Localization of the human gene encoding heterogeneous nuclear RNA ribonucleoprotein G (hnRNP-G) to chromosome 6p12

Summary The gene encoding nuclear RNA ribonucleoprotein G (hnRNP-G), a p43 glycoprotein, has been mapped to human chromosome 6, band p12, by radioactive in situ hybridization.     

Physical and radiation hybrid mapping of canine chromosome 12, in a region corresponding to human chromosome 6p12-q12

The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited.     

The γ-subunit of the rod photoreceptor cGMP-binding cGMP-specific PDE is expressed in mouse lung

The type 6 phosphodiesterase (PDE-6) from retinal rod photoreceptors is an αβγ[in2] heterotetramer. The α-and β-subunits contain catalytic sites for cGMP hydrolysis, whereas the γ-subunits (Pγ) serve as a protein inhibitor of the enzyme. Pγ is believed to be expressed only in photoreceptors. Using RT-PCR, we have amplified the complete coding sequence for Pγ from mouse lung RNA. The expression of Pγ in this tissue may be related to its ability to interact the type 5 phosphodiesterase (PDE-5), which is the predominant cGMP binding protein in lung. We therefore suggest that Pγ may have a wider signaling role in mammalian cells than previousl y appreciated.     

Molecular gene mapping of human aldolase A (ALDOA) gene to chromosome 16

Summary Mapping of human aldolase A (ALDOA) gene was performed by molecular hybridization techniques using a panel of human-mouse cell hybrids and sorted fractions of human metaphase chromosomes besides in situ hybridization. For the purpose, three kinds of DNA probes derived from the coding region (probe-1), the 3 noncoding region (probe-2), and the coding and 3 noncoding regions (probe-3) of human aldolase A cDNA clone, pHAAL116-3, were selectively employed. The results of RNA and DNA blot analyses indicated that the human ALDOA gene is located on chromosome 16. The in situ hybridization experiment also indicated that the ALDOA gene was localized to 16q22–q24.     

The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization

Retinal rod cGMP phosphodiesterase (PDE6 family) is the effector enzyme in the vertebrate visual transduction cascade. Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of alpha and beta subunits. A system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6alphabeta and PDE5, which contain the N-terminal GAFa/GAFb domains, or portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed using gel filtration and sucrose gradient centrifugation. The composition of formed dimeric enzymes was analyzed with Western blotting and immunoprecipitation. Consistent with the selectivity of PDE6 dimerization in vivo, efficient heterodimerization was observed between the GAF regions of PDE6alpha and PDE6beta with no significant homodimerization. In addition, PDE6alpha was able to form dimers with the cone PDE6alpha' subunit. Furthermore, our analysis indicated that the PDE6 GAFa domains contain major structural determinants for the affinity and selectivity of dimerization of PDE6 catalytic subunits. The key dimerization selectivity module of PDE6 has been localized to a small segment within the GAFa domains, PDE6alpha-59-74/PDE6beta-57-72. This study provides tools for the generation of the homodimeric alphaalpha and betabeta enzymes that will allow us to address the question of functional significance of the unique heterodimerization of rod PDE6.     

Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig

Genes located on human chromosome 12 (HSA12) are conserved on pig chromosomes 5 and 14 (SSC5 and SSC14), with HSA12q23.3-->q24.11 harboring the evolutionary breakpoint between these chromosomes. For this study, pig sequence-tagged sites (STS) were developed for nine HSA12 genes flanking this breakpoint. Radiation hybrid (RH) mapping using the IMpRH panel revealed that COL2A1, DUSP6, KITLG, PAH and STAB2 map to SSC5, while PXN, PLA2G1B, SART3 and TCF1 map to SSC14. Polymorphisms identified in COL2A1, DUSP6, PAH, PLA2G1B and TCF1 were used for genetic linkage mapping and confirmed the map locations for these genes. Our results indicate that the HSA12 evolutionary breakpoint occurs between STAB2 and SART3 in a region spanning less than five million basepairs. These results refine the comparative map of the HSA12 evolutionary breakpoint region and help to further elucidate the extensive gene order rearrangements between HSA12 and SSC5 and 14.