ANT-C, DmAntp, Hu, Scx, Ant
AlphaFold produces a per-residue confidence score (pLDDT) between 0 and 100. Some regions with low pLDDT may be unstructured in isolation.
The secondary structure of an N-terminally elongated Antp protein fragment, including both the homeodomain and the YPWM motif, from amino acids -14 to +67 was determined by NMR in solution (this study). Results strongly support the conclusion that the homeodomain is connected through a flexible linker to the main body in the Antp protein and that the minor groove contacts by residues 1-6 are intrinsic to the DNA binding interactions of the Antp protein (this study). The stability and specificity of the DNA binding previously observed for the shorter Antp
homeodomain polypeptide is preserved for the elongated polypeptide.
Sequences of the mammalian thyroid transcription factor 1 (TTf-1) and Antp homeodomains were exchanged to identify regions responsible for DNA binding specificity. Mutations that make the TTf-1 recognition helix identical to that of Antp have no effect on binding specificity. Sequences outside of the recognition helix are shown to play a role in determining binding specificity.
The 1:1 complex of the mutant AntpC39S homeodomain with a 14bp DNA fragment corresponding to the BS2 binding site was studied by NMR spectroscopy in aqueous solution. The AntpC39S protein and the DNA were found to have similar conformations in the free form and in the complex. In the complex, intermolecular 1H-1H Overhauser effects (NOE) are involved in protein-DNA binding.
NMR spectroscopy in solution was used to determine the structure of the Antp homeodomain. It includes 3 well defined helices (residues 10-21, 28-38, and 42-52) and a more flexible fourth helix (53-59). Residues 30-50 form a helix-turn-helx motif like those in various prokaryotic repressors. The fourth helix is unique to the Antp homeodomain.
Click to get a list of regulatory features (enhancers, TFBS, etc.) and gene disruptions (point mutations, indels, etc.) within or overlapping Dmel\Antp using the Feature Mapper tool.
Antp is expressed in a gradient in the ventral nerve cord in the NB5-6 lineage, high anteriorly and low posteriorly, with the anterior limit at segment T1. In both NB5-6A and NB5-6T expression begins at embryonic stage 12 and is maintained in all cells born after this stage.
Antp protein is expressed in a ring near the proximal periphery of the leg disc; the expression region is distal to that of hth and proximal to dac. At embryonic stages, Antp protein is broadly distributed in leg disc primordia, becoming restricted to a more proximal domain as the disc matures.
Protein is detected in the anterior embryonic dorsal vessel. The protein is strongly expressed in four consecutive pairs of cardioblasts corresponding to tin expressing cardioblasts in abdominal segment A1 and the boundary between A1 and A2. There is also weaker expression in tin positive cardioblasts in A2 and thoracic segment 3.
The level of Antp protein (expressed from the + chromosome) is reduced in imaginal discs of Df(3R)SCB-XL2/+ third instar larvae.
Antp protein is expressed in all thoracic imaginal discs in distinct patterns. No significant staining is seen in the eye-antennal disc.
Mutants in the shv region of dpp cause a posterior shift of both the Antp protein expression domain and the first midgut constriction. Furthermore, the Antp domain includes only the anterior portion of the first midgut constriction and no longer extends on either side.
Antp protein is first detected in embryonic stage 13 in the visceral mesoderm. It is expressed in a domain that is 8 nuclei long and is located posterior to and separated from the Scr domain. By stage 14, the two lateral patches expressing Antp protein split. In stage 16, Antp protein expression is seen in the anterior constriction. Later in stage 16, the patches spread out along the anterior/posterior body axis while the midgut constrictions tilt. Finally, during stage 17, the Antp protein-expressing nuclei form four one-nucleus-wide rows.
Antp protein is first detected prior to the germ band retraction stage in the visceral mesoderm of the midgut and the ectoderm of parasegments 5-6.
