Somatic Instability Research Articles

Somatic instability of CTG repeat in myotonic dystrophy.

An unstable expansion of the CTG repeat in the 3' untranslated region of the myotonin protein kinase (MT-PK) gene is the mutation specific for myotonic dystrophy (DM). To examine somatic stability of the repeat, we studied tissue variability of the repeat size. In five DM patients, the restriction fragment containing the repeat region was substantially larger in skeletal muscle than in peripheral blood leukocytes (PBL). In addition, one normal subject showed a size discrepancy in one of the normal alleles by one repeat on the polymerase chain reaction analysis. In most DM patients, the repeat size of native PBL differed from the transformed lymphoblastoid cells after passages. In contrast, various tissues from a congenital DM patient showed a similar size of the expanded repeat, including the transformed lymphoblastoid cells. We conclude that somatic instability of the CTG repeat may cause substantial tissue variability of the CTG repeat size in adult-onset DM, providing a potential mechanism for the variable pleiotropism.

Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype.

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the Ro-1A phenotype were observed in 65,000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360,000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wildtype rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425,000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.

A Tetranucleotide Repeat Mouse Minisatellite Displaying Substantial Somatic Instability during Early Preimplantation Development

The highly variable mouse minisatellite Hm-2 is located on chromosome 9 and consists of GGCA tetranucleotide repeats with alleles containing up to 5000 repeat units. This locus is unstable with a germline mutation rate to new length alleles of at least 3.6% per gamete. Hm-2 also shows substantial somatic instability, producing mutational mosaicism detectable in 20% of adult mice. Analysis of allele dosage in mice carrying somatic mutations, plus studies of mosaicism in mouse embryos and extraembryonic tissues, suggests that somatic mutant alleles preferentially arise during preimplantation development and particularly during the first two cell divisions after fertilization.

Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.

GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis.

GLOBOSA (GLO) is a homeotic gene whose mutants show sepaloid petals and carpelloid stamens. The similarity of Glo mutants to those of the DEFICIENS (DEFA) gene suggests that the two genes have comparable functions in floral morphogenesis. The GLO cDNA has been cloned by virtue of its homology to the MADS-box, a conserved DNA-binding domain also contained in the DEFA gene. We have determined the structure of the wild type GLO gene as well as of several glo mutant alleles which contain transposable element insertions responsible for somatic and germinal instability of Glo mutants. Analyses of the temporal and spatial expression patterns of the DEFA and GLO genes during development of wild type flowers and in flowers of various stable and unstable defA and glo alleles indicate independent induction of DEFA and GLO transcription. In contrast, organ-specific up-regulation of the two genes in petals and stamens depends on expression of both DEFA and GLO. In vitro DNA-binding studies were used to demonstrate that the DEFA and GLO proteins specifically bind, as a heterodimer, to motifs in the promoters of both genes. A model is presented which proposes both combinatorial and cross-regulatory interactions between the DEFA and GLO genes during petal and stamen organogenesis in the second and third whorls of the flower. The function of the two genes controlling determinate growth of the floral meristem is also discussed.

Constitutional ring chromosomes and tumour suppressor genes.

The types of malignancy reported in carriers of constitutional ring chromosomes r(11), r(13), and r(22) are concordant with the chromosomal assignment of tumour suppressor loci associated with Wilms' tumour, retinoblastoma, and meningioma. It is suggested that the somatic instability of ring chromosomes may play a role in this association and that constitutional ring chromosomes may be a source for mapping of tumour suppressor loci with the potential for covering most or all of the human genome. The hypothesis predicts the presence of a locus on chromosome 10 associated with follicular carcinoma of the thyroid, in line with previous cytogenetic findings of rearrangements involving chromosome 10 in thyroid tumours, and a locus on chromosome 22 associated with testicular cancer. Development of neurofibromatoses (NF) that do not fulfil the clinical criteria of neurofibromatosis type 2 (NF2) in carriers with r(22) suggests either the presence of an additional NF locus on chromosome 22 or that ring chromosome mediated predisposition to somatic mutation of a specific tumour suppressor may be associated with atypical development of features usually associated with germline mutations.

Reactivation of Mutator transposable elements of maize by ultraviolet light.

After epigenetic loss of Mutator activity, the family of Mu elements in Zea mays becomes immobile and highly methylated; in addition, Mu9, the presumptive autonomous regulatory element, is transcriptionally silent and its copy number decreases in successive crosses to non-Mutator lines. Spontaneous reactivation, scored as restoration of somatic instability of potentially mutable alleles of Bronze-2, of such cryptic Mutator lines is rare, occurring with a frequency of about 10(-4). Irradiation of pollen with 254 nm ultraviolet light increases reactivation rate in the progeny kernels by up to 40-fold. Accompanying reactivation, the copy number of Mu9 elements increased, two-fold in one line and 20 to 40-fold in a second line. Reactivation may involve direct DNA damage or immediate physiological stress in the treated pollen.

Somatic instability of a Drosophila chromosome.

