Detection of homeobox genes in development and evolution.

The homeobox genes encode a family of DNA-binding regulatory proteins whose function and genomic organization make them an important model system for the study of development and differentiation. Oligonucleotide primers corresponding to highly conserved regions of Antennapediaclass homeodomains were designed to detect and identify homeobox sequences in populations of DNA or RNA by means of the polymerase chain reaction (PCR). Here we present a survey of sequences detected by PCR using an initial set of primers (HoxA and HoxB) based on an early nucleotide consensus for vertebrate Antennapedia-class homeodomains. Several novel sequences are reported from both mouse genomic DNA and RNA from the developing mouse telencephalon. Forebrain-derived clones are similar to the chicken CHox7, Drosophila H2.0, and mouse Hlx genes. PCR also proved to be a rapid method for identifying homeobox sequences from diverse metazoan species. Cloning of three Antennapedia-related sequences from cnidarians provides evidence of ancient roles for homeobox genes early in metazoan evolution.     

Evolutionary conserved modules associated with zinc fingers in Xenopus laevis.

Many DNA-binding proteins that are involved in the differential regulation of gene expression are composed of multiple discrete modules. Association of the homeobox-encoded helix-turn-helix DNA-binding motif with conserved modules, such as the paired box or the POU domain, has led to the definition of structurally and functionally related subfamilies of regulatory proteins. The zinc finger, which is the second major nucleic acid-binding motif characterized to date, defines large multigene families in higher eukaryotes; we have isolated more than 100 Xenopus finger protein-encoding cDNAs and in this study we show that at least 10 of these clones share extensive sequence homologies in a region of more than 200 amino acids in the N-terminal nonfinger portion of the predicted proteins, which is connected to variable finger clusters. We refer to this element as a finger-associated boxes (FAX) domain. Cross-hybridization with human genomic DNA indicates that the finger-associated boxes domain is evolutionary conserved. Northern blot analysis shows that the corresponding genes are differentially expressed in the course of early Xenopus embryogenesis.     

The ovalbumin serpins revisited: perspective from the chicken genome of clade B serpin evolution in vertebrates

Serpin superfamily proteins, most of which are serine protease inhibitors, share an unusual mechanism rooted in their conserved metastable tertiary structure. Although serpins have been identified in isolated members of archea, bacteria, and plants, a remarkable expansion is found in vertebrates. The chicken protein ovalbumin, a storage protein from egg white, lacking protease inhibitory activity, is an historical member of the superfamily and the founding member of the subgroup known as ov-serpins (ovalbumin-related serpins) or clade B serpins. In the human, ov-serpins include 13 proteins involved in the regulation of inflammation, apoptosis, angiogenesis, and embryogenesis. Here, a detailed analysis of the chicken (Gallus gallus) genome identified 10 clade B serpin genes that map to a single approximately 150-kb locus and contain the signature protein sequence of serpins and the gene structure of ov-serpins, with either seven or eight exons. Orthologues of PAI-2 (SERPINB2), MNEI (SERPINB1), PI-6 (SERPINB6), and maspin (SERPINB5) are highly conserved. Comparison with human ov-serpins identified avian-specific and mammal-specific genes. Importantly, a unique model of mammalian ov-serpin evolution is revealed from the comparative analysis of the chicken and human loci. The presence of a subset of ov-serpin genes in zebrafish (Danio rerio) gives insight into the ancestral locus. This comparative genomic study provides a valuable perspective on the evolutionary pathway for the clade B serpins, allowing the identification of genes with functions that may have been conserved since the origin of vertebrates. In addition, it suggests that "newer" serpins, such as ovalbumin, have contributed to vertebrate adaptation.     

Identification of murine nuclear proteins that bind to the conserved octamer sequence of the immunoglobulin promoter region.

Sequence-specific DNA-affinity chromatography was used to purify a nuclear protein from the B-cell leukemia cell line BCL1 that specifically binds to the octamer sequence ATTTGCAT, previously shown to be important in the regulation of immunoglobulin genes. This protein has a molecular mass of approximately 70 kDa and is responsible for the protein-DNA interaction specific to lymphoid cells. Other proteins of molecular mass 80-90 kDa and 50-55 kDa that specifically bind to the octamer sequence were also identified. These results demonstrate that the octamer is recognized by several biochemically distinct nuclear proteins, perhaps to differentially regulate the expression of immunoglobulin genes.