Mapping of multiple mouse loci related to the farnesyl pyrophosphate synthetase gene

The prenyltransferases are a class of enzymes involved in the synthesis of sterol and nonsterol isoprene compounds. We report here the chromosomal mapping of nine loci in the mouse that hybridize to the cDNA for the enzyme farnesyl pyrophosphate synthetase (FPS), a prenyltransferase that catalyzes the synthesis of an intermediate common to both the sterol and nonsterol branches of the isoprene biosynthetic pathway. Mapping was performed with genomic DNA from a mouse-hamster somatic cell hybrid panel, and by linkage analysis with recombinant inbred strains and the progeny of an interspecific backcross. The mapped loci have been designated farnesyl pyrophosphate synthetase-like-1 (Fpsl-1) on mouse Chromosome (Chr) 3; Fpsl-2 on Chr 4; Fpsl-3, Fpsl-4, and Fpsl-5, dispersed on Chr 10; Fpsl-6 on Chr 12; Fpsl-7 on Chr 13; Fpsl-8 on Chr 17; and Fpsl-9 on Chr X. It is presently unclear which of these loci encode active prenyltransferases and which may correspond to pseudogenes. The strongly hybridizing loci provide convenient genetic markers for seven mouse chromosomes.     

Regional localization of the human genes for S-adenosylhomocysteine hydrolase (cen→q131) and adenosine deaminase (q131→qter) on chromosome 20

Summary The gene loci for S-adenosylhomocysteine hydrolase (AHCY) and adenosine deaminase (ADA), two enzymes with related metabolic functions, have both been assigned to human chromosome 20. We have used rodent-human somatic hybrids containing translocations involving human chromosome 20 to more precisely determine the relative locations of the AHCY and ADA loci. Our results assign the AHCY locus to the long arm of chromosome 20, in the region cenq131, and ADA to the region q131qter.     

The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1

We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.     

Regulation of red cell membrane deformability and stability by skeletal protein network

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca2+ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca2+ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and physiologic concentrations of calmodulin. Our data show that Ca2+ concentrations in the range of 1 to 100 microM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca2+ concentrations higher than 100 microM, and calmodulin had no effect. Examination of the the effects of Ca2+ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca2+ and calmodulin on spectrin-protein 4.1-actin interaction.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

Expression of galactose-1-P-uridyltransferase in Chinese hamster × human galactosemia somatic cell hybrids

Lymphocytes from a patient with classic galactosemia (GALT deficiency) were hybridized with a Chinese hamster cell line. Electrophoretic evaluation of GALT in 31 independently derived interspecific hybrid clones failed to demonstrate expression of the human GALT gene even when human chromosome 9 was present. Possible mechanisms for this lack of expression are presented.This work was supported in part by Grants HD-04612 and HD-05615 from the National Institute of Child Health and Human Development.     

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.