Smith, Darrin Paul (1990) Xenopus laevis octamer-binding proteins. PhD thesis, University of Warwick.

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Abstract

The ubiquitous human octamer-binding transcription factor, Oct-1, is believed to regulate the expression of a number of ubiquitously expressed genes. These include genes which are expressed throughout the cell-cycle (eg. snRNA genes) and histone H2B genes, whose expression is tightly coupled to nuclear DNA synthesis at S-phase of the cell-cycle.

I have isolated and completely sequenced two X. laevis homologues of Oct-1. The high degree of relatedness of the two homologues indicates that these are likely to be copies of the same gene, which arose during the theoretical genome duplication event in X. laevis evolution.

X. laevis and human Oct-1 display strong evolutionary conservation (85% Identity over a stretch of 750 amino acids), which presumably means that X. laevis has a similar, if not identical function to human Oct-1. Homology between human and X. laevis does, however, break down shortly before the N terminal end, at a point where alternate splicing is known to occur in hunan Oct-1 (W. Herr, pers. comn.). The full length X. laevis cDNA clone which I have isolated may represent a novel alternately spliced form of Oct-1.

Two octamer-binding proteins have been identified (in band shift assays) in X. laevis oocyte, embryo and tissue extract. Oct-1, and a second, previously unidentified octamer-binding protein which has been termed Oct-R, for octamer-related. Oct-1 does not bind to a degenerate octamer motif most often seen in X. laevis H2B promoters. Oct-R binds more strongly to this degenerate motif than the consensus motif, but only in the context of the H2B promoter, and does not bind either motif in another sequence context. This suggests that Oct-R may have a role in regulation of H2B transcription, although no direct evidence has been obtained.

Since Oct-1 is believed to stimulate the S-phase specific induction of histone H2B gene transcription the possibility that Oct-1 binding activity is cell-cycle regulated is of interest. X. laevis Oct-1 (and Oct-R) binding activity does not appear to be cell-cycle regulated.
Oct-1 and Oct-R are stored in the oocyte (partly in the cytoplasm), in an amount equivalent to at least 80 000 somatic cells. Histone protein and message are stored in the oocyte as part of the mechanism to provide enough histones to keep-up with the high rate of DNA synthesis in early Xenopus development. It is possible that histone gene transcription factors are stored for the same purpose.

By mutation of the octamer motif in the promoter of X. laevis histone H2B gene promoter I have tentatively concluded that the octamer motif is required for the expression of a H2B gene (independently of DNA synthesis) in the oocyte. The H2B gene occurs in association with a H2A gene, as part of a divergently expressed gene pair. The octamer motif may be required for the expression of both H2B and H2A genes. The degenerate octamer motif contained in this H2B promoter does not bind efficiently to Oct-1 in vitro, but binds well to Oct-R, indirectly suggesting that Oct-R is required for the expression of the H2B gene.

A polyclonal antiserum raised against the N terminal domain of X. laevis Oct-1 reacts to proteins other than Oct-1 on Western blots of oocyte and embryo extract. These proteins, which are antigenically related to the N terminal domain of Oct-1, are entirely located in the cytoplasm of the oocyte, and entirely located in the nucleus of somatic cells. These proteins are synthesised during oogenesis, and stored in the oocyte in an amount equivalent to at least 100 000 somatic cells.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Xenopus laevis, Transcription factors
Official Date:	September 1990
Dates:	Date Event September 1990 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Old, R. W.
Sponsors:	Medical Research Council (Great Britain)
Format of File:	pdf
Extent:	xxv, 219 leaves : illustrations
Language:	eng

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