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A cosmid library for human chromosome 8 has been constructed from flow-sorted chromosomes in the vector sCos-1. This library is 85% human and has been arrayed into 210 microtiter plates representing four genome equivalents. Cosmids have been isolated with 10 of 11 probes representing nine different loci from chromosome 8.

Fluoresence in situ hybridization (FISH) was used to localize cosmids to regions of human chromosome 10. A total of 301 cosmids were selected randomly from a flow-sorted human chromosome 10 cosmid library constructed from human × hamster cell line 762–8A and arrayed in microtiter storage dishes. Over 70% (211/301) of the cosmids mapped to unique regions of chromosome 10. About 7% (22/301) produced multiple hybridization signals indicative of chimeric clones or sequences repeated at low copy number. Three cosmids (3/301, or 1 %) hybridized to the centromeric regions of chromosome 10 and one or more other human chromosomes. About 19% (59/301) consisted mostly or entirely of hamster DNA inserts, and about 2% (6/301) appeared to be nonrecombinants.

A new strategy is briefly described for employing nucleic acid probes that are directly labeled with fluorochromes in fluorescence in situ hybridization techniques. These probes will permit the detection, quantitation, and high-precision spatial analysis of multiple DNA sequences along a single chromosome using video-enhanced fluorescence microscopy and digital image processing and analysis. Potential advantages of direct labeled DNA probes for fluorescence in situ hybridization far surpass currently available, indirect DNA probe labeling techniques in ease of use, versatility, and increased signal- to-noise ratio.

Chinese hamster cells (line CHO) were examined for cadmium-induced growth-rate perturbations and chromosomal aberrations. The threshold concentration of Cd++ for the induction of chromosomal aberrations in CHO cells cultured in F-10 medium supplemented with 15% newborn calf serum was determined to be 1 × 10–6 M. CHO cells grown in the presence of nontoxic levels of Cd+ + (2×10-7 m) for 12 wk became resistant to toxic levels (2 x 10–6 m) after this exposure. Metaphase cells in these resistant populations contained only background levels of chromosomal aberrations. CHO cells grown in medium supplemented with several types of serum (fetal calf, newborn calf, and human) at concentrations commonly used for in vitro culture (5–20% v/v) or found in mammalian circulatory systems (50% v/v) differed markedly in Cd+ + tolerance. Cells grown in medium containing 1 × 10–6 MCd++ and supplemented with human or newborn calf serum were slightly protected against Cd++ damage at high serum concentrations (30 and 50% v/v) and accumulated approximately 90 µg Cd++/109 cells in 48 h. Cells growing at low or high concentrations of fetal calf serum were completely protected from 1×10-6 m Cd++ and accumulated approximately 12 µg Cd/109 cells in 48 h. The threshold Cd++ concentration for euploid Chinese hamster cells derived from skin, lung, and kidney tissues was determined to be 2 x 10–δ m. Cadmium does not induce elevated sister chromatid exchange levels. These results demonstrate the necessity for standardized protocols for cytogenetic investigations of Cd++ toxicity and may help to explain the discrepancies between studies of chromosome damage in patients with high Cd++ blood levels.

To assess the functional significance of nuclear pore complexes, we have investigated whether the number of pores per nucleus is determined by such factors as the nuclear volume, nuclear surface area, DNA content, or aspects of nuclear activity. Comparisons were made between cell types chosen to permit observation of differences in nuclear pore number as a function of differences in the other qualities measured. The number of nuclear pores was determined by freeze-etching and measurements of nuclear surface and nuclear volume by electron and light microscopy. Pairs of cell strains in culture that contained different numbers of chromosome sets were investigated to examine the relation of pore number to total DNA content. Tetraploid cells of the rat kangaroo (Potorous tridactylus) have almost exactly twice the number of pores found in the parental diploid strain. However, the pore number in diploid grassfrog (Rana pipiens) cells was only 65% greater than in the parental haploid cells. In addition, a polyploid series of nucleated RBC had a 62% pore number increase with each successive increase in ploidy. Diploid cell strains from the canyon mouse (Peromyscus crinitus) and from the cactus mouse (P. eremicus) were compared to test whether a difference reflecting the 36% additional DNA in cells of the latter, associated with extra heterochromatin, existed. Although both were found to have the same number of pores and nuclear surface area, the cells differed in nuclear volume. These observations suggest that the number of nuclear pores is independent of the total amount of nuclear DNA, the nuclear surface area (and, thus, presumably the fraction of DNA that is bound to the nuclear membrane), the nuclear volume, and the size of the genome. Rather, the number of nuclear pores appears to be associated with some aspect of nuclear metabolic activity, e.g., transcriptional capacity or release of products to the cytoplasm. Further evidence for such a view comes from studies of chick embryo erythroblasts. In these, nuclear pore number was found to be lower in association with the decreasing nuclear transcriptional activity and longer generation times that characterize the successive cell divisions leading to the fully differentiated state. The number of pore complexes reconstructed in the last cell cycles declined in a manner consistent with reutilization of previously formed pores in the absence of new pore synthesis. Challenging this interpretation is the increase in pore number at lower metabolic activity when Xenopus laevis cells are grown at different temperatures. The speculation that pore complexes have a longer half-life in cooler grown Xenopus cells could resolve the discrepancy.

