Conservation of chromosome 1 in turtles over 66 million years.

Fluorescence in situ hybridization of a whole chromosome 1-specific probe from the yellow-bellied slider turtle (Trachemys scripta) to cells from four other species of turtle ranging from a desert tortoise to a loggerhead sea turtle resulted in specific and exclusive hybridization to chromosome 1 in all five species. Previous observations of conservation in the giemsa banding pattern and chromosome morphology and number among turtles are thus extended to the DNA sequence level, revealing a cytogenetic stability of chromosome 1 in these turtles during the past 66-144 million years. This contrasts with the situation for various hominoid species where, in many instances, extensive chromosomal rearrangements have been reported in one third of that time period. Our probe, which was prepared by microdissecting whole chromosomes from embryonic T. scripta fibroblasts and amplifying using DOP-PCR, is the first report of a whole-chromosome FISH probe for any reptile.

Chromosome translocations in turtles: a biomarker in a sentinel animal for ecological dosimetry.

Nonhuman organisms are being exposed to ionizing radiations at radionuclide-contaminated sites around the world. Direct methods are seldom available for measuring biologically relevant doses received by these organisms. Here we extend biological dosimetry techniques, which are much better developed for humans and a few other mammalian species, to a nonmammalian species. Turtles were chosen because a long-lived animal would best serve the need for low-level, chronic exposure conditions. We chose the yellow-bellied slider turtle (Trachemys scripta), which is known to have a maximum life span of at least 22 years. As reported elsewhere, we first isolated an embryonic fibroblast cell line and constructed whole-chromosome-specific DNA libraries for chromosome 1 by microdissection and PCR. A FISH painting probe was prepared and used to establish a dose-response curve for ionizing radiation-induced chromosome interchange aberrations in turtle fibroblasts. This was compared to the dose response for human fibroblasts treated under similar conditions in our laboratory. With respect to induction of chromosome interchange aberrations, human fibroblasts were approximately 1.7 times more sensitive than the T. scripta fibroblasts. To the extent that symmetrical interchanges are persistent over long periods, this approach could eventually provide a measure of the integrated lifetime dose these organisms receive from radionuclides in their environment and give a measure of the extent of relevant genetic damage over that time.

Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.

An ionizing radiation-sensitive CHO mutant cell line: irs-20. IV. Genetic complementation, V(D)J recombination and the scid phenotype.

The genetic defect responsible for hypersensitivity of Chinese hamster ovary (CHO) irs-20 cells to ionizing radiation was found to be recessive in nature and could be complemented to produce wild-type radiosensitivity in irs-20/human hybrids. The radiosensitivities of six hybrid clones were determined based on their colony-forming ability under continuous irradiation at 6 cGy/h. A parallel cytogenetic analysis revealed a concordance between the presence or absence of human chromosome 8 and the resistant or sensitive phenotype. Confirming evidence was obtained using human chromosome 8-specific PCR primers. Positive amplification was obtained in hybrids with wild-type radiosensitivity, while no amplification was obtained in sensitive hybrids. Complementation analysis between radiosensitive CHO irs-20 and murine scid cell lines was carried out to determine whether the defects leading to their ionizing radiation hypersensitivity could be corrected by genetic complementation in the hybrids. Complementation did not occur. A transient V(D)J recombination assay after the introduction of the RAG1 and RAG2 genes indicated that the V(D)J recombination ability of the CHO irs-20 cells was about 10% of that for the CHO wild-type cells for signal join formation with an 80% joining fidelity and only 3% of the parental level for coding join formation. These data show that murine scid and irs-20 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination.

Application of 5-bromo-2'deoxyuridine as a label for in situ hybridization in chromosome microdissection and painting, and 3' OH DNA end labeling for apoptosis.

We have utilized 5-bromo-2'deoxyuridine (BrdU) substituted DNA as a probe for a number of applications including, principally, for chromosome painting by fluorescence in situ hybridization (FISH) but also for DNA end-labeling to detect apoptotic cell death and for filter hybridization. Br-dUTP was used as a substitute for biotin or digoxigenin-dUTP in probe labeling techniques, such as random priming, nick translation, end-labeling or PCR. An especially useful application is that it may be incorporated into probe DNA while cells or plasmids in bacteria are growing in the presence of BrdU. This can be particularly advantageous when large quantities of probe are needed, since the cost per mole of digoxigenin-dUTP or biotin-dUTP is nearly 1000 times that of Br-dUTP. Also, if probe is prepared by growth in BrdU, the difference in cost to prepare equal quantities of labeled DNA is more than 10,000 times greater for biotin-dUTP.

The presence of DNA breaks and the formation of chromatid aberrations after incorporation of 125IdUrd may be necessary but are not sufficient to block cell cycle progression in G2 phase.

Cell progression into mitosis and chromatid aberration frequencies were compared in two Chinese hamster ovary (CHO) cell lines after incorporation of 125IdUrd. Asynchronous, exponentially growing populations of CHO K1 and the DNA repair-deficient, radiation-sensitive CHO irs-20 cells were compared after a 10-min exposure to 14.8 kBq/ml 125IdUrd. Essentially no differences were seen for either end point between the cells of the two cell lines. As the cells in S phase at the time of labeling entered the mitotic cell selection window, the number of mitotic cells of each cell line declined to approximately 60% of the respective unlabeled control. Chromosome analysis of the mitotically selected cells indicated an 125I decay-dependent increase in the number of chromatid aberrations in cells of both cell lines. The appearance of aberrations together with the known rates of production and rejoining of DNA double-strand breaks show that cells are able to progress through G2 phase and into mitosis in the presence of such breaks. The data suggest that DNA damage may be necessary, but is not sufficient to cause a radiation-induced blockade of cell progression through G2 phase.

Comparison of gamma-ray-induced chromosome ring and inversion frequencies.

A method was used to detect chromosome inversions as apparent or false sister chromatid exchanges (SCEs) in the first mitosis after gamma irradiation of human G0 cells. Dose-response relationships for small inversions have not been measured and reported previously, but it has been assumed that these are induced with a frequency equal to that of their easily measured asymmetrical counterpart, the interstitial deletion. Our experiments confirm this expectation. The results also demonstrate, as others have suggested, that in protocols where SCEs have been reported in the first postirradiation mitosis after incorporation of BrdU in the previous cell cycle, the X- or gamma-ray treatment of G0- or G1-phase cells produces virtually no true SCEs.

Breakage of human chromosomes 4, 19 and Y in G0 cells immediately after exposure to gamma-rays.

One of several possible explanations for the fact that the initial number of ionizing radiation-induced DNA double-strand breaks (dsbs) per cell per Gy appears to be approximately 5-10 times greater than the number of excess fragments produced in prematurely condensed chromosomes (PCCs) is that dsbs in DNA tightly associated with protein, such as might be the case for heterochromatin, are held together and not expressed as discontinuities in the PCC assay. To test this idea the breakage frequencies in chromosome 4, 19 and the heterochromatic and more euchromatic portions of the Y chromosome of non-cycling human fibroblasts were measured and compared over a wide range of doses by inducing PCC immediately after irradiation with Cs-137 gamma-rays. Even for doses up to 400 Gy no evidence was seen that Y heterochromatin or large fragments of it remained unbroken. The only significant deviation from the expected initial breakage frequency per Gy per unit length of chromosome, in the dose range where a genome average for all chromosomes could be obtained, was that observed for the euchromatic portion of the Y chromosome (Y p + cen-->q11.2) where breakage was nearly twice that expected. There was no correlation between breakage per unit length and the (A + T)/(G + C) ratio for these chromosomes or regions.

Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.

Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences.