Assignment of a processed mouse Aprt pseudogene to the same chromosome as the functional gene

A novel genetic system has been used to demonstrate that a processed adenine phosphoribosyltransferase (Aprt) pseudogene is located on mouse chromosome 8, which is the same chromosome that carries the functional Aprt gene. A restriction fragment length polymorphism associated with the pseudogene was found to segregate concordantly with chromosome 8 in APRT- mutants of a near-diploid cell line that had lost one copy of the chromosome.     

Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice.

A mouse model was generated to investigate loss of heterozygosity (LOH) events in somatic cells. The adenine phosphoribosyltransferase ( Aprt ) gene was disrupted in embryonic stem cells using a conventional gene targeting approach and subsequently Aprt hetero-zygous and homozygous mice were derived. Aprt homozygous deficient animals were viable though the mendelian inheritance pattern was skewed. On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Aprt heterozygous mice the mean spontaneous mutant frequency at the Aprt locus was 8.7 x 10(-6) while the frequency was 0.8 x 10(-6) at the hypoxanthine phosphoribosyltransferase locus. In order to determine whether LOH events contribute to the high spontaneous mutant frequency at the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded and analysed by allele-specific PCR. In 97 (69%) of these clones the wild-type allele had been lost. Nine of the mutant clones were characterized in more detail using dual-coloured fluorescence in situ hybridization analysis. Five out of six of the mutant clones which arose from an LOH event, based on the PCR assay, contained a duplication of the targeted allele. Therefore, mitotic recombination or chromosome loss followed by duplication of the remaining homologue appears to be the predominant mechanism for the in vivo generation of Aprt mutant T-lymphocytes.     

Analysis of Aprt deficient mutants of Friend erythroleukaemia cells

Cytogenetic analysis of mutants of the Friend Erythroleukaemia cell line deficient in adenine phosphoribosyl transferase (Aprt) was undertaken to ascertain whether non-disjunctional events were involved in the production of this mutation. All mutant clones examined were found to carry two copies of chromosome 8, the chromosome to which Aprt maps. Since the two homologues are distinguishable by silver staining, it was also clear that no mutants contained two copies of one homologue having lost the other. Treatment of mutants with 5-azacytidine failed to reactivate the Aprt locus.     

Gene inactivation as a mechanism for the expression of recessive phenotypes.

A series of Chinese hamster ovary cell hybrids were constructed which were heterozygous at the emtB and chr loci. These loci encode two recessive drug-resistance genes (emetine resistance and chromate resistance, respectively) located on a structurally hemizygous region on the long arm of chromosome 2. These heterozygous hybrids therefore exhibit wild-type sensitivity to both emetine and chromate. Drug-resistant variants were then selected in medium containing either emetine or chromate, and the mechanism of reexpression of the recessive drug-resistant allele was determined by karyotypic analysis of the resultant colonies. In previous studies at these loci we have determined that segregation of the recessive phenotype occurs primarily by (1) the loss of the chromosome 2 carrying the wild-type, drug-sensitive, allele, (2) deletion of the long arm of chromosome 2, or (3) loss of one chromosome 2 followed by duplication of the remaining homologue. However, a small proportion of segregants have also been detected which may have arisen by the mechanisms of de novo gene inactivation or mutation. In this report, hybrids are described which were constructed to allow selection for the retention of the chromosome carrying the wild-type allele and which therefore optimize isolation of these rare segregants. We demonstrate by karyotypic analysis, mutation frequency analysis, and microcell-mediated chromosome transfer that these rare segregants occur primarily by gene inactivation. We also demonstrate a dramatic increase in the proportion of segregants occurring by gene inactivation in two of these hybrids as compared with those previously reported, indicating that this mechanism may be an important mode of phenotype segregation in diploid cells and, therefore, in the development of cancers--such as the childhood tumors retinoblastoma and Wilms tumor--resulting from recessive alleles     

