ABSTRACT
An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlueScript II KS+ (pBSLTR) vector, and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the ORF of the lacZ gene contained in the vector; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 1 x 10(-3) to 1 x 10(-4), the frequency of spontaneously appearing blue colonies not exceeding 10(-9) to 10(-8). The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYC 184) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the beta-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them, gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).
Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/enzymology , Integrases/physiology , Models, Biological , Retroelements/genetics , Transcription Factors/genetics , Animals , Drosophila melanogaster/genetics , Escherichia coli/genetics , Recombinant Proteins/genetics , Recombination, Genetic , Terminal Repeat Sequences/geneticsABSTRACT
A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.
Subject(s)
Drosophila/genetics , Retroelements , Animals , Base Sequence , Blotting, Southern , Codon, Terminator , DNA Primers , Open Reading Frames , Species SpecificityABSTRACT
This article summarizes the results of a ten-year study of genetic instability of a mutator strain of Drosophila melanogaster caused by transposition of the gypsy retrotransposon. The results of other authors working with an analogous system are analyzed. Possible mechanisms are suggested for the interaction of gypsy with the cell gene flamenco that participates in transposition control of this mobile element.
Subject(s)
Drosophila melanogaster/genetics , Mutation , Retroelements/physiology , Animals , Drosophila Proteins/genetics , Evolution, Molecular , Gene Expression RegulationABSTRACT
The distribution of two variants of MDG4 (gypsy) was analyzed in several Drosophila melanogaster strains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutant flamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.
Subject(s)
Drosophila melanogaster/genetics , Retroelements , Animals , Autoradiography , Blotting, Southern , In Situ HybridizationABSTRACT
Distribution of two structural functional variants of the MDG4 (gypsy) mobile genetic element was examined in 44 strains of Drosophila melanogaster. The results obtained suggest that less transpositionally active MDG4 variant is more ancient component of the Drosophila genome. Using Southern blotting, five strains characterized by increased copy number of MDG4 with significant prevalence of the active variant over the less active one were selected for further analysis. Genetic analysis of these strains led to the suggestion that some of them carry factors that mobilize MDG4 independently from the cellular flamenco gene known to be responsible for transposition of this element. Other strains probably contained a suppressor of the flam- mutant allele causing active transpositions of the MDG4. Thus, the material for studying poorly examined relationships between the retrovirus and the host cell genome was obtained.
Subject(s)
Retroelements , Animals , Blotting, Southern , Drosophila melanogaster , Genome , Mutation , Species SpecificitySubject(s)
Drosophila melanogaster/genetics , Retroelements/genetics , Animals , Cells, Cultured , Gene Amplification , Kinetics , TransfectionSubject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Insect Hormones/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Point Mutation , Retroelements , Animals , Base Sequence , Gene Amplification , Homeodomain Proteins , Male , Microfilament Proteins , Molecular Sequence Data , Oligodeoxyribonucleotides , Restriction Mapping , Transcription FactorsABSTRACT
A previously described system of a Drosophila melanogaster mutative strain (MS), which originates from a stable strain (SS), is characterized by genetic instability caused by transposition of the retrotransposon gypsy. New unstable strains were obtained by microinjections of the gypsy transposable copy into SS embryos. In situ hybridization experiments revealed amplification and active transposition of gypsy in SS derivatives. At the same time, introduction of the gypsy transposable copy into another stable strain (208) did not lead to appearance of genetic instability. Genetic instability in the MS system is apparently induced by a combination of two factors: the presence of a gypsy transposable copy and mutation(s) in the gene(s) regulating its transpositions.
Subject(s)
Drosophila melanogaster/genetics , Genome , Retroelements , Animals , Blotting, Northern , Blotting, Southern , Genes, Insect , Mutation , Transformation, GeneticABSTRACT
The copies of mobile element mdg4 (gypsy) were cloned from two different D. melanogaster strains. The first strain (stable) is characterized by small number of mdg4 copies and their constant localization in chromosomes. The second strain (unstable), which was originated from the first one, is characterized by increased number of mdg4 copies and higher frequency of its transpositions. The two copies of mdg4, cloned from stable and unstable strains differ in their structure and represent two different types of mdg4. Southern blot-analysis of structural organization of mdg4 in these two strains showed, that in the stable strain there are mdg4 copies of one type, and in the unstable strain there are both, but only the mdg4 copies of another type were amplified. In was shown by transient-expression experiments, that in spite of the structural differences both types of mdg4 were able to be transcribed. Nevertheless, in flies of stable strain the mdg4 transcripts were not detected.
Subject(s)
DNA Transposable Elements/genetics , Drosophila melanogaster/genetics , Animals , Base Sequence , Blotting, Southern , Cloning, Molecular , Genetic Variation/genetics , Molecular Sequence Data , Restriction Mapping , Species Specificity , Transcription, Genetic/geneticsABSTRACT
The Drosophila melanogaster cultured cells were subjected to stable transformation by cotransfection with two plasmids, one of which conferred G418 resistance and the second contained the Drosophila retrotransposon MDG4 (gypsy) under control of different promoter fragments of the heat shock protein gene hsp70. Transcription of these constructs as well as of the endogenous gypsy was examined in the conditions of heat shock. Active degradation of preexisting MDG4 transcripts is observed after heat shock. Transcription of MDG4 is restored during recovery but its termination and/or 3' end processing becomes aberrant.