The Antp protein domain remains unchanged in homozygous ftz mutant embryos. Embryos homozygous for eve3 showed no Antp staining but there is some staining in embryos homozygous for eve4. Normal homeotic gene function is seen in embryos homozygous for en<up>IO34, en54, en55, wgl-17, opa1, h41, odd5, prd4 and runB102. No Antp gene expression is seen in ftz,prd or opa,prd double mutant embryos and there is normal staining in odd,eve double mutant embryos. The Antp protein domain is normal in hb mutants, extended in width in kni mutants and lacking in KrB206 mutants.
Antp protein is first detected in germ band extended embryos in the presumptive thoracic region. The region extends from the posterior compartment of the labial segment to the anterior compartment of A1. The heaviest staining is in parasegment 4. As the germ band shortens, Antp protein is observed in the ectoderm of posterior T1 and in T2 and T3. As the germ band shortens further, expression diminishes in posterior T3 and appears in the ventral nervous system. With germ band shortening, expression in the ectoderm continues to decrease. Antp protein first appears in the ventral nervous system in 10 pairs of patches in the neurogenic region. Antp protein is present in the ventral nervous system from the posterior part of T1 to the anterior part of A7. At early stages, protein levels are uniform between the thoracic and abdominal segments. As development proceeds protein levels increase in posterior T1, anterior T2 and anterior T3 and diminish in the abdominal segments. Antp protein is also present in some cells of the PNS during germ band retraction. In the thorax, areas of strong Antp protein do not overlap areas of strong Ubx protein expression.
Antp protein is first detected at the onset of germ band retraction. It is limited to the thoracic segments in the epidermis but it is found in all neuromeres in the head, thorax and abdomen. At about 10hr of development, Antp protein levels increase in all neuromeres. This is followed by a rapid disappearance of protein from the neuromeres of the head and abdominal segments. Protein disappears completely from A8 and A9. As a consequence, Antp protein mainly accumulates in the ventral nervous system from posterior T1 to anterior T3 with a gap in posterior T2. Antp protein is also observed in imaginal discs. It is present in the posterior compartment of the 1st leg disc and the anterior compartments of the second and third leg discs. It is expressed most strongly in the proximal regions that will give rise to thoracic structures but is also expressed weakly in a part of the second leg disc that gives rise to the leg. Antp protein is observed in the part of the wing disc that will give rise to thoracic structures of the prescutum.
GBrowse - Visual display of RNA-Seq signalsView Dmel\Antp in GBrowse 2
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Please Note This section lists cDNAs and ESTs that fall within the genomic extent of the gene model, which may include cDNAs and ESTs of genes within introns, or of overlapping genes. Please see GBrowse for alignment of the cDNAs and ESTs to the gene model.
For each fully sequenced cDNA the DGRC maintains various forms of the cDNA (e.g tagged or untagged) in several different host vectors for subsequent cloning and expression in Drosophila and Drosophila cell lines.
Source for identity of: Antp CG1028
Source for merge of: Antp Aus
The posterior signalling centre (PSC) of the lymph gland is specified early in the embryo by Antp.
The modulation of Hox gene activation and repression functions can account for segment-specific morphological differences.
The role of electrostatics in homeodomain-DNA interactions are investigated using techniques based around the use of the Poisson-Boltzmann equation.
In vivo activity of Antp is modified by CkIIα-mediated phosphorylation. Phosphorylation of Antp by CkIIα is important for preventing inappropriate activities of this homeotic protein during embryogenesis.
Effects of overexpression of ANTP-C genes on tarsal segmentation in ss mutants is studied.
Identification: Defined as part of an analysis of the MBT (Malignant Brain Tumor) chromosome which dissected its effects into its component contributive alleles.
Muscle patterning in the mesothoracic segment has a non-autonomous requirement for Antp. Antp has no autonomous mesodermal function in the development and patterning of T2-specific musculature in the embryo. Antp is the homeotic selector gene required for autonomous specification of segmental identity in T3 mesoderm.
