Increase In Chromosome Number Research Articles

Apomixis and sexuality in the Potentilla argentea complex III. Euploid and aneuploid derivatives (including trisomics) of some apomictic biotypes

Three apomictic biotypes belonging to the Potentilla argentea complex formed the basis of this investigation, one of them diploid, one tetraploid and one hexaploid. The effects of chromosome doubling and other changes in chromosome number were studied. Hybrids between the same biotypes were discussed in a previous paper. The diploid was a facultative apomict, but autotetraploids produced by colchicine treatment were totally sexual. This was also true of autotriploids, probably also of trisomics and other aneuploids. Five out of seven possible primary trisomics were obtained; they were morphologically well separated from each other and from the diploid. As shown by the result of test-crosses, facultative apomixis was restored in “recombined diploids” in progenies from triploids and trisomics. This indicates that apomixis is partly dependent on homozygosity for recessive genes, but that also the degree of ploidy as well as chromosomal and genic balance directly influence the mode of reproduction. — In the more strictly apomictic tetraploid (2n= 28) biotype, unreduced egg cells were fertilized in exceptional cases, whereas the chromosome-doubled plants formed some aberrants by fertilization of reduced egg cells. However, an “autotriploid” (2n= 42) formed half of its offspring by fertilization of unreduced and reduced egg cells. Aberrants with 2n= 35 and 2n= 63 were obligate apomicts. — The hexaploid was obligately apomictic, whereas the poorly fertile plants resulting from chromosome doubling gave rise to one plant with further increased chromosome number; thus, even here apomixis was interfered with to some extent. — The concluding discussion also deals with problems of mitotic and meiotic instability, crossing barriers in pseudogamous taxa, and viewpoints on the mechanisms and regulation of apomixis.

Apomixis and sexuality in the Potentilla argentea complex II. Crosses within the complex

The Potentilla argentea complex is mainly built up by apomicts on various levels of ploidy. In a previous paper, the results of crosses with other species were dealt with. Here, an account is given of crosses between taxa within the argentea complex. Most crosses involved a diploid, a tetraploid and a hexaploid biotype. The first-mentioned is a facultative apomict, whereas its autotetraploid is wholly sexual. The tetraploid biotype forms aberrants at a low frequency, whereas the hexaploid seems to be entirely constant. Natural tetraploids may have originated from crosses between the more widely distributed diploid (“P. argentea”) and hexaploid (“P. neglecta”) biotypes. The F1 hybrids were in many cases vigorous but had always a reduced seed set as compared with the parents. Crosses between sexual autotetraploid and apomictic tetraploid resulted in sexual F1 plants, whereas F1 hybrids from crosses between sexual autotetraploid and hexaploid apomicts showed a varying mode of reproduction. From crosses between apomictic tetraploid and hexaploid biotypes, one pentaploid and some heptaploid hybrids were obtained. The heptaploids, resulting from fertilization of unreduced egg cells, were apomictic, although two plants with further increased chromosome numbers were found in in the offspring. The pentaploid hybrid reproduced only by fertilization of unreduced egg cells. This mode of reproduction prevailed even in the next generation, leading to the formation of weak high-polyploid plants.

HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells

Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis.

Cytogenetics and Flow Cytometry-based DNA Quantification in Herreria salsaparilha Martius (Herreriaceae): a Medicinal Species

Summary Adventitious root tips developing from base of cuttings and leaf samples of Herreria salsaparilha were used to determine the karyotype and to estimate DNA amount, respectively. Flow cytometry methodology was used with DAPI (4� ,6-diamidino-2-phenylindole) and PI (propidium iodide) as specific DNA fluorochromes, and leaf samples of Pisum sativum (9.09 pg) as an internal standard. Cytogenetic techniques determined that H. salsaparilha has 29 pairs of homologous chromosomes (2n� 58), 12 acrocentric pairs (length varying from 1.75 to 10.51 mm), 16 submetacentric pairs (1.30 to 3.45 mm), and 1 metacentric pair (2.8 mm). The DAPI- and PI-based flow cytometry analysis revealed that H. salsaparilha has nuclear DNA content 2C� 5.5 pg, equivalent to approximately 5.31� 10 9 base pairs. This karyotype asymmetric type may suggest an evolutionary process with involvement of polyploidization, being justified by the relative increased chromosome number (2n� 58) and fusions.

