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Nucleoid sedimentation experiments were performed to compare the repair capacity for strand breaks of 2 permanent cell lines deficient in excision repair (mei-9, mus-201) with that in repair proficient cells (permanent cell line Kc). After ultraviolet (UV) treatment (10 J m{-2}; 256 nm) the number of breaks detected in the mei-9 and mus-201 cells is lower than in the repair proficient Kc cells. This indicates that the first step of the excision repair process, the incision, is impaired in these mutants. The repair of X-ray induced (10 Gy) breaks, as measured by nucleoid sedimentation, appears normal in the mutants mei-9 and mus-201. To determine the repair capacity of Kc cells (proficient repair) to remove UV induced thymidine dimers from deoxyribonucleic acid (DNA) of the complete genome, several experiments were performed using alkaline sucrose gradients. Kc cells were UV irradiated and after a period of repair incubation (0, 4, 8, 16, 24 and 48) the cells are lysed. The DNA is incubated with T4-endonuclease, and the molecular weight of the DNA is determined. The results show that the removal of dimers is very slow during the first 8 hours after irradiation. After 8 hours, the repair increases and at 24 hours after irradiation 95% of the dimers are removed. It has been shown that in mammalian cells and also in the lower eukaryote Saccharomyces cerevisiae UV induced DNA repair is not randomly distributed over the genome. Evidence has been presented for the preferential repair of actively transcribed genes. In addition to studies on the in vivo induction of mutations in germ line cells and somatic cells in Drosophila repair proficient and deficient (mei-9 and mus-201) strains, repair of UV induced damage in 4 specific genes (GART, RpII, white and Notch) in cell lines derived from these strains, is investigated. GART is a 10 kb gene that is involved in the de novo purine synthesis. The RpII (RNA polymerase II) gene (9.4 kb) encodes for the largest subunit of ribonucl eic acid (RNA) polymerase II. The product of the white gene (6 kb) plays a role in the distribution of pigment in the fly. The Notch gene (34 kb) is expressed during embryogenesis and appears involved in the development of neurogenic tissues.
Transcripts from the GART and the RpII gene were found in poly A+ RNA isolated from the repair proficient Kc cell line, by Northern blotting. In contrast, no expression of the white and Notch genes could be detected. It is found that the active gene GART as well as the inactive genes white and Notch are all repaired to the same extent. 4 hours after UV irradiation norepair is measured. After 8 hours only 20 to 30% of repair is found, 60% after 16 hours and about 80% after 24 hours up to 90% after 48 hours. The repair kinetics for these genes appears independent of the doses studied. The repair of the RNA polymerase II gene was found to be faster. Preliminary data, however, indicate that less pyrimidine dimers are induced in this gene as compared to the other genes investigated. In both excision repair deficient cell lines (mei-9 and mus-201) no removal of thymidine dimers from the Gart genes and Notch genes was found. These results are in agreement with the results obtained with other biochemical methods; ie these cells cannot introduce single strand nicks at thymidine dimers (in total genomic DNA) nor do they show unscheduled DNA synthesis (UDS) after UV irradiation.

Repair kinetics in relation to chromosomal aberration formation:
When irradiated human lymphocytes were posttreated with a repair inhibitor, such as cytosine arabinoside (ara C), the yield of aberrations (dicentrics and fragments) increased. When the ara C treatment was given for 15, 30, 60 or 120 minutes, most of the increase in the frequency of aberration was found to occur in the first 15 minutes. If irradiated lymphocytes were allowed to recover for 30 minutes and then challenged with ara C, there was no increase in the frequency of aberrations indicating that fast repairing lesions lead to the formation of aberrations. Parallel studies with premature chromosome condensation (PCC), in which lymphocytes were irradiated and fused immediately or allowed to repair for 60 minutes in the presence or absence of ara C indicated that about 50% of breaks disappear during this repair, while in the presence of ara C, there was a complete inhibition of repair of breaks. When a nucleoid sedimentation technique (which measures the induction of deoxyribonucleic (DNA) strand breaks) was used under a similar protocol, it was seen that ara C completely inhibited the repair of strand breaks. These results pointed out that fast repairing DNA strand breaks induced by X-rays lead to chromosomal aberrations.

