A modifier of the Bg element of the Bg-rbg transposable element system of maize

A modifier of the Bg autonomous element of the Bg-rbg system of transposable elements has been found in the genotype of the inbred maize line 346. In the presence of this modifier (designated Mbg), the frequency of reversion of mutable allele o2-lf in combination with the Bg-lf element increases by 7–24 times. An increase in the Mbg dosage by three times increases the o2-lf reversion frequency by a factor of about two. The joint presence of Mbg and Bg-lf in the same genotype before meiosis is necessary for the expression of the Mbg modifying effect. The possible nature and mechanism of action of the novel modifier are discussed.     

The frequency of reversion of the opaque2 locus mutable alleles controlled by the Bg-rbg system of mobile elements in maize

Maize lines differing in the frequency of reversion of the opaque2 (o2) mutable alleles controlled by the system of Bg-rbg transportable elements were studied. In the presence of the Bg regulatory element, these alleles can revert to normal. When reversion occurs prior to the first division of the primary endosperm nucleus, either phenotypically normal kernels or whole endosperm revertants (WER) develop. Low frequency of whole endosperm revertant formation may be produced by different genetic mechanisms. The frequency of WER formation was shown to nonlinearly depend on the dose of the Bg-hf regulatory element. A dose increase from two to three failed to cause an essential increase in the number of revertants. The regulatory elements Bg-lf and Bg-hf differed in ability to induce excision of the receptor element at the same dose. The frequency of reversion of the receptive alleles was shown to be regulated by epigenetic mechanisms so that high frequency of reversion of receptive alleles requires preliminary premeiotic association between the regulatory and receptor elements. The inheritance of the maize alleles o2-hf and o2-lf proved to be similar to that an3 mutable alleles in petunia.     

Control of the Frequency of Reversion of the Mutable Alleles at the opaque2 Locus by the Bg-rbg System of Transposable Elements in Maize

Maize lines differing in the frequency of reversion of the opaque2 (o2) mutable alleles controlled by the Bg-rbg system of transposable elements were studied. In the presence of the Bg regulatory element, these alleles can revert to normal. When reversion occurs prior to the first division of the primary endosperm nucleus, phenotypically normal kernels or whole endosperm revertants (WER) develop. It was shown that the low frequency of formation of whole endosperm revertant may be produced by different genetic mechanisms. The frequency of WER formation was shown to nonlinearly depend on the dose of the Bg-hf regulatory element. A dose increase from two to three failed to cause an essential increase in the number of revertants. The regulatory elements Bg-lf andBg-hfdiffered in their ability to induce excision of the receptor element at the same dose. The frequency of reversion of the receptive alleles was shown to be regulated by epigenetic mechanisms so that high frequency of reversion of receptive alleles requires preliminary premeiotic association between the regulatory and receptor elements. The inheritance of the maize alleles o2-hfand o2-lf proved to be similar to that of an3 mutable alleles in petunia.     

A mariner-like element with a 5' lesion in Drosophila simulans

The unstable white-S2 (wS2) allele of the white gene occurred spontaneously in the S2 strain of Drosophila simulans. This mutation was caused by insertion of the submariner element, a mariner-like element with an abnormal tandem duplication of the 5' inverted terminal repeat (ITR). Although it has an incomplete ITR, submariner excises efficiently. The rate of somatic reversion, estimated by the number of eye-color mosaic flies, was 79.9%, and the reversion frequency in the germline was 0.6%. The change to the 5' ITR contributes to make this transposon precise excision.     

Occurrence and behavior of the components of the 02-m(r)-Bg system of maize controlling elements

Summary o2-m(r) is an unstable allele of the O2 locus responding to the regulatory element Bg by somatic reversion. The spontaneous occurrence and the properties of the components of this system of controlling elements have been investigated. The system appears to have some degree of specificity for the O2 locus, because the majority of spontaneous O2 mutations are responsive to Bg. The component at the controlled locus undergoes frequent changes in state, while the Bg element appears more stable. Bg activity was revealed in 11 out of 108 open-pollinated varieties of maize. Most of the newly isolated Bg elements are linked with the O2 locus. The timing of induction of reversion events (which are restricted to mitotic division leading to egg or pollen maturation and to the developing endosperm) appears to correlate with the degree of linkage between Bg and the O2 locus. Germinal reversions of o2-m(r) to wild type give rise with a frequency around 5×10^-4 to unstable phenotypes. Some peculiar features of the o2-m(r)-Bg system of controlling elements are discussed.     

