Enrichment for the globin coding region in a chromatin fraction from chick reticulocytes by endonuclease digestion.

A nuclease-sensitive fraction was obtained from chick reticulocyte chromatin by brief digestion with an endonuclease (DNAase II, deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6). The nuclease-sensitive fraction typically contained less than 1% of the chromatin-DNA but about 50% or more of the nascent chromatin-bound RNA. Hybridization of chick globin complementary DNA to the DNA component of the nuclease-sensitive fraction of reticulocyte chromatin indicated a 3--5 fold enrichment for the globin coding region of the chromatin. The control experiment utilizing DNA from a nuclease-sensitive fraction of chick liver chromatin did not show a comparable enrichment for the globin coding region. This suggests that the endonuclease-effected enrichment for the globin coding region in the nuclease-sensitive fraction of reticulocyte chromatin is to some degree specific for structural genes transcribed in reticulocytes.     

Characteristics of rabbit globin mRNA purification by oligo(dT) cellulose chromatography

We have purified rabbit globin mRNA using oligo(dT)-cellulose and sucrose gradient centrifugation. Both α- and β-globulin mRNA molecules behave heterogeneously with respect to their elution properties during chromatography on oligo(dT)-cellulose. Those fractions eluted at the lowest ionic strength are most active in directing cell-free globin biosynthesis. By making use of hybridization with synthetic [³H]DNA complementary to globin mRNA, we have shown that this technique can be used to quantitate the extent of mRNA purification. Thus, globin mRNA is approximately 90-fold purified from reticulocyte polysomal RNA and originally constituted slightly more than 1% of the polysomal RNA. Since more than 98% of the globin mRNA sequences are bound to oligo(dT)-cellulose, we suggest that most polysomal globin mRNAs contain a poly (A)-rich region and that this region is not of uniform length nor preponderately associated with either the α- or β-globin mRNAs. In addition, we observe that the 9S globin mRNA most resistant to dissociation from oligo (dT)-cellulose is most active in directing globin biosynthesis.     

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.