Abstract
Mammalian spermiogenesis is marked by the initial disruption of the nuclear-histone-DNA complex by the transition proteins for ultimate replacement with protamines. The genes for three of these low molecular weight basic nuclear proteins exist as a single linear array of PRM1, PRM2, and TNP2 on human chromosome 16p13.2. To begin to address the mechanism governing their transcriptional potentiation, a region of approximately 40 kilo-bases of the human genome encompassing these genes was introduced into the germ line of mice. Fluorescence in situ hybridization and Southern analysis showed that this segment of the human genome integrated into independent chromosomal sites while maintaining its fidelity. Transcript analysis demonstrated that the expression of the endogenous mouse protamine Prm1 and Prm2 genes as well as the mouse transition protein Tnp2 gene were expressed along with their human transgene counterparts. The pattern of expression of these transgenic human genes within this multigenic cluster faithfully represented that observed in vivo. In addition, all members of this transgenic gene cluster were expressed in proportions similar to those in human testis. Copy number-dependent and position-independent expression of the transgenic construct demonstrated that the corresponding biological locus was contained within this segment of the human genome. Furthermore, DNase I sensitivity established that in sperm the human PRM1-->PRM2-->TNP2 genic domain was contained as an approximately 28.5-kilobase contiguous segment bounded by an array of nuclear matrix associated topoisomerase II consensus sites. This is the first description of a multigenic male gamete-specific domain as a fundamental gene regulatory unit. A model of haploid-specific gene determination is presented.
Highlights
Protamines are highly basic, sperm-specific nuclear proteins that are characterized by an arginine rich core segment
In contrast to the well characterized in vivo and in vitro {3-globin locus, our understanding of haploid expressed genic domains and how these genes, i.e. PRM1-'>PRM2~TNP2, assume a potentiated conformation is lacking
Creation of Transgenic Mouse Model of the Human PRM1-'>PRM2-'>TNP2 Locus-A 40,255-bp Not! fragment of cloned human genomic DNA from cosmid hP3.1 was microinjected into the pronucleus of fertilized C57BU6J X SJL F2 hybrid eggs of mice
Summary
Protamines are highly basic, sperm-specific nuclear proteins that are characterized by an arginine rich core segment (cf. Refs. 1 and 2). Deletion analysis has shown that the tissue-specific element(s) resides within a 113-bp segment, 5' of the initiator codon of the Prml gene [15] encompassing nucleotides -77 to -37 [16] Their significance remains to be assessed, since both testis-specific and ubiquitous protein factors bind to this segment. The concept that transcriptionally competent chromatin assumed an altered nuclear structure was validated when it was shown that the {3-globin gene acquired a DNase !-sensitive conformation within cells in which it was expressed [17] These studies were extended to the chicken ovalbumin, x, y locus, which was one of the first chromatin domains containing a multigenic loci to be defined [18].
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