Molecular mapping within the mouse albino-deletion complex.

Induced germ-line deletion mutations in the mouse provide a malleable experimental system for in-depth molecular and functional analysis of large segments of the mammalian genome. To obtain an initial bank of molecular probes for the region of mouse chromosome 7 associated with the albino-deletion complex, random anonymous DNA clones, derived from a library constructed from flow-sorted chromosomes, were screened on DNAs from Mus musculus-Mus spretus F1 hybrids carrying large, multilocus, lethal albino deletions. Clones falling within a given deletion interval can easily be recognized because hybridization bands that represent restriction fragment length polymorphisms specific for the mutant (deleted) chromosome inherited from the M. musculus parent will be absent. Among 72 informative clones used as probes, one, which defines the locus D7OR1, mapped within two deletions that are 6-11 centimorgans in length. Submapping of this anonymous clone across a panel of 27 smaller deletions localized D7OR1 distal to a chromosomal subregion important for survival of the preimplantation embryo, proximal to globin [beta-chain (Hbb)], and near the shaker-1 (sh-1) locus. The results of these deletion-mapping experiments were also confirmed by standard three-point linkage analysis. This strategy for selection and rapid mapping of anonymous DNA probes to chromosomal segments corresponding to germ-line deletion mutations should contribute to the generation of more detailed physical and functional maps of genomic regions associated with mutant developmental phenotypes.

Cell cycle-dependent modulation of O6-methylguanine-DNA methyltransferase in C3H/10T1/2 cells.

O6-methylguanine-DNA methyltransferase (MGMT) was measured in partially synchronized cultures of C3H/10T1/2 mouse embryo cells as a function of cell cycle. The degree of synchrony and progression of the cell cycle were monitored by flow cytometry. The MGMT level was significantly reduced prior to the onset of S-phase. This reduction was concomitant with the inhibition of in vivo repair of O6-methylguanine in DNA of S-phase cells as observed earlier. The recovery of the MGMT level paralleled the progression of synchronized cells into G2. S-phase cells purified by cell sorting contained approximately 15% of the MGMT present in G0 or early G1 cells. A comparison of the in vivo repair of O6-methylguanine and MGMT levels suggests that the lack of repair of O6-methylguanine in DNA of the mouse embryo cells is due only in part to a temporal loss of MGMT.

Use of multiparameter analysis to quantitate hematological damage from exposure to a chemical (ethylene oxide).

This study was designed to test the value of a multiparameter approach in evaluating perturbations in bone marrow and peripheral blood elements of mice exposed to ethylene oxide (EtO). Mice exposed to 255 ppm EtO for 5 h/d were removed for analysis after 1, 2, 8, and 14 d (sequential exposure) and 4, 6, 8, and 10 wk (5 d/wk). Prior to sacrifice, blood was removed from the orbital sinus for blood cell counts, hemoglobin determination, and hematocrit. A blood film was made for differential leukocyte counts. Bone marrow was flushed from femurs and tibias and counted, and aliquot were used for stem-cell assay (CFU-S) or flow cytometry (FCM) analysis. One aliquot of marrow was stained with propidium iodide for cell-cycle analysis and another was reacted with fluorescein-conjugated monoclonal antibody for B-cell analysis. The preparations were analyzed for forward and 90 degrees scatter and fluorescence on an Ortho 50H cytofluorograph. Perturbations of peripheral leukocytes occurred after one exposure. After multiple exposures, hematocrit, red-cell number, and hemoglobin were generally depressed, with transient compensatory bursts, and bone marrow cellularity and CFU-S were below normal. However, white-cell numbers fluctuated dramatically during the exposure period. There was a shift in differential toward granulocytes, at times resulting in severely depressed numbers of lymphocytes in the peripheral blood. The FCM analysis showed an early depletion of granulocytes in the bone marrow followed by replacement and a relative lymphocyte deficit, especially pronounced at 10 wk. The B-cell changes reflected general lymphocyte perturbations. Shifts in numbers of cells in S and G/M were observed, consistent with a moderate bone marrow response to cell loss.

Differential sensitivity of a mouse myeloid leukemia cell line and normal mouse bone marrow cells to X-ray-induced chromosome aberrations.