Somatic events unmask recessive cancer genes to initiate malignancy

A heritable mutation predisposes an individual to certain childhood malignancies, such as retinoblastoma and Wilms' tumor. The chromosomal locations of the genes responsible for the predisposition are known by linkage with chromosomal deletions and enzyme markers. A study of these tumors in comparison to the normal constitutional cells of the patients, using enzyme and DNA markers near the predisposing genes, has shown that these genes are recessive to normal wild-type alleles at the cellular level. Expression of the recessive phenotype (malignancy) involves the same genetic events that were observed in Chinese hamster cell hybrids carrying recessive drug resistance genes. In both the experimental and clinical situations, the wild-type allele is most commonly eliminated by chromosome loss with duplication of the mutant chromosome. Simple chromosome loss and mitotic recombination have been documented in both systems. In the remaining 30% of cases, inactivation or microdeletion of the wild-type allele are assumed to be responsible for expression of the recessive phenotype. Osteosarcoma is a common second tumor in patients who have had retinoblastoma. Studies with markers in osteosarcoma show that these tumors also result from unmasking of the recessive phenotype by loss of the normal allele at the retinoblastoma locus, whether or not the patient had retinoblastoma. Subsequent chromosomal rearrangements and amplification of oncogenes that occur in these homozygous tumors provide progressive growth advantage. In other malignancies, in which studies have so far focused on oncogene amplification and chromosomal rearrangements, unmasking of recessive mutations may also be the critical initiating events.     

Post-zygotic breakage of a dicentric chromosome results in mosaicism for a telocentric 9p marker chromosome in a boy with developmental delay

Chromosomal rearrangements resulting in an inverted duplication and a terminal deletion (inv dup del) can occur due to three known mechanisms, two of them resulting in a normal copy region between the duplicated regions. These mechanisms involve the formation of a dicentric chromosome, which undergo breakage during cell division resulting in cells with either an inverted duplication and deletion or a terminal deletion. We describe a mosaic 3 year old patient with two cell lines carrying a chromosome 9p deletion where one of the cell lines contains an additional telocentric marker chromosome. Our patient is mosaic for the product of a double breakage of a dicentric chromosome including a centric fission. Mosaicism involving different rearrangements of the same chromosome is rare and suggests an early mitotic breakage event.     

人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。     

Segregation of recessive phenotypes in somatic cell hybrids: role of mitotic recombination, gene inactivation, and chromosome nondisjunction.

Somatic cell hybrids heterozygous at the emetine resistance locus (emtr/emt+) or the chromate resistance locus (chrr/chr+) are known to segregate the recessive drug resistance phenotype at high frequency. We have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To follow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion (2p-) or a long-arm addition (2q+). Karyotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt+- or chr+-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci, giving rise to homozygous resistant segregants; (iii) inactivation of the emt+ or chr+ alleles; and (iv) loss of the emt+- or chr+-bearing chromosome with duplication of the homologous chromosome carrying the emtr or chrr allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.     

The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm

chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase theta;. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority ( approximately 90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.     

A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants.

Previously we described the dose-response relationship for X-ray-induced mutation of the two homologous alleles of the thymidine kinase (tk) gene in a human lymphoblastoid cell line (Amundson and Liber, 1991). The two alleles were differentially mutable by X-rays, with one allele 6-10 times more mutable than the other. This difference was shown to be due to the virtual absence of the class of slow growth mutants from one allele. In the present report, restriction fragment length polymorphism (RFLP) analyses of informative markers along chromosome 17 have been used to delineate a region of chromosome 17 in which heterozygosity is lost with relatively high frequency among slow growth TK- mutants from the more mutable allele. However, loss of heterozygosity of this region has never been observed in normal growth mutants obtained from the more mutable allele, or in TK- mutants from the other, less mutable, allele. This may indicate the presence of a heterozygous essential gene on chromosome 17 distal to TK1.     

Centromeric inactivation in a dicentric human Y;21 translocation chromosome

A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down’s syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event. Received: 30 January 1997; in revised form: 3 April 1997 / Accepted: 8 May 1997     

Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora

New mutations are found among approximately 20% of progeny when one or both parents carry eas allele UCLA191 (eas(UCLA), easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa3 deficient, linkage group VII), resulting in thin, "transparent" mycelial growth. Other eas alleles fail to produce cya-8 mutant progeny. The recurrent cya-8 mutations are attributed to repeat-induced point mutation (RIP) resulting from a duplicated copy of cya-8+ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas(UCLA)-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas(UCLA), the new mutations arise exclusively in eas(UCLA) nuclei. Mutation of cya-8 is suppressed when a long unlinked duplication is present. Stable cya-8 mutations are effectively eliminated in crosses homozygous for rid, a recessive suppressor of RIP. The eas(UCLA) allele is associated with a long paracentric inversion. A discontinuity is present in eas(UCLA) DNA. The eas promoter is methylated in cya-8 progeny of eas(UCLA), presumably by the spreading of methylation beyond the adjoining RIP-inactivated duplication. These findings support a model in which an ectopic insertion that created a mutation at the target site acts as a locus-specific mutator via RIP.     

