Centromeric index versus DNA content flow karyotypes of human chromosomes measured by means of slit-scan flow cytometry

We have applied slit-scan flow cytometry (SSFCM) to classify human chromosomes according to their centromeric index (CI) and relative DNA content. The resulting bivariate--CI vs. DNA content--distributions shows 14 peaks for normal human chromosomes. Distinct peaks are produced by chromosomes 1, 2, 3, 4 + 5, 6 + 7 + X, 8, 13 + 14 + 15, 16, 17 + 18, 19 + 20, and 21 + 22 + Y. In addition, chromosomes 9 through 12 are resolved into three peaks. The identity of the chromosomes comprising each peak was determined by comparing CI vs. DNA content distributions measured for normal human chromosomes by means of SSFCM with CI and DNA content values measured for human chromosomes with image analysis. The accuracy of CI measurement by SSFCM was verified by measuring CIs for human chromosomes isolated from human/rodent hybrid cell lines containing only a few known human chromosomes. These studies showed CIs measured for human chromosomes 1-19 and 21 to be in close agreement with the CIs calculated by means of image analysis. We further confirmed the chromosome assignments for each peak by showing that the relative volumes of the peaks in the CI vs. DNA content distributions for chromosomes from normal cells are similar to the relative frequencies of chromosomes expected for these peaks based on the peak assignments.     

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.     

Unstable Chromosome Aberrations Do Not Accumulate in Normal Human Fibroblast after Fractionated X-Irradiation

We determined the frequencies of dicentric chromosomes per cell in non-dividing confluent normal human fibroblasts (MRC-5) irradiated with a single 1 Gy dose or a fractionated 1 Gy dose (10X0.1 Gy, 5X0.2 Gy, and 2X0.5 Gy). The interval between fractions was between 1 min to 1440 min. After the completion of X-irradiation, the cells were incubated for 24 hours before re-plating at a low density. Then, demecolcine was administrated at 6 hours, and the first mitotic cells were collected for 42 hours. Our study demonstrated that frequencies of dicentric chromosomes in cells irradiated with a 1 Gy dose at different fractions were significantly reduced if the fraction interval was increased from 1 min to 5 min (p<0.05, χ²-test). Further increasing the fraction interval from 5 up to 1440 min did not significantly affect the frequency of dicentric chromosomes. Since misrejoining of two independent chromosome breaks introduced in close proximity gives rise to dicentric chromosome, our results indicated that such circumstances might be quite infrequent in cells exposed to fractionated X-irradiation with prolonged fraction intervals. Our findings should contribute to improve current estimation of cancer risk from chronic low-dose-rate exposure, or intermittent exposure of low-dose radiation by medical exposure.     

Centromeric index measurement by slit-scan flow cytometry

We report here the application of slit-scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total fluorescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where fluorescence profiles were measured. The centromere for each profile was taken as the point of maximum difference between the measured profile and a standard profile having no centromeric dip. The areas under the profile on either side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportional to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and X + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3-1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.57, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.     

Interstitial deletions of repetitive DNA blocks in dicentric human Y chromosomes

Cytogenetic analysis of aberrant human Y chromosomes was done by fluorescence in situ hydbridization (FISH) with Y specific repetitive DNA probes. It revealed an interstitial deletion of different DNA blocks in two dicentric chromosome structures. One deletion includes the total alphoid DNA structure of one centromeric region. The second deletion includes the total repetitive DYZ5 DNA structure in the pericentromeric region of one short Y arm. Both dicentric Y chromosomes were iso(Yp) chromosomes with break and fusion point located in Yq11, the euchromatic part of the long Y arm. Their phenotypic appearance was abnormal, resembling small monocentric Yq-chromosomes in metaphase plates. Mosaic cell lines, usually included in karyotypes with dicentric Y chromosomes, were not observed. It is assumed that both deletion events suppress the kinetochore activity in one Y centromeric region and thus stabilize its dicentric structure. Local interstitial deletion events had not been described in dicentric human Y chromosomes, but are common in dicentric yeast chromosomes. This raises the question of whether deletion events in dicentric human chromosomes are rare or restricted to the Y chromosome or also represent a general possibility for stabilization of a dicentric chromosome structure in human.     

