V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair.

V(D)J recombination has been examined in several X-ray-sensitive and double-strand break repair-deficient Chinese hamster cell mutants. Signal joint formation was affected in four mutants (xrs 5, XR-1, V-3, and XR-V9B cells, representing complementation groups 1 through 4, respectively) defective in DNA double-strand break rejoining. Among these four, V-3 and XR-V9B were the most severely affected. Only in V-3 was coding joint formation also affected. Ataxia telangiectasia-like hamster cell mutants (V-E5 and V-G8), which are normal for double-strand break repair but are X ray sensitive, were normal for all aspects of the V(D)J recombination reaction, indicating that X-ray sensitivity is not the common denominator but that the deficiency in double-strand break repair appears to be. The abnormality at the signal joints consisted of an elevated incidence of nucleotide loss from each of the two signal ends. Interestingly, in complementation groups 1 (xrs 5) and 2 (XR-1), signal joint formation was within the normal range under some transfection conditions. This suggests that the affected gene products in these two complementation groups are not catalytic components. Instead, they may be either secondary or stochiometric components involved in the later stages of both the V(D)J recombination reaction and double-strand break repair. The fact that such factors can affect the precision of the signal joint has mechanistic implications for V(D)J recombination.     

Seven complementation groups of respiratory syncytial virus temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of the RSN-2 strain of respiratory syncytial virus have been classified into six complementation groups, two of which appeared to be homologous with two of the three complementation groups of the A2 strain described by Wright et al. (P. F. Wright, M. A. Gharpure, D. S. Hodes, and R. M. Chanock, Arch. Gesamte Virusforsch, 41:238--247). Thus seven complementation groups of respiratory syncytial virus, designated A, B, C, D, E, F, and G, have been defined. The frequency and type of mutant isolated varied according to strain; group C was unique to the A2 strain, and groups D, E, F, and G were unique to the RSN-2 strain. The highest complementation indexes were obtained by preincubation for 7 h at permissive temperature, followed by incubation at restrictive temperature for 40 to 50 h in the case of A2 strain mutants or 80 to 90 h for RSN-2 strain mutants. Genetic recombination was not detected.     

Genetic Characteristics of Conditional Lethal Mutants of Vesicular Stomatitis Virus Induced by 5-Fluorouracil, 5-Azacytidine, and Ethyl Methane Sulfonate

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been "cured" of infection by prolonged exposure at the restrictive temperature.     

Identification of New Genes Required for Meiotic Recombination in Saccharomyces Cerevisiae

Mutants defective in meiotic recombination were isolated from a disomic haploid strain of Saccharomyces cerevisiae by examining recombination within the leu2 and his4 heteroalleles located on chromosome III. The mutants were classified into two new complementation groups (MRE2 and MRE11) and eight previously identified groups, which include SPO11, HOP1, REC114, MRE4/MEK1 and genes in the RAD52 epistasis group. All of the mutants, in which the mutations in the new complementation groups are homozygous and diploid, can undergo premeiotic DNA synthesis and produce spores. The spores are, however, not viable. The mre2 and mre11 mutants produce viable spores in a spo13 background, in which meiosis I is bypassed, suggesting that these mutants are blocked at an early step in meiotic recombination. The mre2 mutant does not exhibit any unusual phenotype during mitosis and it is, thus, considered to have a mutation in a meiosis-specific gene. By contrast, the mre11 mutant is sensitive to damage to DNA by methyl methanesulfonate and exhibits a hyperrecombination phenotype in mitosis. Among six alleles of HOP1 that were isolated, an unusual pattern of intragenic complementation was observed.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.     

Genetic studies on temperature-sensitive mutants ofBacillus subtilis bacteriophage SPP 1

A total of 30ts mutants ofBacillus subtilis bacteriophage SPP1 were isolated and subjected to complementation test. On the basis of this test 21 mutants were classified into 4 functional groups; the classification of the remaining 9 mutants was unclear. The frequency of recombination by mutual crossing was determined in representatives of individual groups; this made it possible to place these mutants on a linear map comprising a total length of 7.62 recombination units.     

Qualitative Complementation Test for Temperature-Sensitive Mutants of Herpes Simplex Virus

A simple, rapid, qualitative method for classifying temperature-sensitive mutants of herpes simplex virus into functional complementation groups has been developed. The positive reaction observed in this test reflects the ability of mutant pairs to interact by both complementation and recombination.     

