Possible causes of chromosome instability: comparison of chromosomal abnormalities in cancer cell lines with mutations in BRCA1, BRCA2, CHK2 and BUB1

A large proportion of epithelial cancers show the chromosome-instability phenotype, in which they have many chromosome abnormalities. This is thought to be the result of mutations that disrupt chromosome maintenance, but the causative mutations are not known. We identified cell lines known to have mutations that might cause chromosome instability, and examined their karyotypes. Two cell lines, the breast cancer line HCC1937 and the pancreatic cancer line CAPAN-1, that have mutations respectively in BRCA1 and BRCA2, had very abnormal karyotypes, with many structural and numerical chromosome changes and substantial variation between metaphases. However, two colorectal cancer lines with mutations in BUB1, a spindle checkpoint protein involved in chromosome segregation, had rather simple near-tetraploid karyotypes, with minimal loss or gain of chromosomes other than the endoreduplication event, and minimal structural change. Apart from tetraploidy, these karyotypes were typical of colorectal lines considered to be chromosomally stable. Two lines derived from the same tumour, DLD-1 and HCT-15, with bi-allelic mutation of CHK2, had karyotypes that were typical of near-diploid colorectal lines considered chromosomally stable. The karyotypes observed supported the proposed role for BRCA1 and BRCA2 mutations in chromosomal instability, but showed that the tested mutations in BUB1 and CHK2 did not result in karyotypes that would have been predicted if they were sufficient for chromosomal instability.     

Intergroup Phylogenies in Drosophila as Determined by Comparisons of Salivary Banding Patterns

A salivary gland chromosome phylogeny is presented which summarizes the evolutionary relationships of twenty-two species belonging to the sub-genus Drosophila, and members of the twelve species groups: D. melanica, D. repleta, D. carbonaria, D. polychaeta, D. annulimana, D. robusta, D. carsoni, D. virilis, D. funebris and the "picture-wing," D. mimica and D. crassifemur groups (of Hawaii).-Photographic salivary chromosome maps were prepared for all twenty-two species studied. While the chromosomes of different species belonging to the same group can usually be homologized almost completely, so that construction of intragroup phylogenies is easy, chromosomes of members of different groups are so modified structurally that in most cases only short sections can be fully homologized, and these in only one or two chromosome elements.-Broadly homologous chromosome elements were compared for three species at a time, and on the basis of overlapping homologous sections, or overlapping inversions included within homologous sections, the trio of chromosomes, and the species to which they belonged can often be arranged in a two-step phylogenetic series. Detection of many such ordered trios permits construction of a single phylogenetic scheme encompassing all species.-D. nigromelanica, of the D. melanica group is found to be chromosomally intermediate between the rest of its group and the species belonging to other groups, suggesting that it is the most nearly ancestral member of its group. When trios of species including D. nigromelanica and members of two other species groups are compared, it is found that in twelve of fourteen such comparisons the chromosomes of D. nigromelanica are structurally intermediate between those of the members of the other two species groups, indicating the central position of D. nigromelanica in the phylogeny as a whole.-Available cytological evidence indicates that among the nine continental groups studied, it is the D. robusta group which is chromosomally closest to the Hawaiian "picture-wing" groups. Among the members of the Hawaiian groups it is D. primaeva and D. attigua which are found to be closest to the continental species. This finding tends to confirm the earlier conclusion of Carson and Stalker, based on different evidence, that the above two species were in an ancestral position in the Hawaiian phylogeny.-The relationship of the D. robusta and D. melanica groups demonstrated in this paper, the phylogenies within each of these two groups earlier worked out by Narayanan and by Stalker, and the present geographical distributions of the species within them, require that at least three Asiatic-New World migrations must have occurred during the evolution of the two groups.     

A cytogenetic study of repeated spontaneous abortions.

During a cytogenetic study of spontaneous abortions, successive abortions from 40 couples were karyotyped. The chromosome constitutions of the first and second abortions were found to be highly correlated. In each of 21 instances in which the first abortion was chromosomally normal, the subsequent abortion(s) was normal as well. In nine cases, the two abortions were chromosomally abnormal, and in four of these, both abortions were trisomic. Combined with findings from other studies of consecutive spontaneous abortions, the present data indicate that certain couples are at an increased risk for either repeated chromosomally normal abortions or for repeated trisomic conceptions. The increased risk of trisomy does not seem to be restricted to a particular chromosome, and the magnitude of the risk increase appears to be independent of maternal age.     

