Characterization and localization of the mProx1 gene directly upstream of the mouse α-globin gene cluster: Identification of a polymorphic direct repeat in the 5′UTR

The α-globin major regulatory element (αMRE) positioned far upstream of the gene cluster is essential for the proper expression of the α-globin genes. Analysis of the human and mouse α-globin Upstream Flanking Regions (αUFR) has identified three nonglobin genes in the order Dist1-MPG-Prox1-α-globin. Further characterization of the whole region indicates that the αMRE and several other erythroid DNase HSSs are associated with the transcription unit of the Prox1 gene. In this paper we describe the characterization and localization of the mouse Prox1 cDNA and compare it with its human homolog, the -14 gene, and another human cDNA sequence named hProx1. Our results show a strong conservation between the -14 gene and the mouse Prox1 gene with the exception of the first exon of the mProx1 gene. This exon is absent in the -14 cDNA but is present and conserved in the human Prox1 cDNA, indicating that the human -14/hProx1 gene is alternatively spliced or transcribed. The mProx1 gene encodes a predicted protein of 491 amino acids (aa) whose function is not known. In the 5′UTR of this gene, a 35-bp repeat (VNTR) is positioned, which is highly polymorphic among laboratory inbred mice (Mus domesticus). Our results strongly suggest that the mProx1 VNTR arose during the divergence of M. spretus and M. domesticus. Besides its use in evolutionary studies and positional cloning, the mProx1 VNTR might be invaluable for monitoring the expression of a transgenic mProx1 gene. The cloning of the mProx1 gene will be helpful to analyze its possible role on α-globin as well on MPG expression in the mouse

PDZK1: II. an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells

BACKGROUND: PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells. In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed. Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4. METHODS: After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells. RESULTS: Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD. Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney. In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity. Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate. In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells. CONCLUSION: We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA