Functional analysis of repressor binding sites in the iab-2 regulatory region of the abdominal-A homeotic gene.

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. Previously, the Hunchback (HB) protein has been implicated in setting the anterior expression limit of the UBX homeotic protein in parasegment 6. Here we investigate DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that in contrast to Ubx enhancer elements, both HB and Krüppel (KR) are required to set the ABD-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that HB and KR are direct regulators of this iab-2 enhancer. The single KR site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that KR repression occurs through a local quenching mechanism. We also show that the gap repressor Giant (GT) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. Finally, we show that this iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (PHO). These iab-2 PHO sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the PHO sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer.

The Drosophila activin receptor baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development.

The TGF-beta superfamily of growth and differentiation factors, including TGF-beta, Activins and bone morphogenetic proteins (BMPs) play critical roles in regulating the development of many organisms. These factors signal through a heteromeric complex of type I and II serine/threonine kinase receptors that phosphorylate members of the Smad family of transcription factors, thereby promoting their nuclear localization. Although components of TGF-beta/Activin signaling pathways are well defined in vertebrates, no such pathway has been clearly defined in invertebrates. In this study we describe the role of Baboon (Babo), a type I Activin receptor previously called Atr-I, in Drosophila development and characterize aspects of the Babo intracellular signal-transduction pathway. Genetic analysis of babo loss-of-function mutants and ectopic activation studies indicate that Babo signaling plays a role in regulating cell proliferation. In mammalian cells, activated Babo specifically stimulates Smad2-dependent pathways to induce TGF-beta/Activin-responsive promoters but not BMP-responsive elements. Furthermore, we identify a new Drosophila Smad, termed dSmad2, that is most closely related to vertebrate Smads 2 and 3. Activated Babo associates with dSmad2 but not Mad, phosphorylates the carboxy-terminal SSXS motif and induces heteromeric complex formation with Medea, the Drosophila Smad4 homolog. Our results define a novel Drosophila Activin/TGF-beta pathway that is analogous to its vertebrate counterpart and show that this pathway functions to promote cellular growth with minimal effects on patterning.

Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins.

During early Drosophila embryogenesis, several zygotic gene products act to establish a posttranscriptional activity gradient of the morphogen DPP. Among these molecules, Tolloid, a putative metalloprotease related to BMP-1, enhances DPP function, while SOG, an ortholog of the Xenopus organizer Chordin, inhibits DPP function. Using epistasis tests and a Xenopus secondary axis induction assay, we show that TLD negates the inhibitory effects of SOG/CHD on DPP/BMP-type ligands. In transient transfection assays, we demonstrate that TLD cleaves SOG and that cleavage is stimulated by DPP. We propose that formation of the embryonic DPP activity gradient involves the opposing effects of SOG inhibiting DPP and TLD processing SOG to release DPP from the inhibitory complex.

Characterization of tolloid-related-1: a BMP-1-like product that is required during larval and pupal stages of Drosophila development.

The Drosophila tolloid (tld) gene product belongs to a family of developmentally important proteins that includes bone morphogenetic protein-1 (BMP-1). In Drosophila, tld is required at the blastoderm stage to establish pattern within the dorsal half of the embryo. Genetic analysis suggests that the major function of tld is to augment the activity of the decapentaplegic gene product, a close relative of the TGF-beta superfamily members, BMP-2 and BMP-4. In this report, we describe a new gene called tolloid-related-1 (tlr-1) that maps immediately proximal to tld. Sequence analysis indicates that tlr-1 has a large N-terminal extension relative to tld, but otherwise shows the same general organization of sequence motifs found in tld and other BMP-1 family members. These include a region of similarity to astacin, a crayfish metalloprotease, five copies of a repeat first found in complement proteins C1r and C1s, and two copies of an epidermal growth factor-like sequence. In situ hybridization experiments show that tlr-1 expression partially overlaps tld expression in early embryos, but shows unique transcriptional patterns in late stage embryos that are not seen with tld. In larval stages, both genes are expressed in identical patterns in imaginal discs and in the optic lobes of the brain, but tlr-1 is more abundant than tld. Deletions that eliminate tlr-1 expression cause lethality during larval and pupal stages of development. A small proportion of homozygous mutant flies eclose and show wing veination defects. Transgenic animals in which a tlr-1 cDNA is driven by the tld promoter fail to rescue tld mutations, and extra copies of tld fail to rescue tlr-1 mutations, implying that these genes have evolved functionally distinct features. We propose that tld and tlr-1 arose by gene duplication and that each has evolved independently to acquire distinct tissue specific roles in Drosophila development.

Enhancer point mutation results in a homeotic transformation in Drosophila.