Bacillus subtilis mutants with alterations in ribosomal protein S4.

Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsD1 allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.     

Mutations in Genes Encoding Essential Mitotic Functions in DROSOPHILA MELANOGASTER

Temperature-sensitive mutations at 15 loci that affect the fidelity of mitotic chromosome behavior have been isolated in Drosophila melanogaster. These mitotic mutants were detected in a collection of 168 EMS-induced X-linked temperature-sensitive (ts) lethal and semilethal mutants. Our screen for mutations with mitotic effects was based upon the reasoning that under semirestrictive conditions such mutations could cause an elevated frequency of mitotic chromosome misbehavior and that such events would be detectable with somatic cell genetic techniques. Males hemizygous for each ts lethal and heterozygous for the recessive autosomal cell marker mwh were reared under semirestrictive conditions, and the wings of those individuals surviving to adulthood were examined for an increased frequency of mwh clones. Those mutations producing elevated levels of chromosome instability during growth of the wing imaginal disc were also examined for their effects on chromosome behavior in the cell lineages producing the abdominal cuticle. Fifteen mutations affect chromosome behavior in both wing and abdominal cells and thus identify loci generally required for the fidelity of mitotic chromosome transmission. Mapping and complementation tests show that these mutations represent 15 loci. One mutant is an allele of a locus (mus-101) previously identified by mutagen-sensitive mutants and a second mutant is an allele of the lethal locus zw 10.--The 15 mutants were also examined cytologically for their effects on chromosomes in larval neuroblasts. Taken together, the results of our cytological and genetical studies show that these mutants identify loci with wild-type functions necessary for either maintenance of chromosome integrity or regular disjunction of chromosomes or chromosome condensation. Thus, these mutations define a broad spectrum of genes required for the normal execution of the mitotic chromosome cycle.     

Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.     

A comparison of induced mutation at homologous alleles of the tk locus in human cells.

Using a restriction fragment length polymorphism which can distinguish the two copies of the thymidine kinase (tk) gene in the TK6 human lymphoblastoid cell line, we have identified heterozygous subclones with alternate active alleles. Quantitative mutagenesis studies with X-rays revealed a markedly different response, depending on which homolog carried the active allele. The slopes of the dose-response curves differed by approximately 10-fold for mutation of the two alleles and this relationship held true for several independently isolated cell lines. Only one of the cell lines showed a different response to ethyl methanesulfonate. There were no differences among any of the cell lines at the X-linked hprt locus. Analyses of TK- mutants recovered from these cell lines indicated that the reduced yield of mutants from the one allele may be due, at least in part, to a lack of a specific class of TK- mutant, that is, the slow-growing mutants which have been associated with large-scale mutagenic events.     

Genetics of barley hooded suppression

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

Chromosome studies on a human liposarcoma cell line.

Numerical and structural chromosome analysis of a human retroperitoneal liposarcoma cell line maintained under standard cell culture conditions revealed a very stable hypodiploid mode. If the cells were not trypsinized for several generations, a near-triploid stemline, which was generally a duplication of the hypodiploid mode, emerged. Some chromosomes appeared to be relatively stable pairs (1, 2, 7, 9, and 12), but most had "lost" one homolog or both (4 and 21) or were rearranged into "new" marker chromosomes. Quantitation of the genetic material showed a loss of 12.0 +/- 3.7% per spread. Only one characteristically long marker chromosome, which is present in every cell, could be identified with certainty as a translocation between chromosomes 4 and 11. Several of the marker chromosomes showed interstitial negatively staining regions with the trypsin-Giemsa method.     

Two Sequence-Ready Contigs Spanning the Two Copies of a 200-kb Duplication on Human 21q: Partial Sequence and Polymorphisms

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications.