hydroxyurea and 3-aminobenzamide than M10 or L5178Y. The response to mitomycin C (MMC) and 4-nitroquinoline-l-oxide (4NQO) is the most pronounced in M10, the least in L5178Y and intermediate in LX830. The complementation tests between M10 and LX830 showed that the X-ray survival curves of M10-LX830 hybrid cells were similar to those of M10 or LX830. An MMS-resistant clone was found among MMS-sensitive M10 cells and proved to be just as sensitive to killing by X-rays as the original M10 cells. These results indicate that M10 and LX830 have a common defect in the repair of damage caused by X rays, bleomycin, Ara-C and aphidicolin but a different defect in the repair of MMS-, MMC-, 4NQO-, hydroxyurea and 3-aminobenzamide-damage.

An X-ray-sensitive mutant LX830, isolated from mouse lymphoma L5178Y cells, was examined for chromosomal aberrations induced by γ-rays and 4-nitroquionoline-l-oxide (4NQO), as compared with a methyl methanesulphonate (MMS) and X-ray-sensitive mutant M10 and their parental L5178Y cells. Spontaneous incidence of chromosomal aberrations in LX830 cells was 2.5 times higher than that in L5178Y cells, but lower than that in M10 cells. In the first mitosis of LX830 cells after 1.0 Gy γ-irradiation, the frequencies of chromosome- and chromatid-type aberrations were significantly higher than those in L5178Y cells. In addition, chromatid exchanges, particularly triradials, isochromatid breaks, and chromatid gaps and breaks were markedly enhanced at G1 phase of LX830 cells. These results are very similar to those in M10 cells. After 4NQO treatment (50 ng/ml), chromatid-type aberrations were induced more frequently at late fixation times. The maximal frequency of chromatid-type aberrations in LX830 cells was about 1.7 times higher than that in L5178Y cells, but LX830 cells were less susceptible to 4NQO than M10 cells. These results indicate that there exist some heterogeneity in the response to 4NQO among ionizing radiation-sensitive mouse cell mutants.

The mouse lymphoma L5178Y cell line and its radiosensitive variants M10 and LX830 were examined for DNA synthesis after X-irradiation. The dose-response curves show that the rates of DNA synthesis immediately after exposure are reduced in a dose-dependent fashion and that the extents of reduction in these 3 cell lines are similar to one another. But a difference was observed in the recovery of DNA synthesis with time of incubation. The recovered levels in M10 and LX830 cells were much higher than those in L5178Y cells at high doses of X-rays. These results are discussed in relation to radioresistant DNA synthesis in ataxia telangiectasia cells.