Cytogenetics and mechanisms of spontaneous abortions: increased apoptosis and decreased cell proliferation in chromosomally abnormal villi

Genetic defects of the zygote, such as chromosome aberrations, are the most frequent causes of abnormal embryonic development and spontaneous abortion. However, the underlying mechanisms remain unknown. Chromosome aberrations likely cause changes in placental morphology and function (such as size, shape, vascularity, and the presence of trophoblastic inclusion). We postulated that chromosome aberrations may affect rates of cell proliferation or programmed cell death (apoptosis) during the differentiation of chorionic villi. To address these questions, we evaluated cell proliferation using a monoclonal antibody to Ki-67 (a cell-cycle marker) and apoptosis using the in situ end-labeling method (TUNEL) on paraffin-embedded placental tissues. Tissues were obtained from spontaneous abortions in early gestational periods with normal (11 cases) and abnormal karyotypes (15 cases), as well as eight normal control placentas from elective abortions. Apoptotic cells were found in the stroma of all cases, but were significantly higher in number in the stroma of chromosomally abnormal versus chromosomally normal spontaneous abortions. The apoptotic index of the trophoblasts was not significantly different between groups. Cell proliferation was higher in muscularized blood vessels in chromosomally normal placentas (both elective and spontaneous abortions) versus chromosomally abnormal spontaneous abortions. Cell proliferation was different in the trophoblast and stroma between the groups but to a lesser degree than in blood vessels. The morphological and biological data presented here suggest that: (1) chromosomally abnormal spontaneous abortions may occur because of different mechanisms than chromosomally normal spontaneous abortions, (2) apoptosis of the stromal cells and cell proliferation in blood vessels and stroma play an important role in the differentiation and functioning of villi, and (3) these changes could explain the etiology of spontaneous abortion and growth retardation of chromosomally abnormal embryos.     

Toward a comprehensive genetic analysis of male fertility in Drosophila melanogaster

Drosophila melanogaster is a widely used model organism for genetic dissection of developmental processes. To exploit its full potential for studying the genetic basis of male fertility, we performed a large-scale screen for male-sterile (ms) mutations. From a collection of 12,326 strains carrying ethyl-methanesulfonate-treated, homozygous viable second or third chromosomes, 2216 ms lines were identified, constituting the largest collection of ms mutations described to date for any organism. Over 2000 lines were cytologically characterized and, of these, 81% failed during spermatogenesis while 19% manifested postspermatogenic processes. Of the phenotypic categories used to classify the mutants, the largest groups were those that showed visible defects in meiotic chromosome segregation or cytokinesis and those that failed in sperm individualization. We also identified 62 fertile or subfertile lines that showed high levels of chromosome loss due to abnormal mitotic or meiotic chromosome transmission in the male germ line or due to paternal chromosome loss in the early embryo. We argue that the majority of autosomal genes that function in male fertility in Drosophila are represented by one or more alleles in the ms collection. Given the conservation of molecular mechanisms underlying important cellular processes, analysis of these mutations should provide insight into the genetic networks that control male fertility in Drosophila and other organisms, including humans.     

Quantitative trait loci mapping in F(2) crosses between outbred lines

We develop a mixed-model approach for QTL analysis in crosses between outbred lines that allows for QTL segregation within lines as well as for differences in mean QTL effects between lines. We also propose a method called "segment mapping" that is based in partitioning the genome in a series of segments. The expected change in mean according to percentage of breed origin, together with the genetic variance associated with each segment, is estimated using maximum likelihood. The method also allows the estimation of differences in additive variances between the parental lines. Completely fixed random and mixed models together with segment mapping are compared via simulation. The segment mapping and mixed-model behaviors are similar to those of classical methods, either the fixed or random models, under simple genetic models (a single QTL with alternative alleles fixed in each line), whereas they provide less biased estimates and have higher power than fixed or random models in more complex situations, i.e., when the QTL are segregating within the parental lines. The segment mapping approach is particularly useful to determining which chromosome regions are likely to contain QTL when these are linked.     

