There is considerable interest in understanding how cis-regulatory modifications drive morphological changes across species. Because developmental regulatory genes, including Hox genes, are remarkably conserved, their noncoding regulatory regions are likely sources for variations. Modifications of Hox cis-regulatory elements have potential to alter Hox gene expression and, hence, axial morphologies. In vertebrates, differences in the axial levels of Hox gene expression correlate with differences in the number and relative position of thoracic vertebrae. Variation in cis-regulatory elements of Hox genes can be identified by comparative sequence and reporter gene analyses in transgenic mouse embryos. Using these approaches, we show a remarkable divergence of the Hoxc8 early enhancers between mammals and fishes representing diverse axial morphologies. Extensive restructuring of the Hoxc8 early enhancer including nucleotide substitutions, inversion, and divergence result in distinct patterns of reporter gene expression along the embryonic axis. Our results provide an evolutionary perspective on how the enhancer elements are engineered and support the hypothesis that remodeling of Hox regulatory elements in different species has played a significant role in generating morphological diversity.

Mice lacking transcription factor AP-2α exhibit defects in the formation of the head, body wall, heart, neural tube, eye, and limbs, reflecting important sites of AP-2α expression in the developing embryo. AP-2α is also expressed in the postnatal mammary gland and has been linked to tumor progression and defects in growth regulation in the breast. We have used a transgenic mouse approach to identify tissue-specific cis-acting sequences associated with expression of the human AP-2α gene. Our analysis indicates that multiple elements located throughout the gene contribute to expression in the trigeminal ganglia, spinal cord, mammary gland, and epidermis. A discrete cis-element located within the fifth intron is required for expression in the face and limbs, and we have derived a permanent line of AP-2α: lacZ transgenic mice to assess expression of this latter enhancer throughout morphogenesis. We also introduced this transgene into an AP-2α-null mouse background and detected subtle alterations of its expression within the progress zone and apical ectodermal ridge of the forelimbs. Similar changes in lacZ expression were observed within the zeugopod, and these correlated with defects in radius condensation in AP-2α-knockout mice. Taken together, these findings indicate that cell:cell communication within the forelimb is altered in the absence of AP-2α and reveal novel regulatory potential for AP-2α in limb development.

AP-2 transciription factors are key regulatrs of mouse embryonic development,Aberrant expression of these genes has also been linked to the progression of human breast cancer. Here, we have investigated the role of the AP-2 geme family in the postnatal maturation of the virgin and pregnant mice. Subsequently, AP-2 expression declines during lactation and then is reactivated during involution. The AP-2α and AP-2γ proteins are localized in the ductal epithlium, as well as in the terminal end buds, suggesting that they may infuence growth of the ductalnetwork. We have tested this hypothesis by targeting AP-2α expression to the mouse mammary gland using the MMTV promoter. Ourstudies indicate that overexpresion of AP-2α inhibits mammary gland growth and morphogenesis, and this coincides with a rise in PTHrP expression. Alveolar buding is severly curtailed in transgenic virgin mice, while lobuloalveolar development and functional differentiation are inhibited druing preganancy and lactation, repectiely. Our studies strongly support a role for the AP-2 proteins in regulating the proliferation and differenatation of mammary gland epithelial cells in both mouse and human.

The catalytic subunit (Mr≈124,000) of human DNA polymerase б has been cloned by PCR using poly(A)+ RNA from HepG2 cells and primers designed from the amino acid sequence of regions highly conserved between bovine and yeast DNA polymerase б. The human cDNA was 3443 nucleotides in length and coded for a polypeptide of 1107 amino acids. The enzyme was 94% identical to bovine DNA polymerase б; and contained the numerous highly conserved regions previously observed in the bovine and yeast enzymes. The human enzyme also contained two putative zinc-finger domains in the carboxyl end of the molecule, as well as a putative nuclear localization signal at the amino-terminal end. The gene coding for human DNA polymerase б was localized to chromosome 19.

The 125- and 48-kDa subunits of bovine DNA polymerase б have been isolated by SDSpolyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase б preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nueleotide sequence of a full-length eDNA encoding the catalytic subunit of bovine DNA polymerase б. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123 707. This is in agreement with the molecular weight of 125 000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase б with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase б contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase б, but these regions share little or no homology with the a polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far. The polypeptide also contains two C-terminal clusters of cysteine residues postulated to be DNA binding sites or zinc fingers.