The PCC technique and conventional metaphase chromosome analysis were used to examine the kinetics of X-ray induced primary breaks, their rejoining and formation of dicentrics in human lymphocytes. A dose dependent increase in the frequency of fragments and a linear quadratic increase in the frequency of dicentrics were observed. The frequency of fragments was much higher (factor of about 10) when determined by PCC technique immediately after irradiation in comparison to the frequencies observed in metaphases. For an assessment of repair kinetics irradiated lymphocytes were fused with mitotic Chinese hamster ovary (CHO) cells either immediately or after several recovery times a fter irradiation. It was found that the frequency of chromosome fragments decreased with time whereas the dicentrics were formed very fast and their frequency remained the same, despite the decrease in the number of fragments with increased recovery time. These results confirm our earlier findings using repair inhibitor ara C, which indicated that early repairing lesions (strand breaks) lead to chromosomal aberrations, especially the exchange types.

Chromatin structure in relation to yield of chromosomal aberrations:
We have demonstrated earlier that when lymphocytes were incubated in a medium containing sodium butyrate, which hyperacetylates histones, thus relaxing DNA, prior to X-ray irradiation increased the yield of aberrations. When human lymphocytes were fused to mitotic CHO cells irradiated with X-rays (2 Gy), immediately or after 30 minutes or 60 minutes the frequency of induced breaks fell dramatically with increased condensation. It has been suggested that treatment with hypertonic salt solution suppresses the fast component of repair of radiation induced strand breaks. This should increase the frequency of chromosome breaks. Hypertonic treatment is also expected to condense the chromatin which could prevent accessibility to repair enzymes, thus increasing the frequency of induced breaks. 2 types of experiments were carried out to study the role of hypertonic salt solution on the yield of X-ray induced chromosomal aberrations in lymphocytes. In a fractionation regime, lymphocytes were irradiated in the medium or a hypertonic salt solution (0.3 M sodium chloride), with 1 Gy and allowed to recover for 30 minutes or 90 minutes and irradiated further with 1 Gy. The lymphocytes were fused with mitotic CHO cells and the frequencies of dicentrics and fragments were evaluated in PCCs. Contrary to the expectation, the yield of dicentrics and fragments was reduced in the lymphocytes irradiated in the presence of hypotonic solution. In another experiment, lymphoc ytes were X-ray irradiated with 3 Gy alone, or they were irradiated in the medium or treated with a hypertonic salt solution before and after irradiation, or irradiated in cold isotonic solution and transferred immediately to hypertonic salt solution. After 30 minutes postincubation, lymphocytes were cultured for 48 hours and the frequency of aberrations was evaluated. The frequency of dicentrics was very similar in all treatments except the one in which the lymphocytes were irradiated in hypertonic salt solution, which responded with lower (about 40%) number of dicentrics. These results indicate that, at least in isolated lymphocytes, irradiation under hypertonic conditions reduces the frequencies of aberrations probably due to induced chromatin condensation.

Inducible repair in human lymphocytes:
It has been reported that human lymphocytes subjected to a small conditioning dose of radiation leads to some protection (in terms of yield of chromosomal aberrations) to a challenging higher dose of radiation, a phenomenon termed as adaptive response. 2 series of experiments were carried out with whole blood from healthy volunteers: 1 in which radioisotopes were used to administer the conditioning dose, and the other in which a low dose of X-rays was used as the conditioning treatment. 2 important findings emerged from these experiments: first, in cells that received both the conditioning and the challenge doses, the frequencies of chromosomal aberrations (chromatid and isochromatid deletions) were lower than expected on the basis of additivity of the effects of individual treatments; these results thus support those published from Wolff's laboratory in showing that human lymphocytes can become adapted by prior exposure to low level irradiation so that they become less sensitive to the chromosome breaking effects of X-rays delivered subsequently. Second, the magnitude of reduction in aberration frequencies in the adapted cells varied between the different donors (and also between blood samples from the same donor exposed to different radioisotopes), was generally lower than those observed in the studies from Wolff's laboratory and, with lymphocytes from some donors, no adaptive response could be demonstrated. This finding is consistent with the existence of interindividual differences with respect to the magnitude of the adaptive response, a finding which has received support from the work of Bosi and Olivier!

