BIOLOGICAL OVERVIEW

The paired ( prd) gene, once accorded a low significance rating in comparison with other pair-rule genes, has had its status elevated due to more recent information. Transcription of primary pair-rule genes is controlled directly by gap genes and maternal factors, while secondary pair-rule genes are considered to be regulated by the pair-rule primary genes (in addition to regulation by maternal and gap genes). paired is now thought to be a secondary pair rule gene. As such, it plays a decisive role in the progression of a regulatory hierarchy from pair-rule directed segmentation of the embryo to the subdivision carried out by segment polarity genes specifying positional information within segments.

The most recent analysis shows that early paired expression is determined by a combination of maternal factors and gap genes. The dissection of the paired promoter has revealed that all the other pair-rule genes are regulating the refinement of paired early stripe expression and the transition from an early 8 stripe pattern to a late 14 stripe pattern. A "prd zebra element" was found in the proximal promoter able to direct the expression of seven stripes; a 3' regulatory region regulates the anterior dorsal spot expression (Gutjahr 1993, 1994).

A specific function of paired is the development of the ventral chemosensory organ. Chemosensory organs are involved in gustatory and/or olfactory responses in larvae and/or adult flies. Part of the larval fly's feeding apparatus is a chemosensory structure known as the ventral organ, located near the mouth opening. This chemosensory organ consists of a pair of simple lobes with five small pores. These pores are innervated by the ventral ganglia that connect to the brain via the maxillary nerve.

An experimental manipulation demonstrates the involvement of paired in the development of this organ.Hammerhead ribozymes designed to cleave prd mRNA were placed under the regulation of heat shock promoters and induced during specific periods in the fly's larval development. Late stage embryos were exposed to heat for four to five hours, at a time when paired is expressed in the head. Treatment at this time avoids confusing possible effects with prd early stripe expression in head segments. In essence, the prd transcripts were knocked out by the ribozyme activity, drastically reducing Paired protein expression. The ventral chemosensory organs failed to develop, supporting the notion that paired is crucial for their normal development (Vanario-Alonso, 1995).

paired, whose product ishomologous to the Drosophila Gooseberry and mammalianPax3 proteins, has three general functions: properdevelopment of the larval cuticle, survival to adulthood andmale fertility. Both DNA-binding domains, the conservedN-terminal paired-domain (PD) and prd-type homeodomain (HD), arerequired within the same molecule for all general pairedfunctions, whereas a conserved His-Pro repeat located nearits C terminus is a transactivation domain potentiatingthese functions. The C-terminal moiety of Paired includestwo additional functional motifs: one, also present inGooseberry and Pax3, is required for segmentationand cuticle development; the other, retained only inGooseberry, is necessary for survival. The male fertilityfunction, which cannot be replaced by Gooseberry andPax3, is specified by the conserved N-terminal rather thanthe divergent C-terminal moiety of Paired. It is concludedthat the functional diversification of paired, gooseberryand Pax3, primarily determined by variations in theirenhancers, is modified by adaptations of their codingregions as a necessary consequence of their newly acquiredspatiotemporal expression (Xue, 2001).

With the aid of two alleles of prd, prd-Gsb andprd-Pax3, in which the gsb and Pax3 coding regions wereplaced under the control of the entire prd cis-regulatory region,it has been shown that Prd activity is required in vivoduring at least three distinct developmental stages to ensureproper segmentation of the larval cuticle, postembryonicviability and male fertility. In this study, a series of prd transgenes were constructed thatexpress various versions of the Prd protein, includingtruncations or chimeras of Prd, Gsb and Pax3 under the controlof the complete prd cis-regulatory region. All transgenes weretested for their ability to rescue any of these Prd functions.Thus, this report is the first example of a complete functionalanalysis of the Prd protein under natural conditions (Xue, 2001).

The presence of two DNA-binding domains, PD and HD, inPrd and some other members of the Pax gene family raises thequestion of whether the regulation of any of its target genesrequires the binding of both or only one of its two DNA-bindingdomains. Both mechanisms are compatible with invitro results. In vivo studies show that both PD andHD are absolutely required for Prd function because deletionof either or both of these domains from the prd-Gsb transgeneresults in the complete loss of its ability to rescue the segment-polaritygene activation, cuticular phenotype and lethality ofprd mutants. Moreover, since a point mutation in the PD (i.e., prd-Gsb^P17L)eliminates all Prd functions, the DNA-bindingability of the PD is necessary for the normal functions of Prd.An analogous mutation abolishes DNA binding of the humanPAX5 protein and causes Waardenburg'ssyndrome I when present in PAX3 (Xue, 2001).