Germ line and somatic instability of a white mutation in Drosophila mauritiana due to a transposable genetic element

A spontaneous white mutation recovered in Drosophila mauritiana is unstable and reverts to normal eye color at a frequency greater than 4 per 1,000 X-chromosomes. Germ line reversion occurs at a high rate in D. mauritiana males and in interspecific hybrid females, while the rate is depressed in D. mauritiana females. These events are not restricted to the germ line, as cases of variegated patterns of eye pigmentation, indicating somatic reversion, are recovered at a frequency comparable to that of the male germ line reversion rate. Germ line reversion events are genetically stable, while the somatic variegation patterns are not heritable. The patterns of eye pigment variegation produced suggests that reversion events are occurring throughout development. Whole genome DNA digests blotted and probed with the cloned D. melanogaster white gene indicate that this unstable white mutation in D. mauritiana is associated with an insertion of DNA that is lost upon reversion to wild type, indicating that this DNA insert is in fact a transposable element.     

The origin of footprints of the Tc1 transposon of Caenorhabditis elegans.

Mutations caused by the Tc1 transposon in Caenorhabditis elegans can revert by loss of the element. Usually the transposon leaves behind a 'footprint'--a few nucleotides of one or both ends of the transposon. Two possible explanations for the footprints are: (i) imprecise excision or (ii) interrupted repair. Here I report that in a diploid animal having a homozygous Tc1 insertion the reversion frequency is approximately 10(-4), and a Tc1 footprint is found; however when the corresponding sequence on the homologous chromosome is wild-type, the reversion frequency is 100 times higher, and the reverted sequence is precise. Apparently the footprint results from incomplete gene conversion from the homologous chromosome, and not from imprecise excision of Tc1. These results support the following model: Tc1 excision leaves a double-strand DNA break, which can be repaired using the homologous chromosome or sister chromatid as a template. In heterozygotes repair can lead to reversion; in homozygotes Tc1 is copied into the 'empty' site, and only rare interrupted repair leads to reversion, hence the 100-fold lower reversion rate and the footprint.     

P element excision in Drosophila melanogaster and related drosophilids

Summary The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlike previous assays, this mobility assay permitted the recovery of excision products from plasmids regardless of whether the excision event was precise or imprecise. Both quantitative and qualitative differences between the products of excision in the various species studied were observed. The frequency with which P element excision products were recovered from D. melanogaster was 10-fold greater than from D. virilis and C. procnemis; however, the proportion of all excision events resulting in the reversion of a P-induced mutant phenotype was the same. Virtually all excision products recovered, including those resulting in a reversion of the mutant phenotype, did not result in the exact restoration of the original target sequence. Sequence analysis suggested that duplex cleavage at the 3 and 5 termini of the P element, or their subsequent modification, occurred asymmetrically and interdependently. P element-encoded transposase was not absolutely required for P element excision.     

Germ line and somatic instability of a white mutation in Drosophila mauritiana due to a transposable genetic element

A spontaneous white mutation recovered in Drosophila mauritiana is unstable and reverts to normal eye color at a frequency greater than 4 per 1,000 ×-chromosomes. Germ line reversion occurs at a high rate in D. mauritiana males and in interspecific hybrid females, while the rate is depressed in D. mauritiana females. These events are not restricted to the germ line, as cases of variegated patterns of eye pigmentation, indicating somatic reversion, are recovered at a frequency comparable to that of the male germ line reversion rate. Germ line reversion events are genetically stable, while the somatic variegation patterns are not heritable. The patterns of eye pigment variegation produced suggests that reversion events are occurring throughout development. Whole genome DNA digests blotted and probed with the cloned D. melanogaster white gene indicate that this unstable white mutation in D. mauritiana is associated with an insertion of DNA that is lost upon reversion to wild type, indicating that this DNA insert is in fact a transposable element.     

Effect of the adenine level of a culture medium on the frequency of revertants in haploid yeasts

The frequency of reversion from adenine auxotrophy in the yeast Saccharomyces cerevisiae increases with the decrease of adenine concentration in the medium: for the 8PG-59 strain (a ade2-192 rad2) the reversion frequency is 1.7 X 10(-8), 3.2 X 10(-6) and 1.8 X 10(-5) per cell division, the initial adenine concentrations being 10, 1 and 0.1 mg/l, respectively. An increase of the reversion frequency with the culture age in the stationary phase of growth is demonstrated using an improved method of registration of revertants, with the initial concentration of 0.1 mg/l of adenine in the medium. The reversion frequency was 9.1 X 10(-7) on the 7th day, 1.8 X 10(-6) on the 10th day and 1.8 X 10(-5) on the 14th day.     

