Cytogenetic validation of DS02R1-estimated dose for atomic bomb survivors in Hiroshima and Nagasaki with FISH.

INTRODUCTION: For Hiroshima and Nagasaki survivors, it has not been possible to calculate individual doses from the cytogenetic data and compare them with the physically estimated doses. This is because the cytogenetic studies used solid Giemsa staining which only provides the percent of cells bearing at least one stable-type aberration (most of the unstable-type aberrations had already disappeared), and a gamma-ray dose plus a 10-times neutron dose was used to integrate the data for both cities. OBJECTIVES: To compare the FISH-derived gamma-ray dose with the DS02R1-derived gamma-ray dose after correcting for a contribution of the neutron dose. It was also an attempt to determine if the frequency of stable-type aberrations had remained unchanged after the exposure. METHODS: Stable exchange-type aberration data was obtained using the 2-color FISH method from 1,868 atomic bomb survivors in Hiroshima and Nagasaki. The collected frequency was first extended to a genome-equivalent frequency. Then, by using known induction rates of exchange-type aberrations in vitro caused by neutrons and gamma-rays, respectively, and the mean relationship between the neutron and gamma-ray doses in the DS02R1 estimates for the survivors, the gamma-ray effect was estimated from the total yield of translocations. RESULTS: It was found that over 95% of individual cytogenetic gamma-ray doses fell within the expected range of plus/minus about 1 Gy from the DS02R1 dose and the mean slope for the linear regression was 0.98, which reassures us of the validity of the DS02R1 study. CONCLUSIONS: The present results demonstrate the validity of the individual DS02R1 doses, and that the frequency of stable-type aberrations in blood lymphocytes did not decay over the years, and thus is useful for retrospective dose evaluations of exposures which took place in the distant past.

Chromosome aberrations do not persist in the lymphocytes or bone marrow cells of mice irradiated in utero or soon after birth.

Mice were exposed at various ages to 1 Gy or 2 Gy of X rays, and translocation frequencies in peripheral blood T cells, spleen cells, and bone marrow cells were determined with FISH painting of chromosomes 1 and 3 when the animals were 20 weeks old. It was found that the mean translocation frequencies were very low (< or =0.8%) in mice exposed in the fetal or early postnatal stages. However, with the increase in animal age at the time of irradiation, the frequency observed at 20 weeks old became progressively higher then reached a plateau (about 5%) when mice were irradiated when > or =6 weeks old. A major role of p53 (Trp53)-dependent apoptosis for elimination of aberrant cells was not suggested because irradiated fetuses, regardless of the p53 gene status, showed low translocation frequencies (1.8% in p53(-/-) mice and 1.4% in p53(+/-) mice) compared to the frequency in the p53(-/-) mother (7.4%). In contrast, various types of aberrations were seen in spleen and liver cells when neonates were examined shortly after irradiation, similar to what was observed in bone marrow cells after irradiation in adults. We interpreted the results as indicating that fetal cells are generally sensitive to induction of chromosome aberrations but that the aberrant cells do not persist because fetal stem cells tend to be free of aberrations and their progeny replace the pre-existing cell populations during the postnatal growth of the animals.

Clonally expanded T-cell populations in atomic bomb survivors do not show excess levels of chromosome instability.

Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.

Cefoselis, a beta-lactam antibiotic, easily penetrates the blood-brain barrier and causes seizure independently by glutamate release.

