No abstract

Major histocompatibility complex (MHC) class I molecules bind peptides that are delivered from the cytosol into the endoplasmic reticulum by the MHC-encoded transporter associated with antigen processing (TAP). Peptide capture by immature heterodimers of class I heavy chains and beta 2-microglobulin may be facilitated by their physical association with TAP. A genetic defect in a human mutant cell line causes the complete failure of diverse class I heterodimers to associate with TAP. This deficiency impairs the ability of the class I heterodimers to efficiently capture peptides and results from loss of function of an unidentified gene or genes linked to the MHC.

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y 3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y 7 , has 352 amino acids and shares 23-30% amino acid identity with the P2Y 1 -P2Y 6 purinoceptors. The P2Y 7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 ؎ 6 nM), much less for UTP and ADP (ϳ1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y 7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y 7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y 7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y 7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y 7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.The widespread occurrence of metabotropic receptors for extracellular ATP has long been inferred from physiological and pharmacological evidence (1). A number of such G proteincoupled ATP receptors have been characterized and a consensus on their nomenclature has termed all of these as P2Y purinoceptors (to be individually named P2Y 1 to P2Y n ), regardless of previous terminology such as P 2U or P 2T for subclasses thereof (2). The first such receptors to be characterized by DNA cloning and expression were the P2Y 1 receptor (where UTP is inactive) (3) and the P2Y 2 receptor (ATP and UTP are equally active) (4). True species homologues (or orthologues) of P2Y 1 have since been obtained, e.g. bovine (5) and human (6), and of P2Y 2 , e.g. from human airway epithelium (7) or human erythroleukemia (HEL) 1 cells (8). Further types identified by cloning have been the P2Y 3 receptor (UDP Ͼ ADP Ͼ ATP) (9) and P2Y 4 (UTP Ͼ Ͼ ATP, and more strongly related to P2Y 2 ) (10, 11). Further novel P2Y receptors have recently been identified from their cDNAs from chicken activated T lymphocytes (12) and rat vascular smooth muscle cells (13) and designated P2Y 5 and P2Y 6 receptors. Previously we have demonstrated at least three P2 purinoceptors on the hematopoietic cell line, HEL cells, by intracellular calcium mobilization and by photoaffinity labeling (8). Here we report the molecular cloning and characterization of one of these, a novel P2 purinergic receptor designated P2Y 7 .

No abstract

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.