Skewed X Chromosome Inactivation and Trisomic Spontaneous Abortion: No Association

Several studies suggest that highly skewed X chromosome inactivation (HSXI) is associated with recurrent spontaneous abortion. We hypothesized that this association reflects an increased rate of trisomic conceptions due to anomalies on the X chromosome that lead both to HSXI and to a diminished oocyte pool. We compared the distribution of X chromosome inactivation (XCI) skewing percentages (range: 50%–100%) among women with spontaneous abortions in four karyotype groups—trisomy (n = 154), chromosomally normal male (n = 43), chromosomally normal female (n = 38), nontrisomic chromosomally abnormal (n = 61)—to the distribution for age-matched controls with chromosomally normal births (n = 388). In secondary analyses, we subdivided the nontrisomic chromosomally abnormal group, divided trisomies by chromosome, and classified women by reproductive history. Our data support neither an association of HSXI with all trisomies nor an association of HSXI with chromosomally normal male spontaneous abortions. We also find no association between HSXI and recurrent abortion (n = 45).     

Characterization of deletions in common wheat induced by an Aegilops cylindrica chromosome: detection of multiple chromosome rearrangements

An Aegilops cylindrica chromosome induces terminal deletions of chromosomes in wheat as identified by C-banding. We are constructing high-density physical maps of wheat chromosomes and have detected additional chromosome rearrangements. Among 63 lines with chromosomal subarm deletions in group 7 chromosomes, 7 lines (11.1%) were shown to harbor additional chromosome rearrangements. Two other lines were also omitted from the physical mapping because of the nature of the breakpoint calculations. The presence or absence of chromosome-specific restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) markers indicated that additional interstitial deletions are present in 3 lines (4.8%) with deletions in the short chromosome arms and in 4 lines (6.3%) with deletions in the long chromosome arms. We also used chromosome pairing analysis of F₁ plants of deletion lines with double ditelosomic lines of Chinese Spring wheat to detect small terminal deletions. The deletion of the most distal 1% of chromosome arm 7AL was associated with a pairing reduction of 60%.     

Specific break points in chromosomally abnormal human fibroblast subpopulations.

Common chromosome break points were found in a banding study of 25 chromosomally abnormal fibroblast clones derived from normal individuals and patients exposed to environmental carcinogens. Of 81 break points, 38% appeared to be in terminal bands. This specificity did not appear to be the consequence of the biased recognition of exchanges. In addition, specificities for break points at exact interstitial loci were detected. The presence of clones in cultures grown in a wide variety of conditions, the finding of two identical clones in two independent lines from the same biopsy, and the absence of any obvious continuing instability in lines support the view that chromosomally abnormal fibroblasts occur in vivo.     

Aneuploid epididymal sperm detected in chromosomally normal and Robertsonian translocation-bearing mice using a new three-chromosome FISH method

We present a new method to detect epididymal sperm aneuploidy (ESA) in mice using simultaneous fluorescence in situ hybridization (FISH) with DNA probes specific for mouse chromosomes X, Y and 8. The method was applied to Robertsonian (Rb) translocation (8.14) heterozygotes and homozygotes as well as the chromosomally normal B6C3F1. The sex ratios of sperm did not differ from the expected 1∶1 and the hybridization efficiencies were ≈99.7% for over 60 000 sperm analyzed. Mice heterozygous for Rb (8.14) produced about tenfold higher rates of sperm with chromosome 8 hyperhaploidy than did Rb (8.14) homozygotes or chromosomally normal mice, while frequencies of sperm with hyperhaploidies for chromosomes X and Y were unaffected in all three lines of mice. Hyperhaploid frequencies obtained with the ESA method were consistent with those of the previous testicular FISH method and were validated by published data obtained by conventional cytogenetic analyses (meiotic metaphase II and first cleavage). Thus, the mouse three-chromosome ESA assay together with the previously developed aneuploidy assay for human sperm constitute a promising pair of interspecific biomarkers for comparative studies of the genetic and physiologic mechanisms of the induction and persistence of aneuploidy in male germ cells. Edited by: T. Hassold     

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non-redundant groups of cDNAs were obtained. Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region. We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis. To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.     

Polyploid abnormalities in day 3 and day 5 merino sheep embryos

The chromosome complement was assessed in Merino sheep embryos collected at 3 and 5 days after the onset of oestrus. Donor ewe treatments were: untreated, or immunized against androstenedione (day 3); and untreated, or treated with follicle-stimulating hormone (FSH), or treated with FSH plus immunization against androstenedione (day 5). No significant differences in the frequency of chromosomally abnormal embryos between treatment groups within each age group were observed, so the data have been combined. Euploid abnormalities were observed in 10.8% of the day-3 embryos (4/37), with the abnormalities being one 1n, one 3n and two 5n. Embryos with euploidy (10%) were also observed at day 5, with three 1n/2n mosaics and a 3n embryo present in a sample of 40. These data suggest that chromosomally aberrant embryos are not lost before day 5 of development.     

