Cross sensitivity of mutator strains to physical and chemical mutagens.

Ten different mutator strains of Saccharomyces cerevisiae were tested for cross sensitivity to two alkylaitng agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), to determine if any of them are defective in the repair systems which normally deal with damage caused by these agents. For one of the mutators, namely mut2-1, it was shown by genetic analysis that mutator activity and MMS sensitivity are both controlled by the same gene. Two mutants, mut2-1 and mut7-1, were found to be sensitive to MMS but normal to ultraviolet and gamma-rays. Another group is represented by mut1, mut6 and mut8 which are not sensitive to any of the mutagens tested so far. Mutator strain mut2-1 was also shown not to be significantly altered for levels of UV-induced forward and reverse mutations. These observations lend support to the idea of multiple repair systems that deal with DNA damage caused by different agents and also show that mutator activity can often result from the loss of normal cellular repair systems.     

Modeling the periodically-changing predictable response to mutagen exposure in CBA/LacY mice in a successive inbred generations]

A hypothesis on the genetic determination of periodic fluctuations of the sensitivity to the mutagen thioTEPA in successive inbred generations of mice has been earlier put forward. This study was the initial stage of testing this hypothesis. The mouse strain CBA/LacY was divided into two substrains, which differed in the rate of generation change. As a result, two colonies of isogenic mice differing by 10-12 generations with respect to the inbred age were obtained. Both the rate and range of variations in the mutagen sensitivity (four generations per period of the cycle and 20-40% of cells with chromosome aberrations after the standard dose of 2.5 mg/kg of thioTEPA, respectively) in 19 generations of the "fast" substrain agreed with earlier data. The response of the "slow" substrain corresponded to the expected response of the "fast" substrain after the given number of generations. In the mice of generations F142 and F146 that lived simultaneously and differed in thioTEPA sensitivity, the effects of the carcinogen benzo[a]pyrene (BaP) were significantly different. The levels of these effects corresponded to the levels of the responses to thioTEPA. The data obtained agree with the hypothesis tested.     

Initial locomotor sensitivity to cocaine varies widely among inbred mouse strains

Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant‐induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC.     

Preferential segregation of marker chromosomes 14 and 18 in mouse recombinant inbred strains derived from the KE and CBA/Kw strains

The segregation pattern of chromosomes 14 and 18 were analyzed in recombinant inbred strains of mice developed from KE and CBA/Kw strains. The analysis was possible owing to the fact that the C-band on chromosomes 14 of the CBA/Kw strain and that of chromosome 18 of the KE strain show size polymorphism: while the CBA/Kw mice have a small sized C-band on chromosome 14, the KE mice show small C-bands on chromosome 18. Chromosomes were identified by G-banding and FISH. The results show that the chromosomes with small centromeric chromatin segregate preferentially.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

Spontaneous mutagenesis in bone marrow cells in the CBA and CBA/H-T6 mouse strains

Comparative analysis of cytogenetic characteristics in bone marrow cells of the mouse lines CBA and CBA/H-T6 has been carried out. It was shown that translocation T6 effects the apparatus of cell division and can cause additional cytogenetic abnormalities.     

Analysis of oocyte quality in recombinant inbred mouse strains developed from KE and CBA strains that differ in fertilization efficiency

Recombinant inbred (RI) mouse strains were developed from reciprocal crosses between two inbred strains differing in the proportion of fertilized ova (CBA, 100%; KE, 77%), to analyse the underlying factors. A correlation (r = 0.83, P < 0.01) between fertilization efficiency within 22 RI strains and after mating RI females with KE males proved that oocyte quality was involved. The following oocyte parameters were analysed in RI and progenitor strains: time of meiotic maturation, rapidity of enzymatic removal of egg investments, and proportion of fertilized ova with supplementary spermatozoa in the perivitelline space. Among the RI strains, high incidence of supplementary spermatozoa was correlated with lower efficiency of fertilization (r = -0.58, P < 0.05) and with slow meiotic maturation (r = -64, P < 0.01), suggesting that delayed maturation may affect oocyte ability of being fertilized by the first penetrating spermatozoon. However, significant correlations were also found between characters which coexist within the progenitor strains, but are not likely to be physiologically related; this suggests that RI strains have inherited large blocks of progenitor genomes, not disrupted by recombination. The strain distribution pattern (SDP) of the analysed traits revealed CBA-like, KE-like, and intermediate phenotypes, indicating that they are polygenically determined. No linkages were found between the studied traits and 12 enzymatic markers. However, the SDP for fertilization efficiency showed a preponderance of non-matching strains (15/19) in relation to agouti locus; the known instability of this chromosome region makes it possible that a putative linkage was disrupted by recombination when RI strains were created.     