Spontaneous Transformation of Cultured Mouse Bone Marrow–Derived Stromal Cells

Bone marrow-derived stromal cells have engendered interest because of their therapeutic potential for promoting tissue vascularization and repair. When mononuclear cells isolated from mouse bone marrow were cultured in DMEM supplemented with 10% fetal bovine serum, cell populations arose that showed rapid proliferation and loss of contact inhibition. These cells formed invasive soft tissue sarcomas after i.m. injection into nude or scid mice. I.v. injection resulted in the formation of tumor foci in the lungs. The tumors were transplantable into syngeneic immunocompetent mice. Direct injection of cultured cells into immunocompetent mice also resulted in tumor formation. Karyotype analysis showed that increased chromosome number and multiple Robertsonian translocations occurred at passage 3 coincident with the loss of contact inhibition. The remarkably rapid malignant transformation of cultured mouse bone marrow cells may have important implications for ongoing clinical trials of cell therapy and for models of oncogenesis.

Extra divisions and nuclei fusions in microspores from Brassica allohexaploid (AABBCC) × Orychophragmus violaceus hybrids

Abnormal meiosis and microspore development and related defective mutants have often been reported in plants and wide hybrids. Here extra divisions and nuclei fusions were observed to occur in microspore nuclei of partial hybrids between synthetic Brassica hexaploid (2n = 54, AABBCC) and another crucifer Orychophragmus violaceus (2n = 24). Abnormal spindle were formed and chromosomes were separated into several nuclei of variable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As a consequence, more than eight mini-microspores of different sizes were produced by one tetrad. Genomic in situ hybridization results indicated that no chromosome replication occurred during such divisions. In some tetrads, the four nuclei were fused to form one large cell with increased chromosome number. The extra divisions or fusions appeared only in some flower buds of one plant, some anthers in the same buds, or even in individual cells of tetrads. The possible mechanisms behind these cytological phenomena are discussed.

Loss of DNA Polymerase ζ Causes Chromosomal Instability in Mammalian Cells

Rev3L encodes the catalytic subunit of DNA polymerase zeta (pol zeta) in mammalian cells. In yeast, pol zeta helps cells bypass sites of DNA damage that can block replication enzymes. Targeted disruption of the mouse Rev3L gene causes lethality midway through embryonic gestation, and Rev3L-/- mouse embryonic fibroblasts (MEFs) remain in a quiescent state in culture. This suggests that pol zeta may be necessary for tolerance of endogenous DNA damage during normal cell growth. We report the generation of mitotically active Rev3L-/- MEFs on a p53-/- genetic background. Rev3L null MEFs exhibited striking chromosomal instability, with a large increase in translocation frequency. Many complex genetic aberrations were found only in Rev3L null cells. Rev3L null cells had increased chromosome numbers, most commonly near pentaploid, and double minute chromosomes were frequently found. This chromosomal instability associated with loss of a DNA polymerase activity in mammalian cells is similar to the instability associated with loss of homologous recombination capacity. Rev3L null MEFs were also moderately sensitive to mitomycin C, methyl methanesulfonate, and UV and gamma-radiation, indicating that mammalian pol zeta helps cells tolerate diverse types of DNA damage. The increased occurrence of chromosomal translocations in Rev3L-/- MEFs suggests that loss of Rev3L expression could contribute to genome instability during neoplastic transformation and progression.

