A novel diagnostic method based on DNA strand displacement.

A novel approach for the detection of specific DNA or RNA sequences based on branch migration and DNA strand displacement is described. A partially double-stranded probe complex is prepared with a detectable label on one of the two strands and incubated with analyte molecules under hybridization conditions. The analyte molecules hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labelled DNA strand from the complex. Initial characterization of the assay indicates that both qualitative and quantitative information about analytes present in a sample can be obtained. The strand displacement assay is more sensitive to sequence alterations in the analyte than is a hybridization assay and can be promoted by rec A protein at 37 degrees C. Finally, a method for preparing probe complexes by cloning in a single-stranded DNA vector is also described.

Strand displacement applied to assays with nucleic acid probes.

This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.

SCE induction is uncoupled from mutation induction in mammalian cells following exposure to ethylnitrosourea (ENU).

It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that "S-dependent" processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE-inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log-phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU-induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined.

Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro: result of an IAEA-coordinated programme.

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored. It is concluded that using controlled experimentals conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.

Mammalian in vivo and in vitro cytogenetic assays: a report of the U.S. EPA's gene-tox program.

This report presents an assessment made by the U.S. Environmental Protection Agency Gene-Tox Program's Work Group on mammalian cytogenetics of the clastogenic effects of chemicals in in vivo and in vitro mammalian cell assays. This assessment is based on information provided by the Environmental Mutagen Information Center, Oak Ridge National Laboratory, with the proviso that the experimental protocol used in these papers was adjudged to be acceptable by standards outlined by the Work Group. Some data were accepted as "qualitative only" because the protocol used was fairly close to that proposed as suitable. Using these criteria, 177 papers were selected for review. 6 assays were reviewed: bone marrow (32 papers, 31 chemicals), spermatogonial (10 papers, 10 chemicals), spermatocyte (25 papers, 25 chemicals), oocyte or early embryo (18 papers, 19 chemicals), in vitro cell culture (30 papers, 66 chemicals), and leukocyte (66 papers, 53 chemicals). Each assay was considered separately, and comparisons were then made between them for their similarities or differences in producing a positive or negative clastogenic effect of a particular chemical or chemical class. A large proportion of the available cytogenetic data was not suitable for inclusion in the final data base because of poor experimental design or unsatisfactory reporting of the information. It was not possible to recommend any one assay for determining potential clastogenicity because each had its own particular advantages and limitations and provided unique information. For demonstrating in vivo effects, the bone-marrow assay is probably the simplest and most economical. If only in vitro exposures were considered, leukocytes or cultured mammalian cell lines would be suitable. However, there are advantages to using leukocytes because they are a synchronous population, at least through their cell division, and because of the ready availability of human cells. In general, there was good agreement between clastogenicity and carcinogenicity.

Presumptive evidence for the absence of functional germ cell chimerism in the marmoset.

Presumptive evidence for functional germ cell chimerism in the marmoset was evaluted by calculating the offspring sex ratio of young derived from animals in which the blood chimerism status was known. In the four possible mating combinations involving chimeric and non-chimeric marmosets, there was no apparent deviation from the anticipated 1:1 sex ratio. Analysis of 2,500 primary spermatocytes from ten known blood chimeras failed to reveal a single cell in which an unequivocal XX bivalent could be identified. The combined breeding and cytogenetic data offer presumptive evidence against functional germ cell chimerism in the marmoset.

X-ray stage sensitivity of mouse oocytes and its bearing on dose-response curves.

A detailed cytogenetic study of maturing mouse oocyte radiosensitivity was performed. Oocytes were collected at various intervals ranging from 1.5 days to 28.5 days after irradiation with 50, 100, 200, and 300 R of acute X-rays. The observed sensitivity to chromatid aberration induction varied greatly over this time span. Sensitivity was lowest at the shortest time interval before ovulation and gradually increased up to 9.5 days; it then remained constant until insufficient numbers of oocytes could be collected. The data were analyzed in three ways. First, the data from all time intervals at each dose were pooled; second the data from the least sensitive time intervals, at each dose, were pooled, and third, the data from the period of uniform sensitivity, at each dose, were pooled. Dose-response regression analyses were done on these pooled data and the best fits obtained were to the models Y = a + bD + cD2 and Y = a + cD2 for both deletions and interchanges. This result is interpreted as indicating that the aberrations result from a predominantly two-track process. The cytogenetic data were compared to specific-locus mutation induction data in comparable oocyte stages, and qualitative similarity in dose-response characteristics were observed. This similarity is interpreted to mean that both events result from the same mechanism, and that the large dose-rate effect, observed for both events, is a reflection of the two-track component in the dose-response curves.

Studies on chemically induced dominant lethality. III. Cytogenetic analyses of TEM-effects on maturing dictyate mouse oocytes.

A comparative cytogenetic study was done on metaphase-I oocytes and first cleavage mitoses following treatment of young mature female mice with triethylenemelamine (TEM). The ova were collected at intervals ranging from 12 h to 10.5 days after single intraperitoneal injection of TEM. Very few structural aberrations were seen in the metaphase-I cells after TEM treatment. There was, however, a very clear effect on the female genome when first cleavage mitoses were analyzed. The aberrations seen were principally chromatid deletions and interchanges, and their frequency varied with dose and the time between treatment and mating.

The production of chromosome aberrations in various mammalian cells by triethylenemelamine.

