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Structure and expression of mammalian zinc finger genes

Cunliffe, Vincent Trevor; (1990) Structure and expression of mammalian zinc finger genes. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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The zinc finger domain is a sequence-specific DNA binding motif that was originally identified in the Xenopus laevis transcription factor IIIA (TFIIIA), and subsequently observed in the Kruppel segmentation gene of Drosophila melanogaster and the yeast transcription factor ADR1. Such extensive phylogenetic conservation of zinc finger structures in these regulatory genes suggested that genes containing them might also serve important regulatory functions during mammalian development. In this study an originalities encoding a sequence conserved between TFIIIA, Kruppel, and ADR1 was used to screen mammalian cDNA libraries with the objective of identifying zinc finger genes selectively expressed during human B-lymphocyte and mouse germ cell development. A large number of cDNA clones were isolated from a human B-lymphocyte library. The sequence of a 1.8kb cDNA, ZFP36-1.8, revealed that its corresponding gene contained a minimum of 14 contiguous zinc fingers and an N-terminal non-finger region. At high stringency of hybridisation in Southern analysis this cDNA identified a large family of 20-30 highly related genomic DNA EcoRI fragments. Pulsed field gel analysis suggested that these fragments were clustered in the genome. The subfamily of sequences defined by ZFP36-1.8 was differentially transcribed in a wide range of cell types, although the transcription patterns in Northern analyses did partially overlap. Zinc finger cDNA clones were also isolated from an 11.5 day post coitum (p.c.) mouse urogenital ridge library. One was derived from a 2.4kb mRNA encoding a protein with a block of 18 zinc finger domains and an N-terminal region of 79 amino acids rich in acidic residues. Such acidic domains are a characteristic feature of the activation domains of many transcription factors. The corresponding gene, Zfp-35, was shown to be a single copy gene at high stringency of hybridisation. The 2.4kb mRNA was selectively expressed in adult testis by comparison with embryonic gonads. The expression of this mRNA was analysed in a variety of adult tissues, whole testes of prepuberal adult XY mice, and germ cells isolated at different stages of development from XY testes. Combined with in situ hybridisation to mRNA in sections from adult XY testes, these studies showed that a large increase in Zfp-35 expression was restricted to spermatocytes at the pachytene stage of meiotic prophase. Cosmid clones covering the Zfp-35 gene were characterised. The first exon of the 2.4kb transcription unit contained 53 nucleotides of the 5' untranslated region, exon 2 contained the remaining 5' untranslated region plus the first 17 codons of the open reading frame, and the third exon corresponded to the remainder of the mRNA including the acidic and 18 zinc finger domains of the open reading frame. The cognate gene could be identified in rat, human, whale, and horse genomic DNA. In situ hybridisation to mouse metaphase chromosomes revealed that Zfp-35 localised to a region of chromosome 18 encompassing bands B3 to C.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structure and expression of mammalian zinc finger genes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Zinc finger genes
URI: https://discovery.ucl.ac.uk/id/eprint/10111107
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