Expression of the erythropoietin gene.

Abstract

Injection of cobalt into rats resulted in erythropoietin (EPO) mRNA accumulation in the kidney. The same response was obtained upon bleeding. No EPO mRNA was detected in the spleen, salivary gland, or thymus following cobalt injection or bleeding. In some animals, but not in others, EPO mRNA was also expressed in the liver in response to cobalt injection. Time course studies showed that message appearance begins sometime between 3 and 6 h after cobalt injection. This correlated very well with the EPO concentration in the circulation; EPO levels in the circulation were the same as those of controls at 3 h but increased to six-to sevenfold that of controls by 6 h after cobalt injection. The mature EPO mRNA in the rat and mouse comigrated with the 18S rRNA, indicating that it is about 1,850 nucleotides in length.Abundant evidence indicates that, in the adult mammal, the kidney is a major source of erythropoietin (EPO) (2,5,10,11,17,18 These questions can now be addressed more directly by the use of cloned mouse EPO probes (15). We have induced EPO appearance in the circulation by cobalt injection (7) or bleeding and have monitored both EPO levels in the serum and EPO mRNA transcription in various tissues as a function of time. The data indicate that there is accumulation of the EPO mRNA in the kidney after stimulation and that, except for the liver, no other tissue examined shows the presence of the mRNA.MATERIALS AND METHODS Animals and cells. Male Long Evans rats, 2 to 4 months old (Charles Rivers Breeding Laboratories Inc., Wilmington, Mass.), were used in these experiments. In the experiments that used CoCl2, the rats were injected with 1 ml of 75 mM CoCl2 solution in saline subcutaneously in the back and killed by carbon dioxide asphyxiation at specified time intervals. Controls were injected with saline and killed immediately. In the bleeding experiments, 3 ml of blood (1.5 % of body weight) was drawn by cardiac puncture, and the animals were killed 10 h later. The kidneys, livers, thymuses, salivary glands, and spleens were dissected out and frozen in a dry ice-ethanol bath. Once frozen, the organs were stored at -70°C until RNA was extracted from them.The Assay of EPO. Where EPO levels in the serum were measured, blood was obtained by cardiac puncture and allowed to clot for 48 h at 4°C, and serum was collected after centrifugation. EPO was assayed in the serum by radioimmunoassay (19). The assays were done on four different volumes of serum, each one in triplicate.RNA preparation. Frozen tissues were crushed to a powder and homogenized in 4 M guanidine thiocyanate, using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, N.Y.). RNA was extracted by the method of Chirgwin et al. (3), as modified by Feramisco et al. (6). Cell pellets, in contrast to tissues, were simply suspended in 4 M guanidine thiocyanate, and the liberated DNA was sheared by repeatedly taking the lysate up in a syringe and ejecting through an 18-gauge needle before proceeding with the RNA extraction.After precipitation with e...

Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

“…The major control of oxygen-dependent EPO formation has been shown to operate at the level of its messenger ribonucleic acid (mRNA) [3,4,43]. Increases in EPO mRNA levels upon hypoxia are at least in part mediated by increased transcription of the EPO gene [44].…”

Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats

“…The rats were made anemic by phlebotomy (a blood volume equal to 1% of the body weight, ie 16% of the total blood volume was removed). 32,33 Twenty-four hours later, the animals were bled for Epo measurement.…”

Loss of heterozygosity and reduced expression of the CUTL1 gene in uterine leiomyomas

“…The order 7q22-D7S658-D7S501-D7S496-D7S471-D7S486-7q31 was determined using YAC contig analysis, pulsed-®eld gel electrophoresis, and radiation hybrid mapping (Goldberg et al, 1988;Schuster et al, 1989). D7S518-D7S666-D7S515 were placed between the two previously mentioned sets of markers using somatic cell hybrid analysis and genetic linkage mapping (Beru et al, 1986;Semenza et al, 1991b). Although a YAC contig has been established surrounding these markers these three markers were suciently close that their order along the chromosome could not be discerned.…”