Flamenco, a Gene Controlling the Gypsy Retrovirus of Drosophila Melanogaster

Gypsy is an endogenous retrovirus of Drosophila melanogaster. It is stable and does not transpose with detectable frequencies in most Drosophila strains. However, we have characterized unstable strains, known as MG, in which it transposes at high frequency. These stocks contain more copies of gypsy than usual stocks. Transposition results in mutations in several genes such as ovo and cut. They are stable and are due to gypsy insertions. Integrations into the ovo(D1) female sterile-dominant mutation result in a null allele of the gene and occurrence of fertile females. This phenomenon, known as the ovo(D1) reversion assay, can be used to quantitate gypsy activity. We have shown that the properties of MG strains result from mutation of a host gene that we called flamenco (flam). It has a strict maternal effect on gypsy mobilization: transposition occurs at high frequency only in the germ line of the progeny of females homozygous for mutations of the gene. It is located at position 65.9 (20A1-3) on the X chromosome. The mutant allele present in MG strains is essentially recessive. Flamenco seems to control the infective properties of gypsy.     

A P Element Containing Suppressor of Hairy-Wing Binding Regions Has Novel Properties for Mutagenesis in Drosophila Melanogaster

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.     

Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.     

The Non-Random Pattern of Insertion of IS2 into the hemB Gene of Escherichia coli

The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non-random manner into a 240 bp segment at the 5′ end of the gene (region I). Twenty-one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3′ terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3′ terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.     

Transposition and gene disruption in the male germline of the mouse

We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation‐site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)‐trap transposon vectors, were outcrossed to wild‐type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics. genesis 30:82–88, 2001. © 2001 Wiley‐Liss, Inc.     

The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects.

Mutations in the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of a number of tissue-specific mutations caused by insertion of a gypsy retrotransposon. The su(Hw) gene encodes a zinc finger protein which binds to a 430 bp region of gypsy shown to be both necessary and sufficient for its mutagenic effects. su(Hw) protein causes mutations by inactivation of enhancer elements only when a su(Hw) binding region is located between these regulatory sequences and a promoter. To understand the molecular basis of enhancer inactivation, we tested the effects of su(Hw) protein on expression of the mini-white gene. We find that su(Hw) protein stabilizes mini-white gene expression from chromosomal position-effects in euchromatic locations by inactivating negative and positive regulatory elements present in flanking DNA. Furthermore, the su(Hw) protein partially protects transposon insertions from the negative effects of heterochromatin. To explain our current results, we propose that su(Hw) protein alters the organization of chromatin by creating a new boundary in a pre-existing domain of higher order chromatin structure. This separates enhancers and silencers distal to the su(Hw) binding region into an independent unit of gene activity, thereby causing their inactivation.     

Genetic Evidence That the Ovo Locus Is Involved in Drosophila Germ Line Sex Determination

Zygotically contributed ovo gene product is required for the survival of female germ cells in Drosophila melanogaster. Trans-allelic combinations of weak and dominant ovo mutations (ovoD) result in viable germ cells that appear to be partially transformed from female to male sexual identity. The ovoD2 mutation is partially suppressed by many Sex-lethal alleles that affect the soma, while those that affect only the germ line fail to interact with ovoD2. One of two loss-of-function ovo alleles is suppressed by a loss-of-function Sex-lethal allele. Because ovo mutations are germ line dependent, it is likely that ovo is suppressed by way of communication between the somatic and germ lines. A loss-of-function allele of ovo is epistatic to germ line dependent mutations in Sex-lethal. The germ line dependent sex determination mutation, sans fille, and ovoD mutations show a dominant synergistic interaction resulting in partial transformation of germ line sexual identity. The ovo locus appears to be involved in germ line sex determination and is linked in some manner to sex determination in the soma.     