Screen for new mutations on the 2nd chromosome involved in indirect flight muscle development in Drosophila melanogaster.

An extensive ethylmethanesulfonate mutagenesis of Drosophila melanogaster was undertaken to isolate the stronger alleles of 3 indirect flight-muscle mutations. We isolated 17 strong mutant lines, with nearly complete penetrance and expressivity, using direct screening under polarized light, from more than 1700 mutagenized chromosomes. On complementation, we found 11 of these 17 mutant lines to be alleles of 3 indirect flight-muscle mutations (Ifm(2)RU1, 3 noncomplementing lines; ifm(2)RU2, 6 alleles; ifm(2)RU3, 2 alleles) of the previously isolated 8 complementation groups (Ifm(2)RU1to ifm(2)RU8). In addition, we found 6 new complementation groups with strong defects in adult-muscle morphology; we named these ifm(2)RS1 to ifm(2)RS6. All mutant lines were mapped by meiotic recombination, and 5 of the 6 new complementation lines were mapped using chromosome deficiencies. ifm(2)RS1 maps to a region that harbors ifm(2)RU4 (a mutation that was isolated previously); however, theses are not alleles because each complements the other mutation, and the mutant-muscle phenotype is very different. We used direct screening under polarized light to find recessive mutations; although this method was labor intensive, it can be used to identify recessive genes involved in myogenesis, unlike screens for flightlessness or wing-position defects. This screen identifies regions on the second chromosome that harbor probable genes that are likely expressed in the mesoderm and are thought to be involved in myogenesis. This screen has generated valuable resources that will help us to understand the role of many molecular players involved in myogenesis.     

THE CONTRIBUTION OF GENE MOVEMENT TO THE “TWO RULES OF SPECIATION”

The two “rules of speciation”—the Large X‐effect and Haldane's rule—hold throughout the animal kingdom, but the underlying genetic mechanisms that cause them are still unclear. Two predominant explanations—the “dominance theory” and faster male evolution—both have some empirical support, suggesting that the genetic basis of these rules is likely multifarious. We revisit one historical explanation for these rules, based on dysfunctional genetic interactions involving genes recently moved between chromosomes. We suggest that gene movement specifically off or onto the X chromosome is another mechanism that could contribute to the two rules, especially as X chromosome movements can be subject to unique sex‐specific and sex chromosome specific consequences in hybrids. Our hypothesis is supported by patterns emerging from comparative genomic data, including a strong bias in interchromosomal gene movements involving the X and an overrepresentation of male reproductive functions among chromosomally relocated genes. In addition, our model indicates that the contribution of gene movement to the two rules in any specific group will depend upon key developmental and reproductive parameters that are taxon specific. We provide several testable predictions that can be used to assess the importance of gene movement as a contributor to these rules in the future.     

Sperm aging in the male and cytogenetic anomalies. An animal model

Summary Superovulated females, outbred ICR Swiss, were inseminated naturally with spermatozoa aged 6–20 days in the male genital tract by bilateral ligation of the corpus epididymis. Females inseminated by the males (also ICR Swiss) mating at 3-day intervals prior to ligation and those mated to sham-operated males served as controls. A total of 1167 fertilized one-cell zygotes of which 868 were at metaphase first cleavage were recovered 33–35 h post-HCG. First-cleavage divisions were analyzed in 631, or 72%, of all zygotes at metaphase and late prophase. The gametic origin of chromosome anomalies was determined on the basis of differential condensation of the chromosomes. The sex ratio of the zygotes was unaffected by aging sperm. In 321 zygotes from the controls the frequency of trisomy was 3.1%, monosomy 2.2%, triploidy 0.93%, and structural rearrangements 0.31%. Aging of sperm for 6 days did not significantly alter the number of heteroploid zygotes recovered. For the periods beyond day 6 and in the combined experimental series there was a highly significant increase in the number of all chromosome anomalies compared with controls. In the experimental zygotes, the incidence of trisomy was 9.7%, monosomy 7.4%, triploidy 3.9%, and structural anomalies 2.6%. There were also significant differences between control and experimental in the origin of the anomalies. The male genome was implicated significantly more often than the female in the origin of trisomies in the experimental series compared with the controls. It was also the direct source of all the structural anomalies and the majority of the triploids in the experimentals, where 8 of 9 were a result of diploid sperm. Therefore, the over-riding mechanism involved in the production of chromosomally anomalous offspring from aging sperm seems to be altered conditions of competition between chromosomally balanced and unbalanced sperm. Additionally, chromosomally normal sperm in an aging population may be affected in some aspects of their physiology so that they create preferential loss of maternally derived chromosomes leading to monosomy or nuclear fragmentation. The implications of these findings for the etiology of human chromosome anomalies are discussed.     

