Molecular characterization of hprt mutants induced by low- and hgh-LET radiations in human cells

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequenceprobes for regions of the X-chromosome which are linked to hrpt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30–48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14–54% of the observed changes. In the case of mutants induced by [^125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rae events.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.     

Efficient gene targeting by homologous recombination in rat embryonic stem cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat.     

The thymidine-kinase locus of friend erythroleukaemic cells: 1. Mutation rates and properties of mutants

Spontaneous mutation at the thymidine-kinase locus in clone 707 of the Friend cell lines has been examined. The rate of mutation in BrdU resistance was found to be 2.6 × 10^?6 cell^?1 generation^?1. The rate of reversion to HAT resistance was found to vary from 1.1 × 10^?7 to 2.85 × 10^?6 cell^?1 generation^?1 in 4 BrdU-resistant clones. Of 14 mutant clones assayed for thymidine-kinase activity only one had greater than 13% the activity of wild-type cells. Fluctuation analysis showed that mutations occurred spontaneously and were not induced by the selective agent.The mutation rate at the thymidine-kinase locus is several orders of magnitude greater than those reported for several other cell lines. There does not appear to be a general genetic instability in this cell line as the mutation rate at the HGPRT locus is similar to those found in other established cell lines.It is suggested that the high forward mutation rate at the thymidine-kinase locus may be due to only one functional thymidine-kinase allele being present in cells of this alone.     

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

The nature of mutants induced by ionising radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by gamma-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene

DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation.     

A comparison of mutation induction at the tk and hprt loci in human lymphoblastoid cells; quantitative differences are due to an additional class of mutations at the autosomal tk locus

X-Rays, ethyl methanesulfonate and ICR-191 induced 2 classes of trifluorothymidine-resistant mutants at the autosomal tk locus in human lymphoblastoid cells. These classes were differentiated by their growth rates; some mutants grew with a normal doubling time of 14-18 h (tk-NG), while others grew much more slowly, with doubling times of 21-44 h (tk-SG). Only mutants with normal growth rates were observed at the X-linked hprt locus; the frequencies of mutations induced at hprt were equal to those induced for tk-NG mutants. Thus, more mutations overall (by up to a factor of 6) were induced at tk than at hprt. These results are discussed in relation to recent studies in rodent cells, in which much greater mutation frequencies were observed at autosomal loci.     

Sequential genetic modification of the hprt locus in human ESCs combining gene targeting and recombinase-mediated cassette exchange

Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line.     

Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.     

Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro

Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor beta and gamma loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system.     

Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides

The repair of point mutations in hprt gene by single-stranded oligonucleotides represents a model to test targeted nucleotide exchange. We studied the concurrent nucleotide exchange of two or three nucleotides in the hprt deficient hamster cell line V79-151. The used oligonucleotides resulted in mismatches at two (151, 159) or three (151, 144, and 159) hprt positions. The hprt point mutation at position 151 was repaired in about 2/10(6) cells as shown by hprt sequencing in clones surviving HAT selection. The second nucleotide exchange at hprt position 159 was found in 7% of these HAT selected clones. Using oligonucleotides resulting in three mismatches, 29% of the clones showed nucleotide exchanges at the two hprt positions (151, 144) and about 4% at three positions (151, 144, and 159). These results indicate that single-stranded oligonucleotides can generate two or three nucleotide exchanges in a mammalian chromosomal gene.     

Hprt mutations and karyotype abnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients

In vivo mutations at the locus for hypoxanthine phosphoribosyl transferase (hprt) were studied in 6-thioguanine (TG)-resistant T-lymphocytes clones from healthy male and female subjects and ovarian carcinoma patients treated with melphalan. Southern blot analysis of 108 clones showed alterations in 14% (4/29) of the colnes from healthy males, 4.3% (2/47) of the clones from healthy females and 3.1% (1/32) of the clones from melphalan-treated patients. 2 of the 7 abnormal clones had a total deletion of the hprt gene; the others had partial deletions. Karyotype analysis of 82 clones revealed 1 clonal abnormality in 29 mutant clones from healthy males (3.6%). Loss or structural aberration of 1 X-chromosome occurred in 6% of the clones from healthy females. The frequency of karyotypic abnormalities (excluding those affecting one of the X-chromosomes) was significantly higher in clones from patients (37%) as compared to healthy females (5.9%). No aberration was found to affect the hprt locus at Xq27 in any of the 82 clones studied.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

Transfection,Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.     

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.     

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.     

Regulation of actin and other proteins in the differentiation of myeloid leukemic cells.

