Cell-Free Transcription of Mammalian Chromatin: Transcription of Globin Messenger RNA Sequences from Bone-Marrow Chromatin with Mammalian RNA Polymerase

Abstract

A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin. The messenger RNA sequences are detected by measurement of the ability of the transcribed RNA to hybridize with globin complementary DNA. The globin complementary DNA is synthesized by the enzyme from avian myeloblastosis virus, RNA-directed DNA polymerase, with purified globin messenger RNA as template. The specificity of the globin complementary DNA in annealing reactions was verified by preparing DNA complementary to liver messenger RNA and showing that the globin and liver complementary DNAs are specific for their own messenger RNAs. Both DNA-dependent RNA polymerase II from sheep liver and RNA polymerase from Escherichia coli can transcribe globin messenger RNA sequences from rabbit bone-marrow chromatin; however, the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized. Neither polymerase can transcribe globin messenger RNA sequences from rabbit-liver chromatin. This cell-free assay system should be useful in searching for mammalian transcriptional regulatory factors.In an attempt to understpd, the regulation of expression of the hemoglobin genes in mammalian erythroid cells, particularly in the human genetic disease, beta thalassemia, we have developed a mammalian cell-free mRNA-synthesizing system. The approach is similar to that of Axel, Cedar and Felsenfeld (1) and of Gilmour and Paul (2) except that these two groups have used Escherichia coli polymerase to transcribe avian or mammalian chromatin while our approach has been to establish a fully mammalian system. The use of a mammalian cell-free system should permit a search for regulatory factors that might be involved in mRNA transcription.In this first paper we describe the mammalian cell-free transcriptional system and its use in synthesizing globin mRNA sequences from bone-marrow chromatin; in addition, we examine the hybridization specificity of globin complementary DNA (cDNA), and we compare mammalian with E. coli RNA polymerase. MATERIALS AND METHODSPreparation of Chromatin. Rabbits (2-4 lbs; 1-2 kg) were exsanguinated, and the bone marrow and liver were Abbreviations: cDNA, complementary DNA; TKM buffer, 50 mM Tris HCl (pH 7.9)-25 mM KCI-5 mM MgCl2; TGMED buffer, 50 mM Tris HCl (pH 7.9)-30% glycerol-10 mM 2-mercaptoethanol-0. 1 mM EDTA-0. 1 mM dithiothreitol.removed and stored at -20°until used. The isolation of chromatin was similar to that described by Spelsberg and Hnilica (3). The rabbit tissues were defatted and/or deveined, and homogenized with a Teflon-glass homogenizer in 10 volumes of 0.5 M sucrose in TEKM buffer (50 mM Trist HCI, pH 7.9-25 mM KCl-5 mM MgCl2). The 10% homogenate was filtered through two layers of cheesecloth and centrifuged at 5000 X g for 10 min. The 5000 X g pellet, which consists of nuclei contaminated with cellular debris, was suspended in 2.0 M sucrose in TKM buffer; th...

Regulation of Gene Expression in Eucaryotes

“…Chromatin was reconstituted from the purified DNA, histones, and nonhistone of either reticulocytes or liver and then transcribed with bacterial RNA polymerase. The fractional yield of globin se quences fo r native reticulocyte chromatin was 0.019% in agreement with values reported by Axel et al (68) and Steggles et al (85). The reconstituted chromatin containing reticulocyte nonhistone had a similar template activity to the native chromatin and a fractional yield of globin sequences in the range of 0.007 to 0.02%.…”

In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase

“…As a step toward analysis of the components and their mechanisms in transcriptional regulation, cell-free systems of R N A synthesis involving isolated chromatin as well as nuclei have been extensively studied. Many of these studies suggest that chromatin and nuclei appear to retain their in vivo template specificities in transcription of specific genes (Axel et al, 1973;Gilmour and Paul, 1973;Astrin, 1973;Shih et al, 1973;Steggles et al, 1974;Rymo et al, 1974;Jacquet et al, 1974;Wilson et al, 1975;Gilmour et al, 1975;Marzluff and Huang, 1975;Stein et al, 1975;Tsai et al, 1976). Recent reports on the use of mercurated nucleotide triphosphate as the substrate for RNA synthesis in isolated nuclei or chromatin and purification of the Hg-tagged RNA by sulfhydryl affinity column have been met with interest (Smith and Huang, 1976;Crouse et al, 1976;Beebee and Butterworth, 1976;Biessmann et al, 1976).…”

“…tailedv analysis of less abundant m R N A species.' Another approach involves analysis of in vitro R N A products with highly radiolabeled probes prepared from m R N A by reverse transcriptase or from virions of DNA viruses (Axel et al, 1973; Paul, 1973: Astrin, 1973; Shih et al, 1973;Steggles et al, 1974; Wilson et al, 1975; Gilmour et al, 1975;Marzluff and Huang, 1975; Stein et al, 1975;Tsai et al, …”

Cell‐free Transcription of Human Bone Marrow Chromatin

“…These data are very similar to those published by Axel, Cedar Hybridization of rabbit globin (Hb) cDNA and rabbit liver (Liv) cDNA with rabbit globin (Hb) mRNA and rabbit liver (Liv) mRNA. For details see Steggles et al17…”