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.     

Polymorphism and differentiation of rainbow trout Y chromosomes.

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.     

Genetic tests for autosomal non-disjunction and chromosome loss in mice

Two new genetic methods for detecting autosomal non-disjunction and chromosome loss in mice are described. Both methods involve the use of marker genes and Robertsonian translocations, the latter present only in tester parents, to detect events in chromosomally normal mice. With the Rb method, the tester parent carries one or more Robertsonian translocations heterozygously; with the MBH method the tester parent carries two Robertsonian translocations showing monobrachial homology. The high rates of meiotic non-disjunction in the tester mice provide gametes with specific extra or missing chromosomes which, at fertilization, can allow the survival of a proportion of the zygotes lacking or carrying an extra specific chromosome from tested chromosomally normal parents. The Rb method has been assessed for X-ray-induced chromosome 1 loss and non-disjunction in mature oocytes and also for such chromosome 1 loss from the maternal pronuclei of 1-cell zygotes. The MBH method has been assessed for X-ray-induced chromosome 1 loss in male postmeiotic cells and for non-disjunction in spermatocytes. Both methods proved effective in detecting chromosome 1 loss. A single case of the much rarer non-disjunctional event was also found. As applied, both methods compared favourably with the numerical sex chromosome anomaly (NSA) method and have considerable potential for further development.     

Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction.

While it is known that all chromosomes are susceptible to meiotic nondisjunction, it is not clear whether all chromosomes display the same frequency of nondisjunction. By use of multicolor FISH and chromosome-specific probes, the frequency of disomy in human sperm was determined for chromosomes 1, 2, 4, 9, 12, 15, 16, 18, 20, and 21, and the sex chromosomes. A minimum of 10,000 sperm nuclei were scored from each of five healthy, chromosomally normal donors for every chromosome studied, giving a total of 418,931 sperm nuclei. The mean frequencies of disomy obtained were 0.09% for chromosome 1; 0.08% for chromosome 2; 0.11% for chromosome 4; 0.14% for chromosome 9; 0.16% for chromosome 12; 0.11% for chromosomes 15, 16, and 18; 0.12% for chromosome 20; 0.29% for chromosome 21; and 0.43% for the sex chromosomes. Data for chromosomes 1, 12, 15, and 18, and the sex chromosomes have been published elsewhere. When the mean frequencies of disomy were compared, the sex chromosomes and chromosome 21 had significantly higher frequencies of disomy than that of any other autosome studied. These results corroborate the pooled data obtained from human sperm karyotypes and suggest that the sex chromosome bivalent and the chromosome 21 bivalent are more susceptible to nondisjunction during spermatogenesis. From these findings, theories proposed to explain the variable incidence of nondisjunction can be supported or discarded as improbable.     

Molecular genetic approaches to the study of cellular senescence.

Normal cells in culture exhibit limited division potential, which is used as a model for cellular aging. In contrast, tumor-derived, carcinogen- or virus-transformed cells are capable of dividing indefinitely (immortal). Fusion of normal with immortal human cells yielded hybrids having limited life span, indicating that cellular senescence is a dominant phenotype and that immortality is recessive. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. In order to identify the chromosomes and genes involved in growth regulation, that had been modified in immortal cells, we used the technique of microcell fusion to introduce either a normal human chromosome 11 or 4 into cell lines representative of the different complementation groups. Chromosome 11 had no effect on the in vitro life span of the different immortal human tumor lines. However, when a normal human chromosome 4 was introduced into cell lines assigned to complementation group B, the cells lost the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. These results suggest that a gene(s) on human chromosome 4 has been modified in immortal cell lines assigned to complementation group B, to allow escape from senescence. They also provide evidence for a genetic basis for cellular aging.