Genetic Map of Bacteriophage α

Temperature-sensitive mutants of phage alpha were obtained by means of various mutagens and assigned to 25 complementation groups. Temperature-sensitive mutants belonging to 21 complementation groups and a mutant giving turbid plaques were used to perform two- and three-factor crosses. Seventeen of the cistrons and the turbid mutant were shown to belong to the same linear linkage group, which showed no signs of circularity. The remaining four unlinked cistrons showed peculiarities in their recombination properties. Genes which are known to be expressed earlier apear to be grouped together in a terminal segment of the linkage group.     

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

Summary A total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR^-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.     

Assembly of the mitochondrial membrane system. Genetic complementation of mit- mutations in mitochondrial DNA of Saccharomyces cerevisiae.

A method has been devised to test intergenic complementation of mutations in the mitochondrial DNA of Saccharomyces cerevisiae. The test is based on the observation that diploids issued from pairwise crosses of certain mit- mutants with deficiencies in cytochrome oxidase, or coenzyme QH2-cytochrome c reductase, acquire high levels of respiratory activity shortly after zygote formation. Under our experimental conditions neither biochemical complementation, interallelic complementation, nor recombination has been found to contribute to any significant extent toward the respiration measured in the diploids at early times. The test has been used to study the number of complementation groups represented by a large number of mit- mutants. Results of pairwise crosses of mutants in the oxi 1, oxi 2, oxi 3, cob 1, and cob 2 loci indicate that complementation occurs between the oxi and cob loci between different oxi loci but not between the two cob loci. The five loci have, therefore, been assigned to four different complementation groups.     

Temperature-Sensitive Mutants of Adenovirus Type 12 Defective in Viral DNA Synthesis

Ten temperature-sensitive (ts) mutants of adenovirus type 12 which produce plaques at 31 but not at 38.5 C have been isolated after mutagenesis with nitrosoguanidine or nitrous acid. The mutants have been classified into six separate complementation groups. DNA-DNA hybridizations have shown that at 38.5 C the ts 401 and 406 mutants of groups B and E, respectively, synthesized less than 10% of the normal level of viral DNA. The two mutants were also defective in the production of late proteins at the nonpermissive temperature, as shown by fluorescent-antibody tests and analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Genetic recombination between the ts viruses 401 and 406 has been demonstrated; the recombination frequency for the wild-type virus production was 17.7%. Both mutants induced an increase in thymidine kinase activity at 38.5 C. Moreover, the two viral DNA-defective mutants shut off host DNA synthesis at the restrictive temperature. It is striking that at 38.5 C ts virus 401 transformed two to eight times more hamster cells than the wild-type virus, whereas ts virus 406 transformed at a frequency similar to the wild-type virus.     

Menage á trois: Double strand break repair,V(D)J recombination and DNA-PK

All organisms possess mechanisms to repair double strand breaks (dsbs) generated in their DNA by damaging agents. Site-specific dsbs are also introduced during V(D)J recombination. Four complementation groups of radiosensitive rodent mutants are defective in the repair of dsbs, and are unable to carry out V(D)J recombination effectively. The immune defect in Severe Combined Immunodeficient (scid) mice also results from an inability to undergo effective V(D)J recombination, and scid cell lines display a repair defect and belong to one of these complementation groups. These findings indicate a mechanistic overlap between the processes of DNA repair and V(D)J recombination. Recently, two of the genes defined by these complementation groups have been identified and shown to encode components of DNA-dependent protein kinase (DNA-PK). We review here the three fields which have become linked by these findings, and discuss the involvement of DNA-PK in dsb rejoining and in V(D)J recombination.     

Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants.

Temperature-sensitive (ts) mutants in a number of complementation groups of herpes simplex virus type 1 (HSV-1) are deficient in DNA polymerase induction at the restrictive temperature. Twenty-two mutants in 15 complementation groups were tested for sensitivity to phosphonoacetate (PAA), a compound that inhibits HSV replication in vivo and the DNA polymerase in vitro. One mutant, tsD9, was resistant to PAA (Pr), whereas all others were sensitive. Revertants of tsD9 to the ts+ phenotype simultaneously lost PAA resistance. Additional Pr mutants were isolated from ts mutants belonging to several complementation groups of HSV-1. Double mutants (ts Pr phenotype) were used in three-factor recombination analyses to locate the PAA locus on the genetic map at a position indistinguishable from the ts lesion in tsD9. In all cases, resistance or sensitivity to PAA in vivo was correlated with resistance or sensitivity of DNA polymerase in vitro. These data are compatible with the temperature-sensitive lesion of tsD9 and the determinant of PAA sensitivity both residing in the structural gene for DNA polymerase.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Mutation Induction by Difunctional Alkylating Agents in NEUROSPORA CRASSA