Antp 5' UTR acts as an internal ribosome entry site (IRES), the Antp 5' UTR inserted between a Ecol\CAT and Ecol\lacZ dicistronic gene shows IRES activity in transgenic flies. The IRES exhibits high degree of developmental regulation.
One of a class of genes with TATA-less promoters that have the conserved DPE sequence.
Amino acids in the N-terminal arm of the homeodomain, as well as at position 54 of the homeodomain, control the DNA binding specificity of the homeodomain. The DNA-binding specificity of a single homeodomain is conferred by several determinants.
Region of the Antp homeodomain responsible for internalisation is mapped to the third helix and a 16 amino acid long peptide corresponding to this region translocates across biological membranes, enters the cytoplasm and is conveyed to the nucleus (FBrf0076807). Internalisation does not required specific interactions with a chiral receptor or the formation of a charged pore by an α-helical conformation of the peptide.
The dose-response dynamics of the antenna disc exposed to genes that transform antenna to leg-like structures are determined. Varying the duration and temperature of heat shock over the course of the sensitive period is used to assess the timing of changes in sensitivity of the antenna-to-leg transformation. Varied sensitivities are found both spatially and temporally.
Chromosome homologies of Muller's element D (J chromosome in the Paleartic species and XR chromosome arm in Nearctic species) and of element E (O chromosome in the Paleartic species and 2 chromosome in Nearctic species) have been confirmed by single copy probes in the species of the obscura group and in D.melanogaster.
A phylogenetic analysis of the Antp-class of homeodomains in nematode, Drosophila, amphioxus, mouse and human indicates that the 13 cognate group genes of this family can be divided into two major groups. Genes that are phylogenetically close are also closely located on the chromosome, suggesting that the colinearity between gene expression and gene arrangement was generated by successive tandem gene duplications and that the gene arrangement has been maintained by some sort of selection.
Expression of Antp in C.elegans demonstrates the specificity of function of the Drosophila and C.elegans Hox proteins is conserved in an assay to control the anterior versus posterior migration of Q-cell decendents. The Drosophila protein can substitute the normal function of the C.elegans protein in three different cell-fate decisions.
Heat shock induction of Antp can cause a leg bristle transformation preceding the morphological antenna to leg transformation. This uncoupling of cell differentiation from morphogenesis suggests separate mechanisms may be involved in the determinative events underlying these processes.
Heat shock induced expression of mouse Hox genes in Drosophila embryos deficient for homeotic genes demonstrates that functional hierarchy is a universal property of the homeobox genes. Correlations exist between the expression patterns of the mouse Hox genes along the antero-posterior body axis of mice and the extent of their effect along the antero-posterior body axis of flies.
A 16 amino acid peptide corresponding to Antp helix 3 with the N-terminal glutamate residue deleted is capable of translocating through biological membranes.
Systematic characterisation of DNA sequence recognition properties reveals that Antp, Ubx and Dfd protein homeodomain regions binds preferentially to a core sequence which differs from the binding sequence of Abd-B. Antp and Ubx homeodomains display indistinguishable preferences outside the core, while Ubx differs.
Homeoproteins Ubx and abd-A act through the same downstream element to differentially regulate Antp P1 promoter activity. This demonstrates that regulatory specificity can result from differences in activity than targetting.
Ligation mediated PCR procedure has been used to quantitate the accessibility of restriction sites in the chromatin fibre of Abd-B expressed in the wing and eye-antennal disc. Inactivation is not accompanied by substantial change in the accessibility of the chromatin fibre.
Ectopic expression of dpp eliminates Scr and Antp expression, attenuating abd-A expression, inducing Ubx, dpp, wg and tsh expression in the visceral mesoderm and inducing lab expression in the apposing endoderm. The result is failure of all of the morphogenetic events except formation of midgut constriction 2.
NMR experiments are used to determine the structure of the entire complex.
trx exerts its effects by positively regulating homeotic gene expression, but Ubx, Antp, abd-A, Abd-B, Scr and Dfd all have different tissue-specific, parasegment-specific and promoter-specific reductions in expression in a trx mutant background.
Antp homeodomain differs at only 5 amino acid positions from that of Scr : using ectopically expressed Antp::Scr fusion proteins, the specificity of Antp protein was shown to be determined by four specific amino acids in the flexible N-terminal arm of the homeodomain.
NMR experiments are used to collect data on intra and intermolecular conformational constraints as input for the structure calculations of the bound protein and the entire complex, and the NMR structure of the DNA-bound mutated Antp homeodomain.
The homologs of Antp, ftz, Scr, Dfd, Ama, bcd, zen, pb and lab, but not zen2 are all present in D.pseudoobscura.pseudoobscura, in the same linear order and similarly spaced along the chromosome as in D.melanogaster.
Comparative analysis of the homeobox sequences reveals the subdivision of the Antp-type homeobox genes into three classes early in metazoan evolution, one includes Abd-B, the second includes abd-A, Ubx, Antp, Scr, Dfd and ftz, and the third includes zen, zen2, pb and lab.
The N terminus of the homeodomain is critical for determining the specific effects of the Antp and Scr homeotic proteins in vivo, though other parts of the protein do have a role. The N terminal part of the homeodomain has been observed, in crystal structures and in NMR studies in solution, to contact the minor groove of the DNA.
Dfd and Antp have distinct DNA binding specificities that correlate with their different regulatory functions in embryos. Antp P1 promoter is repressed in embryonic cells by the Ubx protein and activated by the Dfd::Ubx fusion protein.
Distamycin and the Antp homeodomain peptide compete for their DNA binding sites. This demonstrates that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.
A Ecol\lacZ reporter gene construct carrying Antp P1 promoter fragment was used to identify the regulatory elements controlling spatial and temporal expression of the Antp gene. The fusion gene construct integrated into the endogenous gene Antp. The position of integration was crucial for the expression pattern of the reporter gene. No autoregulatory function of the Antp protein on Antp P1 transcription was identified.
Connectin expression examined in Antp mutant, and is reduced in T2 and T3 but unaffected in T1.
The PNS has been studied in embryos homozygous for Antp with antibodies that label specific sensory organs. The effect of ectopic expression of Antp was investigated on the normal development of sensory organs in the embryonic PNS.
Antp has been compared in D.melanogaster, D.virilis and D.subobscura. The overall gene structure was similar in that the intron/exon sequences were conserved. Protein coding potential was perfectly conserved around the C-terminal homeodomain, well conserved in the N-terminal region and more variable in between. The large size of the gene may reflect the high number of control elements necessary for its proper expression, and the conservation of transcript complexity suggests functional requirements for the different protein forms.