CBXC: a set of recombinant inbred strains between CBA/Ca and BALB/c

A new set of nine recombinant inbred strains designated CBXC-1 to CBXC-9 has been developed from a cross between CBA/Ca female and BALB/c male mice. All of the strains have been brother x sister mated for more than 20 generations, and have been characterized at eight electrophoretic, one immunological and two coat colour loci at which the progenitor strains differ. This set of strains should be useful in investigating the genetics of any characters which differ between the two progenitor strains.     

Differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains

Respiratory syncytial virus (RSV) is a prominent cause of airway morbidity in children under 1 yr of age. It is assumed that host factors influence the severity of the disease presentation and thus the need for hospitalization. As a first step toward the identification of the underlying genes involved, this study was undertaken to establish whether inbred mouse strains differ in susceptibility to pneumonia virus of mice (PVM), the murine counterpart of RSV, which has been shown to accurately mimic the RSV disease of children. With this purpose in mind, double-chamber plethysmography and carbon monoxide uptake data were collected daily for 7 days after inoculation of PVM in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values reflected the success of viral replication in the lungs and revealed a pattern of continuous variation, with resistant, intermediate, and susceptible strains. The results suggest that SJL (resistant) and 129/Sv (susceptible) strains should be used in crossing experiments aimed at identifying genes controlling pneumovirus replication by the positional cloning approach. Similarly, crossing experiments using BALB/c or C57BL/6 (resistant) and DBA/2 or 129/Sv (susceptible) will allow the identification of the genes involved in the control of pulmonary inflammation during pneumovirus infection.     

The immune response of inbred mouse strains to DNP-BGG

The immune response of six inbred mouse strains (SJL, A, C57BL/6, CBA, BALB/c, and DBA/1) to DNP₅₆BGG was tested under three separate immunization schedules: 1 Μg DNP-BGG in 1 mg Al(OH)₃ adjuvant, 50 Μg DNP-BGG in 1 mg A1(OH)₃ adjuvant, and 1 Μg DNP-BGG in complete Freund's adjuvant. Individual serum samples were titered using a modified Farr assay. It was found that the first schedule allowed classification of the mice into responder (SJL, A) and nonresponder (C57BL/6, CBA, BALB/ c, DBA/1) strains. The second schedule produced quantitative as well as qualitative differences among the strains and allowed classification of the mice into higher-responder (SJL, A), intermediate-responder (C57BL/6, CBA, BALB/c), and low-responder (DBA/1) categories. When complete Freund's adjuvant was used in the third schedule, the differences among strains became insignificant. The sera from each strain were pooled and assayed for relative antibody affinity and IgM content. Both of these parameters were dependent largely on the dose of antigen and type of adjuvant used, rather than on the particular mouse strain being studied. The mechanism of adjuvant action, and possible cell interactions in the genetic control of the immune response, are discussed.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Effect of a single severe stress on behaviour of males and females in the mouse inbred strains CBA/Lac and C57BL/6J

The effect of single severe stress in the form of forced swimming on the behavior of males and females in the mouse inbred strains CBA/Lac and C57BL/6J were examined in the open field test. Measurements were carried out within two hours after the stress exposure (Trial 1) and repeated 2 hours thereafter (Trial 2). Intact males and females of the both mouse strains which tested in the open field twice too were used as control. An increased latency was found until first escape from the center both in males and females of the CBA/Lac strain within two hours after the end of forced swimming. This parameter was still high in females in the Trial2. Four out of seven behavior parameters were changed in females of the C57BL/6J strain two hours after the stress exposure, but their behavior was similar to control in the Trial 2. The males of the C57BL/6J strain demonstrated the least changed behavior in the open field test after the stress exposure with the exception of increased number of grooming in the Trial 1. Further on, a detailed analysis of repeated testing in the open field within intact and stressed mice of both strains was performed. This comparison allowed revealing hereditary and gender peculiarities in the mouse behavior after single severe stress exposure. The results are discussed in respect to the possible genetically inherent increased traitanxiety in females of C57BL/6J strain and the state of anxiety in females of CBA/Lac strain.     