Cefoselis is a widely used beta-lactam antibiotic, but occasionally induces seizures and convulsion in elder and renal failure patients. However, beta-lactams are known not to pass through the blood-brain barrier (BBB). In this study, we examined the BBB penetration of cefoselis in normal and renal failure rats by means of brain microdialysis. Cefoselis was dose-dependently appeared in brain extracellular fluid in proportion to its blood level. The elimination constant from brain extracellular fluid (apparent) was slightly lower than that from blood. These results indicated that cefoselis might penetrate the BBB or be discharged by a certain transport system. In contrast to the result of cefoselis, cefazolin, a leading drug of cephalosporins, could not be detected in the brain extracellular fluid after an intravenous injection. In renal dysfunction rats, the elimination half-lives of cefoselis from both blood and brain were extensively prolonged. This would be one of responsible factors inducing seizures seen in patients. However, the additional factor, such as decrease in brain function related to aging, would be involved in seizures in patient received cefoselis, because an extremely high dose was required to induce seizures even in renal failure rats. A local administration of cefoselis into the hippocampus through the microdialysis probe caused a striking elevation of extracellular glutamate, with a minimum increase in gamma-aminobutyric acid (GABA). However, a systematic cefoselis administration via the tail vein did not elevate extracellular glutamate and GABA concentrations in the hippocampus of renal failure rats that exhibited marked seizures. These results suggested that not the stimulation of glutamate release, but the blockade of GABA receptors might be responsible for the seizure induced by cefoselis.

Human fetuses do not register chromosome damage inflicted by radiation exposure in lymphoid precursor cells except for a small but significant effect at low doses.

Human fetuses are thought to be highly sensitive to radiation exposure because diagnostic low-dose X rays have been suggested to increase the risk of childhood leukemia. However, animal studies generally have not demonstrated a high radiosensitivity of fetuses, and the underlying causes for the discrepancy remain unidentified. We examined atomic bomb survivors exposed in utero for translocation frequencies in blood lymphocytes at 40 years of age. Contrary to our expectation of a greater radiosensitivity in fetuses than in adults, the frequency did not increase with dose except for a small increase (less than 1%) at doses below 0.1 Sv, which was statistically significant. We interpret the results as indicating that fetal lymphoid precursor cells comprise two subpopulations. One is small in number, sensitive to the induction of both translocations and cell killing, but rapidly diminishing above 50 mSv. The other is the major fraction but is insensitive to registering damage expressed as chromosome aberrations. Our results provide a biological basis for resolving the long-standing controversy that a substantial risk of childhood leukemia is implicated in human fetuses exposed to low-dose X rays whereas animal studies involving mainly high-dose exposures generally do not confirm it.

Prediction of clonal chromosome aberration frequency in human blood lymphocytes.

We recently conducted a large-scale screening for clonal aberrations among atomic bomb survivors and proposed a model for the gross clonal composition of blood lymphocytes. Here we show an application of the model indicating that the number, m,of clones detectable by cytogenetic methods in an individual is predictable by the equation m= (1.8 + 6.4FG) x FP x n/500, where FG represents the estimated translocation frequency in the 46 chromosome set, FP is the observed translocation frequency with FISH or other methods, and nis the number of cells examined. Application of the equation to the results of seven other reports gave close agreement between the observed and calculated numbers of clones. Since the model assumes that clonal expansion is ubiquitous, and any translocation can be the constituent of a clone detectable by cytogenetic means, the vast majority of observed clonal expansions of these somatic cells are likely the result of random-hit events that are not detrimental to human health. Furthermore, since our model can predict the majority of clonal aberrations among Chernobyl workers who were examined 5-6 years after irradiation, clonal expansion seems to occur primarily within a few years after exposure to radiation, most likely being coupled with the process of recovery from radiation-induced injury in the lymphoid and hematopoietic systems.

Estimating the number of hematopoietic or lymphoid stem cells giving rise to clonal chromosome aberrations in blood T lymphocytes.