Identification of essentially derived varieties with molecular markers: an approach based on statistical test theory and computer simulations

Genetic similarities (GS) based on molecular markers have been proposed as a tool for identification of essentially derived varieties (EDVs). Nevertheless, scientifically reliable criteria for discrimination of EDVs and independently derived varieties with GS estimates are scanty, and implementation into practical breeding has not yet taken place. Our objectives were to (1) assess the influence of chromosome number and length, marker density, and distribution, as well as the degree of polymorphism between the parental inbreds on the distribution of GS between parental inbreds and their progenies [GS(P1,O)] derived from F2 and different backcross populations and (2) evaluate these factors with regard to the power for distinguishing F2- versus BC1- and BC1- versus BC2-derived lines with molecular markers. We developed an approach based on statistical test theory for the identification of EDVs with molecular markers. Standard deviations and overlaps of distributions of GS(P1,O) of F2-, BC1-, and BC2-derived lines were smaller with (1) increasing chromosome number and length, (2) increasing marker density, and (3) uniformly instead of randomly distributed markers, approaching a lower boundary determined by the genetic parameters. The degree of polymorphism between the parental inbreds influenced the power only if the remaining number of polymorphic markers was low. Furthermore, suggestions are made for (1) determining the number of markers required to ascertain a given power and (2) EDV identification procedures.

Integrative Study on Chromosome Evolution of Mammals, Ants and Wasps Based on the Minimum Interaction Theory

There is well-known evidence that in many eukaryotes, different species have different karyotypes (e.g.n =1–47 in ants and n=3–51 in mammals). Alternative (fusion and fission) hypotheses have been proposed to interpret this chromosomal diversity. Although the former has long been accepted, accumulating molecular genetics evidence seems to support the latter. We investigated this problem from a stochastic viewpoint using the Monte Carlo simulation method under the minimum interaction theory. We found that the results of simulations consistently interpreted the chromosomal diversity observed in mammals, ants and wasps, and concluded that chromosome evolution tends to evolve as a whole toward increasing chromosome numbers by centric fission. Accordingly, our results support the fission hypothesis. We discussed the process of chromosome evolution based on the latest theory of the molecular structure of chromosomes, and reconfirmed that the fission burst is the prime motive force in long-term chromosome evolution, and is effective in minimizing the genetic risks due to deleterious reciprocal translocations and in increasing the potential of genetic divergence. Centric fusion plays a biological role in eliminating heterochromatin (C-bands), but is only a local reverse flow in contrast to the previously held views.

Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. Conventional banding techniques, FISH and SKY

Permanent Sertoli cell lines provide an ideal system for the in vitro analysis of function and responsiveness to biochemical/hormonal factors of this particular cell type. In general, cytogenetic analyses of cell lines often reveal remarkable chromosomal changes that may be associated with functional characteristics. In the present study we investigated the mouse Sertoli cell line TM4 by C-banding, silver staining, FISH and spectral karyotyping (SKY). A highly increased chromosome number (average 85–95) as well as five stable marker chromosomes were detected by the conventional staining techniques. SKY identified the markers as a translocation chromosome T(1;3), isochromosomes 11 and 18 and two different-sized microchromosomes. The results show the usefulness of combining SKY and conventional banding methods for the evaluation of chromosome alterations in widely used cell lines.

The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages.

The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.

Characters of Peppermint Cultivars (Mentha x piperita L.) with Different Chromosome Numbers

Polyploidization is a widespread method used in peppermint breeding to increase the chromosome numbers and to restore the fertility as a prerequisite for combination breeding and the induction of new genetic diversity. The use of these methods in breeding new varieties often results in progenies with increased chromosome numbers and characters divergent in comparison with the parents. Ten peppermint cultivars were classified into two groups according to their chromosome numbers: Group 1 with normal chromosome numbers 2 n = 66–72, Group 2 with increased chromosome numbers 2 n = 90–96. Cultivars with the higher chromosome numbers showed a more vigorous growth, the generative development was accelerated, the essential oil content and its content of menthone, isomenthone and limonene were increased and the content of menthol, menthofuran, 1,8-cineole and viridiflorol was lowered.