In Drosophila, the misexpression or altered activity of genes from the bithorax complex results in homeotic transformations. One of these genes, abd-A, normally specifies the identity of the second through fourth abdominal segments (A2 to A4). In the dominant Hyperabdominal mutations (Hab), portions of the third thoracic segment (T3) are transformed toward A2 as the result of ectopic abd-A expression. Sequence analysis and deoxyribonuclease I footprinting demonstrate that the misexpression of abd-A in two independent Hab mutations results from the same single base change in a binding site for the gap gene Krüppel protein. These results establish that the spatial limits of the homeotic genes are directly regulated by gap gene products.

Elements of the Drosophila bithorax complex that mediate repression by Polycomb group products.

The Polycomb (Pc) group genes encode repressors that restrict expression of homeotic genes to precise domains along the anterior-posterior (A-P) axis. We describe germ-line transformation constructs, containing portions of the bxd, iab-2, or iab-3 regulatory regions of the bithorax complex (BX-C), that are controlled by Pc group products in embryos. There are multiple BX-C elements that mediate Pc group control, which we call Pc group response elements (PREs), and they can work when removed from the normal BX-C context. These constructs are each regulated by the genes Polycomb (Pc), extra sex combs (esc), Enhancer of zeste (E[z]), polyhomeotic (ph), Polycomb-like (Pcl), Sex comb on midleg (Scm), Posterior sex combs (Psc), and Additional sex combs (Asx), consistent with multiple Pc group products acting together. Depending upon context, a PRE from the iab-3 region can restrict expression in different A-P positions. Thus, PREs are not specialized for particular parasegments, suggesting that Pc group products do not directly specify A-P boundaries of homeotic expression. Instead, the results support the idea that Pc group products provide stable memory or imprinting of boundaries which are initially specified by gap and pair-rule regulators.

The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1.

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-beta family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-beta superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.

Sequence homology of clindamycin resistance determinants in clinical isolates of Bacteroides spp.

The sequence homology of clindamycin resistance (Clnr) determinants was studied in 16 Clnr Bacteroides strains. The isolates were surveyed for plasmid content, homology with the Clnr determinant of pBFTM10, and ability to transfer Clnr. The Clnr DNA probes used in the Southern hybridizations were pBFTM10 and a plasmid derivative containing an EcoRI fragment of pBFTM10 cloned into Escherichia coli. A total of 13 of 16 Clnr strains also carried tetracycline resistance, and 15 of 16 Clnr Bacteroides isolates showed homology with the Clnr determinant of pBFTM10. These data suggest that the previously characterized Clnr determinant of pBFTM10 is widely distributed in nature and may be found on either a plasmid or the chromosome. The Clnr Bacteroides fragilis strain which lacked homology with pBFTM10 also had different transfer properties; thus, more than one type of Clnr determinant may exist in Bacteroides spp.

Hybridization studies reveal homologies between pBF4 and pBFTM10, Two clindamycin-erythromycin resistance transfer plasmids of Bacteroides fragilis.

Two clindamycin-erythromycin resistance transfer factors of Bacteroides fragilis, pBF4 and pBFTM10, were analyzed for regions of DNA homology. Although the plasmids were derived from different clinical isolates of B. fragilis and have different sizes, they showed homology in the clindamycin-erythromycin resistance region; no homology could be detected outside of this region.

Characterization of pBFTM10, a clindamycin-erythromycin resistance transfer factor from Bacteroides fragilis.

Bacteroides fragilis TMP10, which is clindamycin-erythromycin resistant (Clnr) and tetracycline resistant (Tetr), contains several plasmids and is capable of transferring drug resistance markers to suitable recipients. We were able to separate a 14.6-kilobase self-transmissible Clnr plasmid, pBFTM10, from the other plasmids of TMP10 in a tetracycline-sensitive recipient strain, B. fragilis TM4000. All Clnr transconjugants acquired an unaltered pBFTM10 and became plasmid donor strains. Transfer is proposed to occur by conjugation since it required to cell-to-cell contact of filter matings and was insensitive to DNase, but sensitive to chloroform treatment of donor cells. The efficiency of transfer of pBFTM10 in a Tets background (TM4003) was not affected by pretreatment of donor cells with clindamycin. A spontaneously occurring Clns derivative, pBFTM10 delta 1, suffered a deletion of DNA, which included a 4.4-kilobase EcoRI fragment. A complex interaction between the autonomous plasmid pBFTM10 and a tetracycline transfer element also present in strain TMP10 was observed since pretreatment of this donor with tetracycline or clindamycin resulted in a marked increase in transfer of both tetracycline and clindamycin resistance.