Quantifying the proliferative capacity of long-term hematopoietic stem cells in humans is important for bone marrow transplantation and gene therapy. Obtaining appropriate data is difficult, however, because the experimental tools are limited. We hypothesized that tracking clonal descendants originating from hematopoietic stem cells would be possible if we used clonal chromosome aberrations as unique tags of individual hematopoietic stem cells in vivo. Using FISH, we screened 500 blood T lymphocytes from each of 513 atomic bomb survivors and detected 96 clones composed of at least three cells with identical aberrations. The number of clones was inversely related to their population size, which we interpreted to mean that the progenitor cells were heterogeneous in the number of progeny that they could produce. The absolute number of progenitor cells contributing to the formation of the observed clones was estimated as about two in an unexposed individual. Further, scrutiny of ten clones revealed that lymphocyte clones could originate roughly equally from hematopoietic stem cells or from mature T lymphocytes, thereby suggesting that the estimated two progenitor cells are shared as one hematopoietic stem cell and one mature T cell. Our model predicts that one out of ten people bears a non- aberrant clone comprising >10% of the total lymphocytes, which indicates that clonal expansions are common and probably are not health-threatening.

Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats.

The herbicide paraquat is an environmental factor that could be involved in the etiology of Parkinson's disease. We have previously shown that paraquat penetrates through the blood-brain barrier and is taken up by neural cells. In this study, we examined the in vivo toxic mechanism of paraquat to dopamine neurons. GBR-12909, a selective dopamine transporter inhibitor, reduced paraquat uptake into the striatal tissue including dopaminergic terminals. The subchronic treatment with systemic paraquat significantly decreased brain dopamine content in the striatum and slightly in the midbrain and cortex, and was accompanied by the diminished level of its acidic metabolites in rats. When paraquat was administered through a microdialysis probe, a transitory increase in the extracellular levels of glutamate, followed by long-lasting elevations of the extracellular levels of NO(x)(-) (NO(2)(-) plus NO(3)(-)) and dopamine were detected in the striatum of freely moving rats. This dopamine overflow lasted for more than 24 h after the paraquat treatment. Dopamine overflow was inhibited by N(G)-nitro-L-arginine methyl ester, dizocilpine, 6,7-dinitroquinoxaline-2,3-dione and L-deprenyl. The toxic mechanism of paraquat involves glutamate induced activation of non-NMDA receptors, resulting in activation of NMDA receptor-channels. The influx of Ca(2+) into cells stimulates nitric oxide synthase. Released NO would diffuse to dopaminergic terminals and further induce mitochondrial dysfunction by the formation of peroxynitrite, resulting in continuous and long-lasting dopamine overflow. The constant exposure to low levels of paraquat may lead to the vulnerability of dopaminergic terminals in humans, and might potentiate neurodegeneration caused by the exposure of other substances, such as endogenous dopaminergic toxins.

Detection of stable chromosome aberrations by FISH in A-bomb survivors: comparison with previous solid Giemsa staining data on the same 230 individuals.

PURPOSE: To evaluate the relative abilities of the solid Giemsa staining (conventional) and fluorescence in situ hybridization (FISH) methods in the detection of stable chromosome aberrations in the peripheral blood lymphocytes of A-bomb survivors. MATERIALS AND METHODS: Lymphocytes from a total of 230 A-bomb survivors for whom prior chromosome aberration data had been obtained by the conventional method were recently examined afresh using FISH in which chromosomes 1, 2 and 4 were painted with composite probes. RESULTS: It was found that the early use of the solid Giemsa staining method had allowed the detection of translocations with a mean frequency of 73% of the value for the genome-equivalent translocation frequency (F(G)) that was now obtained using FISH. The disparity may at least in part be due to the reciprocal exchange of seemingly identical amount of chromosome material; such exchanges can escape detection by the conventional method but can be readily identified using FISH. CONCLUSION: It has previously been established that the conventional method can detect about 20% of radiation-induced translocations as abnormal monocentric chromosomes. Present results indicate that an additional 50% can be detected if proper karyotyping is conducted and the remaining 30% are not likely to be detected unless FISH or banding methods are used. Thus, solid Giemsa staining accompanied by karyotyping may not be quite as unsuitable as is generally assumed for retrospective biodosimetry analyses, which deal mainly with stable aberrations.

