Doctoral Dissertations

Date of Award

5-1990

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Raymond A. Popp

Committee Members

Audrey Stevens, Kai-Un Lee, Gene Rinchik, Siwo R. DeKloet

Abstract

Oxygen association-dissociation and stability analyses were performed on mouse hemoglobins with amino acid substitutions (induced by END) in an alpha globin (α 89, His to Leu) and a beta globin (β 60, Val to Glu). Mice of the Hbag2 and Hbbs2 haplotypes contain the variant alpha chain 5m and beta chain βs2 respectively and both chains have an elevated oxygen affinity. The beta globin chain βs2 is also moderately unstable in isopropanol. Oxygen association P50 values for the nine (Hbbg2/Hbbg2;Hbbs/Hbbs) X (Hbaa/Hbaa;Hbbs/Hbbs) F2 genotypes can be grouped into five classes which are linearly related and dependent on mutant globin gene dose. Physiologically, both globin variants have an identical oxygen affinity, but at the molecular level. Alpha chain 5m has a 30 percent higher oxygen affinity than beta βs2 globin. Mice of the Hbag2/Hbag2;Hbbs/Hbbs, Hbaa/Hbaa;Hbbs2/Hbbs2 and Hbag2/Hbag2;Hbbs2/Hbbs2 F2 genotypes have slightly elevated red blood cell counts. Both natural and induced alpha globin variants can be used to study alpha globin gene expression during development of the mouse. Adult alpha globin is synthesized in large amounts, first in nucleated erythrocytes of yolk sac origin and later in non-nucleated cells derived from the fetal liver, spleen and bone marrow. Isoelectric focusing analysis of hemoglobins shows that the ratios of alpha chain 1 to chain 5m in Hbag2 mice and alpha chain 1 to chain 4 in Hbac mice change from day 11.5 to 16.5 of gestation in adult hemoglobin of nucleated erythrocytes while no change occurs in the ratios of these chains in adult hemoglobin of non-nucleated red cells. The percent ratios of the two different alpha chains are different for Ell, Elll, and adult hemoglobins of both murine haplotypes. The observed differences in alpha globin gene expressin during development in mice may be a result of either a temporal change in alpha globin gene transcription or post-translational competitive affinities among the globins in assembling hemoglobin tetramers. Gene conversion has been reported for the a1 and a2 alpha globin genes of four inbred strains of mice. The adult alpha globin loci (Hba) of Inbred strains of (M. musculus musculus) mice are heterogeneous and this is also true for the "Skive" (M. musculus musculus) mouse. The a1 and a2 adult alpha globin genes of the Skive mouse were cloned and subcloned into EMBL/4 and pBluescript-ll-SK+, respectively, and sequenced. The a1 and a2 genes of the Skive mouse encode alpha globin chains 2 and 1, respectively. The 5' untranscribed region of the a1 gene has two unique polymorphic nucleotide sites and the 3' untranscribed region of the a2 gene has six unique polymorphic sites. Nucleotide site polymorphisms in mice are clustered In the 5' and 3' untranscribed regions an in the second coding block. One third of these sites are shared among two or more of the ten known a1 and a2 gene region sequences (the second exon of the a1 gene of Skive, Hbac and Hbab mice and the 5' untranscribed region of Skive, Hbac and Hbaf mice). These regions of shared sequence homology may have resulted from gene conversion or multiple reciprocal crossing over within and between the a1 and a2 genes or may have resulted from simple sequence divergence from an ancestral gene. The a1 and a2 genes of Skive and Hbac mice share the same sequence homology regions. Skive and Hbac (SWR/J) mice derive their ancestry from two separate geographical locations which implies that their alpha globin gene sequences diverged from a related haplotype.

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