The cytogenetic effects of triethylenemelamine (TEM) were studied using five different mammalian tissues. Treatments of 0.1 and 0.2 mg/kg TEM on differentiating mouse spermatogonia and bone marrow cells showed no significant differences in the frequency of chromosomal aberrations produced in these two tissues. At higher doses, however, the sensitivites of the two tissues appear to be different. The frequency of aberrations varies with time after treatment, with the greatest amount occurring at the latter fixation times. Results of an experiment on primary spermatocytes indicated a correlation between the frequency of chromosome aberrations and DNA replication. Human peripheral leukocytes were utilized in an attempt to clarify the cell-stage specificity of TEM-induced chromosome aberrations. Cultures were treated with TEM prior to PHA stimulation (G0), as well as various time intervals after stimulation (late G,1 S, and G2). The most sensitive stages of the cell cycle to aberration induction were later G1 and S, with chromatid aberrations the predominant type. A very low yield of chromosome damage was observed with the G0 and G1 treated stages. The experiments described tend to support the view that TEM is most effective at inducing aberrations when an intervening round of DNA replication has occurred.

Radiation-induced chromosome aberrations in somatic and germ cells of the male marmoset.

The induction of chromosome aberrations by low LET radiations was studied in peripheral lymphocytes and spermatogonial stem cells of the male marmoset. The data showed that there was not significant difference in the sensitivity of the lymphocytes whether they were irradiated in vitro or in vivo, but the frequency of heritable translocations recovered in the primary spermatocytes were considerably lower than that calculated to occur in the lymphocytes. The data are used to make estimates of human genetic risk from radiation based on limited interspecific comparisons.

X-ray-induced chromosome aberrations in mouse dictyate oocytes. II. Fractionation and dose rate effects.

Slit-dose experiments were done on maturing dictyate oocytes to determine if the magnitude of the first dose influenced the "rejoining time" of radiation-induced chromosomal lesions. A total dose of 400r was split into various combinations with varying fractionation intervals. The data derived from analyzing interchanges indicate that there is no difference in the rejoining time whether the first dose was 100, 200, or 300r. It thus appears that the radiation dose in the ranges studied does not significantly alter the rate of repair of the chromosomal lesions. This conclusion is contrary to that which has been propounded to explain the nonlinear dose curves obtained for specific locus mutations. Chronic 60Co gamma-ray exposures were given to female mice over an 8-day period. The exposures were delivered during the period of peak sensitivity, i.e., 8-16 days prior to ovulation. The doses given were 117, 240, 348, and 483r. The aberration yields observed were dramatically lower than for comparable doses of acute X rays even when the RBE of gamma rays compared with X rays is taken into account. The large drop in yields at the low dose rates is interpreted as resulting from a large two-track component in the acute curve, and as being independent of effects on repair systems.

Practical evaluation of mutagenicity data in mammals for estimating human risk.

Chrosomal aberrations are known to constitute a significant portion of the genetic risk from practically all mutagenic agents. These effects are diverse in both nature and in the stage of the germ cells' life cycle at which they are produced. Arguments are made pointing out many of the problem areas in our understanding the significance of chromosomal aberrations in relation to genetic risk. Data are summarized that offer explanations as to (1) why so few chromosomal effects are recovered after treating spermatogonial stem cells, (2) how chromosome damage and dominant lethality are correlated when postmeiotic germ-cell stages are treated with MMS, and (3) why so few reciprocal translocations are recovered after irradiation of oocytes.

Studies on chemically induced dominant lethality. II. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes.

Young adult female mice were injected intravenously with either 50- or 100- mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, but at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMS-induced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike later spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.

X-Ray-induced translocations in spermatoginia. II. Fractionation in mice.

The dose-response curve for reciprocal translocations induced by X-rays in spermatogonial stem cells, and observed in primary spermatocytes of mice, is "hump-shaped", with a maximum yield at about 600 R. To test the hypothesis that the decrease in yield with increasing dose above 600 R is a consequence of the different sensitivities of cells in different stages of the cell cycle to both cell killing and chromosome aberration induction, several fractionation experiments were carried out. A total dose of 2800 R was given in repeated doses of 400 R, separated by 8-week intervals. The yield of translocations is that expected for additivity; for example, the yield at 1600 R is approximately equal to that for four separate 400-R doses. When a total dose (500 R) which gives a translocation yield on the ascending part of the dose-response curve is given as two equal fractions separated by intervals of 30, 90, or 150 min, the translocation yield decreases with increasing interval. However, when a total dose (1000 R) which would give a translocation yield on the descending part of the dose-response curve is given in two equal fractions separated by intervals of from 30 min to 6 weeks, the response is different; the translocation yield increases with intervals up to 18 h, then decreases with intervals up to 4 weeks, and finally increases again to a yield equal to additivity with an interval of 6 weeks. These changes in translocation yield with changes in interval between the two doses are explained in terms of the differential sensitivity of cells to killing and aberration induction in the different phases of the cell cycle, and by assuming that the cells surviving the first dose and repopulating the testis have different cycle characteristics from normal cells.

X-ray-induced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships.

Structural chromosome aberrations were analyzed in superovulated metaphase-I oocytes of the mouse, Mus musculus, at various times after a single acute dose of 200 R of X-rays. The aberrations seen were of the chromatid type, i.e., chromatid interchanges, isochromatid deletions and chromatid deletions. The aberration frequency was low during the interval 24 h to 5 days between irradiation and ovulation; peak frequency was reached when irradiation was given 14 days prior to ovulation. A dose-response study was made 14 days prior to ovulation at doses of 50, 100, 200, 300 and 400 R. A curve of these data indicated that a significant two-track component was present for both interchanges and deletions. Centromere staining revealed that symmetrical and asymmetrical interchanges occurred at approximately equal frequency and also that the asymmetrical equivalent of crossing-over was induced at a measurable frequency.