High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.     

Aberrant pre-mRNA maturation is caused by LINE insertions into introns of the white gene of Drosophila melanogaster.

Insertional mutagenesis screens have provided thousands of mutant alleles for analysing genes of varied functions in Drosophila melanogaster. We here document mechanisms of insertional mutagenesis by a LINE element, the I factor, by determining the molecular structure of RNAs produced from two alleles of the white gene of D.melanogaster, wIR1 and wIR6. These alleles result from insertion of the I factor into introns of the gene. We show that sequences present within the element direct aberrant splicing and termination events. When the I factor is inserted within the white first intron it may lead to the use of a cryptic 3' splice site which does not contain the dinucleotide AG. This splicing gives rise to a chimeric messenger RNA whose synthesis is controlled differently in tissues where the mutated gene is expressed. When the I factor is inserted within the white last intron it induces synthesis of truncated mRNAs. These results provide, for the first time, mechanisms for I factor insertional mutagenesis. They are discussed in the more general context of RNA processing in Drosophila and the evolution of eukaryotic gene introns.     

Sequence and heterogeneity in the 5S RNA gene cluster of Drosophila melanogaster.

The sequence of the entire 5S RNA gene of Drosophila melanogaster was determined by sequencing collectively 23 copies contained in a cloned fragment of Drosophila DNA and by sequencing individually four subcloned gene copies. A repetitive heptamer (GCTG CCT) present in variable numbers immediately following the coding sequence, is responsible for the length heterogeneity in the spacer region. Some of the gene copies contain a nucleotide change in the coding region which results in a new site for the restriction enzyme Mn1 I. The variant 5S RNA produced by these gene copies has not been detected in vivo. Two other single nucleotide variations were identified in the spacer region.     

A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.

The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of Tpl we used a rapid combination of transposon tagging, chromosome microdissection and PCR to clone the P element that had transposed into the Tpl region. That P element is located within the gene for a new and unique member of the RNA helicase family. This new helicase differs from all others known by having glycine-rich repeats at both the amino and carboxyl termini. Curiously, genetic analysis shows that the P element inserted into this gene is not responsible for the Tpl mutant phenotype. We present possible explanations for these findings.     

Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition

The Tetrahymena thermophila ribosomal DNA (rDNA) replicon contains dispersed cis-acting replication determinants, including reiterated type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4. Here, we show that TIF4, previously implicated in cell cycle-controlled DNA replication and rDNA gene amplification, is the T. thermophila origin recognition complex (TtORC). We further demonstrate that TtORC contains an integral RNA subunit that participates in rDNA origin recognition. Remarkably, this RNA, designated 26T, spans the terminal 282 nts of 26S ribosomal RNA. 26T RNA exhibits extensive complementarity to the type I element T-rich strand and binds the rDNA origin in vivo. Mutations that disrupt predicted interactions between 26T RNA and its complementary rDNA target change the in vitro binding specificity of ORC and diminish in vivo rDNA origin utilization. These findings reveal a role for ribosomal RNA in chromosome biology and define a new mechanism for targeting ORC to replication initiation sites.     

A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene.

A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.