Date of Award

2002

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

Stem cell factor (SCF) encoded by the Steel locus plays an essential role in hematopoiesis, gametogenesis and melanogenesis during both embryonic and adult life. We have characterized a new spontaneous mutant of the Steel locus in mice designated Sl 20J , which arose in the breeding colony at Jackson Laboratories and display a white belly spot. Molecular analysis of the Steel gene transcripts in Sl 20J mice by RT-PCR, revealed a tandem duplication of exon 3. We hypothesized that impaired recombination (IR) resulting in exon duplication was the mechanism of this mutation. A PCR strategy using primers in exon3 was designed to test this hypothesis. A ∼7kb product generated from DNA isolated from Sl 20J mice but not amplified from wild-type DNA and sequence analysis of the amplified PCR product supports IR as the mechanism of this mutation. Further phenotypic analysis was undertaken. Timed pregnancies documented fetal demise in Sl 20J /Sl 20J embryos after 14.5 dpc and analysis of 14.5 dpc viable embryos demonstrated a significant reduction in fetal liver cellularity and erythroid CFU-E. In-situ alkaline phosphatase staining revealed a complete lack of germ cells at 13.5 dpc. For functional studies, both mutant and wild-type Steel cDNAs were cloned into a mammalian expression vector and transfected into CHOK1 cells. In functional assays, Sl 20J mutant protein induced significantly less proliferation of c-kit+ M07e cells compared with wild-type SCF and did not bind to the receptor c-kit. In summary, we report the identification of a novel Steel gene mutant in mice due to a rare mechanism of impaired recombination. Analysis of the expressed mutant protein demonstrated defective receptor binding suggesting a conformation change affecting receptor interaction and among Steel mutants unique since most reported lethal alleles are large deletions.

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