Combined metaphase, interphase cytogenetic, and flow cytometric analysis of DNA content of pediatric acute lymphoblastic leukemia

Eleven pediatric acute lymphoid leukemia patients were investigated for chromosomal aneuploidy by interphase cytogenetics using chromosome specific (peri)centromeric probes for all the somatic and sex chromosomes. Results were compared with metaphase cytogenetic and flow cytometric derived DNA aneuploidy data. Experiments performed on normal human cells using chromosome specific (peri)centromeric probes indicated that disomy could be recognized in a range of 89.1+/-2.7% (12.9)-96.8+/-0.2% (0.9) for the somatic chromosomes and in 98.1+/-0.4% (1.3) for the sex chromosomes. Using the cutoff level of the mean false monosomy and trisomy in the control cells +2 S.D., chromosome loss or gain for the somatic chromosomes could be revealed beyond a clonal ratio of 3.6-13.2% and 1.1-6.8%, respectively. The same value for the sex chromosomes was 3.5% and 0%, respectively. In 5 of 11 patients the leukemic cells proved to be diploid with all three methods at both gross DNA and chromosome levels. Interphase cytogenetics revealed chromosome loss or gain in all of the remaining six patients, however, the metaphase analysis indicated numerical aberration in only two patients. In one of them only the increased chromosome number could have been detected without identifying the chromosomes involved and in the other one the two methods indicated trisomy for a different chromosome. Flow cytometric data showed aneuploidy in three of the six aneuploid leukemia patients. The results suggest that interphase cytogenetics might be more accurate compared with flow cytometry and metaphase analysis to reveal aneuploidy.

Combined metaphase, interphase cytogenetic, and flow cytometric analysis of DNA content of pediatric acute lymphoblastic leukemia

Eleven pediatric acute lymphoid leukemia patients were investigated for chromosomal aneuploidy by interphase cytogenetics using chromosome specific (peri)centromeric probes for all the somatic and sex chromosomes. Results were compared with metaphase cytogenetic and flow cytometric derived DNA aneuploidy data. Experiments performed on normal human cells using chromosome specific (peri)centromeric probes indicated that disomy could be recognized in a range of 89.1 ± 2.7% (12.9)–96.8 ± 0.2% (0.9) for the somatic chromosomes and in 98.1 ± 0.4% (1.3) for the sex chromosomes. Using the cutoff level of the mean false monosomy and trisomy in the control cells +2 S.D., chromosome loss or gain for the somatic chromosomes could be revealed beyond a clonal ratio of 3.6–13.2% and 1.1–6.8%, respectively. The same value for the sex chromosomes was 3.5% and 0%, respectively. In 5 of 11 patients the leukemic cells proved to be diploid with all three methods at both gross DNA and chromosome levels. Interphase cytogenetics revealed chromosome loss or gain in all of the remaining six patients, however, the metaphase analysis indicated numerical aberration in only two patients. In one of them only the increased chromosome number could have been detected without identifying the chromosomes involved and in the other one the two methods indicated trisomy for a different chromosome. Flow cytometric data showed aneuploidy in three of the six aneuploid leukemia patients. The results suggest that interphase cytogenetics might be more accurate compared with flow cytometry and metaphase analysis to reveal aneuploidy. Cytometry (Comm. Clin. Cytometry) 34:87–94, 1998. © 1998 Wiley-Liss, Inc.

Phylogenetics of the slipper orchids (Cypripedioideae, Orchidaceae): Nuclear rDNA ITS sequences

