A deletion map of the human Y chromosome was constructed by testing 96 individuals with partial Y chromosomes for the presence or absence of many DNA loci. The individuals studied included XX males, XY females, and persons in whom chromosome banding had revealed translocated, deleted, isodicentric, or ring Y chromosomes. Most of the 132 Y chromosomal loci mapped were sequence-tagged sites, detected by means of the polymerase chain reaction. These studies resolved the euchromatic region (short arm, centromere, and proximal long arm) of the Y chromosome into 43 ordered intervals, all defined by naturally occurring chromosomal breakpoints and averaging less than 800 kilobases in length. This deletion map should be useful in identifying Y chromosomal genes, in exploring the origin of chromosomal disorders, and in tracing the evolution of the Y chromosome.
In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism.The GLUT4 glucose transporter is expressed predominantly in adipose and muscle tissues, where it accounts for the bulk of insulin-stimulated glucose uptake (12,84,95). In the presence of insulin, GLUT4 is redistributed from an intracellular compartment to the plasma membrane, where it facilitates the diffusion of glucose into the cell (15,35,73,77,102). Another glucose transporter isoform, GLUT1, is also expressed in fat and muscle tissues and is present at high levels in many other cell types and in cultured cell lines. A large proportion of GLUT1 is present on the plasma membrane even in the absence of insulin. Thus, while both GLUT1 and GLUT4 recycle at the plasma membrane, only GLUT4 recycling is characterized by significant intracellular sequestration, resulting from a slow rate of exocytosis, in the absence of insulin. Insulin increases the rate of GLUT4 exocytosis, with little or no decrease in its rate of endocytosis, so that in adipocytes the proportion of GLUT4 at the cell surface increases from Ͻ10% in the absence of insulin to 35 to 50% in its presence (41,55,85,113,114).Characterization of the intracellular insulin-responsive GLUT4-containing compartment is complicated by the fact that GLUT4 resides in several morphologically distinct locations within the cell. Ultrastructural studies have shown that GLUT4 is present in tubulovesicular structures distinct from lysosomes, as well as in a perinuclear compartment that is in close vicinity to the trans-Golgi network (39,(96)(97)(98). Recent work demonstrates that approximately 40 to 45% of intracellular GLUT4 localizes in a transferrin receptor (TfnR)-positive endosomal compartment, while 50 to 60% is in a second, TfnR-negative compartment; it is GLUT4 in this TfnR-negative compartment that is rapidly mobilized upon insulin addition (1,32,46,55,57,65,76). In primary adipocytes, this TfnR-negative ...
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