Cell line ML-1 was established from a myelogenous leukemia of an RFM mouse. The ML-1 cells and in vitro normal mouse bone marrow cells were analyzed to determine if there was a differential sensitivity to X-ray-induced chromosome aberrations in G1 cells and/or differences in postirradiation cell cycle progression. Cells identified as being in G1 at the time of irradiation by their staining pattern after replication in 5-bromodeoxyuridine were analyzed for all types of chromosomal aberrations following X-ray doses of 0.5, 1.0, 1.5, and 2.0 Gy. ML-1 cells showed a greater sensitivity to the induction of both chromosome-type aberrations (dicentrics and terminal deletions) and chromatid-type aberrations (exchanges and deletions) compared to normal mouse bone marrow cells, which only contained chromosome-type aberrations. The presence of chromatid-type aberrations in the ML-1 cells and not normal bone marrow cells suggested a differential progression through the cell cycle for the two cell types after irradiation. Mitotic index and flow cytometric analyses were performed and showed that both cell types have a delay in progression from G2 into mitosis, but only the normal mouse bone marrow cells have a delay in progression from G1 into S, as well as delayed progression through the S phase following X-irradiation. These results indicate that the ML-1 leukemia cells have an increased radiosensitivity. This may be due to a defect in their ability to respond to DNA damage as evidenced by their lack of a G1- and S-phase delay which allows normal cells an increased time to repair DNA damage before replication. These same characteristics have been observed in ataxia telangiectasia cells and may well represent a general feature of cells with increased radiosensitivity.

Application of flow cytometry to detection and characterization of Legionella spp.

Flow cytometry, using fluorescein-bound specific antibodies and propidium iodide, was shown to be effective in detecting Legionella spp. in cooling tower waters. The procedure was quicker and less labor intensive than fluorescent microscopy. The use of these procedures also identified qualitative differences, perhaps related to infectivity, in Legionella populations.

The use of projections for dimensionality reduction of flow cytometric data.

In order to visualize multiparameter flow cytometric measurements it is desirable to reduce the dimensionality of the data to two while preserving important features of each data pattern. We describe a method that projects two-parameter data onto a straight line that forms an angle theta with one of the original parameter axis. The angle is selected interactively or by principal component analysis. The procedure is applied on-line or off-line to pairs of parameters of the original data set that has been collected in LIST mode.

Monoclonal antibodies directed against rat tracheal epithelial cells transformed in vitro.

Cloned hybridoma cell lines were obtained by fusion of murine myeloma cells with spleen lymphocytes of either F344 rats or BALB/c mice immunized against the malignant F344 tracheal cell line, 2-10-1. Monoclonal antibodies were selected for their ability to bind to the immunizing cell line and not to normal tracheal epithelial cells. Results from quantitative binding assays indicated that such monoclonal antibodies recognized six epitopes. Each epitope was detected on four other malignant tracheal epithelial cell lines, and was also expressed during early preneoplastic cell passages (i.e. before the cells acquired the ability to produce carcinomas in vivo). Quantitative differences in epitope expression between non-tumorigenic and tumorigenic passages of individual cell lines could not be detected for five of the epitope groups. However, two monoclonal antibodies, recognizing the same epitope, did show quantitative binding differences between non-tumorigenic and tumorigenic cell passages on three of the five cell lines tested. Our results show that carcinogen-altered tracheal cell populations can be distinguished from non-altered cells (by our current assay methods) by use of monoclonal antibodies. The suggest that the antigen expression is an early event associated with the transformation of rat tracheal epithelial cells in culture.

The randomization test applied to flow cytometric histograms.

The randomization test is used to test the hypothesis that two groups of flow cytometric histograms consist of samples from the same probability density function. The hypothesis is tested channel-by-channel. This non-parametric method does not require any assumptions as to the probability density functions involved and is therefore applicable to histograms from many different biological systems. Moreover, it allows for very few histograms per group so that the hypothesis can be tested on the basis of a small number of experiments. The procedure is implemented in PASCAL on a mini computer which is connected to a flow cytometer.

Parametric analysis of histograms measured in flow cytometry.

Flow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.

Immunofluorescent analysis of murine leukemia virus-infected cells by flow microfluorometry.

Flow microfluorometric assay with a Cytofluorograf model 4801 in combination with immunofluorescence was applied to the quantitative assay of cells exogenously infected with mouse leukemia viruses and to the chemical induction of virus in AKR cells. The Cytofluorograf provides sensitivity equal to the visual method and is capable of assaying up to 5000 cells/sec with specificity equivalent to that of the direct visual immunofluorescence assay, thereby providing a large, statistically valid sampling in a fraction of the time required by visual counting. Flow microfluorometry also provides a method of quantitatively resolving fluorescent cell populations on the basis of relative size and degree of fluorescence.

Effects of a warm gutta-percha technique on the lateral periodontium.

The microanatomy of lateral periodontal tissues was examined after root canal obturation by a warm gutta-percha technique had been accomplished. Inflammatory responses were slight and of shot duration. The use of hot instruments for the condensation of the filling material did not appear to endanger the integrity of the lateral periodontium.

Effect of cell physiological state on infection by rat virus.

Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with (3)H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated (3)H-thymidine at the time of infection.