Cypripedioideae (Orchidaceae) have been the subject of numerous taxonomic treatments with conflicting interpretations of relationships among the five genera and the 150–170 species. We have produced nuclear ribosomal ITS nucleotide sequences for nearly 100 slipper orchid species and used parsimony analysis to investigate their relationships. Our results demonstrate that each genus, as currently circumscribed, is monophyletic (Mexipedium andSelenipedium being represented by a single taxon). LikerbcL data, ITS sequences placeMexipedium sister toPhragmipedium. Relationships at the sectional level inPaphiopedilum are largely as described byCribb. However, the division ofPaphiopedilum into subgg.Brachypetalum andPaphiopedilum is not supported; subg.Brachypetalum is paraphyletic to subg.Paphiopedilum. Phragmipedium species are divided into the same three major clades as in the taxonomic scheme ofMcCook. The plicate-leaved genera,Cypripedium andSelenipedium, are successive sister groups to the rest of the subfamily, confirming generally held opinions that they display plesiomorphic characters compared to the conduplicate-leaved genera. A survey of karyotypes in the context of the ITS tree reveals a general trend toward increased chromosome number, probably brought about by centric fission. These data also accord with a previously suggested biogeographic hypothesis of a widespread Northern Hemisphere distribution, followed by range fragmentation due to Miocene cooling.

Introgressive hybridization between a native and an introduced species:Viola lutea subsp.Sudetica versusV. Tricolor

A hybrid swarm betweenViola lutea subsp.sudetica (2n=50, native species) andViola tricolor (2n=26, introduced species) originated in the 1970’s in the Krkonose Mts. Analyses of chromosome numbers, reproductive biology, morphology, and ecology gave the following results: (1) Compared to the plants found in the 1970’s, the number of colour morphs have decreased and the types now prevailing in the field are morphologically closer toV. lutea subsp.sudetica, forming a continuum. (2) In the field, hybrids having approximately the same chromosome number as the primary hybrids are most common. Some plants of the hybrid swarm have certain characters unknown to their parents. (3) In the field, autogamous types similar toV. tricolor were found. (4) A limited number of plants from the field and culture have higher somatic chromosome numbers thanV. lutea subsp.sudetica; there was a tendency towards increasing chromosome numbers in their progeny (up to 2n=62). These plants have some new morphological characters (a small proportion of hexacolpate pollen) not found in the parents (nor in the other hybrids with prevailing tetracolpate and pentacolpate pollen grains) and higher pollen fertility in comparison to the other hybrids. These plants also have the highest germination rate. (5) There is a tendency for chromosome numbers to decline in the progeny of open pollinated hybrid plants in the lowland experimental graden. (6) The phenology of the plants in the mountain grasslands and the lowland garden is different; the parents behave in a totally contrasting manner. (7) In the field at least some hybrids extend outside the geographical and ecological ranges of the parental species, invading new communities. (8) Seeds ofV. tricolor do not show any dormancy and germinate in the year of production; most of theV. lutea subsp.sudetica seeds germinate during the spring of the following year. Hybrids with intermediate chromosome numbers had both types of germination strategy. The germination rate of intermediates with high chromosome numbers was even higher than that ofV. tricolor.

Variation in chromosome number in the seedling progeny of a somaclone of Paspalum dilatatum

The somaclone, C39, derived by tissue culture from the obligate apomict Paspalum dilatatum cv Raki (2n=50), had 50 chromosomes and a karyotype apparently identical to Raki. SC2 seedlings of C39 showed a high degree of phenotypic variation which was often associated with increased chromosome numbers, but some of the variant seedlings were karyotypically indistinguishable from Raki or C39. Plants with increased chromosome numbers exhibited a high degree of intraplant chromosome variation (aneusomaty). In one of the SC2 seedlings, the chromosome number of root tip cells varied from 58 to 82 and in several other seedlings the range was more than 10. The results suggested that the ability to form seed apomictically was much reduced in C39 and that this plant showed some capacity for sexual reproduction and the resulting seedlings, with a chromosome number of about 70, were genetically unstable. Of 11 SC2 seedlings examined cytologically, 6 did not produce any viable seed. Seedlings grown from seed of the remaining 5 plants showed that aneusomaty persisted in the SC3 generation. SC3 seedlings which were phenotypically similar to their maternal parent showed a similar range of chromosome numbers to that parent. Some of the SC3 seedlings exhibited an even wider range of chromosome numbers (e.g.56-136), and these plants were all dwarfs.

Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

We attempted to generate homozygous dhfr-ts (dihydrofolate reductase-thymidylate synthase) knockouts in virulent Leishmania major, an asexual diploid protozoan parasite. Transfection of a neo (neomycin phosphotransferase) targeting fragment yielded heterozygous replacement lines with high efficiency. However, second transfections with a hyg (hygromycin B phosphotransferase) targeting fragment in the presence of metabolites shown to rescue homozygous knockouts in attenuated Leishmania did not yield the expected dhfr-ts- thymidine auxotrophs obtained previously with attenuated lines. Molecular karyotype, Southern blot, and flow cytometric DNA content analysis of clonal transfectants revealed three classes: (i) genomic tetraploids, containing two wild-type dhfr-ts chromosomes and one neo and one hyg replacement chromosome; (ii) aneuploid trisomic lines with one wild-type dhfr-ts and one neo and one hyg replacement chromosome; (iii) diploids bearing homologous integration of the targeting fragment without replacement. Aneuploid and tetraploid lines predominated. This confirms the common impression that natural populations of Leishmania are often aneuploid. The remarkable ability of these parasites to undergo and tolerate changes in chromosome number suggests a general method for testing whether genes are essential for growth in vitro, as the ability of Leishmania to simultaneously undergo homologous gene replacement while retaining wild-type genes by increasing chromosome number provides a diagnostic and positive experimental result. Our results show that virulent Leishmania require at least one copy of dhfr-ts and argue that DHFR-TS plays an unanticipated role in addition to its role in the de novo synthesis of thymidine. These results also have implications for genetic tests of the organization of Leishmania populations.

Mode, tempo and pattern of evolution in subterranean mole rats of the Spalax ehrenbergi superspecies in the quaternary of Israel

The Spalax ehrenbergi superspecies in Israel involves 4 chromosomal species (2n = 52, 54, 58 and 60) displaying active ecological speciation throughout the Quaternary, matching climatic diversity. The mode of speciation was presumably peripatric, initiated chromosomally in small peripherally isolated populations. The emergence of the new chromosomal species was followed by a gradual genic accumulation of positive assortative mating, based on premating ethological species-specific isolating mechanisms involving olfaction, vocalization, seismic (vibrational), aggression and mating (through bacular variation) cues. Speciation was gradual, not punctuational, budding sequential daughter species with increasing chromosome numbers and heterozygosity, in a trend along an ecological gradient of increasing aridity towards the desert. The adaptationist programme is indispensable in understanding the evolution of Spalax ehrenbergi. The evolution of Spalax links micro- and macroevolution, corroborating the Synthetic Theory of Evolution. The evolutionary model of Spalax is potentially rich and its future advancement can enrich evolutionary theory.

Dimethylformamide-Induced Changes in the Radiation Survival of Low- and High-Passage Intestinal Epithelial Cells (IEC-17) in Vitro

The effects of dimethylformamide (DMF) on the radiation response of low- and high-passage intestinal epithelial cells (IEC-17) were examined. The IEC-17 cell line, a rat intestinal epithelial cell line, exhibited a bimodal response to X radiation. The sensitive fraction, which was attributed to a stem cell-like component, had a D0 of 0.90 Gy. The resistant fraction, thought to be the expression of a more mature component, exhibited a D0 of 2.00 Gy. Treatment using a putative cell differentiating agent, N,N-dimethylformamide, increased the resistant fraction of the population from 35 to 80%, suggesting that DMF treatment (100 mM) increased the proportion of mature cells in the IEC-17 cell population. In addition, extended age in culture (greater than 100 passages) resulted in altered morphology, decreased doubling time, increased chromosome number, and loss of anchorage dependence, all features characterizing spontaneously transformed high-passage IEC-17 cells. These high-passage cells also exhibited a bimodal response to X radiation; the sensitive fraction had a D0 of 0.80 Gy while the resistant fraction D0 was 1.50 Gy. DMF increased the resistant fraction from 35 to 55% of the population. Results suggested that the different radiosensitivities of the subpopulations remained throughout the spontaneous transformation of high-passage IEC-17 cells.