Structure and chromosomal localization of the gene encoding Pur-alpha, a sequence-specific single-stranded DNA-binding protein in human and mouse.
Item
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Title
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Structure and chromosomal localization of the gene encoding Pur-alpha, a sequence-specific single-stranded DNA-binding protein in human and mouse.
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Identifier
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AAI9510689
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identifier
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9510689
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Creator
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Ma, Zhi-Wei.
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Contributor
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Adviser: Edward M. Johnson
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Date
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1994
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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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Pur-alpha is a sequence-specific single-stranded DNA-binding protein with affinity for an element present in several eukaryotic origins of DNA replication and gene regulatory regions. I have cloned and sequenced the cDNAs for human and mouse Pur-alpha (hPur-alpha and mPur-alpha, respectively). There is an extraordinary degree of conservation between hPur-alpha (322 aa) and mPur-alpha (321 aa), and there are only two amino acid (aa) differences between them. One Pur-alpha region of 22 aa, termed the "psycho" motif, possesses significant homology to counterparts in the SV40 large T-antigen, several other transforming proteins of DNA tumor viruses and certain cellular proteins in yeast and human cells that may also be involved in the initiation of DNA replication.;Multiple mRNA species are homologous to Pur-alpha cDNA with similar pattern in several human cell lines and tissues. I have attempted to distinguish between alternate processing of the same pre-mRNA from a single gene, the presence of a related genes, or both. I have isolated human genomic clones for Pur-alpha and another family member, Pur-beta, which contains cDNA sequence similar to that of Pur-alpha. Based on Southern analysis of human genomic DNA and genomic clones, both human pur-alpha and pur-beta are demonstrated to be single copy genes. Pur-alpha, expressed as a GST-fusion protein, specifically binds to the hypophosphorylated form of the retinoblastoma protein, Rb, with an affinity at least as high as that of SV40 large T-antigen. Binding of Pur-alpha to Rb alters the conformation of the Pur-alpha complex with its DNA recognition element as seen under standard gel shift conditions.;The pur-alpha gene is localized to chromosome 5q31 by fluoresence in situ hybridization (FISH) of genomic DNA probes on human metaphase chromosome spreads and by Southern analysis of human/hamster somatic hybrid cells containing single human chromosome. The site 5q31 is frequently altered in nonlymphocytic leukemia and in other cancers. Results of the experiments suggest that Pur-alpha may be involved in the cell cycle control of DNA replication.
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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.