Specificity of interactions between the individual gene locus and the structure of chemical mutagens

Summary Relative frequencies of visible mutations (egg colour mutants) induced by two similar mutagenic agents of the mustard group, nitromine (methyl-bis--chloroethyl-amine N-oxide) and N. M.-alanine (N-bis--chloroethyl-alanine), have been studied in the silkworm. The solution of either agents was injected into the wild male moths, which were then mated to double recessivepere/pere female moths. (Normal eggs are black,pere/pere andpe/pe eggs are white andre/re eggs, red.) Eggs produced from these matings were examined for their colour and the mutation rates were estimated by the ratio of uncoverings at the marked loci to the total pigmented eggs.Analysis of the results has revealed that the ratio of the mutation rates at thepe-locus to those at there-locus varies markedly according to whether nitromine or N. M.-alanine is injected. When nitromine was used the ratios ofpe-aberrants tore-aberrants were 1.06 and 1.58, while in the case of N. M.-alanine the ratios were 0.59 and 0.60. In many other experiments in which moths were treated with nitromine, thepe/pe ratio was consistently greater than 1 and often as high as 2. The same was true for experiments with irradiated male moths. These results have been discussed in relation to possible specific interactions between chemical mutagens and chromosome loci.     

Susceptibility and resistance to poliovirus-induced paralysis of inbred mouse strains.

Susceptibility to human poliovirus-induced disease in different inbred mouse strains was analyzed after intracerebral inoculation of two mouse-adapted type 2 polioviruses, the attenuated W-2 strain and the virulent Lansing strain. In contrast to inoculation with the Lansing strain, which was invariably lethal, inoculation with the W-2 strain defined three groups of mice with high, intermediate, or low disease incidence. Those in the high-disease-incidence group, the DBA/1J and DBA/2J mice, exhibited a high level of virus replication in the spinal cord by day 2 postinfection, with no detectable neutralizing-antibody response. Mice in the intermediate- and low-incidence groups had lower levels of virus replication in the spinal cord and/or produced neutralizing antibodies. No correlation was observed between H-2 haplotype and the extent of virus replication, production of neutralizing or enzyme-linked immunosorbent assay-detectable antibodies, or T-cell-proliferative response. However, mice of the H-2k haplotype manifested a low incidence of disease.     

Responses of inbred mouse strains to infection with intestinal nematodes

Comparisons were made of the immune and inflammatory responses of four strains of inbred mice to infection with the intestinal nematodes Trichinella spiralis and Nippostrongylus brasiliensis to determine whether genetically determined 'high responsiveness' to infection, seen most clearly in intestinal responses, is independent of the parasite concerned and necessarily correlated with protection. The time course of infection was followed by counting adult worms at intervals after infection. Mucosal mast cells and Paneth cell numbers were determined as indices of the intestinal inflammatory response. Levels of IgG2a and IgG1 antibodies and of the cytokines IFN-gamma and IL-5 released from in vitro-stimulated mesenteric node lymphocytes were measured to assess type 1 and type 2 responses. NIH and CBA mice were the most resistant to T. spiralis and N. brasiliensis respectively, resistance in each case being correlated with the most intense intestinal inflammatory responses. C57BL/10 (B10) and B10.BR were the least resistant to T. spiralis, but were as resistant as CBA to N. brasiliensis, despite their intestinal inflammatory responses to both parasites being much lower than the other two strains. Mice infected with T. spiralis made the expected switch from a type 1 (IFN-gamma) to a type 2 (IL-5) response between days 2 and 8, and there were no significant differences in levels of these cytokines between the strains. In contrast, when infected with N. brasiliensis, CBA showed an IFN-gamma response at day 4, all strains switching to IL-5 by day 8 and NIH mice releasing the greatest amount of IL-5. The results indicate that the "high responder" phenotype to intestinal nematode infection is in part determined by host characteristics, but is also determined by the parasite concerned--seen most clearly by the differences between NIH and CBA when infected with T. spiralis and N. brasiliensis. The fact that "low responder" B10 background mice were more resistant to N. brasiliensis than "high responder" NIH implies that each parasite elicits a particular pattern of protective host responses, rather than parasites being differentially susceptible to the same response profile.     

A quantitative analysis of the cytotoxic action of chemical mutagens

A quantitative hypothesis is developed to explain the cytotoxic action of chemical mutagens on eukaryotic cells. The hypothesis forms an extrapolation of previously developed concepts used to explain the effect of ionizing radiation and the cytotoxic action of UV light. The crucial potentially lethal lesion is assumed to be a DNA double-strand lesion which may be an interstrand cross-link or a pair of DNA single-strand alkylations, for example. The effect of repair processes is included in the analytical equation derived to describe cell survival. The analysis of several sets of cell survival data for different chemical mutagens is used to demonstrate the applicability of the hypothesis. The logical extension of the hypothesis permits a division of chemical mutagens into 4 separate classes on the basis of the mechanisms proposed for the cytotoxic activity, and the relative importance of the risk associated with low-level exposure to each class is discussed. The hypothesis is amenable to further experimental verification.