Translocation specificity of the Tn3 element: characterization of sites of multiple insertions

247 independent events involving insertion of the TN3 transposable element into a 4 kb constructed plasmid (pTU4) of partially known DNA sequence were studied by restriction endonuclease mapping, and 65 of these insertion sites were examined further by DNA sequence analysis. Our results show that the previously proposed regional specificity for Tn3 insertion is associated with a strong preference for AT-rich segments as insertion sites. Moreover, multiple insertions of the Tn3 occurred at certain AT-rich nucleotide positions, and 23 of 26 independent insertion events at a single nucleotide position were found to be in the same orientation. A region of the recipient plasmid showing major homology with the terminal 18 bp of Tn3 was identified in the vicinity of an 11 nucleotide segment that included three insertional hot spots and 36 independent insertions. Our results indicate that the site and orientation of insertion of Tn3 are at least partly determined by the primary nucleotide sequence of the recipient genome, and suggest that insertional hot spots may result from the combined effects of AT richness plus homology of the recipient genome with the terminal sequences of Tn3.     

Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer.

The cut locus is a complex gene whose function is necessary for specification of a number of cell types, including the external sensory organs. The cut wing class of mutations of the cut locus are homozygous viable and lack tissue from the wing margin, which is normally composed of external sensory organs and noninnervated bristles. Expression of cut was examined in the developing wings of wild-type and mutant pupae using an antiserum against Cut protein. Cut is expressed in all of the external sensory organs of the wing and the noninnervated bristles of the posterior margin. The cut wing class of mutations prevents Cut expression specifically in the wing margin mechanoreceptors and noninnervated bristles, apparently preventing neural differentiation. The transformed cells die soon after differentiation would have occurred. We identify an enhancer, located about 80 kb upstream of the cut gene promoter, that confers expression in the cells of the mechanoreceptors and noninnervated bristles from a heterologous promoter. The 27 gypsy retrotransposon insertions that prevent expression in these margin cells, all occur between this enhancer and the promoter. These gypsy insertions probably interfere with the interaction between the enhancer and the cut gene promoter.     

Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor with an Mr of 78,000. It is required for the binding and uptake of ferrichrome and is the receptor for bacteriophages T5, T1, phi 80, and UC-1 as well as for colicin M. The fhuA gene was cloned into pBR322, and the recombinant plasmid pGC01 was mutagenized by the insertion of 6-base-pair TAB (two amino acid Barany) linkers into CfoI and HpaII restriction sites distributed throughout the coding region. A library of 18 TAB linker insertions in fhuA was generated; 8 of the mutations were at CfoI sites and 10 were at HpaII sites. All mutations inserted a hexamer that encoded a unique SacI site. A large deletion in fhuA was also isolated by TAB linker mutagenesis. Except for the deletion mutant, all of the linker insertion mutant FhuA proteins were found in the outer membrane in amounts similar to those found in the wild type. Five of the linker insertion mutants were susceptible to cleavage by endogenous proteolytic activity: a second FhuA-related band that migrated at approximately 72 kilodaltons could be detected on Coomassie blue-stained gels and on Western blots (immunoblots) by using a carboxy terminus-specific anti-peptide antibody. Receptor functions were measured with the mutated genes present in a single copy on the chromosome. Some of the receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected. Several mutants were altered in their sensitivities to the lethal agents. TAB linker insertions after amino acids 69 and 128 abolished all receptor functions. Phage T5 id not bind to these mutant FhuA proteins in detergent extracts. The deletion mutant was also defective in all FhuA functions. Sensitivity to the lethal agents of cellsl that expressed mutant FhuAs with insertions after amino acids 59 and 135 was reduced by several orders of magnitude. Insertion at other selected sites decreased some or all receptor functions only slightly. An insertion after amino acid 321 selectively eliminated ferrichrome growth promotion. Finally, a strain carrying a mutant fhuA gene on the chromosome in which the linker insertion occurred after amino acid 82 showed a tonB phenotype. These subtle perturbations that were introduced into the FhuA protein resulted in changes in its stability and in the binding and uptake of its cognate ligands.     

Mobile Element Insertions Causing Mutations in the Drosophila Suppressor of Sable Locus Occur in Dnase I Hypersensitive Subregions of 5''-Transcribed Nontranslated Sequences

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.     

High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.