The observation that prd-GsbdeltaP and prd-GsbdeltaH cannotcomplement for any function of Prd implies that the PD andHD must be present in the same Prd molecule, presumablybecause each Prd function requires the recognition of at leastone composite DNA target site.In agreement with these findings, Prd proteins unable to bindDNA as a result of single amino acid substitutions in either thePD or HD can no longer activate the ectopic expression of Prd-targetgenes when expressed ubiquitously under the control ofthe heat-shock promoter nor will these mutant proteinsperform any Prd in vivo function when expressed under thecontrol of some of prd enhancers.In addition, a composite Prd target site has been identified inthe even-skipped enhancer whose mutation in either the PD orHD binding portion dramatically reduces Prd binding activityboth in vitro and in vivo. The finding thatthe PD and HD cannot complement in trans for any functionof Prd agrees with some observations obtained with mutanttransgenes in vivo, but contradictsresults obtained in vitro, and in vivowhen the two Prd mutant proteins are expressed under heat-shockcontrol. Taken together, theseresults imply that the PD and HD of Prd may interact with theirDNA targets cooperatively and that this cooperativity can occurin trans only if the proteins are produced at concentrationsmuch higher than those occurring naturally (Xue, 2001 and references therein).

The PRD repeat, which encodes a 20-30 amino acid His-Prorepeat, was discovered in an attempt to verify predictions ofthe gene network hypothesis in a search for protein-codingdomains of prd. The PRD repeat is foundin a number of Drosophila early developmental genes,including bicoid and daughterless, but its invivo function remained unknown. Previous experiments in cellculture systems have shown that the PRD repeat is part of atransactivation domain thatis necessary to drive ectopic expression of Prd-target genesunder the control of ubiquitously expressed Prd. Other studies, however, have suggested that the PRD repeatis not essential for in vivo functions of Prd. The Prd protein whose PRDrepeat has been deleted in prd-PrddeltaPRD is still able to performall in vivo functions of Prd, which implies that the PRD repeatis not absolutely required for Prd function. However, the factthat one copy of prd-PrddeltaPRD exhibits significantly reducedefficiency in its ability to rescue the lethality and male sterilityof prd mutants indicates that the PRD repeat greatly facilitatesthese Prd functions. This conclusion is corroborated andextended by the results obtained with prd-Gsb+PRDtransgenes, which demonstrate that the PRD repeat enhancesthe viability as well as the cuticle function of Prd. Thus, thePRD repeat is an important transactivation domain thatfacilitates all functions of Prd (Xue, 2001).

Previous work has demonstrated that Prd, Gsb and Pax3proteins are, at least partially, functionally equivalent. When expressed under thecontrol of the entire cis-regulatory region of prd, both Gsb andPax3 can activate Prd-target genes necessary for the generationof wild-type cuticle, while Gsb is able to rescue prd mutantsto adulthood. These results strongly suggested that theacquisition of cis-regulatory regions rather than the divergenceof their coding regions is the primary evolutionary mechanismresponsible for the functional diversification of prd, gsb andPax3 genes. However, although Gsb and Pax3 can substitutefor most Prd functions, they do so at considerably reducedefficiencies: this indicates that these proteins had to adapttheir new functions for optimal performance by subsequentmutations producing the observed divergence of the Prd, Gsband Pax3 proteins. The result of thisprocess of adaptation has been studied by examining the functional differencesbetween these proteins when expressed as evolutionary allelesunder the same cis-regulatory region (Xue, 2001).

The results lead to the idea that, in addition to the PRDrepeat, two motifs or domains are present in the C-terminalportion of Prd, on whose functions the formation of wild-typelarval cuticle and survival to adulthood depend. Although nosignificant similarity has been found among the primarysequences of the C-terminal moieties of Prd, Gsb and Pax3, themotif required for implementing wild-type cuticle is shared byall three proteins. In contrast, the motif necessary for Prd’sviability function is retained only in Gsb, presumably assecondary or tertiary protein structure. It should be stressedthat at least two independent functions of Prd are required forviability, one of which, Pax3, is able to perform even better thanGsb. However, Pax3 is unable tosubstitute for one of the viablity functions of Prd and evenexerts a dominant-negative effect on it.In agreement with this postulate, combining the results withthose obtained with two weak prd alleles encoding truncatedPrd proteins, allows the motifs for the cuticle andviability functions to be mapped within the C terminus of Prd (Xue, 2001).