Chromosomal aberrations induced by restriction endonucleases:
Among the DNA lesions induced by ionising radiations, DNA double strand breaks (DSB) appear to be the most important one leading to chromosomal aberrations. We have provided both biochemical and cytological evidence for this conclusion. When CHO cells are permeabilised with inactivated Sandai virus and treated with restriction endonucleases (RE) (which exclusively induces DNA DSBs), chromosomal aberrations are induced in a pattern similar to that induced by ionising radiation, namely chromosome type in G1 and chromatid type in G2 and a mixture in the S phase. When CHO cells were treated with the restriction enzyme Alu I and posttreated with ara C, the frequency of aberrations increased indicating the ara C can inhibit rejoining of RE induced DSBs. Ara C has a similar influence on X-ray induced breaks. This indicates that X-ray induced DSBs respond in a similar way as RE induced DSBs to ara C.

Influence of incorporated bromodeoxyuridine (BrdU) on the induction of chromosomal aberrations and sister chromatid exchanges (SCE) and cleaving of DNA by REs differing in their recognition sequences especially the number and postion of thymidine nucleosides was investigated.

For induction of SCEs, it was found that the efficiency of RE did not depend on the nature of the initial cleavage, blunt and cohesive breaks were equally effective in inducing SCEs, whereas blunt ends were more effective in inducing chromosomal aberrations. It was found that mid S phase was the most sensitive phase for the induction of chromosomal alterations. Restriction pattern analysis of DNA from BrdU (100%) substituted and unsubstituted nuclei following Alu I digestion, showed some inhibition at 100% level, which may be responsible for less efficient induction of SCEs by this RE. Some increase in the frequency of induced chromosomal aberrations BrdU substituted cells was found which may indicate that incorporated BrdU may interfere with the repair of the induced strand breaks, since BrdU substitution was not found to significantly influence the induction of strand breaks by different REs.

Dose response relationships for the induction of mutations at the hypoxanthine guanine phosphoribosyltransferase (HPRT) and sodium/potassium adenosine triphosphatase (Na/K-ATPase) loci have been determined. After treatment the cells were propagated for expression of the induced mutations and subsequently seeded for selection of mutants. After 8 days of selection mutant colonies were fixed, stained and counted.

Consequences of the presence of the human excision repair cross complementing (ERCC-1) gene in repair deficient 43-3B cells:
The human gene restores the sensitivities to deoxyribonucleic (DNA) damaging agents of the repair deficient rodent cells to normal levels. Also the frequency of induced mutations after treatment with ultraviolet (UV) returnals to the level of rodent wild type cells. The data suggested that the ERCC-1 gene largely corrects all impaired functions in the defective 43-3B cells and that therefore the ERCC-1 gene is probably homologous with respect to function to the defective gene in 43-3B cells.

Characterisation of the UV sensitive mutant VH-1:
The dose repose relationships for mutations induced by UV in VH-1 cells were linear. Mutation induction at the sodium/potassium adenosine triphosphatase locus was 4-fold enhanced and at the HPRT-locus, 7-fold enhanced. These increases are lower than might have been expected on the basis of the approximately 10-fold enhanced UV sensitivity with regard to survival.
V-H1 cells were also compared with V79 wild type cells and with 2 UV sensitive Chinese hamster ovary (CHO) mutants of the same complementation group with respect to induction to unscheduled DNA synthesis (UDS) by UV. The level of UDS in V-H1 is only slightly reduced compared to wild type cells while the 2 other mutants of the same complementation group show only the background level of UDS. These data show that a phenotypic heterogeneity exists within the second complementation group of UV sensitive mutants. It is hypothesised that t he repair gene of this complementation group has more than 1 functionally important domain or that the gene is involved in preferential repair of active genes.