FISH examination of lymphocytes from Mayak workers for assessment of translocation induction rate under chronic radiation exposures.

PURPOSE: To estimate the translocation-induction rate under chronic exposure conditions by measuring chromosome aberration frequencies in lymphocytes from Mayak nuclear workers using fluorescence in situ hybridization (FISH). MATERIALS AND METHODS: Lymphocytes were examined from 27 nuclear workers at the Mayak Production Association and two control individuals using FISH with probes for chromosomes 1, 2 and 4. Official doses derived from worker film-badge records varied from 0 to 8.50 Gy. RESULTS: The mean (+/-SD) genome-equivalent translocation frequency (F(G)) was 2.30 (+/-0.75)% in the zero-dose group (n = 7), and Poisson regression analysis provided the best-fit equation of F(G)(%) = 2.96(+/-0.39) + 0.69(+/-0.14)D + 0.12(+/-0.05)A, where D is the film-badge-derived dose (Gy), and A is age centred at 67 years. The induction rate would increase to nearly 1% Gy(-1) if the radiation dose to bone marrow, one of the major organs for lymphocytes and where their precursor cells reside, is considered. CONCLUSION: The estimated induction rate in vivo appeared substantially smaller than linear coefficients estimated from various in vitro studies.

Stable chromosome aberrations in atomic bomb survivors: results from 25 years of investigation.

Frequencies of stable chromosome aberrations from more than 3,000 atomic bomb survivors were used to examine the nature of the radiation dose response. The end point was the proportion of cells with at least one translocation or inversion detected in Giemsa-stained cultures of approximately 100 lymphocytes per person. The statistical methods allow for both imprecision of individual dose estimates and extra-binomial variation. A highly significant and nonlinear dose response was seen. The shape of the dose response was concave upward for doses below 1.5 Sv but exhibited some leveling off at higher doses. This curvature was similar for the two cities, with a crossover dose (i.e. the ratio of the linear coefficient to the quadratic coefficient) of 1.7 Sv (95% CI 0.9, 4). The low-dose slopes for the two cities differed significantly: 6.6% per Sv (95% CI 5.5, 8.4) in Hiroshima and 3.7% (95% CI 2.6, 4.9) in Nagasaki. This difference was reduced considerably, but not eliminated, when the comparison was limited to people who were exposed in houses or tenements. Nagasaki survivors exposed in factories, as well as people in either city who were outside with little or no shielding, had a lower dose response than those exposed in houses. This suggests that doses for Nagasaki factory worker survivors may be overestimated by the DS86, apparently by about 60%. Even though factory workers constitute about 20% of Nagasaki survivors with dose estimates in the range of 0.5 to 2 Sv, calculations indicate that the dosimetry problems for these people have little impact on cancer risk estimates for Nagasaki.

Carrier-mediated processes in blood--brain barrier penetration and neural uptake of paraquat.

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringer's solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

Localization of the pituitary adenylate cyclase-activating polypeptide receptor and its mRNA in the rat adrenal medulla.

We examined the localization of the pituitary adenylate cyclase-activating peptide (PACAP) receptor (PAC1-R) and its mRNA with immunocytochemistry and in situ hybridization, respectively. PAC1-R immunoreactivity and its transcript were detected in both chromaffin cells and ganglion cells but not detected in the adrenal cortex. In addition, strong PAC1-R immunoreactivity was found beneath the plasma membrane of the immunoreactive medullary cells. Electron microscopic immunocytochemistry revealed that PAC1-R was predominantly expressed in adrenaline-containing cells. This report supports the notion that PACAP is an activator and modulator of catecholamine secretion as well as synthesis in the adrenal medulla.

The F value for chromosome aberrations in atomic bomb survivors does not provide evidence for a primary contribution of neutrons to the dose in Hiroshima.

Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) proposed that the ratio of interchromosomal to intrachromosomal exchanges, termed the F value, can be a cytogenetic fingerprint of exposure to radiations of different linear energy transfer (LET). Using published data, they suggested that F values are over 10 for low-LET radiations and approximately 6 for high-LET radiations. Subsequently, as F values for atomic bomb survivors were reported to be around 6, Brenner suggested that the biological effects of atomic bomb radiation in Hiroshima are due primarily to neutrons. However, the F values used for the survivors were means from individuals exposed to various doses. As the F-value hypothesis predicts a radiation fingerprint at low doses, we analyzed our own data for the survivors in relation to dose. G-banding data for the survivors showed F values varying from 5 to 8 at DS86 doses of 0.2 to 5 Gy in Hiroshima and around 6 in Nagasaki with no evidence of a difference between the two cities. The results are consistent with our in vitro data that the F values are invariably around 6 for X and gamma rays at doses of 0.5 to 2 Gy as well as two types of fission-spectrum neutrons at doses of about 0.2 to 1 Gy. Thus, apart from a possible effect at even lower doses, current data do not provide evidence to support the proposition that the biological effects of atomic bomb radiation in Hiroshima are caused mainly by neutrons.

S values are not a signature for a significant contribution of neutrons to the radiation dose received by atomic-bomb survivors.

PURPOSE: It has been proposed previously that the ratio of complete to incomplete translocations as seen by fluorescence in situ hybridization (FISH), the S value, can be a cytogenetic fingerprint of exposure to radiation of different qualities. Results from a previous study suggested that the S value is approximately 10 for sparsely ionizing radiations such as X- and gamma-rays, and 2 for densely ionizing radiations. Based on FISH data of atomic-bomb (A-bomb) survivors, which showed an S value of 3.25, a significant neutron component to A-bomb radiation was suggested. To examine the possibility, the present in vitro study was conducted using X-rays. MATERIALS AND METHODS: Human blood lymphocytes were exposed to X-rays and first metaphases were examined with FISH using DNA probes for chromosomes 1, 2 and 4. RESULTS: The S value was 3.16 for X-rays, which differs from approximately 10 as reported previously, and not larger than the 3.25 obtained from the blood lymphocytes of A-bomb survivors. CONCLUSIONS: S values seem to vary among laboratories even after exposure of cells to sparsely ionizing radiations. Data from this study show that S values are not a signature for a significant contribution of neutrons to the radiation dose received by A-bomb survivors in Hiroshima.

Structural significance of azaheterocyclic amines related to Parkinson's disease for dopamine transporter.

We have evaluated the neuronal uptake of 12 neutral and quaternary azaheterocyclic amines that are possible candidates for idiopathic Parkinson's disease via dopamine transporter of striatal synaptosomes. The double-reciprocal plots for dopamine transporter obtained from Wistar rat and C57BL/6 mouse synaptosomes with N-methyl-4-phenylpyridinium cation (MPP+) as a substrate were identical to each other. Neutral beta-carbolines and tetrahydroisoquinolines were unfavorable substrates for dopamine transporter. The quarternization of these compounds strikingly increased the affinity for dopamine transporter with 2-10 times greater Km and 10 times smaller Vmax values than MPP+. Although catechol tetrahydroisoquinolines were weak substrates, their quarternization reduced their original properties as substrates for dopamine transporter. These results provide both topographic and electrogenic information of azaheterocyclic amines for the dopamine transporter-mediated influx. The intramolecular distance between the N-atom and the centroid of the benzene ring could be an important factor for the recognition of binding site of dopamine transporter, and an adequate net charge similar to dopamine would be further required for translocation into the cells.

Endogenously occurring beta-carboline induces parkinsonism in nonprimate animals: a possible causative protoxin in idiopathic Parkinson's disease.

