Pierre Spierer

Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster

A chromosomal walk is described that covers 315 X 10(3) base-pairs of DNA from the 87DE region of the third chromosome of Drosophila melanogaster. The walk includes the DNA for the rosy and Ace loci, which code for xanthine dehydrogenase and acetylcholinesterase, respectively. Several dispersed repetitive elements were encountered in the walk. In every case, their positions in the chromosome differed in different strains, and so they are all presumed to be transposable elements. Several rearrangement breakpoints have been localized within the walk, including the break for In(3R) Cbx+R1 (87E1, 2-89E1, 2). One breakpoint fusion fragment of this inversion was isolated to jump from 87E into the cluster of homeotic genes of the bithorax complex, at 89E1-4.

The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader

The Ace locus of Drosophila melanogaster has been mapped at the molecular level. cDNA clones from the locus have been isolated and their sequence determined, confirming that Ace forms the structural gene for acetylcholinesterase (AChE). The cDNAs have a 1950 nucleotide open reading frame from which the complete amino acid sequence of AChE has been deduced. The Drosophila enzyme is found to have extensive homology to the known sequence of Torpedo AChE. Ace cDNAs have an unusual structure with a long 5′ leader and several short upstream open reading frames.

SU(VAR)3‐7, a Drosophila heterochromatin‐associated protein and companion of HP1 in the genomic silencing of position‐effect variegation

An increase in the dose of the Su(var)3‐7 locus of Drosophila melanogaster enhances the genomic silencing of position‐effect variegation caused by centromeric heterochromatin. Here we show that the product of Su(var)3‐7 is a nuclear protein which associates with pericentromeric heterochromatin at interphase, whether on diploid chromosomes from embryonic nuclei or on polytene chromosomes from larval salivary glands. The protein also associates with the partially heterochromatic chromosome 4. As these phenotypes and localizations resemble those described by others for the Su(var)2‐5 locus and its heterochromatin‐associated protein HP1, the presumed co‐operation of the two proteins was tested further. The effect of the dose of Su(var)3‐7 on silencing of a number of variegating rearrangements and insertions is strikingly similar to the effect of the dose of Su(var)2‐5 reported by others. In addition, the two loci interact genetically, and the two proteins co‐immunoprecipitate from nuclear extracts. The results suggest that SU(VAR)3‐7 and HP1 co‐operate in building the genomic silencing associated with heterochromatin.

Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha‐like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha‐subunits is also indicated by the failure of an ALS‐beta‐galactosidase fusion protein to bind alpha‐bungarotoxin on blots in contrast to vertebrate endplate alpha‐subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5′ leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system.

Molecular mapping of genetic and chromomeric units in Drosophila melanogaster

We have used a set of overlapping cloned segments defining a 315 kb (X 10(3) base-pairs) region of Drosophila melanogaster chromosomal DNA to map the sequences associated with the polytene band-interbands (chromomeric units) and with the lethal complementation groups contained within this region. The molecular map positions of the 13 +/- 1 chromomeric units from the 87D5-6 to 87E5, 6 region of the third chromosome were determined by in situ hybridization of selected segments to the polytene chromosomes. The length of the largest chromomeric unit within the 315 kb region is approximately 160 kb, while that for the smallest is less than 7 kb and may be as short as 3 kb. By mapping the breakpoints of deletions within the 315 kb region, we have located its 12 lethal complementation groups, which include the genes coding for acetylcholinesterase (Ace) and xanthine dehydrogenase (rosy). Comparison of the two molecular maps indicates a one-to-one topographical correlation between the genetic and chromomeric units.

Drosophila SETDB1 Is Required for Chromosome 4 Silencing

Histone H3 lysine 9 (H3K9) methylation is associated with gene repression and heterochromatin formation. In Drosophila, SU(VAR)3–9 is responsible for H3K9 methylation mainly at pericentric heterochromatin. However, the histone methyltransferases responsible for H3K9 methylation at euchromatic sites, telomeres, and at the peculiar Chromosome 4 have not yet been identified. Here, we show that DmSETDB1 is involved in nonpericentric H3K9 methylation. Analysis of two DmSetdb1 alleles generated by homologous recombination, a deletion, and an allele where the 3HA tag is fused to the endogenous DmSetdb1, reveals that this gene is essential for fly viability and that DmSETDB1 localizes mainly at Chromosome 4. It also shows that DmSETDB1 is responsible for some of the H3K9 mono- and dimethyl marks in euchromatin and for H3K9 dimethylation on Chromosome 4. Moreover, DmSETDB1 is required for variegated repression of transgenes inserted on Chromosome 4. This study defines DmSETDB1 as a H3K9 methyltransferase that specifically targets euchromatin and the autosomal Chromosome 4 and shows that it is an essential factor for Chromosome 4 silencing.

Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, we observe a specific bloating of the male X chromosome, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose.

Genes and loops in 320,000 base-pairs of the Drosophila melanogaster chromosome

We have mapped the DNA sequences bound to the nuclear scaffold along 320,000 base-pairs of a genetically well-defined region of the Drosophila chromosome. We have found that the domains delimited by the scaffold attachment regions are heterogeneous in size (ranging from 26,000 to 112,000 base-pairs in this interval), and that the attachment sites are within unique sequences as judged by blot hybridization. We also found that looped domains contain up to five, or even eight, unrelated genes including, in some cases, more than one transcribed gene. The loop organization unravelled here in cultured cells does not correspond to the banding pattern seen in salivary gland polytene chromosomes.

The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster.

A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging. Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene was constructed with the cDNA and found to have the effect opposite from that of the mutation, namely, to suppress variegation. Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins. We discuss the link established here between a deubiquitinating enzyme and epigenetic silencing processes.

Binding of 50 S ribosomal subunit proteins to 23 S RNA of Escherichia coli

Each of the 50 S ribosomal subunit proteins of Escherichia coli was tested independently in two laboratories for its ability to bind specifically to 23 S RNA. Four new RNA-binding proteins, L1, L3, L4 and L13 were identified in this way. Consistent with earlier work, proteins L2, L6, L16, L20, L23 and L24 were found to interact directly and independently with 23 S RNA as well. No binding of L17 was detected, however, contrary to previous reports, and the results for L19 were variable. The molar ratio of protein and RNA in each complex was measured at saturation. Significant differences in binding stoichiometry were noted among the various proteins. In addition, saturation levels were found to be influenced by the state of both the RNA and the proteins.