Null loss-of-function alleles result in embryonic lethality. Animals succumb at the end of embryogenesis and show homeotic transformations in the larval cuticle of the first, second and third thoracic segments. Specifically the cuticle derived from parasegments 4 and 5 are transformed to a more anterior identity such that the posterior of the first thorax produces fragments of mouth hook material on its dorsal surface presumably owing to a new posterior labial identity, whereas the anterior of the second thorax resembles the first thorax. The anterior of the third thoracic segment is weakly transformed toward a T1-like identity. The posterior of T2 is presumably T1 like as there are no gnathal structures seen in this compartment. There are also partial loss-of-function mutations which allow survival into the larval, pupal and adult stages. Those that allow adult survival produce animals in which the anterior of the dorsal mesothorax shows a transformation to prothorax. There are no other apparent defects associated with these lesions. Those 'leaky' mutants which die in the pupal and larval stages show similar parasegmental transformations as the null alleles, except that only the parasegment 4 to 3 homeosis is generally apparent. Animals which survive to the pupal stage fail to evert their anterior spiracles resulting in a blunt appearance of the anterior pupa. This same phenotype is seen in genotypes which survive to the adult stage. These partial mutants in many cases are associated with chromosome rearrangements notably deletions which approach the locus from its distal end. Moreover these mutations have been shown to complement fully other seemingly null mutations. Subsequent molecular analyses have shown that these results are accounted for by the presence of two promoters, one, P1, distal to the other, P2. The partial mutants affect the ability of the P1 promoter to initiate transcription, while the complementing lesions inactivate P2. Null mutants affect the transcription unit and protein encoding portion of the gene which is common to both promoters. X-ray induced somatic clones of Antp- cells demonstrate that the locus is required in the adult for the proper development of the dorsal pro and mesothorax, and legs. The former is reduced in size presumably reflecting an anteriorward transformation while the latter are transformed to antennae. Thus Antp+ function is required in the embryo and adult in parasegments 4 and 5 to prevent more anterior segmental identities, specifically those normally found in the anterior thorax and head. The Antp locus was initially recognized by virtue of several striking dominant gain-of-function alleles. Thirteen of these transform the antenna of the adult into a mesothoracic leg (Antp49, AntpB, AntpYu, AntpPW, AntpLC, AntpR, AntpWu, Antp50, AntpRM, Antp73b, AntpCB, Antp72j and AntpNs). Three of these also have effects on the orbit of the eye and the vibrissal region of the ventral head (AntpRM, Antp72j and AntpNs). There are also two dominant alleles (AntpCtx and AntpW) which transform portions of the head capsule (dorsal and posterior) and the eye to a dorsal mesothoracic identity. In some cases this includes the production of wing tissue in the eye. Finally, a unique dominant AntpHu produces bristles on the normally bald propleurae just ventral to the mesothoracic spiracle. This latter phenotype has been interpreted as the production of sternopleural bristles on the propleurae and thus a T1 to T2 transformation. With the exception of AntpNs and Antp72j all these dominant lesions are associated with recessive lethality and gross chromosome rearrangements.
All the mutant breakpoints fall in the interval between the distal and proximal promoters. The dominant gain-of-function phenotype results from the misregulation of the P2 promoter by position affect or by the production of novel transcripts initiated in the newly juxtaposed sequences and spliced to the downstream Antp coding sequences. Both events result in the ectopic accumulation of the Antp protein product in the eye-antennal disc where the normal head repressive function of the gene causes the observed alteration. The recessive lethality associated with these lesions falls into the partially deficient category mentioned above. That is, these lesions show complementation with the P2 specific (Antp1 and Antp23) mutations and in general show only strong parasegment 4 --> parasegment 3 transformations. However, there is a gradient of this affect among the breakpoints. Those closest to P1 and furthest from P2 are the weakest, whereas those close to P2 show the strongest phenotype and earlier lethal phase. This same result is obtained with breakpoint mutations in the P2-to-P1 interval which are not associated with a dominant phenotype. Therefore this interval likely contains sequences necessary for the proper regulation of the P2 promoter.
Exon D, common to mRNAs from both transcription units, and devoid of AUG codons, can mediate initiation of translation by internal ribosome binding in cultured Schneider cells.
Antp gene product is not required for salivary gland development, at least up until the cuticle forms.
Tissue culture experiments show that the homeobox of the Antp protein can confer transport into the nucleus on GTP binding protein that would not normally be nuclear.
Secondary structure of an N-terminally elongated Antp protein fragment, including both the homeodomain and the YPWM motif, from amino acids -14 to +67, has been determined by NMR in solution. Results strongly support that the homeodomain is connected through a flexible linker to the main body in the Antp protein and that the minor groove contacts by residues 1-6 are intrinsic to the DNA binding interactions of the Antp protein.
tsh expression is modulated by Antp. Scr and lab are expressed ectopically in embryos deficient for tsh and Antp. Antp and Ubx act independently of tsh for the determination of trunk identity, although tsh is essential for the complete function of the trunk homeotic genes.
