Conservation of the paired domain in metazoans and its structure in three isolated human genes.

Sequences homologous to the paired domain of Drosophila melanogaster have been conserved in species as distantly related as nematodes, sea urchins, or man. In particular, paired domains of three human genes, HuP1, HuP2 and HuP48, have been isolated and sequenced. Together with four Drosophila paired domains, they fall into two separate paired domain classes named according to their Drosophila members, paired--gooseberry and P29 class. The P29 class includes the mouse Pax 1 and the human HuP48 gene which are nearly identical in their sequenced portions and hence might be true homologues. In addition to the paired domain, the two human genes HuP1 and HuP2 share the highly conserved octapeptide HSIAGILG with the two gooseberry genes of Drosophila. Possible functions of the paired domain are discussed in the light of a predicted helix-turn-helix structure in its carboxy-terminal portion.     

A new Pax gene,Pax-9, maps to mouse Chromosome 12

Members of the Pax gene family have recently been shown to play important roles in mouse embryogenesis. Of eight so far characterized Pax genes, three have been associated with mouse developmental mutants. Here we report the cloning of a new Pax gene, Pax-9. Most of the DNA sequence encoding the highly conserved paired domain has been determined and compared with previously known paired domains. This comparison classifies Pax-9 as a member of the same subgroup as Pax-1/undulated. By analysis of the segregation of a Pax-9 restriction fragment length polymorphism and a large number of simple sequence length polymorphisms in an interspecific C57BL/6 x Mus musculus mollosinus backcross, Pax-9 was mapped close to the D12Nds1 locus on the proximal part of Chromosome (Chr) 12.     

Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system

The murine genome contains multiple genes with protein domains homologous to the Drosophila paired box, present in certain segmentation genes. At least one of these murine paired box (Pax) genes is associated with a developmental mutation. This report, in conjunction with the accompanying paper, describes a second member of this gene family, Pax2, that is also expressed during embryogenesis. Two overlapping cDNA clones were isolated and sequenced. At least two forms of the Pax2 protein can be deduced from the cDNA sequence. In addition to the highly conserved paired domain, an octapeptide sequence is located downstream. Expression of Pax2 is primarily restricted to the developing embryo in the excretory and central nervous systems. The transient nature of Pax2 expression during kidney organogenesis correlates with polarization and induction of epithelial structures and may indicate an important morphogenetic role for this gene.     

The Gene for PAX7, a Member of the Paired-Box-Containing Genes, Is Localized on Human Chromosome Arm 1p36

The murine Pax-7 gene and the cognate human gene, formerly designated HuP1, are members of the multi-gene paired-box-containing class of developmental regulatory genes first identified in Drosophila. By analysis of somatic cell hybrids segregating human chromosomes, the gene encoding PAX7 was localized to human chromosome 1. Fluorescence in situ hybridization confirmed this assignment and allowed mapping of the gene to the terminal region of the short arm (1p36) of the chromosome. Additionally, these results confirm the extensive homology between human chromosome 1p and the distal segment of mouse chromosome 4, extending from bands C5 through E2.     

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.     

Identification of essential amino acid changes in paired domain evolution using a novel combination of evolutionary analysis and in vitro and in vivo studies

Pax genes are defined by the presence of a paired box that encodes a DNA-binding domain of 128 amino acids. They are involved in the development of the central nervous system, organogenesis, and oncogenesis. The known Pax genes are divided into five groups within two supergroups. By means of a novel combination of evolutionary analysis, in vitro binding assays and in vivo functional analyses, we have identified the key residues that determine the differing DNA-binding properties of the two supergroups and of the Pax-2, 5, 8 and Pax-6 subgroups within supergroup I. The differences in binding properties between the two supergroups are largely caused by amino acid changes at residues 20 and 121 of the paired domain. Although the paired domains of the Pax-2, 5, 8 and the Pax-6 group differ by >19 amino acids, their distinct DNA-binding properties are determined almost completely by a single amino acid change. Thus, a small number of amino acid changes can account in large part for the divergence in binding properties among the known paired domains. Our approach for selecting candidate sites responsible for the functional divergence between genes should also be useful for studying other gene families.     

Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland.

Several mouse genes designated 'Pax genes' contain a highly conserved DNA sequence homologous to the paired box of Drosophila. Here we describe the isolation of Pax8, a novel paired box containing clone from an 8.5 day p.c. mouse embryo cDNA library. An open reading frame of 457 amino acids (aa) contains the 128 aa paired domain near the amino terminus. Another conserved region present in some other paired box genes, the octapeptide Tyr-Ser-Ile-Asn-Gly-Leu-Leu-Gly, is located 43 aa C-terminal to the paired domain. Using an interspecies backcross system, we have mapped the Pax8 gene within the proximal portion of mouse chromosome 2 in a close linkage to the surf locus. Several developmental mutations are located in this region. In situ hybridization was used to determine the pattern of Pax8 expression during mouse embryogenesis. Pax8 is expressed transiently between 11.5 and 12.5 days of gestation along the rostrocaudal axis extending from the myelencephalon throughout the length of the neural tube, predominantly in two parallel regions on either side of the basal plate. We also detected Pax8 expression in the developing thyroid gland beginning at 10.5 days of gestation, during the thyroid evagination. In the mesonephros and metanephros the expression of Pax8 was localized to the mesenchymal condensations, which are induced by the nephric duct and ureter, respectively. These condensations develop to functional units, the nephrons, of the kidney. These data are consistent with a role for Pax8 in the induction of kidney epithelium. The embryonic expression pattern of Pax8 is compared with that of Pax2, another recently described paired box gene expressed in the developing excretory system.     

DNA-binding characteristics of cnidarian Pax-C and Pax-B proteins in vivo and in vitro: no simple relationship with the Pax-6 and Pax-2/5/8 classes

Cnidarians are the simplest animals in which distinct eyes are present. We have previously suggested that cnidarian Pax-Cam might represent a precursor of the Pax-6 class. Here we show that when expressed in Drosophila imaginal discs, Pax-Cam chimeric proteins containing the C-terminal region of EY were capable of eye induction and driving expression of a reporter gene under the control of a known EY target (the sine oculis gene). Whilst these results are consistent with a Pax-6-like function for Pax-Cam, in band shift experiments we were unable to distinguish the DNA-binding behaviour of the Pax-Cam Paired domain from that of a second Acropora Pax protein, Pax-Bam. The ability of a Pax-Bam/EY chimera to also induce eye formation in leg imaginal discs, together with the in vitro data, cast doubt on previously assumed direct relationships between cnidarian Pax genes and the Pax-6 and Pax-2/5/8 classes of bilateral animals.     

The functional conservation of proteins in evolutionary alleles and the dominant role of enhancers in evolution.

Drosophila paired- embryos can be rescued to viable adults by the evolutionary alleles prd-Gsb and prd-Pax3, which express the Drosophila Gooseberry and mouse Pax3 proteins under the control of the paired cis-regulatory region. As prd-Gsb uncovers a prd function involved in the proper abdominal segmentation of adults, evolutionary alleles, defined and constructed in this manner, may often be weak and thus serve to discover hitherto unknown functions of a gene. Our findings show that the Gooseberry and Pax3 proteins have conserved most or all functions of the related Drosophila Paired protein although their C-terminal halves appear unrelated in sequence but not in 3-D structure essential for function. It follows that the acquisition of new cis-regulatory regions rather than the divergence of the C-terminal coding regions has been the primary device for the functional diversification of the Drosophila genes paired and gooseberry and the mouse Pax3 gene. The operation of this mechanism in insects as well as vertebrates suggests a major role in evolution.