Human testis-specific lactate dehydrogenase-C (LDHC) gene-related sequences are located with the LDHA gene on chromosome 11. The LDHB gene is on chromosome 12. Chromosomes 1, 2, 4, 9, and 10 appear to contain LDHA gene-related sequences, whereas the X chromosome and chromosome 13 possess LDHB gene-related sequences.

The two National Laboratories at Livermore and Los Alamos have played a prominent role in the development and application of flow cytometry and sorting to chromosome classification and purification. Both laboratories began to receive numerous requests for specific human chromosomal types purified by flow sorting for gene library construction, but these requests were difficult to satisfy due to time and personnel constraints. The Department of Energy, through its Office of Health and Environmental Research, has a long-standing interest in the human genome in general and in the mutagenic and carcinogenic effects of energy-related environmental pollutants in particular. Hence, it was decided in 1983 to use the flow construct chromosome-specific gene libraries to be made available to the genetic research community. The National Laboratory Gene Library Project was envisioned as a practical way to deal with requests for sorted chromosomes, and also as a way to promote increased understanding of the human genome and the effects of mutagens and carcinogens on it. The strategy for the project was developed with the help of an advisory committee as well as suggestions and advice from many other geneticists. 4 refs., 2 tabs.

Chinese hamster cells (line CHO) were examined for cadmium-induced growth rate perturbations and chromosome aberrations. Experimental protocols were designed to simulate conditions of cellular Cd/sup + +/ exposure both in vivo and in vitro. CHO cells grown in the presence of nontoxic levels of Cd/sup + +/ (2 x 10/sup -7/ M) for 12 wk became resistant to toxic levels (2 x 10/sup -6/ M) after this exposure. Metaphase cells in these resistant populations contained only background levels of chromosome aberrations. This suggests that individuals exposed to constant or intermittent low levels of Cd/sup + +/ may be more resistant to the toxic effects of this metal and may not show chromosome aberrations as readily as nonexposed individuals. CHO cells grown in medium supplemented with several types of serum (fetal calf, newborn calf, and human) at concentrations commonly used for in vitro culture (5 to 20 percent v/v) or found in mammalian circulatory systems (50 percent v/v) differed markedly in Cd/sup + +/ tolerance. Cells grown in medium containing 1 x 10/sup -6/ M Cd/sup + +/ and supplemented with human or newborn calf serum were slightly protected against Cd/sup + +/ damage at high serum concentrations (30 and 50more » percent v/v) and accumulated approximately 90 ..mu..g Cd/sup + +//10/sup 9/ cells in 48 h. Cells growing at low or high concentrations of fetal calf serum were completely protected from 1 x 10/sup -6/ M Cd/sup + +/ and accumulated approximately 12 ..mu..g Cd/sup + +//10/sup 9/ cells in 48 h. These results demonstrate the necessity for standardized protocols for cytogenetic investigations of Cd/sup + +/ toxicity and should help to explain discrepancies between studies of chromosome damage in patients with high blood levels of cadmium.« less

The gene encoding the E1α subunit of branched chain α-ketoacid dehydrogenase was mapped to human chromosome . 19.32P-labeled human Elα cDNA was hybridized with DNA derived from flow-sorted human chromosomes; it hybridized exclusively with that from chromosome 19

The cytogenetic effects of exposure to crude shale oil by either skin painting or intraperitoneal injectional and transplacental exposure of embryos to water derived from surface retort processing of shale oil were analyzed. Exposure to crude shale oils from two sources, Paraho and Occidental, by skin painting had essesntially no effect on the frequency of chromosomal aberrations in bone marrow cells. Intraperitoneal injection of the Paraho crude shale oil increased the frequency of chromosome damage in bone marrow cells at all three doses tested (0.5 ml, 1.0 ml, and 2.0 ml/kg). Metaphase analysis of cells from embryos at day 12 gestation from females that had been exposed to 1% Paraho retort water ad libitum from day 1 of gestation indicated that clastogenic compounds present in this water can cross the placenta and induce chromosomal damage in embryonic cells.