We report here that a point mutation in the gene which encodes the heterochromatin-specific nonhistone chromosomal protein HP-I in Drosophila melanogaster is associated with dominant suppression of position-effect variegation. The mutation, a G-to-A transition at the first nucleotide of the last intron, causes missplicing of the HP-1 mRNA. This suggests that heterochromatin-specific proteins play a central role in the gene suppression associated with heterochromatic position effects.The partitioning of eukaryotic chromosomes into regions which differ in their degrees of compaction has long been appreciated. Most of the transcriptionally active chromatin appears to decondense after mitotic telophase into euchromatin, but a substantial fraction of chromosomal material remains condensed as heterochromatin. Heterochromatin replicates relatively late in the cell cycle and, in tissues which undergo polytenization, the heterochromatin may be underreplicated.The potential of heterochromatin formation to result in transcriptional inactivation is inferred from two genetic phenomena: Barr-body formation (Lyonization) in mammalian females and position-effect variegation in a variety of organisms (reviewed in ref. 1). In both cases chromosomal regions which are euchromatic under some circumstances assume the morphology of heterochromatin. The condensed structure observed in these cases is strongly correlated with transcriptional inactivity.In Drosophila, the genetic dissection of heterochromatin is aided by the availability of numerous rearrangements which lead to variegated expression of euchromatic genes that have come to be relocated near the heterochromatic breakpoint. A number of loci have been identified which, when mutated, act as dominant modifiers of such variegating position effects (2-7). Many of these loci are believed to encode chromatin proteins or factors that modify chromatin structure (see refs. 8 and 9 for recent reviews).A heterochromatin-specific chromosomal protein called HP-1 has been identified and characterized in D. melanogaster (10, 11). A cDNA encoding this protein has been cloned (10), and the gene has been localized to cytological position 29A on the polytene chromosome map. In this report, we provide the sequence of the gene$, identifying exon and intron boundaries, and present molecular evidence that a point mutation at one boundary, causing missplicing of the HP-1 pre-mRNA, is associated with dominant suppression of heterochromatic position effect. This indicates a requirement for HP-1 protein in generating normal heterochromatin structure. MATERIALS AND METHODSDrosophila Stocks. Su(var)205/In(2LR)CyO and the iso-2nd line (marked with b It rl) were obtained from T. Grigliatti (University of British Columbia, Vancouver). Flies were cultured in half-pint plastic bottles at room temperature, using a cornmeal-based medium supplemented with dried bakers' yeast.Northern Blot Analysis. Total nucleic acids were purified from several flies essentially according to the method of Meyerowitz and H...

The transport of Fe(III)-siderophore complexes and vitamin B 12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membranederived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB. This process requires direct physical contact between the outer membrane receptor and TonB. Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonBdependent outer membrane receptors as being essential for productive energy transduction. In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB. Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact. This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors. This possibility was supported by the finding that TonB carboxylterminal truncations that retained Gln-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction. These studies indicated that the final 48 residues of TonB were essential to this physical interaction. This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction. Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex. The effects of amino acid substitutions at several other TonB sites were also examined.Escherichia coli and other gram-negative bacteria scavenge Fe(III)-bearing siderophores and vitamin B 12 from the environment through a set of ligand-specific, high-affinity transport proteins displayed at the cell surface (outer membrane receptors). The subsequent release of ligand from the periplasmic face of the outer membrane receptor is an active process, dependent upon energy derived from the cytoplasmic membrane (4, 14). The spatial separation of transport events from their energy source necessitates a mechanism for energy transfer. This need is met by TonB, a cytoplasmic membrane protein that spans the periplasmic space to transduce cytoplasmic membrane-derived energy to the outer membrane receptors (most recently reviewed in reference 5).The requirement of TonB for active transport at the outer membrane was evident in early experiments, in which mutations at the tonB locus were found to abolish transport of Fe(III)-complexed siderophores and vitamin B 12 , as well as the transport of certain colicins and the irreversible adsorption of bacteriop...

The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the 0r54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (>100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for activation, and (iii) they activate the or54 RNA polymerase.We have characterized the NtrC protein from a photosynthetic bacterium, Rhodobacter capsulatus, which represents a metabolically versatile group of bacteria found in aquatic environments. We have shown that the R. capsukitus NtrC protein (RcNtrC) binds to two tandem sites that are distant from promoters that it activates, nifAI and nifA2. These tandem binding sites are shown to be important for RcNtrC-dependent nitrogen regulation in vivo. Moreover, the conserved nucleotide-binding fold of RcNtrC is required to activate nifAI and nifA2 but is not required for DNA binding of RcNtrC to upstream activation sequences. However, nifAl and nifA2 genes do not require the Cr54 for activation and do not contain the highly conserved nucleotides that are present in all or54-type, EBP-activated promoters. Thus, the NtrC from this photosynthetic bacterium represents a novel member of the class of bacterial EBPs. It is probable that this class of EBPs is more versatile in prokaryotes than previously envisioned.Enhancer-binding proteins (EBPs) are present in a wide range of bacteria and activate the expression of genes required for diverse cellular processes, including nitrogen assimilation, the development of cell polarity, pathogenicity, and biological nitrogen fixation (46). Members of the EBP family characteristically activate transcription from promoters recognized by the alternative sigma factor u54 (36,42), and the binding sites for EBPs are located distal to the promoter, usually at sites greater than 100 bp upstream from the transcription start (13,35). Like many eukaryotic activators, prokaryotic EBPs are composed of several domains which are modular in structure, since in some cases the activation and DNA-binding functions may be separated (see reference 49 for a review). All members of the EBP family share a highly conserved central domain which includes a nucleotide-binding fold (46) and a C-terminal domain which includes a helix-turn-helix motif required for DNA recognition (14,44). Several activators are members of two-component regulatory systems (e.g., NtrC, DctD, FlbD, and HoxA) and possess an amino-terminal regulatory domain which is phosphorylated at a conserved aspartate in response to an environmental stimulus (32, 47; for reviews, see references 50, 62, and 66). This phosphorylation induces ATPase activity (2, 70) and probably oligomerization (43,53,71), which are considered prerequisites in the activation process.The enteric NtrC was the first prokaryotic EBP to b...

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R. capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1-lacZ and nifA2-lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen-deficient conditions. This nitrogen-controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON-activated promoters.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.