Globin reticulocyte

Some of this differential expression is achieved by having different regions of chromatin available for transcription in cells from various tissues. For example, the DNA containing the P-globin gene cluster is in active chromatin in the reticulocyte but in inactive chromatin in muscle cells. All the factors involved in the determination of active chromatin have not been elucidated. The presence of nucleosomes and of complexes of histones and DNA (see Chapter 36) certainly provides a barrier against the ready association of transcription fac-... 

Inhibitory constants for inhibition of natural globin mRNA translation in a rabbit reticulocyte lysate system. For the tri-, tetra-, and pentaphosphate series, each value for Kx was normalized by dividing with the value for Kj for the cap analog standard for the series m7Gp3G, m7Gp4G, and m7GpsG, respectively. 

During p-globin synthesis in normal reticulocytes the sequoice his-atg-pro occurs at position 165-167. How many high-energy phosphate bonds are required to insert these 3 amino acids into the p-globin polypeptide during translation ... 

Accumulation of heme in reticulocytes can regulate globin synthesis by indirectly inacti-vati ng eIF-2. Which of the following steps is most directly affected by this mechanism ... 

A well studied example for control at the level of eIF-2 is the regulation of protein biosynthesis in erythroid cells (review Chen and London, 1995). A decrease in the heme concentration in reticulocytes leads to inhibition of globin synthesis at the level... 

In a further experiment we assayed for the presence of a cap structure on the mRNAs for both Inhibitors I and II by competitive inhibition by 7-methyl-guanosine 5 -monophosphate (m G p) of the in vitro translation of these messengers. Concentrations of 40 pM m G p inhibited by 50% the in vitro translation of total tomato leaf poly(A)" " mRNA (Fig. 7A). This level is 40-fold lower than that required to similarly inhibit rabbit globin mRNA translated in a rabbit reticulocyte lysate (17) and 4-fold lower than that required to inhibit the same mRNA in a wheat germ system (18). It was of interest that the translation of Inhibitor I is inhibited to 50% by 20 pM m G p while 50% inhibition of Inhibitor II requires less than 10 pM (Fig. 7B). The basis of this difference is not understood but... 

Heme synthesis is controlled primarily by 8-aminolevulinate synthase (ALA synthase). There are two mechanisms of control, and each involves a process that affects the concentration of the enzyme. First, the half-life of ALA synthase, as shown by experiments in rat liver, is very short (60-70 min). Like many mitochondrial proteins, ALA synthase is encoded by nuclear genes, synthesized on cytoplasmic ribosomes, and the enzyme is translocated into the mitochondria. The second and main regulating factor is the inhibition of ALA synthase by hemin. Hemin differs from heme in that the Fe atom is in the Fe3+ oxidation state. Heme spontaneously oxidizes to hemin when there is no globin to form hemoglobin. Hemin serves a second function in the regulation of hemoglobin synthesis in reticulocytes. It controls the synthesis of globin. 

Globin is synthesized in reticulocytes (see Chap. 1, Prob. 1.1). which have no nucleus and therefore cannot utilize transcriptional and other potential modes of control. Control of globin synthesis from the pool of globin-enriched mRNA is geared to the concentration of hemin (Fe(III)-protoporphyrin]. which has the ability to inactivate a translational inhibitor of protein synthesis. The inhibitor is a protein kinase that phosphorylates and inactivates one of the initiation factors involved in initiation of translation. When the concentration of hemin is high, it binds to a regulatory subunit of the kinase and. as a result, initiation of globin synthesis can proceed. 

Transcriptional control of globin synthesis in reticulocytes is not possible because transcription does not occur in these cells. Does this mean that the overall control of globin synthesis is completely lacking an aspect of transcriptional control ... 

No. Prior to the formation of the reticulocyte from its precursor cells during the process of erythropoiesis (Chap. 1), there must have been a stage of preferential transcription of the globin genes to yield the globin-enriched mRNA of the reticulocyte. 

The protein kinase H RI (heme regulated eIF-2 kinase) was first identified in studies on the regulation of protein biosynthesis in erythroid cells. A decrease in the heme concentration in reticulocytes leads to inhibition of globin synthesis at the level of translation. This regulation mechanism ensures that only so much globin is produced as is heme available. If the level of heme drops, then HRI becomes activated. The activated HRI phosphorylates the eIF-2a subunit, which in turn shuts off protein biosynthesis (Fig. 1.48). The mechanism of regulation of HRI kinase by heme is not well understood. Heme binding sites have been identified on the N-terminus and the kinase domain of HRI. 

Ann O Rexia has a hypochromic anemia, which means that her red blood cells are pale because they contain low levels of hemoglobin. Because of her iron deficiency, she is not producing adequate amounts of heme. Consequently, elF2 is phosphorylated in her reticulocytes and cannot activate initiation of globin translation. 

Why does an anemia result from iron deficiency When an individual is deficient in iron, the reticulocytes do not have sufficient iron to produce heme, the required prosthetic group of hemoglobin. When heme levels are low, the eukaryotic initiation factor eIE2 (see Fig. 16.22) is phosphorylated, and inactive. Thus, globin mRNA cannot be translated because of the lack of heme. This results in red blood cells with inadequate levels of hemoglobin for oxygen delivery, and an anemia. 

Thus, 3 -amino-3 -deoxy-2, 3 -ieco-adenosine and 3 -deoxy-2, 3 -5eco-inosine were prepared, and the former was used for the synthesis of a seco-puromycin analogue, which was not capable of inhibiting protein synthesis in reticulocyte systems using globin mRNA (88T6419). 

The evidence that ceruloplasmin (Cp) (E.C. 1.12.3,1) is a direct molecular link between copper and iron metabolism is summarized. Copper deficiency results in low plasma Cp and iron, reduced iron mobilization, and eventually anemia, even with high iron storage in the liver. Cp controls the rate of iron uptake by transferrin. Transferrin plays a key role in the availability of iron for the biosynthesis of hemo-globin in the reticulocytes. The ferroxidase activity of Cp results in the reduction of free iron ion generating a conr centration gradient from the iron stores to the capillary system, thus promoting a rapid iron efflux in the reticuloendothelial system. It has been confirmed both in vivo and in the perfused liver that lOr M Cp specifically induces a rapid rise in plasma iron. 

The poly(A) tracts contained in these presumptive mRNA molecules are generally 150-200 nucleotides in length and have a base composition of greater than 85 adenine. Evidence has also been presented for the occurrence of an adenylate-rich sequence in 10 S rabbit reticulocyte globin mRNA (Lim and Canellakis, 1970). RNA transcribed from vaccinia virus DNA has also been shown to contain a large poly( A) sequence localized at the 3 end of the molecule (Kates, 1970). Poly (A) tracts... 

Scale on right side of figure. (A) Globin-like material from a batch of 20 oocytes. The cells had been injected with duck reticulocyte 9 S RNA (dissolved in injection medium at a concentration of 1.25 mg/ml) and incubated in medium containing [ H]-... 

Tables I and II catalog the wide variety of mRNA fractions which have been faithfully translated in the rabbit reticulocyte lysate and the preincubated S30 of Krebs II cells without the addition of homologous tissue fractions. In addition, the mRNA s for mouse and rabbit globin have been translated in the preincubated mouse (and rat) liver system (Sampson et al., 1972 Sampson and Borghetti, 1972). Rabbit globin, immunoglobulin light chain, and collagen mRNA s have been translated in the Xenopus eggs and oocytes (see Chapter 4, this volume). The most obvious interpretation of these results is that messenger-specific initiation factors do not exist in animal cells and that there is no restriction on the range of mRNA s that a tissue can translate.

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