A cloned I-factor is fully functional in Drosophila melanogaster

Summary I-R hybrid dysgenesis in Drosophila melanogaster occurs in female progeny of crosses between reactive strain females and inducer strain males, and is controlled by transposable elements called I-factors. These are 5.3 kb elements that are structurally similar to mammalian LINE elements and other retroposons. We have tested the activity of an I-factor directly, by introducing it into the genome of a reactive strain, using P-element mediated transformation. It confers the complete inducer phenotype on the reactive strain, and can stimulate dysgenesis when transformed males are mated with reactive females. It has transposed in the transformed lines, and we have cloned one of the transposed copies. This is the first time that it has been possible to demonstrate that a particular retroposon is transposition proficient, and to compare donor and transposed elements. We propose a mechanism for I-factor transposition based on these results, and the coding capacity of these elements. We have been unable to detect either autonomous transposition of a complete I-factor from a plasmid injected into reactive strain embryos, or transposition of a marked I-factor when co-injected with a complete element.     

Colonization of heterochromatic genes by transposable elements in Drosophila

As a further step toward understanding transposable element-host genome interactions, we investigated the molecular anatomy of introns from five heterochromatic and 22 euchromatic protein-coding genes of Drosophila melanogaster. A total of 79 kb of intronic sequences from heterochromatic genes and 355 kb of intronic sequences from euchromatic genes have been used in Blast searches against Drosophila transposable elements (TEs). The results show that TE-homologous sequences belonging to 19 different families represent about 50% of intronic DNA from heterochromatic genes. In contrast, only 0.1% of the euchromatic intron DNA exhibits homology to known TEs. Intraspecific and interspecific size polymorphisms of introns were found, which are likely to be associated with changes in TE-related sequences. Together, the enrichment in TEs and the apparent dynamic state of heterochromatic introns suggest that TEs contribute significantly to the evolution of genes located in heterochromatin.     

The generation of cloned Drosophila melanogaster

We report here the first successful use of embryonic nuclear transfer to create viable adult Drosophila melanogaster clones. Given the generation time, cost effectiveness, and relative ease of embryonic nuclear transplant in Drosophila, this method can provide an opportunity to further study the constraints on development imposed by transplanting determined or differentiated nuclei.     

The expression and genomic organization of randomly selected cloned Drosophila melanogaster genes

A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences.     

The structure of heterochromatic DNA is altered in polyploid cells of Drosophila melanogaster.

DNA sequences within heterochromatin are often selectively underrepresented during development of polyploid chromosomes, and DNA molecules of altered structure are predicted to form as a consequence of the underrepresentation process. We have identified heterochromatic DNAs of altered structure within sequences that are underrepresented in polyploid cells of Drosophila melanogaster. Specifically, restriction fragments that extend into centric heterochromatin of the minichromosome Dp(1;f)1187 are shortened in polyploid cells of both the ovary and salivary gland but not in the predominantly diploid cells of the embryo or larval imaginal discs and brains. Shortened DNA molecules were also identified within heterochromatic sequences of chromosome III. These results suggest that the structure of heterochromatic DNA is altered as a general consequence of polyploid chromosome formation and that the shortened molecules identified form as a consequence of heterochromatic underrepresentation. Finally, alteration of heterochromatic DNA structure on Dp(1;f)1187 was not correlated with changes in the variegated expression of the yellow gene located on the minichromosome.     

X-chromosome inversion in Drosophila melanogaster accompanied by a mosaic-type position effect: an analysis using cloned DNA fragments

DNA fragments carrying the genes affected by position effect variegation in the In(1LR)pn2a chromosome were revealed. In situ hybridizations demonstrate preservation of specific DNA sequences in the polytene chromosome regions containing the inactivated genes. It was shown that both euchromatic-heterochromatic boundaries in the In(1LR)pn2a chromosome contain the mobile element B104 (roo).     

Inactivation of Reporter Genes by Cloned Heterochromatic Repeats of Drosophila melanogaster is Accompanied by Chromatin Compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter genemini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of themini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-whitehave been found.     

Somatic synapsis of eu- and heterochromatic regions of Drosophila melanogaster chromosomes

Quantitative cytogenetical analysis has been used to study the synapsis of D. melanogaster neuroblast mitotic chromosomes from normal females, flies with heterozygous deletions, duplications or inversions in the heterochromatic regions of chromosome 2 and in triploid females. In all these genotypes chromocentric fusion of heterochromatic regions of heterologous chromosomes is observed. Eu- and heterochromatic regions of homologous chromosomes are intimately paired at the same time during the cell cycle. The structural rearrangements lead to reduced frequencies of chromocentric association as well as of homologous synapsis compared with the frequencies in the wild-type. The results obtained are discussed with respect to the general problem of the homologous interaction of chromosomes and the significance of heterochromatin for these processes.     

Transposable elements as artisans of the heterochromatic genome in Drosophila melanogaster

Over 50 years ago Barbara McClintock discovered that maize contains mobile genetic elements, but her findings were at first considered nothing more than anomalies. Today it is widely recognized that transposable elements have colonized all eukaryotic genomes and represent a major force driving evolution of organisms. Our contribution to this special issue deals with the theme of transposable element-host genome interactions. We bring together published and unpublished work to provide a picture of the contribution of transposable elements to the evolution of the heterochromatic genome in Drosophila melanogaster. In particular, we discuss data on 1) colonization of constitutive heterochromatin by transposable elements, 2) instability of constitutive heterochromatin induced by the I factor, and 3) evolution of constitutive heterochromatin and heterochromatic genes driven by transposable elements. Drawing attention to these topics may have direct implications on important aspects of genome organization and gene expression.     

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

Pairing-dependent mislocalization of a Drosophila brown gene reporter to a heterochromatic environment.

We describe the precise positioning of a reporter gene within heterochromatin where it may be silenced. A transposition of the 59E-60A region into pericentric heterochromatin ensnares distal 59E-60A via somatic pairing. The frequency with which a brown (bw) reporter gene in 59E is silenced is influenced by chromosomal configurations. Silencing occurs only when the bw+ reporter is unpaired due to heterozygosity with a deficiency, where the frequency of bw+ reporter expression is correlated with the extent of bw gene and flanking sequence present. Surprisingly, the frequency of pairing between the transposition in heterochromatin and distal 59E observed cytologically is indistinguishable from the frequency of pairing of homologous chromosomes at 59E in wild-type larval brains, regardless of configuration. Therefore, bringing a susceptible reporter gene into close proximity with heterochromatin does not necessarily affect its expression, but local pairing changes resulting from altered chromosomal configurations can lead to silencing. We also find that an ensnared distal copy of bw that is interrupted by a heterochromatic insertion enhances silencing. This demonstrates that bw can be simultaneously acted upon by pericentric and distal blocks of heterochromatin.