Molecular and genetic analysis of the gene encoding the A subunit of lactate dehydrogenase in the mouse

Author

Jane E. Mendel

Date of Award

8-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Edward G. Bernstine

Committee Members

David Housman, Liane Russell, Pater Lalley, Francis Kenny

Abstract

In higher vetebrates, the genes encoding the A and B subunits of lactate dehydrogenase (E.G. 1.1.1.27, LDH) show complex patterns of developmental and tissue-specific regulation.

In order to determine whether the genes encoding LDH in mammals are controlled by feedback regulation, a mouse stock, 46DFiOD, having a recessive lethal deletion of Ldh-1, the mouse gene encoding the LDH-A subunit, was identified in a screen of several presumptive deletion carriers affected at a closely linked marker, p, on mouse chromosome 7. Analysis of LDH activity in tissues of this stock provided no evidence for feedback regulation of mouse LDH genes.

A molecular analysis of Ldh-1 was initiated by using a partial LDH-A cDNA, obtained from Dr. Kenneth Fong, to isolate full-length LDH-A cDNA and genomic clones.

Cloning of expressed LDH-A genomic sequences was complicated by the presence of several LDH-A pseudogenes. A 14 kilobase pair (Kb) EcoRl restriction fragment having homology to mouse LDH-A cDNA was localized to mouse chromosome 7 using Southern analysis of DNA from a somatic-cell hybrid mapping panel. Southern analysis of DNA from offspring of a cross between 46DFiOD, the Ldh-1 deletion stock, and Mus spretus, a species having a polymorphism for the 14 Kb restriction fragment was used to further localize this fragment to the chromosomal region containing Ldh-1. Genomic clones containing that fragment were shown to direct the synthesis of mouse LDH-A following transfection into Chinese hamster cells.

Sequence analysis of a portion of the LDH-A genomic clone showed that Ldh-1 has a promoter region that is G-C rich, lacks TATAA and CAAT boxes, and contains consensus sequences for Spl binding and cAMP induction.

The 46DFiOD deletion was also characterized for the involvement of nearby markers using the 46DFiOD x Mus spretus offspring. Xld-1 and LHβ are not included in the chromosomal segment missing in 46DFiOD, while the Saa gene complex, and 24.2, a probe from a syntenic region of human chromosome 11, are included in that segment.