Although prd-Gsb rescues prdmutants to viable adults, all males are sterile. Since wild-typemales transgenic for two copies of prd-Gsb are fertile, it is concluded that prd has a function required for male fertility.Moreover, since prd-Gsb includes the entire cis-regulatory regionof prd, its failure to rescue male fertilitymust be caused by the inability of Gsb to replace this functionof the Prd protein. Since Prd and Gsb share a highly conservedN-terminal portion consisting of two DNA-binding domains,the PD and HD, it seemed plausible to map this functionaldifference to their divergent C termini. Surprisingly, however,the protein-domain-swapping experiments indicate that theconserved N-terminal rather than the divergent C-terminalportion is the determinant for this particular function of Prd.Therefore, it is suggested that at least one specific Prd target site,recognized by Prd but not Gsb, is involved in male fertility.The male fertility function of Prd is controlled by a specificprd enhancer uncovered in prd mutants by a prd rescueconstruct that lacks 5 kb of the downstream regulatory region. Consistent with this interpretation, aprd transgene that expresses Prd merely under the control ofthis 5 kb regulatory region is able to confer fertility to prd-Gsbmales mutant for prd. Malescompletely deficient for this fertility function of prd have noaccessory glands, while accessory glands begin to form in prdmutant males rescued by prd-Gsb, but stop development at aseverely reduced size. These findings are in agreement with the hypothesisthat new functions evolve primarily through the acquisition ofnew enhancers during gene duplication and that the adaptation of the protein issecondary and a necessary consequence of its expression in thenewly acquired context of this function (Xue, 2001).

These results further imply that the C-terminal portions of Prdand Gsb, though divergent in their primary sequences, are stillqualitatively the same. Hence, the validity ofamino acid similarity as a general measure of functionalequivalence in homologous proteins can be questioned.Instead, it has been proposed that this measure offunctional equivalence should be replaced by calculations of the mutual entropybetween two protein sequences, a more precise statisticalmeasure that takes into account the probability by whichcertain amino acids are replaced by others (Xue, 2001 and references therein).

GENE STRUCTURE

Bases in 5' UTR - 264

Exons - two

Bases in 3' UTR - 350

PROTEIN STRUCTURE

Amino Acids - 613

Structural Domains

The Paired protein has a bipartite paired domain (PD), a central paired class homeodomain and a C-terminal PRD (His-Pro sequence) repeat (Frigerio, 1986). The Paired domain is a region of homology shared by certain homeodomain proteins called Paired homeodomains. The Paired repeat, is embedded in a proline rich transactivation domain (Cai, 1994).

Pax-3, the vertebrate Paired homolog, contains two structurally independent DNA-binding domains: a paired-domain and a homeodomain. Their functionalinterdependence has been suggested by the analysis of the Sp-delayed (Spd) mouse mutant, in which a glycine to arginine substitutionat position 9 of the paired-domain abrogates DNA binding by both domains. This glycine is located in the beta-turn portion of abeta-hairpin motif; the requirement for this structure was investigated by mutagenesis at this and neighboring positions. At position9, only substitution with proline increases DNA binding by the paired-domain and homeodomain above the level observed with the Spdarginine mutation, suggesting that the beta-turn is necessary for the function of both DNA-binding domains. Alanine scanningmutagenesis also identifies a number of flanking residues important for DNA binding by both domains, emphasizing the requirement ofthe beta-hairpin for the interaction of Pax-3 with DNA. These mutations reduce binding by thehomeodomain at the monomeric level and do not impair dimerization on a TAAT(N)2ATTA consensus motif. In contrast, thewild-type paired-domain prevents dimerization on consensus motifs with 3-base pair spacing of the typeTAAT(N)3ATTA. Importantly, both the deleterious effect of the Spd mutation on homeodomain DNA binding and the loss ofdimerization on TAAT(N)3ATTA motifs can be transferred to a heterologous homeodomain from the human phox protein. Moreover,the presence of the paired-domain affects sequence discrimination within the 3-base pair spacer in this context. These analysesestablish that the beta-hairpin motif is essential for paired-domain and homeodomain DNA binding, and suggest a novel mechanism bywhich the paired-domain can influence sequence specificity of the homeodomain within the Pax-3 polypeptide (Underhill, 1997).

date revised: 28 February 2001  

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