Meiotic and Mitotic Behavior of Dicentric Chromosomes in SACCHAROMYCES CEREVISIAE

Meiotic recombination between a circular and a linear chromosome in Saccharomyces cerevisiae has been investigated. The circle was a haploid-viable derivative of chromosome III constructed by joining regions near the two chromosome ends via a recombinant DNA construction: (HMR/MAT-URA3-pBR322-MAT/HML) and was also deleted for MAL2 (which therefore uniquely marks a linear chromosome III). Recombination along chromosome III was measured for eight intervals spanning the entire length of the circular derivative. Only 25% of all tetrads from a ring/rod diploid contained four viable spores. These proved to be cases in which there was either no recombination along chromosome III or in which there were two-strand double crossovers or higher order crossovers that would not produce a dicentric chromosome.--At least half of the tetrads with three viable spores included one Ura+ Mal+ spore that was genetically highly unstable. The Ura+ Mal+ spore colonies gave rise to as many as seven genetically distinct, stable ("healed") derivatives, some of which had lost either URA3 or MAL2. Analysis of markers on chromosome III suggests that dicentric chromosomes frequently do not break during meiosis but are inherited intact into a haploid spore. In mitosis, however, the dicentric chromosome is frequently broken, giving rise to a variety of genetically distinct derivatives. We have also shown that dicentric ring chromosomes exhibit similar behavior: at least half the time they are not broken during meiosis but are broken and healed during mitosis.--The ring/rod diploid can also be used to determine the frequency of sister chromatid exchange (SCE) along an entire yeast ring chromosome. We estimate that an unequal number of SCE events occurs in approximately 15% of all cells undergoing meiosis. In contrast, the mitotic instability (and presumably SCE events) of a ring chromosome is low, occurring at a rate of about 1.2 X 10(-3) per cell division.     

Mutagenic radioadaptation in a human lymphoblastoid cell line

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.     

The persistence of lymphocytes with dicentric chromosomes following whole-body X irradiation of mice

Thirty-six male C57B1/6 mice were X-irradiated whole body with 3 Gy to generate lymphocytes with dicentric chromosomes to study the persistence of these lymphocytes in the spleen and peripheral blood to estimate the life span of mature B- and T-cells. Peripheral blood and spleen were removed from groups of four mice immediately after radiation exposure and on Days 1, 3, 7, 14, 28, 56, and 112 thereafter. Peripheral blood lymphocytes were cultured with phytohemagglutinin to stimulate T-cell division, and splenic lymphocytes were cultured with either lipopolysaccharide or phytohemagglutinin to stimulate B- or T-cell division, respectively. The initial frequencies of dicentric chromosomes with accompanying fragments observed in splenic T-cells (0.44), splenic B-cells (0.43), and peripheral blood lymphocyte cultures (0.48) initiated on Day 0 were not significantly different. For both splenic and peripheral blood T-lymphocytes, the frequency of cells containing dicentric chromosomes declined in an exponential manner following irradiation, with a 50% reduction in frequency occurring 14 days after exposure. In contrast, the frequency of B-cells containing dicentric chromosomes remained stable through Day 7 but then declined precipitously between Day 7 and Day 14 and remained relatively stable, although slightly above baseline, through Day 112 post-exposure. For both B- and T-cells, less than 5% of the cells contained a dicentric chromosome with accompanying fragments at Day 112. These data indicate that B- and T-lymphocytes with dicentric chromosomes show different decay kinetics and suggest that they may possess different life spans.     

Condensation anomalies and exclusion in micronuclei of rearranged chromosomes in human fibroblasts cultured in vitro

Anomalies of chromatin condensation, such as fragmentation, uncoiling and pulverization, were observed in XP9UV25, a xeroderma pigmentosum fibroblast clone in which a high proportion of cells carried an end-to-end dicentric chromosome, dic (5;16) (p15.2;q24), that gives rise during propagation in culture to a variety of dicentric and monocentric derivatives. The coiling anomaly affected exclusively part of a rearranged chromosome, in particular the region previously involved in breakage events. The heterochromatic 16q region, which is a preferential breakpoint in the formation of dicentric and monocentric derivatives, was consistently the limit of the uncoiled or pulverized regions. This observation suggests that the anomalous chromatin behavior could derive from alteration of a region relevant for the correct condensation of the chromosome. In XP9UV25 the frequency of nuclei with associated micronuclei increased with time in culture, in parallel with that of mitoses with dicentric chromosomes. In situ hybridization with DNA probes specific for chromosomes 5 and 16 revealed hybridization signals in about 40% of micronuclei. Since the frequency of micronuclei is about ten times less than that of dicentrics, it is probable that only the rearranged chromosomes undergoing coiling anomalies are excluded in micronuclei.     