Characterisation of the X-ray sensitive mutants V-15B, V-C8, V-C4 and E5:
Despite that V-15B cells are approximately 8-fold more sensitive to X-rays than wild type cells the mutation induction was not significantly enhanced. Mutants from the same complementation group have been shown to depend on epigenetic alterations as they can be induced to revert by 5-azacytidine. We found that reversion of V-15B cannot be induced by 5-azacytidine. Despite the fact that V-G8, V-C4 and V-E5 belong to the same complementation group, different levels of spontaneous chromosomal aberrations were observed. For V-G8 these frequencies were similar to that observed in wild type cells whereas an increase of about 2-fold and 6-fold was found for V-E5 and V-C4 respectively. In all 3 mutants the frequencies of X-ray induced aberrations were higher in comparison to wild type V79 cells in both G1 and G2 cells. The types of aberrations induced in G1 cells were mainly of the chromosome type in the normal cells, whereas in all 3 X-ray sensitive mutants, both chromosome exchanges and chromatid exchanges were found to occur within the same cell. These levels and patterns of chromosomal aberrations are similar to those observed in cells derived from patients with ataxia telangiectasia. As the biochemical characteristics of these cells also agree with those observed for ataxia telangiectasia, our mutants from the new complementation group are the first rodent repair deficient mutants which phenotypically resemble ataxia telangiectasia cells.

Characterisation of the mytomycin C (MMC) sensitive mutant V-H4:
The level of spontaneous chromosome aberrations is almost 2-fold increased in V-H4 and the level of chromosome abnormalities induced by treatment with MMC or cis-diamminedichtoroplatinum (cis-DDP) is 2 to 3 fold higher in this mutant than in the wild type cells. Preliminary results indicate that V-H4 is rather hypomutable after treatment with (8-MOP) plus near ultraviolet (UVA), 4-nitroquinoline-N-1-oxide (4NQO) or ethyl methanesulphonate (EMS).

This project studies the biochemical aspects of deoxyribonucleic acid (DNA) repair induced by radiation, in normal cells as well as radiosensitive mutants, in relation to biological endpoints such as cell killing, induction of gene mutations, and chromosomal aberrations. Radiosensitive cell lines in which the radiosensitivity is complemented by the introduction of a cloned repair gene will also be investigated.
Emphasis will be put on:
the role of chromatin structure in the distribution and repair of damage induced by radiation;
the nuclear localisation of the various steps of the repair process;
the structure of DNA repair patches using inhibitors of DNA synthesis.

Our results suggest that ultraviolet (UV) induced repair synthesis is not confined to the nuclear matrix, as has been shown for replication and transcription. The efficiency of repair may be based on a sliding mechanism as has been proposed for incision of bulky damage by the ultraviolet radiation (UVR) ABC enzymes in Escherichia coli. The occurrence of clustered repaired sites within DNA loops is in favour of repair enzymes processively operating along DNA molecules. In fact a substantial fraction of incisions was observed at interdimer distance which is consistent with a processively acting repair process. In the presence of hydroxyurea, ligation or polymerisation following the incision step was delayed leading to single strand breaks within DNA molecules. It is clear that in human fibroblasts certain domains within the chromatin are more rapidly repaired than the bulk of chromatin. These domains are located proximal to the nuclear matrix end and comprise transcriptionally active DNA. We have shown that the heterogeneity in distribution of repaired sites in chromatin loops correlates with the heterogeneity in removal of pyrimidine dimers from the genome. Yet it is more likely that the preferential repair of nuclear matrix associated DNA observed in normal cells exposed to low UV dose, reflects p referential repair of another important type of photoadduct namely the 6-4 photoproduct (6-4 PP) since repair incorporation initially after irradiation can be almost exclusively attributed to repair of 6-4 pyrimidine dimer (PD). Our data suggest that repair of 6-4 PP is subject to the same regime as removal of PD ie preferentially directed towards repair of transcriptionally active DNA. The absence of preferential repair at high UV dose may be due to a sufficient local disruption of chromatin structure to allow efficient interaction of repair enzymes with bulk chromatin. Our findings suggest the existence of 2 independently operating pathways directed towards repair of PD in either active or inactive chromatin Xeroderma pigmentosum group c (XP-C) cells have lost the capacity to repair inactive chromatin, but are still able to repair active chromatin. The reverse situation may exist in cells from Cockayne's syndrome (CS) patients, which appear to be unable to perform efficient repair of active genes. Although direct evidence is lacking repair of PD in active achromatin may be mediated by the transcription process itself. The experiments with XP-C cells indicate that the preferential repair of PD from active genes confers considerable UV resistance to confluent cells in the absence of efficient repair of 6-4 PP. In growing cells UV resistance seems to be provided both by preferential repair of PD from active genes and efficient removal of 6-4 PP from the genome overall, pointing to a strong cytotoxic potency of 6-4 PP. The absence of preferential repair of PD in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene concomitant with a high frequency of mutations in this locus in UV sensitive hamster V-H1 cells points to a dominant role of PD in mutagenesis. However recent data obtained with revertants of V-H1 cells showed the absence of PD repair in the HPRT gene, but mutation frequencies similar to wild type V79. This phenomenon is not well understood at the moment, but indicates a role of 6-4 PD in mutagenesis as well.