Mutations of genes in the polycomb group (esc, E(z), Pc, ph-p, ph-d, Scm, Pcl, Sce, Asx, Psc, pho and Antp) cause abnormal segmental development due to the ectopic expression of abd-A and Abd-B. Embryos lacking both maternal and zygotic Antp product were generated to determine abd-A and Abd-B expression patterns.
Homeodomains of the human thyroid transcription factor 1 and Antp have similar amino acids in their recognition helix but recognise different sequences. Amino acid residues outside of the recognition helix play an important role in the determination of DNA binding specificities.
The interaction of the Antp homeodomain with mutated Antp binding sites has been studied using assays in yeast. Base pair 7 on the Antp recognition helix is important for Antp binding, base pairs 8 and 9 influence recognition.
A probe of the Antp gene of D.subobscura hybridized to the chromosomes equivalent to the E element of Muller's terminology in the 9 species of the obscura group. This result is consistent with the idea that single copy genes do not move around the genome and that chromosomal elements have conserved their genetic identity during evolution.
Enhancer trapping methods have been used to identify regulatory elements and corresponding genes that are influenced by the homeotic Antp gene product.
Ecol\lacZ reporter gene constructs have been used to demonstrate that the effect Pc has on Antp P1 regulatory sequences diminishes the shorter the sequences are. the bithorax complex responsive elements repressing Antp P1 transcription in the CNS are located within the -1.9 to +2.1 kb region, and the epidermis-specific repressing elements are located in the intron region +2.1 to +7.8 kb. Additional sequences located between 10 and 35 kb upstream of P2 are required to regulate P2 promoter transcription.
Heterodimer analysis was used to determine the DNA binding properties of 68 amino acid Antp homeodomain. Results suggest that the Antp homeodomain interacts in vitro as a monomer with the DNA target sites used. Methylation and ethylation studies indicated that the homeodomain closely approaches the target DNA from one side in a region extending across 3 phosphate backbones.
The patterns of transcription from the two Antp promoters during embryogenesis have been analysed.
Data for the determination of the complete three dimensional structure, structure calculations and salient features of the homeodomain have been collected and compared to prokaryotic repressor proteins. The structure of the homeodomain contains a helix from residues 10--21, a helix-turn-helix motif from residues 28--52 and a flexible fourth helix from residues 53--59. The helices enclose a well defined molecular core of hydrophobic amino acid side chains.
Homeotic gene products can be ectopically expressed in regions where they are normally down regulated causing no phenotypic consequence in the epidermis.
Genetic and developmental investigations indicate that the Antp locus has two promoters: P1 and P2, that are separable by deletions.
Ectopic expression of mouse Hox-2.2, an Antp-like gene, in an Antp+ genetic background causes variable levels of embryonic lethality and homeotic transformations of head segments in larvae. The phenotypic effect of Antp ectopic expression differs from mouse Hox-2.2 ectopic expression in that there is little effect on the first thoracic segment. Ectopic expression of mouse Hox-2.2 has induced a transformation of antennae to leg in adults but no obvious morphological changes in the adult cuticular structures of the thoracic and abdominal segments.
DNAse I footprinting of ftz binding sites near the two Antp promoters identifies a consensus sequence including ATTA, as does the consensus sequence for en, eve and bcd binding sites. DNA bending is proposed as an explanation for the presence of a shared motif between proteins with divergent recognition helices. The ATTA would not directly contact amino acid side chains of the recognition helix, but would be necessary for bending the DNA around the homeodomain, perhaps facilitating protein-DNA contacts.
Genetic background variation influencing expression of the Antp homeotic phenotype was examined by reciprocally crossing the Antp73b locus into Antp+ strains and their genetically heterogeneous parent strain. The parent strain, Antp73b, has a small patch of untransformed antennal tissue remaining on the homeotic femur. The size of this patch was used as an assay for background variation.