The regulation of cytoplasmic proteins in mutants of mouse myeloid leukemic cells, differing in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein (MGI) and the steroid inducer dexamethasone, was analyzed using SDS-polyacrylamide gel electrophoresis of ³⁵S-methionine-labeled proteins. Before induction, no consistent differences in the pattern of cytoplasmic proteins were found between clones with different capabilities to differentiate.Four MGI⁺D⁺ clones, which are induced by MGI for Fc and C3 rosettes, the synthesis and secretion of lysozyme, and the formation of mature macrophages and granulocytes, all showed the same nine prominent changes in cytoplasmic proteins after induction. Five of these changes were either an increase or a decrease in proteins present in uninduced cells; four proteins appeared to be newly synthesized. One of the proteins that increased after induction was identified as actin. The pattern of cytoplasmic proteins from MGI-induced MGI⁺D⁺ clones more closely resembled that of normal peritoneal macrophages and granulocytes than the pattern of the uninduced clones. The relationship of these protein changes to cell differentiation was further substantiated by the finding that MGI⁺D^? cells, which can be induced by MGI for Fc and C3 rosettes and lysozyme, but not for mature cells, showed only four cytoplasmic protein changes which were quantitatively less than those found for MGI⁺D⁺ clones. An MGI^?D^? clone which was not inducible for any differentiation-associated properties by MGI showed no alteration in protein synthesis. Thus in all the clones studied, there was a correlation between the number and extent of protein changes and the degree of MGI-induced differentiation.In MGI⁺D⁺ clones, some of the differentiation-associated properties induced by MGI can be induced by the steroid hormone dexamethasone. Of the nine protein changes induced by MGI, six were also induced by dexamethasone, and no changes were induced by dexamethasone which were not also induced by MGI. These results, which were also shown by two-dimensional polyacrylamide gel electrophoresis, indicate that in cells which can respond to both MGI and dexamethasone, the proteins induced by dexamethasone were a subset of those induced by MGI.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI⁺D⁺, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI⁺D^?, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI⁺D^? compared to the MGI⁺D⁺ clones. The MGI⁺D^? clones produced an unusual polyunsaturated C_20:5 fatty acid at 28°C, whereas the MGI⁺D⁺ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI⁺D^? clones than of the MGI⁺D⁺ clones. The cells of MGI⁺D⁺ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus.

Accurate identification of Populus clones and cultivars is essential for effective selection, breeding, and genetic resource management programs. The unit of cultivation and breeding in poplars is a clone, and individual cultivars are normally represented by a single clone. Microsatellite DNA markers of 10 simple sequence repeat loci were used for genetic fingerprinting and differentiation of 96 clones/cultivars and varieties belonging to six Populus species (P. deltoides, P. nigra, P. balsamifera, P. trichocarpa, P. grandidentata, and P maximowiczii) from three sections of the genus. All 96 clones/cultivars could be uniquely fingerprinted based on their single- or multilocus microsatellite genotypes. The five P. grandidentata clones could be differentiated based on their single-locus genotypes, while six clones of P. trichocarpa and 11 clones of P. maximowiczii could be identified by their two-locus genotypes. Twenty clones of P. deltoides and 25 clones of P. nigra could be differentiated by their multilocus genotypes employing three loci, and 29 clones of P. balsamifera required the use of multilocus genotypes at five loci for their genetic fingerprinting and differentiation. The loci PTR3, PTR5, and PTR7 were found to be the most informative for genetic fingerprinting and differentiation of the clones. The mean number of alleles per locus ranged from 2.9 in P. trichocarpa or P. grandidentata to 6.0 in P. balsamifera and 11.2 in 96 clones of the six species. The mean number of observed genotypes per locus ranged from 2.4 in P. grandidentata to 7.4 in P. balsamifera and 19.6 in 96 clones of the six species. The mean number of unique genotypes per locus ranged from 1.3 in P. grandidentata to 3.9 in P. deltoides and 8.8 in 96 clones of the six species. The power of discrimination of the microsatellite DNA markers in the 96 clones ranged from 0.726 for PTR4 to 0.939 for PTR7, with a mean of 0.832 over the 10 simple sequence repeat loci. Clones/cultivars from the same species showed higher microsatellite DNA similarities than the clones from the different species. A UPGMA cluster plot constructed from the microsatellite genotypic similarities separated the 96 clones into six major groups corresponding to their species. Populus nigra var. italica clones were genetically differentiated from the P. nigra var. nigra clones. Microsatellite DNA markers could be useful in genetic fingerprinting, identification, classification, certification, and registration of clones, clultivars, and varieties as well as genetic resource management and protection of plant breeders' rights in Populus.