A mitotically unstable chromosome, detectable because of mosaic expression of marker genes, was generated by X-ray mutagenesis in Drosophila. Nondisjunction of this chromosome is evident in mitotic chromosome preparations, and premature sister chromatid separation is frequent. The mosaic phenotype is modified by genetic elements that are thought to alter chromatin structure. We hypothesize that the mitotic defects result from a breakpoint deep in the pericentric heterochromatin, within or very near to the DNA sequences essential for centromere function. This unique chromosome may provide a tool for the genetic and molecular dissection of a higher eukaryotic centromere.

Plant-transposable elements and gene tagging.

While most genes are lined up on chromosomes like pearls on a string, one group of genes, socalled transposons or transposable elements, are highly mobile. Transposons frequently alter their chromosomal location and consequently often induce mutations, by integrating into other genes and destroying their structural integrity. An additional feature of transposon-induced mutations is their somatic instability. It is by this property that the presence of transposable elements is recognized. While the organism develops, the transposable element leaves its site of integration within the gene and hence the function of that gene is restored in this cell as well as in its progeny. These excision events result in a mosaic tissue of mutant and revertant (wild-type) cells. Such mosaic or variegation patterns strike the eye, provided genes involved in coloration were affected by the transposable element.

Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation

The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km(r)) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km(r) by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km(r); then the Km(r) progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km(r) gene. Initial tests for TMV resistance indicated possible linkage between Km(r) and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30-40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km(r) and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km(r) gene. Linkage between Km(r) and the N gene in this plant line has been verified in each of two additional backcross generations.

Molecular genetics of the fragile-X syndrome: a novel type of unstable mutation

Fragile-X syndrome, the most common inherited form of mental retardation, has a very unusual mode of inheritance. The disease is caused by a multistep expansion, in successive generations, of a polymorphic CGG repeat localized in a 5' exon of FMR-1, a gene of unknown function. Two main mutation types have been categorized. Premutations are moderate expansions of the repeat and do not cause mental retardation. Full mutations are found in affected individuals and involve larger expansions of the repeat, with abnormal methylation of the neighboring CpG island. The full mutations demonstrate striking somatic instability and extinguish expression of FMR-1. Premutations are changed to full mutation only when transmitted by a female with a frequency that increases up to 100% as a function of the initial size of the premutation. Direct detection of the mutations provides an accurate test for pre- and postnatal diagnosis of the disease, and for carrier detection. A similar unstable expansion of a trinucleotide repeat occurs in myotonic dystrophy.

Moderate instability of the trinucleotide repeat in spino bulbar muscular atrophy.

Increased length of a protein-coding CAG repeat within the androgen receptor gene appears to be the only type of mutation responsible for spino-bulbal muscular atrophy (SBMA or Kennedy disease). We have analysed a large 4-generation SBMA family and found that the mutant allele was unstable upon transmission from parent to child, with a documented variation from 46 to 53 repeats and a tendency to increase in size (7 increases and a single decrease in 17 events), which appeared stronger upon transmission from a male than from a female. Our results suggest also limited somatic instability of the abnormal allele, with observable variation of up to 2-3 repeats. This indicates that the behavior of the CAG repeat is similar to that observed for small premutations in the fragile X syndrome, or small abnormal alleles in myotonic dystrophy, two diseases which are caused by expansion of an unstable trinucleotide repeat.

Genome scanning of higher eukaryotes by two-dimensional DNA typing using micro- and minisatellite core probes

Electrophoretic separation of DNA restriction fragments in two dimensions according to two independent criteria, i.e., size and basepair sequence, allows all restriction fragments of DNA molecules of up to 5 million bp, such as the Escherichia coli genome, to be resolved in a single gel. For more complex genomes, hybridization analysis of the two-dimensional separation patterns is necessary to obtain interpretable data. Here we describe a detailed protocol for two-dimensional DNA typing of mammalian (human and cattle) and plant (tomato) genomic DNA using micro- and minisatellite core probes. Spot patterns comprising 100–800 spots were obtained. This method of simultaneously scoring hundreds of genetic loci has various applications e.g., in mapping genomes, defining DNA markers for hereditary traits by genetic linkage analysis, and detecting somatic instability.

Molecular analysis of the loss of somatic instability in the bz2::mu1 allele of maize.

Multiple genetic and epigenetic changes were detected within one plant generation at the bz2::mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10(-4) per gamete. One germinal excision event was found that contained an 8 bp deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75-77 bp deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC 1 (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.

Plant transposable elements.