Chromosome abnormalities in tuberous sclerosis

Summary In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.     

Radiation-induced DNA content variability in mouse sperm

Mouse sperm collected from the cauda epididymidis 35 days after acute testicular X-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to X-ray-induced DNA content variability. In the range between 0 and 600 rad the dose dependence of the square of CV of the DNA content variability, delta CV2D, is described by delta CV2D = Bx + Cx2, with 0 less than or equal to B less than or equal to 0.23 X 10(-2) and C = (0.44 +/- 0.06) X 10(-4). The dose x is measured in rad and delta CVD is expressed in percent. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artificially enriched in one population.     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations

This report describes a fluorescence in situ hybridization approach to chromosome staining that facilitates detection of structural aberrations and allows discrimination between dicentric chromosomes and symmetrically translocated chromosomes. In this approach, selected whole chromosomes are stained in one color by hybridization with composite probes whose elements have DNA sequence homology along the length of the target chromosomes. In addition, all chromosomes are counterstained with a DNA specific dye so that structural aberrations between target and non-target chromosomes are clearly visible. Discrimination between dicentric chromosomes and symmetrical translocations is accomplished by hybridization with a second probe that is homologous to DNA sequences found in the centromeric region of all chromosomes. The centromeric marker is visualized in a different color, so that the number of centromeres per aberrant chromosome can be rapidly determined in the microscope by changing excitation and fluorescence filters.by H.F. Willard     

The influence of DNA content and nuclear volume on the frequency of radiation-induced chromosome aberrations in Bufo species

The frequency of chromosome aberrations was compared in X-irradiated blood lymphocytes of Bufo marinus, B. calamita and B. pardalis which have similar chromosome numbers (2n=22) and karyotypes but differ in chromosomal DNA content and nuclear volume. — For each species the yield, per cell of centric exchange aberrations (dicentrics+rings) and of deletions (interstitial and terminal) increased approximately as the 1.5th power of the dose. — The 55% higher chromosomal DNA content of B. pardalis compared with both B. marinus and B. calamita resulted in the same increase in the frequency of deletions as a 55% increase in radiation dose, approximately doubling the yield. Both factors probably lead to a similar increase in the frequency of primary lesions from which the deletions are derived. — In contrast, an increase in chromosomal DNA content did not result in a higher yield of dicentric and ring exchanges, probably because the greater nuclear volume of B. pardalis (twice that of the other species) offset the potential increase in exchanges by increasing the average distance between chromosomes and chromosome arms. The data support the hypothesis that, in order to be involved in exchange, chromosome regions must be close together at the time of irradiation. The frequency of deletions is unaffected by changes in nuclear volume.     

Mode of ATM-dependent suppression of chromosome translocation

It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.     

The induction of chromosome aberrations during the course of radiation therapy for morbus Hodgkin

In a patient with Morbus Hodgkin, structural aberrations of the chromosome type in peripheral lymphocytes were analyzed during radiation therapy (accumulated target dose 44.6 Gy: 22 fractions of 1.8 Gy each and 2 fractions of 2.5 Gy each at the end of the therapy). The blood was sampled about 5 min after a fraction and/or 24, 48, or 72 h thereafter. The frequency of dicentric chromosomes:acentric fragments:centric ring chromosomes is 37:14:1 throughout the therapy. Independent of the time of blood sampling after a fraction, the distributions of dicentrics and acentrics are overdisperse and represent negative binomial distributions. The yields from these aberrations, as determined during the course of radiotherapy, are best fitted to a linear-quadratic function with a negative quadratic term. The two dose-effect curves (blood sampling about 5 min and 24 to 72 h after a fraction) of dicentrics and acentrics do not differ significantly. Up to an accumulated target dose of about 20 Gy the percentages of cells with chromosome aberrations increase to about 48 to 65% and, at this level, remain constant until the end of therapy.     

Lack of Spontaneous Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER

Neural ganglia of wild type third-instar larvae of Drosophila melanogaster were incubated for 13 hours at various concentrations of BUdR (1, 3, 9, 27 micrograms/ml). Metaphases were collected with colchicine, stained with Hoechst 33258, and scored under a fluorescence microscope. Metaphases in which the sister chromatids were clearly differentiated were scored for the presence of sister-chromatid exchanges (SCEs). At the lowest concentration of BUdR (1 microgram/ml), no SCEs were observed in either male or female neuroblasts. The SCEs were found at the higher concentrations of BUdR (3, 9, And 27 micrograms/ml) and with a greater frequency in females than in males. Therefore SCEs are not a spontaneous phenomenon in D. melanogaster, but are induced by BUdR incorporated in the DNA. A striking nonrandomness was found in the distribution of SCEs along the chromosomes. More than a third of the SCEs were clustered in the junctions between euchromatin and heterochromatin. The remaining SCEs were preferentially localized within the heterochromatic regions of the X chromosome and the autosomes and primarily on the entirely heterochromatic Y chromosome.--In order to find an alternative way of measuring the frequency of SCEs in the Drosophila neuroblasts, the occurrence of double dicentric rings was studied in two stocks carrying monocentric ring-X chromosomes. One ring chromosome, C(1)TR94--2, shows a rate of dicentric ring formation corresponding to the frequency of SCEs observed in the BUdR-labelled rod chromosomes. The other ring studied, R(1)2, exhibits a frequency of SCEs higher than that observed with both C(1) TR94--2 and rod chromosomes.     

C-banding in 6x-Triticale xSecale cereale L. hybrid cytogenetics

Summary The meiotic behaviour of F₁ hybrids of hexaploid Triticale that differed in their genotypic or chromosomic constitution, and diploid rye, was investigated. Meiotic analysis were done by Feulgen and C-banding staining methods. A differential desynaptic effect in the hybrids was detected and explained in terms of genetic differences in pairing regulators. The high homoeologous pairing (A-B wheat chromosomes and wheat-rye chromosomes) observed in the hybrids can be explained in terms of an inhibition of the effect of a single dose of thePh allele of the 5B chromosome produced by two doses of the 5R chromosome. The higher homoeologous pairing detected in the hybrid 188 x Canaleja could be the overall result of the balance between thePh diploidizing system (1 dose), the pairing promoter of the 5R chromosome (2 doses) and that of the 3D chromosome (1 dose coming from the parental line Triticale with the substitution 3R by 3D).     

Distribution of the various radiation-induced chromosomal rearrangements in relation to the dose and sampling time

The quantitative analysis of the chromosome rearrangements detected in 2128 R-banded metaphases, obtained from gamma-irradiated human lymphocytes after 48 to 96 h in culture is reported. Depending on the culture time, and possibly on the dose of radiation (from 1 to 3 Gy), the most frequent type of rearrangement was either dicentrics or reciprocal translocations. In first generation mitoses, the frequency of cells without rearrangement ranged from 0.66 to 0.18, and the mean number of rearranged chromosomes per cell from 0.79 to 3.28. The dose-response curve follows a quadratic function for dicentric aberration yields, but not for other rearrangements.     

Stabilization of dicentric chromosomes in Saccharomyces cerevisiae by telomere addition to broken ends or by centromere deletion.

We introduced CEN6 DNA via integrative transformation into the right arm of chromosome II in a haploid Saccharomyces cerevisiae strain thus creating a dicentric chromosome. The majority of the transformed cells did not grow into colonies as concluded from control transformations with mutated CEN6 DNA. Five percent of the initial transformants with the wild-type centromere gave rise to well growing cells. We analysed the probable fate of the dicentric chromosome in two transformants by electrophoretic separation of chromosome sized DNA and by hybridizations with chromosome II DNA probes. We found two different mechanisms which generated cells lacking dicentric chromosomes. The first mechanism is breakage of the chromatid between the two-centromeres and healing of the new ends to functional telomeres thus creating progeny cells with the chromosome II information split into two genetically stable new chromosomes one carrying CEN2 and the other CEN6. The second mechanism is loss of the resident CEN2 by a 30-50 kb deletion event which resulted in a genetically stable but shortened chromosome II. Both mechanisms operated in the two transformants studied.