The project aims to isolate and characterise repair deficient mutants from mammalian cell cultures. Rodent cells (V79 and Chinese hamster ovary (CHO) cells are mutagenised in order to induce mutations in genes which are involved in deoxyribonucleic acid (DNA) repair. Via replica plating clones are identified which are sensitive to DNA damaging agents.

During the course of this project a large number of repair deficient mutants have been obtained from CHO cells. The V79 cell line appears to be very suitable for the isolation of repair deficient mutants. The complementation analysis has been completed for 9 of the 13 repair deficient mutants and 2 new complementation groups have been identified. 2 for ultraviolet (UV) and 1 for X-rays. Especially rewarding was the finding that V-H4 is homologous with Fanconi's anaemia complementation group A. As presently conditions have been found which permit an efficient transfection of DNA into V79 CHO cells, this opens the exciting possibility of isolating the human gene which is defective in Fanconi's anaemia. The isolation of revertants from V-H1, which are still phenotypically different from wild type, will be important for the study of the function of the excision repair cross complementing (ERCC-2) gene of complementation group 2 which has been recently isolated.

Amherst-M56i wild type males are irradiated (15 Gy) and mated with repair proficient (mei+) females, excision repair deficient females (mus-201) and postreplication repair deficient (mei-41) females.
Only mutations induced in mature sperm are collected. The mutants are analysed and classified according to genetic and cytogenetic characteristics in intragenic mutations and gross chromosomal aberrations (multilocus deletions, translocations, inversions) Deoxyribonucleic acid (DNA) from whole body white mutants from the first class (intragenic mutations) was analysed by molecular techniques including restriction enzyme pattern, S1 protection experiments and, when neccesary, sequencing.

A comparison of all the data indicate, that due to the defect in the excision repair of mus-201 females, among the recovered X-ray induced mutations relatively more intragenic deletions and less multilocus deletions are present than in repair proficient condition. The reverse appears true after recovery in postreplication repair deficient mei-41 females.

Vermilion mutants were induced by exonuclease (ENU) or ethyl methanesulphonate (EMS) treatment. Mutant alleles were cloned in phage lambda using the recombinational screening method or cloned directly in M13 vectors after amplification in vitro polymerase chain reaction (PCR). Single stranded plasmid or M13 deoxyribonucleic (DNA) was then used for the sequence determination using a set of oligonucleotide primers.

ENU induced and 28 EMS induced mutants were analysed. 3 ENU and 1 EMS mutant represent double mutations. Transition mutations were the most prominent sequence change after ENU treatment.
Both guanine cytosine-adenine thymine (GC-AT) and AT-GC transition mutations can be explained by mispairing of the 06-ethylguanine and 04-ethylthymine adducts with thymine and guanine, respectively. The AT-TA transversions in case of ENU can be explained by the 02-ethylthymine adduct. However, AT-TA transversions are also induced by EMS although EMS treatment does not result in 02-ethylthymine adducts. Most likely the transversion mutations results from alkylation of the base nitrogen atoms. Comparison of the alterations in F1 and F2 mutants did not show significant differences. The strong contribution of 06-ethylguanine adduct to the mutagenicity of both ENU and EMS possibly explains the absence of distinct differences between the type of mutations observed in F1 and F2 mutants.

