Subtractive cloning of an erythroid-specific transcription factor and other murine erythroleukemia cell genes.
Item
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Title
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Subtractive cloning of an erythroid-specific transcription factor and other murine erythroleukemia cell genes.
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Identifier
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AAI9315490
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identifier
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9315490
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Creator
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Miller, Ira J.
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Contributor
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Adviser: James Bieker
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Date
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1993
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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The process of erythroid cell specialization involves the coordinate control of structural, enzymatic and regulatory genes. The most popular murine model of erythroid differentiation is the Friend murine erythroleukemia (MEL) cell, which undergoes terminal maturation upon chemical induction. We used the Friend model to ask what genes are expressed in erythroid cells and not in a macrophage cell line by performing subtractive hybridization. We isolated ten novel sequences and eight previously known nonglobin genes that are differentially expressed. We concentrated on the most narrowly expressed transcripts based on Northern blot analysis of tissue RNAs. The most erythroid-specific clone in the group is a 1.4kb transcript with a heterogeneous startsite, expressed only in the spleen and bone marrow- the physiologic organs of adult murine hematopoiesis. It is not expressed in T-cell, B-cell monocyte or macrophage lines, but is present at low levels in mast cell lines. We propose that it encodes a transcription factor on the basis of the deduced aminoacid sequence. The carboxy terminal contains three TFIIIA-like (cys){dollar}\sb2{dollar}-(his){dollar}\sb2{dollar} zinc finger motifs highly homologous to the those of the Kruppel family of DNA-binding proteins, prompting us to name it EKLF- for Erythroid Kruppel-like Factor. The amino 2/3 of the EKLF is acidic and proline rich, both features of functional transcriptional activating domains. On the basis of its homology to Sp1, EKLF is a candidate gene for a previously described factor that binds to "CAC" sites within erythroid specific promoters.;Two other novel sequences displayed provocative homologies. One of them contains a repeated motif found in several guanosine-binding proteins and in the Purkinje cell specific protein L7, and it may play a role in second messenger signaling. On Northern blots, it detects transcripts of 4kb in brain, heart and ovary, and 2.5kb in the heart. The other is a 1,567 nucleotide transcript encoding a protein homologous to the acidic nuclear X-linked lymphocyte-regulated protein. The MEL gene is also present in T-cell line EL-4 and at low levels in most tissues.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