Transposable elements were first detected in maize by Barbara McClintock and reported in the 1950s. These were revealed by experiments that were designed for a cytogenetic study involving the short arm of chromosome 9. It was in the progeny of plants undergoing the chromosomal type of breakage—fusion—bridge cycle that a burst of somatic instability and mutations appeared. Initially, McClintock focused on the breakage events occuring at a specific locus between waxy (wx) gene and the centromere on the short arm of chromosome 9. She termed this “the standard locus of Ds (Dissociation).” However, these breakage events required the presence of another autonomous element, which she termed the activator (Ac). She noted cases in which Ds activity disapeared from the standard locus and reappeared elsewhere in the genome. In addition, somatic instability was observed at several gene loci. Such mutator activity was also under the control of the Ac. McClintock made an operational distinction between genes and transposable elements. Genes occupy fixed loci and transposable elements can move from one location to another. Because the frequency and temporal occurrence of somatic instability were under the control of the Ds state and Ac dose, respectively, she called these “controlling elements.” McClintock (1951) also characterized another family of transposable elements which she termed Supprssor-mutator (Spm), which was independently isolated and characterized by Peterson (1961), but termed Enhancer (En).

Monitoring genomic alterations with a panel of oligonucleotide probes specific for various simple repeat motifs

Germline and somatic instability of the human genome was studied, using synthetic oligonucleotides specific for simple repeat motifs. The following probes were used: (GTG)5, (GACA)4, (GATA)4, (CT)8, (TTAGGG)3, (GT)8, (GAA)6 and (GGAT)4. Each of them is unique with respect to the target regions recognized in the genome. Thus compilation of the various fingerprint data provides a complex map of the genome (and its deviations). While the fingerprints of differentiated somatic tissues never showed any alterations, in tumor tissues (namely gliomas) many changes could be detected. Most of the latter reflect secondary karyological aberrations. In nearly one third of the gliomas, drastically amplified and apparently monomorphic DNA fragments were identified. This marker should make it possible to deal with causal pathogenetic mechanisms as well as novel diagnostic strategies.

Floral homeotic mutations produced by transposon-mutagenesis in Antirrhinum majus.

To isolate and study genes controlling floral development, we have carried out a large-scale transposon-mutagenesis experiment in Antirrhinum majus. Ten independent floral homeotic mutations were obtained that could be divided into three classes, depending on whether they affect (1) the identity of organs within the same whorl; (2) the identity and sometimes also the number of whorls; and (3) the fate of the axillary meristem that normally gives rise to the flower. The classes of floral phenotypes suggest a model for the genetic control of primordium fate in which class 2 genes are proposed to act in overlapping pairs of adjacent whorls so that their combinations at different positions along the radius of the flower can specify the fate and number of whorls. These could interact with class 1 genes, which vary in their action along the vertical axis of the flower to generate bilateral symmetry. Both of these classes may be ultimately regulated by class 3 genes required for flower initiation. The similarity between some of the homeotic phenotypes with those of other species suggests that the mechanisms controlling whorl identity and number have been highly conserved in plant evolution. Many of the mutations obtained show somatic and germinal instability characteristic of transposon insertions, allowing the cell-autonomy of floral homeotic genes to be tested for the first time. In addition, we show that the deficiens (def) gene (class 2) acts throughout organ development, but its action may be different at various developmental stages, accounting for the intermediate phenotypes conferred by certain def alleles. Expression of def early in development is not necessary for its later expression, indicating that other genes act throughout the development of specific organs to maintain def expression. Direct evidence that the mutations obtained were caused by transposons came from molecular analysis of leaf or flower pigmentation mutants, indicating that isolation of the homeotic genes should now be possible.

Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors.

Deficiens (defA+) is a homeotic gene involved in the genetic control of Antirrhinum majus flower development. Mutation of this gene (defA-1) causes homeotic transformation of petals into sepals and of stamina into carpels in flowers displaying the 'globifera' phenotype, as shown by cross sections and scanning electronmicroscopy of developing flowers. A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted 'flower specific' cDNA library. The identity of this cDNA with the defA+ gene product has been confirmed by utilizing the somatic and germinal instability of defA-1 mutants. According to Northern blot analyses the defA+ gene is expressed in flowers but not in leaves, and its expression is nearly constant during all stages of flower development. The 1.1 kb long mRNA has a 681 bp long open reading frame that can code for a putative protein of 227 amino acids (mol. wt 26.2 kd). At its N-terminus the DEF A protein reveals homology to a conserved domain of the regulatory proteins SRF (activating c-fos) in mammals and GRM/PRTF (regulating mating type) in yeast. We discuss the structure and the possible function of the DEF A protein in the control of floral organogenesis.

Effect of mass selection for seed density in populations derived from Avena abyssinica x A. sativa amphidecaploids

Seed of two heterogeneous amphidecaploid Avena populations (NY79143 and NY79148) were separated into three groups based on seed density using a commercial gravity table. Bulk populations were evaluated in a field trial to test the effect of selection for seed density on agronomic characteristics, while chromosome behavior was studied on similarly selected individual plants grown in a coldframe. Selection for seed density was associated with improved agronomic characteristics for individual plants grown in coldframes but the treatment effect was not significant in the field trial. Population and treatment effects were significant for chromosome number. Mean chromosome numbers were higher in NY79148 than in NY79143, and increased with selection for seed density. Populations did not differ for mean somatic instability but did show interactive population*treatment effects. Several generations of selection for seed density is suggested.