Drosophila repair deficient mutants were characterised biochemically in primary cell cultures, ie somatic cells. In contrast, most of the genetic effects of these mutants have been studied in cells of the germ line. To improve the correlation between these 2 levels it was decided to investigate the effects of repair deficient mutants on the mutation induction in somatic cells.

Female larvae of repair deficient mutant strains, heterozygous for an X-linked recessive white eye mutation w/w+ were X-irradiated. The emerging adult flies were scored for (induced) clones of white spots (ommatidia) in the eyes. These spots arise as the result of mutations (point mutation,deletion), complete/partial chromosome loss and recombination events. The number and size of the clones was used to estimate mutation rates.

The frequency of spontaneously occuring small spots is significantly higher in mei-41 females (compared to mei+). This increase is not observed among the larger spots. After X-irradiation, especially the induction of the larger spots is enhanced in mei-41 females. This effect appears stronger after irradiation in anoxic than in oxic conditions. These results differ from those obtained in excision repair deficient (mei-9) females; in these females the spontaneous and induced frequency of both types of spots is increased. In both cases (mei-41 and mei-9) the induced frequency of spots is much higher in these w/w+ heterozygous females than in w+/Y hemizyous males

The last result indicates that recombination events especially are increased after X-irradiation in repair deficient conditions. The fact that this increase in mei-41 females is stronger in anoxic conditions may indicate that in these flies, recombinational events result from the misrepair of X-ray induced base damage.

A series of mutants of Drosophila melanogaster have been reported that are deficient in repair of ultraviolet (UV) induced deoxyribonucleic acid (DNA) damage. Although the mutants alter in general the frequency of X-ray induced genetic damage, no repair defects could be demonstrated yet for X-ray induced DNA damage. Also the detected increases and decreases in the frequencies of X-ray induced genetic damage were relatively small. The direct selection of X-ray hypersensitive mutants was considered but was supposed to be complicated as X-rays are known to induce a wide range of DNA lesions. Instead bleomycin was chosen as the selection agent as it produces primarily DNA strand breaks which are also an important type of lesion induced by X-ray irradiation.

Males flies were mutagenised with exonuclease (ENU) and mated to attached-X females. The F1 males were crossed individually to attached-X females and the F2 cultures were treated with bleomycin. The absence or decreased frequency of males in the F2 indicates a potential bleomycin sensitive mutant.

In total 13807 mutagenised X-chromosomes were screened. Based on the dose of ENU used for the induction and the number of X-linked genes, it can be calculated that (assuming a random induction of mutation) each gene was mutated 4 times. 5 mutants have been isolated that shows a slight hypersensitivity to bleomycin.

The frequency of recovered bleomycin hypersensitive mutants is low compared to that of methyl methanesulphonate (MMS) hypersensitive mutants. As bleomycin induces far less different types of DNA damage than MMS, it is to be expected that mutations at fewer loci will confer bleomycin hypersensitivity. Presumably only a small number of loci are involved in the repair of DNA strand breaks

The mobile element P can be used to clone specific genes. Mutations due to the insertion of a P-element in a gene, will provide a 'tag' which can be used to isolate the deoxyribonucleic acid (DNA) sequences of the mutated gene. To clone DNA repair genes, mutations have to be made in these genes with P-element.

Dysgenic flies are generated in which the mobile element P is transposing at high frequency in germ line cells. Dysgenic males carrying a Pm balancer chromosome are crossed to C(1)DX females. The F2 Pm males are mated individually to mus-201 (excision repair deficient methyl methanesulphonate (MMS) hypersensitive) females for 2 days after which the parental flies are transferred to new culture vials. MMS is added to the first vial. The vials are scored for the presence or absence of Pm flies in the F3. If a P-induced mus-201 is present on the Pm chromosome, only Pm+ flies will survive the MMS treatment.
A total of 27,033 second chromosomes and no P-induced mus-201 mutation were detected.

In Drosophila it is evident that many spontaneous mutations and chromosome aberrations are the result of transposition of mobile elements. Since spontaneous genetic endpoints form a convenient frame of reference for physically induced deoxyribonucleic acid (DNA) damage and chemically induced DNA damage, it is important to study the effect of mutagenic agents on mobile elements and the process of transposition. One of the systems to study such interactions in Drosophila is the transposition of P-elements. However, since there exists considerable heterogeneity among the strains carrying P-elements, these strains have to be characterised in detail.

We studied a number of particular P-strains, MR-strains, that are characterised by the fact that the P-activity in principle is limited to 1 isolated chromosome only. Functional P-elements are 2.9 kb long, including an intact 2.4 kb Acc I fragment.

The results show that the genetic effects in MR-h12/Cy are correlated with 1 complete P-element only, complete P-elements exist without any genetic activity and since the activity of complete P-elements in the same P-strain can vary in activity, the differences in genetic activity of the different MR-strains cannot directly be related to the number of complete elements.

Spontaneous hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations in lymphocytes:
In order to improve our understanding of spontaneous mutants in human lymphocytes, use was made of the cloning method for genetic alterations in the HPRT gene. Mutant frequencies (Mf) were determined in 28 nonsmokers and 23 smokers. Mean Mf values and standard deviations for the 2 groups were 10.0 +/- 10.7E-6 and 11.1 +/- 15.7E-6 respectively. The effect of smoking was not statistically significant. For the pooled data of the 2 groups it could be calculated that Mf values increased significantly with age (range 19 to 60 years) with a rate of 1.8% per year.

A total of 31 mutants from 10 normal nonsmoking donors was used to determine the spectrum of deoxyribonucleic acid (DNA) sequence alterations in the coding region of the HPRT gene. For this purpose messenger ribonucleic acid (mRNA) from expanded HPRT mutant clones was converted into complementary deoxyribonucleic acid (cDNA) which was then used for DNA sequence analysis after polymerase chain reaction (PCR) amplification. In 12 of 31 mutants a single base pair substitution was observed. These were equally divided in transitions and transversions. Furthermore, 5 frame shift mutations and 2 small deletions of 9 and 12 base pairs were found. In 12 of 31 mutants 1 or 2 exons were completely or partially deleted in the HPRT mRNA, probably because of a mutation in a splice acceptor site of the HPRT gene.

HPRT mutant frequencies in patients receiving a low dose of gamma rays:
Blood samples were collected from 13 nuclear medicine patients undergoing a ventriculographic examination of heart function with technetium-99 m. Mean Mf values plus standard deviations for pretreatment and posttreatment samples were 21.2 +/- 19.9E-6 and 11.1 +/- 7.2E-6 respectively. The decrease of 52% in the mutant frequency after the nuclear medicine procedure proved to be statistically significant (probability less than 0.03). This result does not confirm that Seifert et al who concluded that technetium tre
The induction of structural chromosomal changes forms a significant portion of the genetic damage produced by ionizing radiation. For human risk estimates one has to extrapolate from experiments with laboratory animals such as the mouse and, to a limited extent, primates, to the human situation. During 1990 different aspects of translocation induction in spermatogonial stem cells were studied. In the rhesus monkey (Macaca mulatta) the effects of combined treatments with FSH (54 I.U./kg/week) and X-rays (1 Gy) were examined. An insignificant decrease of 30% in the frequency of induced translocations was recorded for follicle-stimulating hormone (FSH) pretreated animals. Comparison of these translocation data with studies on cell killing in the same monkeys show that the ratio between the probabilities that radiation induced basic lesions kill a cell or produce translocations is about 10 to 1. This value of 10 is very similar to that observed for the mouse or calculated on theoretical grounds.

All data so far obtained in the rhesus monkey (dose-effect relationship, dose-rate effect, the effect of radiation quality, etc.) suggest that testicular repopulation after radiation damage in rhesus monkeys is mainly responsible for the observed differences between mouse and monkey. There is also evidence available that the recovery of radiation damage in the rhesus monkey is comparable to that seen in steel (Sl) and dominant spotting (W) mutations in the mouse. Preliminary data obtained by us point to a recovery of translocations from 3 Gy irradiated Wv/+ (viable allele of dominant spotting) and Slcon/Slcon (contrasted allele of steel) male mice which is comparable to the low frequencies observed in the rhesus monkey.


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