The genetic characterization of ad-3 mutants of Neurospora crassa induced by two carcinogenic difunctional akylating agents, 1,2,4,5-diepoxypentane (DEP) and 1,2,7,8-diepoxyoctane (DEO), has shown that point mutations at the ad-3B locus have similar complementation patterns. In addition to the induction of point mutations, DEP induces a low frequency (7.5%) of multilocus deletions, whereas DEO induces an extremely high frequency (42.0%). The distribution of the different classes of ad-3 mutants and the frequency of multilocus deletion mutants among DEP-induced mutants are not significantly different from those induced by the monofunctional alkylating agents EI, EMS and ICR-177 at comparable forward-mutation frequencies. Moreover, the frequencies of DEP-induced ad-3B mutants showing allelic completion or having nonpolarized complementation patterns are similar to those of ad-3B mutants induced by monofunctional agents. It is suggested, therefore, that the mechanism of mutation-induction by DEP in N. crassa is similar to that of monofunctional alkylating agents. Mutation-induction by DEO probably results both from the mechanism of action of monofunctional alkylating agents and from inter-strand cross-linkage of the DNA molecular by the two functional epoxy groups.     

Control of lysogeny and a genetic map of the temperate phage SM of Pseudomonas aeruginosa

76 mutants with impaired ability to lysogenize host cells were isolated in SM phage after mutagenesis using several chemical mutagens. By means of complementation test, these mutants were distributed into two groups, cI and cII. The mutants of the cI group were similar phenotypically to the cI mutants of phage lambda defective in synthesis of repressor. The mutants of the cII group establish and support the lysogenic state in infected cells with very low frequency. Temperature-sensitive mutants belonging to 13 complementation groups and nonlysogenizing mutants of the cI and cII groups were used in genetic mapping of SM phage. Mutual positions of markers and relative distances between them were determined by the method of two-factorial crosses. The greatest distance equal to 20 units of recombination was determined between ts 88 marker and one of early genes marked with ts 105 mutation. The genes cI and cII are closely linked to each other and also to ts 105 marker and are situated at one end of the genetic map.     

Isolation and preliminary characterization of temperature-sensitive mutants of Newcastle disease virus.

Temperature-sensitive (ts) mutants of Newcastle disease virus have been isolated and characterized genetically (complementation), biochemically (RNA synthesis) and biologically (fusion from within and hemadsorption). Fifteen of these mutants have been divided into five complementation groups. Groups A (five mutants) and E (one mutant) are ts for RNA synthesis (RNA-) as well as for the other functions. Group B contains four RNA+ mutants of which one is ts for fusion, one for hemadsorption and two for neither function. Group C contains one RNA+ mutant which is a poor cell fuser. Group D contains two RNA+ mutants which are ts for fusion. In addition, two noncomplementing mutants (group BC) fail to complement both group B and group C mutants while exhibiting complementation with mutants in groups A, D, and E.     

Complementation analysis by somatic hybridisation and genetic crosses of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Summary Fusion complementation experiments between nitrate reductase (NR) deficient lines CNX 20, 27, 82 and 103 of Nicotiana plumbaginifolia were performed with the already characterized N. plumbaginifolia mutants nx 1, 24 and 21, belonging respectively to the complementation groups cnx A, B and C. CNX 20 and 82 were identified as belonging to the group of cnx A. CNX 27 complemented with NX 1 and NX 21 but not with NX 24 indicating another B type. The fourth line, CNX 103 showed complementation with CNX 20, NX 21 and NX 24, revealing a fourth cnx complementation group, cnx D, that until now has not been described in higher plants. Genetic crosses inside respectively the NIA and the CNX group, and between NIA and CNX confirmed the fusion complementation results, and showed allelism for the nia mutants     

DNA-negative temperature-sensitive mutants of herpes simplex virus type 1: patterns of viral DNA synthesis after temperature shift-up.

Temperature-sensitive mutants of herpes simplex virus type 1 belonging to four DNA- complementation groups exhibited two distinct patterns of viral DNA synthesis after shift-up to the nonpermissive temperature. In cultures infected with mutants belonging to complementation groups A, C, and D, little or no viral DNA was synthesized after shift-up. In cultures infected with a mutant in complementation group B, nearly normal amounts of viral DNA were synthesized after shift-up.