Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification.

The origins of specificity in gene expression are a central concern in understanding developmental control. Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specific transcription factors to core RNA polymerase II. Here, we identified the Drosophila homologs of 23 human mediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4). Clonal analysis indicates a general role for dTRAP80 necessary for cell viability. The dTRAP240 gene is also essential, but cells lacking its function are viable and proliferate normally. Clones reveal localized developmental activities including a sex comb cell identity function. This contrasts with the ubiquitous nuclear accumulation of dTRAP240 protein in imaginal discs. Synergistic genetic interactions support shared developmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a common complex. Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissue identities, as a functional partner of these mediator proteins.

Homeotic proboscipedia cell identity functions respond to cell signaling pathways along the proximo-distal axis.

To better understand how the different cell identities composing a segment are attributed and coordinated under the control of a single homeotic selector gene, we examined dose-sensitive homeotic phenotypes associated with gain-of-function and loss-of-function mutations of the homeotic gene proboscipedia (pb; Hox-A2/-B2). We then employed dose-sensitive segment and cell identity phenotypes resulting from ectopic proboscipedia expression to screen for other interacting loci. We find that pb, as well as the homeotic loci Ultrabithorax, Sex combs reduced and Antennapedia, respond to positional information along the proximo-distal axis. This response for pb implicates at least two signal transduction pathways, those involving Ras1 and Notch.

Point mutations within and outside the homeodomain identify sequences required for proboscipedia homeotic function in Drosophila.

The Drosophila homeotic gene proboscipedia (pb) encodes a homeodomain protein homologous to vertebrate HoxA2/B2 required for adult mouthparts formation. A transgenic Hsp70-pb (HSPB) element that rescues pb mutations also induces the dominant transformation of antennae to maxillary palps. To identify sequences essential to PB protein function, we screened for EMS-induced HSPB mutations leading to phenotypic reversion of the HSPB transformation. Ten revertants harbor identified point mutations in HSPB coding sequences. The point mutations that remove all detectable phenotypes in vivo reside either within the homeodomain or, more unexpectedly, in evolutionarily nonconserved regions outside the homeodomain. Two independent homeodomain mutations that change the highly conserved Arginine-5 in the N-terminal hinge show effects on adult eye development, suggesting a previously unsuspected role for Arg5 in functional specificity. Three additional revertant mutations outside the homeodomain reduce but do not abolish PB+ activity, identifying protein elements that contribute quantitatively to pb function. This in vivo analysis shows that apart from the conserved motifs of PB, other elements throughout the protein make important contributions to homeotic function.

Ras1-mediated modulation of Drosophila homeotic function in cell and segment identity.

Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homologue) provoke dose-sensitive defects. These were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes were the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of pb activity both in normal and ectopic cell contexts, while the Ras1-antagonist Gap1 has an opposite effect. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. Our data suggest that the modulation occurs by a mechanism independent of transcriptional control of the homeotic loci themselves, or of the Ras1/Gap1 genes. Taken together our data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation.

A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function.

The Drosophila homeotic gene proboscipedia (pb: HoxA2/B2 homolog) is required for adult mouthparts development. Ectopic PB protein expression from a transgenic Hsp70-pb minigene (HSPB) results in transformation of adult antennae to maxillary palps. In contrast, most tissues appear refractory to PB-induced effects. To study the basis of homeotic tissue specificity we are isolating and studying mutations that modify dominant HSPB-induced phenotypes. One HSPB point mutation (Arg5 of the homeodomain to His) removes homeotic activity in the mouthparts and antennae, but provokes a dose-sensitive eye loss. We show that eye loss can be induced by PB proteins that no longer effectively bind to DNA. The dose-sensitive eye loss thus appears to be mediated by specific, context-dependent protein-protein interactions.

Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control.

The autonomous selector capacity of the homeotic proboscipedia (pb) gnee of the Drosophila Antennapedia Complex was tested. We introduced into the germline a P element containing a transcriptional fusion of a mini-gene for pb (normally required for formation of the labial and maxillary palps of the mouthparts) and the Hsp70 promoter. Uninduced expression of this Hsp70:pb element (HSPB) directs a novel, fully penetrant dominant transformation of antennae toward maxillary palps. Gene dosage experiments varying the number of HSPB elements indicate that the extent of the dominant transformation depends on the level of PB protein. At the same time, expression from the transgene also rescues recessive pb mutations. Finally, HSPB function may override the dominant antennal transformations caused by Antennapedia (Antp) mutations in a dose-sensitive manner, directing a switch of the antennal disc-derived appendage from ectopic leg to ectopic maxillary palp. This switch correlated with strikingly reduced ANTP protein accumulation when PB concentrations exceeded a genetically defined threshold level. These observations support a crucial role for quantitative aspects of pb function in determining segmental identity, including cross-regulatory events involved in this determination.

Ectopic expression of the Drosophila homeotic gene proboscipedia under Antennapedia P1 control causes dominant thoracic defects.

A deletion mutation in the Antennapedia Complex of Drosophila melanogaster, Df(3R)SCBXL2, induces both dominant and recessive loss-of-function phenotypes. The deletion is associated with diminished function of proboscipedia (pb), a homeotic gene required for mouthparts formation. Df(3R)SCBXL2 also has associated dominant thoracic defects related to diminished expression of the homeotic Antennapedia (Antp) gene copy on the homologous chromosome. This is shown to be a consequence of ectopic pb expression in the thorax. Newly juxtaposed Antp sequences provide the pb gene on the deletion bearing chromosome with a second promoter, Antp P1, in addition to its own. Ectopic pb protein expression occurs under Antp P1 control, by alternate splicing, and results in diminished accumulation of Antp protein in the imaginal disc cells where Antp P1 is normally expressed. The analysis of this mutant chromosome thus demonstrates that pb protein is capable of participating in the negative regulation of a more posteriorly expressed homeotic gene, as well as serving a homeotic "selector" function in the head.

Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia.

Mutations of the homeotic gene proboscipedia (pb) of Drosophila cause striking transformations of the adult mouthparts, to legs or antennae. We report here an analysis of the gene structure of pb. Coding sequences across a 34 kb interval yield, by alternative splicing, four identified mRNA forms which differ immediately upstream of the homeobox. As a consequence, the homeodomain is expected to reside in four different contexts in the predicted protein isoforms. Mammalian homologs of pb, human HOX-2H and murine Hox-2.8, were identified based on the similarities of their homeodomains (95% identity) and several other conserved motifs. Examination of a collection of pb mutant alleles with antisera directed against the N-terminal region, the center or the C-terminal region of the protein showed that, surprisingly, several partial loss-of-function pb alleles appear to generate partially functional proteins truncated at their C-termini. This suggests that a significant portion of the protein contributes quantitatively to pb function, but is partially dispensable. Finally, evolutionary considerations suggest that pb may be one of several ancient genes which preceded the process yielding the modern homeotic gene complexes.

Rescue and regulation of proboscipedia: a homeotic gene of the Antennapedia Complex.

The extraordinary positional conservation of the homeotic genes within the Antennapedia and the Bithorax Complexes (ANT-C and BX-C) in Drosophila melanogaster and the murine Hox and human HOX clusters of genes can be interpreted as a reflection of functional necessity. The homeotic gene proboscipedia (pb) resides within the ANT-C, and its sequence is related to that of Hox-1.5. We show that two independent pb minigene P-element insertion lines completely rescue the labial palp-to-first leg homeotic transformation caused by pb null mutations; thus, a homeotic gene of the ANT-C can properly carry out its homeotic function outside of the complex. Despite the complete rescue of the null, the minigene expresses pb protein in only a subset of pb's normal domains of expression. Therefore, the biological significance of the excluded expression pattern elements remains unclear except to note they appear unnecessary for specifying normal labial identity. Additionally, by using reporter gene constructs inserted into the Drosophila genome and by comparing pb-associated genomic sequences from two divergent species, we have located cis-acting regulatory elements required for pb expression in embryos and larvae.

The proboscipedia locus of the Antennapedia complex: a molecular and genetic analysis.

The homeotic proboscipedia (pb) locus of the Antennapedia Complex (ANT-C) directs the differentiation of adult labial and maxillary structures. Loss-of-function pb alleles show a transformation of adult mouth parts to legs and affect maxillary palp morphology. We have identified the pb transcription unit by inducing and analyzing a series of pb null chromosomal breakpoints. In addition, we describe a variegating pb phenotype associated with two rearrangement breakpoints. Having identified the pb locus, we describe the expression of its RNA and protein products. Unlike the other homeotic genes of the ANT-C, pb has no obvious role in embryonic development. Nevertheless, pb protein is detected during embryogenesis in nuclei of the labial and maxillary lobes and in part of the mandibular segment. In this respect, pb expression parallels the early segment-specific expression of neighboring, embryonically essential homeotic genes. Accumulation of pb protein is also detected in mesodermal cells and in a unique subset of nuclei throughout the central nervous system. We also describe a transcription unit very close to pb, which is expressed dorsally during embryogenesis in a pattern resembling that of the nearby zygotic lethal zerknüllt (zen) locus. This transcription unit has been shown to contain a homeo box and has been designated z2. Surprisingly, we find that individuals deleted for both the pb and z2 transcription units survive to adulthood and produce normal larval cuticular structures. Thus, pb and z2 show similarities to neighboring ANT-C genes in their expression patterns, yet these similarities are not manifested in comparable loss-of-function embryonic phenotypes.

Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons.

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster.

The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.

Termination of transcription in the mouse alpha-amylase gene Amy-2a occurs at multiple sites downstream of the polyadenylation site.

We have delimited the region of transcription termination in the alpha-amylase gene Amy-2a. Mapping of in vitro elongated nascent transcripts to Amy-2a restriction fragments indicates that transcription terminates in a region between 2.5 and 4 kb downstream of the polyadenylation site. These runoff transcription experiments, combined with S1 nuclease mapping of nuclear transcripts at steady state, suggest that transcription termination occurs at multiple sites.

The structure of tRNA 5 Lys from Drosophila melanogaster.

The nucleotide sequence of Drosophila melanogaster tRNA 5 Lys is pGCCCGGAUAm2GCUCAGDCGGDAGAGCA psi psi GGACUsU*UUt6A*A psi CCAAGGm7GDm5CCAGGGTm psi CAm1AGUCCCUGUUCGGGCGCCA. The sU* is probably 5-methylcarboxymethyl-2-thiouridine and t6A* is a mixture of modified derivatives of t6A including t6A itself and a component sensitive to treatment with cyanogen bromide. This tRNA 5 Lys is 95% homologous to the rabbit liver tRNA 5 Lys.

Studies of gene control regions. III. Binding of synthetic and modified synthetic lac operator DNAs to lactose repressor.

Chemically synthesized lactose operator DNA was tested for binding with lactose repressor protein. These operator DNAs were found to (1) bind specifically to lactose SQ repressor as measured by release of binding with the inducing ligand isopropyl-beta-D-thiogalactoside, (2) have dissociation half-lives of 37 seconds (21 base-paired duplex) and 46 seconds (26 base-paired duplex) and (3) have dissociation half-lives with x86 repressor of 9 minutes (21 base-paired duplex) and 18 minutes (26 base paired duplex). Modified operators containing 5-bromodeoxyuridine and deoxyuridine at specific sites were also prepared. These analogs bound both repressors about as tightly as the wild type sequence.