In Chicken Leukemia Cells Globin Genes Are Fully Transcribed but Their RNAs Are Retained in the Perinucleolar Area (original) (raw)
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The poly (A)-containing nuclear RNA from sor for fi globin mRNA only. In addition, it was dimethylsulfoxide-induced Friend leukemia shown by electrophoretic analysis that the cells was fractionated by acrylamide gel complex of RNA molecules not resolved by electrophoresis in denaturing conditions and sucrose gradient centrifugatlon (1 IS) com- analyzed for a and fi globin RNA sequences. prises sequences of decreasing size (0.34, The results Indicate that nuclear RNA 0.28, and 0.26 X l0� daltons), which might contains one species of large-size RNA (0.6 be the precursors of a and fi globin x l0� daltons), which is the putative precur- mRNA. I N EUKARYOTES gene transcription is temporally and physically remote from translation. Indeed only a minor proportion of the nuclear transcripts become available for translation in the cytoplasm and long after their production.' Nuclear nonribosomal transcripts (heterogenous RNA, HnRNA) are produced in vast excess over cytoplasmic messenger RN...
Blood, 1979
The poly (A)-containing nuclear RNA from dimethylsulfoxide-induced Friend leukemia cells was fractionated by acrylamide gel electrophoresis in denaturing conditions and analyzed for alpha and beta globin RNA sequences. The results indicate that nuclear RNA contains one species of large-size RNA (0.6 X 10(6) daltons), which is the putative precursor for beta globin mRNA only. In addition, it was shown by electrophoretic analysis that the complex of RNA molecules not resolved by sucrose gradient centrifugation (11S) comprises sequences of decreasing size (0.34, 0.28, and 0.26 X 10(6) daltons), which might be the precursors of alpha and beta globin mRNA.
Genes & Development, 2006
We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine -globin locus during erythroid differentiation. Murine fetal liver cells were separated into distinct erythroid maturation stages by fluorescence-activated cell sorting, and the nuclear position of the locus was determined at each stage. We find that the -globin locus progressively moves away from the nuclear periphery with increasing maturation. Contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells,  major -globin expression begins at the nuclear periphery prior to relocalization. However, relocation of the locus to the nuclear interior with maturation is accompanied by an increase in  major -globin transcription. The distribution of nuclear polymerase II (Pol II) foci also changes with erythroid differentiation: Transcription factories decrease in number and contract toward the nuclear interior. Moreover, both efficient relocalization of the -globin locus from the periphery and its association with hyperphosphorylated Pol II transcription factories require the locus control region (LCR). These results suggest that the LCR-dependent association of the -globin locus with transcriptionally engaged Pol II foci provides the driving force for relocalization of the locus toward the nuclear interior during erythroid maturation.
Journal of Cellular Biochemistry, 2009
The spatial organization of a 250 Kb region of chicken chromosome 14, which includes the alpha globin gene cluster, was studied using in situ hybridization of a corresponding BAC probe with nuclear halos. It was found that in non-erythroid cells (DT40) and cultured erythroid cells of definite lineage (HD3) the genomic region under study was partially (DT40 cells) or fully (HD3 cells) associated with the nuclear matrix. In contrast, in embryonic red blood cells (10-day RBC) the same area was located in the crown of DNA loops surrounding the nuclear matrix, although both globin genes and surrounding house-keeping genes were actively transcribed in these cells. This spatial organization was associated with the virtual absence of RNA polymerase II in nuclear matrices prepared from 10-day RBC. In contrast, in HD3 cells a significant portion of RNA polymerase II was present in nuclear matrices. Taken together, these observations suggest that in embryonic erythroid cells transcription does not occur in association with the nuclear matrix.
The identification of globin messenger ribonucleic acid in newt erythropoietic cells
Biochemical Journal, 1979
Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9 S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts ofthe different globin mRNA subspecies. Globin mRNA activity was also evident in the 25 S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25 S poly(A)+-RNA fraction contained equimolar amounts of 9 S globin mRNA and 26S rRNA. Translation of the 25 S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength. Abbreviations used: SDS, sodium dodecyl sulphate; poly(A)+-RNA, polyadenylated RNA. Vol. 183 Materials and Methods Anaemia was produced in splenectomized newts by injecting 0.5-1.Omg of acetylphenylhydrazine dissolved in phosphate-buffered amphibian saline (Rugh, 1948). After the appearance of early erythroid cells, each animal was given an intraperitoneal injection of 50-150pCi of [3H]adenosine (specific radioactivity 33 Ci/mmol; New England Nuclear Corp., Boston, MA, U.S.A.) and, after 4-26h, was decapitated and bled into 12ml of ice-cold heparinized (100 units/ml) amphibian saline (Rugh, 1948). The blood cells were collected by centrifugation at 750g for 5min at 4°C and washed three times in saline before further processing. In most experiments, three to five animals were used, with a total yield of approx. 1 x 106-5 x 106 erythroid cells. Preparation ofpolyribosomes and extraction ofpolyribosomal RNA Washed cells were suspended in 2.0-2.5 ml of icecold lysis buffer [0.05 M-Tris/HCI (pH 7.5), 0.04M-KCI, 2.5mM-MgCI2 and 50,ug of dextran sulphate/ ml] containing 0.67vol. of rat liver postmicrosomal supernatant (S3) prepared as described by Blobel & Potter (1966). In the absence of crude rat liver supernatant, lysis of erythroid cells was always accompanied by rapid degradation of polyribosomes and
Erythroid cell-specific determinants of alpha-globin mRNA stability
Molecular and Cellular Biology, 1994
Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring a2-globin mutant, aConstant Spring (CS). The CS mutation is a single-base change in the translation termination codon (I!AA-WCAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal a-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the a-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal
Modifications of RNA processing modulate the expression of hemoglobin genes
Proceedings of the National Academy of Sciences, 1996
The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC) to analyze the mechanism of silencing of the embryonic e-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state oftheir adult 1-globin genes. In hAEC, which express primarily the ,-globin gene, quantitative PCR analysis shows that 3-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the y-globin gene are low, with slight differences among the three exons. Although e-globin is not expressed, e-globin RNA transcripts are detected, with exon I levels comparable to that of y-globin exon I and much higher than e-exons II and III. As expected, in K562 cells that express high levels of e-and y-globin, e-and y-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells 3-mRNA levels are very low but j3-exon I levels are much higher than that of exons I or III. Moreover, all or most of the