To examine whether simple beta-carbolines induce parkinsonian-like symptoms in vivo via N-methylation, the simple beta-carbolines norharman (NH), 2-mono-N-methylated norharmanium cation (2-MeNH+), and 9-mono-N'-methylnorharman (9-MeNH) were systematically administered to C57BL/6 mice for 7 days. These substances induced bradykinesia with reduction of locomotion activity. NH or 2-MeNH+ decreased dopamine (DA) contents to 50-70% of values in controls in the striatum and midbrain. 9-MeNH potently decreased not only DA but also serotonin content in various regions. Immunohistochemical examination revealed that the numbers of tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta of NH- and 9-MeNH-treated mice were diminished to 76 and 66% of values in control mice, respectively. The formation of a toxic metabolite, 2,9-di-N,N'-methylated norharmanium cation (2,9-Me2NH+), was 14 and eight times higher in the brain of mice receiving 9-MeNH than that in NH- and 2-MeNH+-treated mice, respectively. In cultured mesencephalic cells from rat embryo, 2,9-Me2NH+ selectively killed TH-positive neurons only at a lower dose but was toxic to all neurons at higher doses. Thus, the excess formation of 2,9-Me2NH+ would induce nonspecific neurotoxicity. These results indicated that 9-indole nitrogen methylation should be the limiting step in the development of the toxicity. NH, a selective dopaminergic toxin precursor, is sequentially methylated to form 2,9-Me2NH+, which could be an underlying factor in idiopathic Parkinson's disease.

Three types of recombinant human granulocyte colony-stimulating factor have equivalent biological activities in monkeys.

Three types of rhG-CSF are commercially available (non-glycosylated: filgrastim, glycosylated: lenograstim and N-terminal mutated: nartograstim). It has been reported that higher in vitro or in vivo efficacy was found in glycosylated or N-terminal mutated rhG-CSF than in non-glycosylated rhG-CSF. We reported that glycosylated or N-terminal mutated rhG-CSF showed equal efficacy to non-glycosylated rhG-CSF in vivo. In this study, we carried out a direct comparison of pharmacokinetics and pharmacological effects of three rhG-CSFs. We used commercially obtained rhG-CSF products whose activities are guaranteed by the manufacturers. Monkeys have been selected as the experimental animals because of their close relationship to humans concerning drug disposition and daily doses were in accordance with the clinical use of rhG-CSFs. Normal cynomolgus monkeys were given 1.5 or 5 micrograms/kg of rhG-CSF either intravenously or subcutaneously for 5 consecutive days. After intravenous injection, the serum concentration-time profiles of nartograstim were almost identical to those of filgrastim at both doses but the concentrations after lenograstim administration decreased faster. Following subcutaneous administration, no marked differences were observed between the three rhG-CSFs, although lenograstim showed lower serum concentrations than both filgrastim and nartograstim. In spite of some small differences in the pharmacokinetics of the three rhG-CSFs, the pharmacodynamics were identical.

Estimation of minimal size of translocated chromosome segments detectable by fluorescence in situ hybridization.

Apparent non-reciprocal translocations are commonly observed using fluorescence in situ hybridization. We hypothesize that these are 'hidden' reciprocals due to one translocated segment being too small to detect. Assuming that the translocation breakpoints distribute randomly, the proportion of reciprocal to non-reciprocal exchanges can be used to estimate the minimal detectable size of translocated segments. To estimate segment size in this study, cytogenetic data for 120 A-bomb survivors were used. Among 2295 aberrant metaphases, 1629 exhibited reciprocal translocations and 666 non-reciprocal. Of the non-reciprocal translocations, 501 showed only a painted chromosome segment, translocated to an unpainted chromosome with centromere, and 165 showed only an unpainted chromosome segment, translocated to a painted chromosome with centromere. On the basis of the above two assumptions, we obtained the most likely estimates for minimal detectable sizes: 11.1 +/- 0.8 Mb for the painted and 14.6 +/- 0.6 Mb for the unpainted chromosomes. The implications of these findings are discussed.