ammonium ͉ mouse mutants ͉ Rhesus deficiency

We previously have described a mouse model for polycystic kidney disease (PKD) caused by either of two mutations, kat or kat 2J , that map to the same locus on chromosome 8. The homozygous mutant animals have a latent onset, slowly progressing form of PKD with renal pathology similar to the human autosomal-dominant PKD. In addition, the mutant animals show pleiotropic effects that include facial dysmorphism, dwarfing, male sterility, anemia, and cystic choroid plexus. We previously fine-mapped the kat 2J mutation to a genetic distance of 0.28 ؎ 0.12 centimorgan between D8Mit128 and D8Mit129. To identify the underlying molecular defect in this locus, we constructed an integrated genetic and physical map of the critical region surrounding the kat 2J mutation. Cloning and expression analysis of the transcribed sequences from this region identified Nek1, a NIMA (never in mitosis A)-related kinase as a candidate gene. Further analysis of the Nek1 gene from both kat͞kat and kat 2J ͞kat 2J mutant animals identified a partial internal deletion and a single-base insertion as the molecular basis for these mutations. The complex pleiotropic phenotypes seen in the homozygous mutant animals suggest that the NEK1 protein participates in different signaling pathways to regulate diverse cellular processes. Our findings identify a previously unsuspected role for Nek1 in the kidney and open a new avenue for studying cystogenesis and identifying possible modes of therapy. P olycystic kidney disease (PKD) is the most common inherited nephropathy in humans (1). PKD is characterized by massive renal enlargement associated with the growth of fluidfilled intrarenal cysts. Genetic studies of inherited PKD in humans and animal models clearly have shown that mutations at multiple loci with different modes of inheritance result in various forms of PKD (2-7). In recent years, major advances have been made toward understanding the genetics of PKD. In humans, the PKD1 and PKD2 loci that cause autosomal-dominant PKD (ADPKD) and the murine orck locus that causes disease pathology similar to the human autosomal-recessive PKD (ARPKD) have been cloned. The PKD1 gene product, polycystin-1, encodes an integral membrane glycoprotein with significant homology to membrane proteins involved in cell-cell and͞or cell-matrix interactions (8). The PKD2 gene product, polycystin-2, is also an integral membrane protein with homology to the voltage-activated Ca 2ϩ channel ␣ 1E and the Na ϩ voltagedependent channels (9). Analysis of the primary sequence of the orck gene product reveals a protein containing 10 copies of an internally repeated, 34-aa (tetratricopeptide) motif that is found in a number of cell-cycle control proteins (10). Although the primary structure has predicted certain roles for these three proteins, their function still remains largely unknown. However, because patients with mutations in either PKD1 or PKD2 have a similar clinical phenotype and recent in vitro studies have shown that polycystin-1 and polycystin-2 interact via their Cterminal ...

Human lymphocyte lysates prepared by detergent treatment of intact, normal resting cells contain ribonucleases that are insensitive to many inhibitors commonly used with eucaryotic cells. Phenol-extracted ribonucleic acid (RNA) obtained directly from unfractionated cytoplasm is sometimes degraded, but after fractionation of the cytoplasmic material by sucrose density gradient centrifugation, the polyadenylated RNA, in particular, is inevitably destroyed. An extensive survey of ribonuclease inhibitors, undertaken as a consequence, indicated that the complexes formed between the oxovanadium ion and the four ribonucleosides were unique in their ability to suppress lymphocyte nuclease activity. It proved possible to isolate intact ribosomal RNA and polyadenylated messenger RNA from lymphocyte cytoplasm fractionated on sucrose gradients when 2.5 mM each of the four ribonucleosidevanadyl complexes was used throughout the procedure. The data showed that the size distribution of poly(A)-bearing RNA remained unchanged, with a peak at ~16 S under denaturing conditions, regardless of whether the mRNA was originally associated with polysomes or was nonpolysome bound. The cytoplasmic RNAs were completely free of contamination by either intact nuclear RNA or nuclear fragments. Furthermore, exogenous globin mRNA mixed with lymphocytes and reisolated together with endogenous cytoplasmic polyadenylated RNA was fully translatable only when ribonucleoside-vanadyl complexes were employed during the preparation. The use of this inhibitor should therefore be considered for all tissues in which ribonucleases impede isolation of intact RNA. e isolation of intact RNA from most animal cells relies on the use of exogenous ribonuclease inhibitors. Many substances including diethyl pyrocarbonate, polyvinyl sulfate, heparin, bentonite, macaloid, an assortment of ribonucleotides, sodium dodecyl sulfate, and proteinase K have been employed

Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rh null patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rh null red cells.