The influence of deficiencies in DNA-repair on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

MR is a frequently occurring mutator in Drosophila melanogaster inducing mutation by the incorporation of insertion sequences. In the presence of MR a mutation at the singed (sn) locus induced by MR, reverts to wild-type at a high frequency of 1.7%. This reversion system which presumably involves the removal of an insertion element, was used to study the effects of defective DNA repair. Thus, reversion frequencies were compared in progeny of flies with mei-9, deficient for excision repair, mei-41, deficient for post-replication repair, or with both mei-9 and mei-41. The data show that under conditions of defective DNA repair, the frequency of MR-mediated reversion, is consistently decreased in comparison to repair-proficient conditions. This effect is explained by assuming that defective repair interferes with some steps in the process of reverse mutation involving the removal of insertion sequences. The observed reduction in reversion frequency may well result from selective elimination of cells in which the reversion process has not been completed.     

Repair of P element ends following hybrid element excision leads to recombination in Drosophila melanogaster

P elements are thought to replicate themselves starting with the association of the left and right ends, followed by a cut-copy-paste process. An abnormal form of this process has been shown to occur when the associated left and right ends come from sister elements rather than from the same element, leading to formation of a 'hybrid element.' These ends can insert nearby in the genome to produce recombination, with associated structural changes. We have previously increased the frequency of such 'hybrid element insertion' by combining end-deleted elements in trans in a genotype with a left-end on one chromosome and a right-end on the homologous chromosome. Although many recombinants produced by this genotype have structural changes expected with insertion, nearly 50% of the predicted insertional recombinants contain no structural change. We present evidence using RFLP markers closely linked to the end-deleted elements that in these cases the P element ends dissociate before insertion, and are subsequently ligated together following a process analogous to synthesis-dependent strand annealing. The results suggest that broken ends containing P elements are resolved by the same repair process as ends not containing P elements, and that such repair from hybrid element events may occur in the majority of cases.     

Physiological role of phosphorylation of the cyclic AMP response element binding protein in rat cardiac nuclei

We determine whether the cyclic AMP signal transduction pathway affects phosphorylation of cyclic AMP response element binding protein and increases muscle gene expression in the heart. Elevation of cyclic AMP results in phosphorylation of the binding protein which is detected using an antibody specific for the phosphorylated, but not the unphosphorylated, form. The protein is present, but not phosphorylated, within the nuclei of myocytes in intact neonatal rat hearts and in high-density cultures. It is not expressed in low-density cultures. Increasing the amount of phosphorylated cyclic AMP with either isoproterenol or forskolin also increases the frequency and force of the beating. The phosphorylated form of the response element binding protein is visible in the nuclei by 10 min and persists for 2 h of drug treatment. A 1.5-fold increase in skeletal α-actin and α-myosin gene expression is detected after 48 h of isoproterenol treatment. However, blockage of beating with a calcium channel blocker (verapamil) in the presence of cyclic AMP results in a similar increased gene expression. This suggests that muscle gene expression can be regulated directly by the cyclic AMP pathway, probably via phosphorylation of the cyclic AMP response element binding protein but independent of contractile activity. Received: 1 December 1995 / Accepted: 2 May 1996     

The mechanism of the mutagenic action of hydroxylamine XIII. Reversion of phage MS2 amber mutants in the presence of hydroxylamines.

The replication of the phage MS2 in the presence of either hydroxylamine (HA) or O-methylhydroxylamine (OMHA) (mutagenesis in vivo) results in an increase in the reversion frequency of two amber mutations in the maturation protein. When acting on the extracellular phage (mutagenesis in vitro) the mutagens do not affect the reversion frequency. The most probable mode of mutagenic action of the hydroxylamines on the vegetative MS2 phage involves the enzymic formation of modified precursors and their incorporation into RNA.     

Involvement of phosphoinositide turnover in tracheary element differentiation in Zinnia elegans L. cells

Mesophyll cells of Zinnia elegans L., cultured in the presence of phytohormones, will transdifferentiate and undergo programmed cell death to become tracheary elements, thick-walled cells of the xylem. This system is a model system for study of plant cell development and differentiation. We report that a high concentration of extracellular Ca(2+) is necessary during the first 6 h of culturing for tracheary elements to form. Extracellular Ca(2+) is still required at later times, but at a much lower concentration. When cells transdifferentiate in adequate Ca(2+), microsomal phospholipase C activity increases and levels of inositol 1,4,5-trisphosphate rise at about hour 4 of culturing. The production of inositol 1,4,5-trisphosphate appears to be important for tracheary element formation, since inhibitors of phospholipase C inhibit both inositol 1,4,5-trisphosphate production and tracheary element formation. Pertussis toxin, an inhibitor of GTP-binding proteins, inhibits transdifferentiation and eliminates inositol 1,4,5-trisphosphate production. Tracheary element formation was not completely abolished by inhibitors that eliminated inositol 1,4,5-trisphosphate production, suggesting the involvement of other pathways in regulating transdifferentiation.     

Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.     

Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 x 10-2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

Kinetic studies and characteristics of a modifier of Ca2+-stimulated ATPase

1. The thermostable modifier increases the Mg2+-stimulated ATPase with a parallel decrease of Ca2+-stimulated ATPase. 2. The modifier inhibited Ca2+-stimulated ATPase by diminishing the velocity without any significant effect on the apparent affinity of binding of Ca2+. 3. The inhibition of Ca2+-stimulated ATPase was independent of Ca2+ concentration and could not be reversed by increasing Ca2+ levels. 4. Monovalent cations and calmodulin increased the Mg2+-stimulated ATPase in the presence of the modifier.     

Relative effects of segregation and recombination on the evolution of sex in finite diploid populations

The mechanism of reproducing more viable offspring in response to selection is a major factor influencing the advantages of sex. In diploids, sexual reproduction combines genotype by recombination and segregation. Theoretical studies of sexual reproduction have investigated the advantage of recombination in haploids. However, the potential advantage of segregation in diploids is less studied. This study aimed to quantify the relative contribution of recombination and segregation to the evolution of sex in finite diploids by using multilocus simulations. The mean fitness of a sexually or asexually reproduced population was calculated to describe the long-term effects of sex. The evolutionary fate of a sex or recombination modifier was also monitored to investigate the short-term effects of sex. Two different scenarios of mutations were considered: (1) only deleterious mutations were present and (2) a combination of deleterious and beneficial mutations. Results showed that the combined effects of segregation and recombination strongly contributed to the evolution of sex in diploids. If deleterious mutations were only present, segregation efficiently slowed down the speed of Muller''s ratchet. As the recombination level was increased, the accumulation of deleterious mutations was totally inhibited and recombination substantially contributed to the evolution of sex. The presence of beneficial mutations evidently increased the fixation rate of a recombination modifier. We also observed that the twofold cost of sex was easily to overcome in diploids if a sex modifier caused a moderate frequency of sex.     

The effect of two chemical mutagens ENU and MMS on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

The effects of two mutagens ENU and MMS characterized by different alkylation patterns have been studied on the reversion of an MR-induced singed mutation to wild-type. Reversion of this unstable singed mutation under the influence of MR is assumed to represent the removal or transposition of an insertion element. Since MR acts primarily in spermatogonia, the mutagens were fed to 1st instar larvae. Recessive lethal tests were carried out simultaneously to calibrate for the mutagenic effectiveness of the chemicals. For both powerful mutagens, it was observed that the frequency of reversion remained far below of what would have been expected on the basis of the mutagenic effectiveness, as registered in the lethal tests. Thus 1 mM ENU, 5 mM and 10 mM MMS did not affect the reversion frequency at all, and with 3 mM ENU only a doubling of the reversion frequency was observed, despite a 5-fold increase in the lethal frequency. The threshold at 1 mM EMU and the low effectiveness of 3 mM on the reversion process are taken as an indication that ENU affected the transposition process in an indirect manner, rather than the excision events themselves. The data obtained with Drosophila are consistent with the microbial observations in that mutation involving removal or transposition of an insertion element is not affected by mutagenic treatments. This finding may have consequences for the evaluation of induced genetic damage on the basis of the spontaneous load of genetic detriment in man.

An incidental observation was that non-MR Cy larvae exhibited greater sensitivity to the induction of recessive lethals by MMS than MR-individuals.