Application of recombinant DNA techniques to structure-function studies of equine protein hormones.

The research involves the use of recombinant DNA techniques to study the structure and function of equine protein hormones. Specifically, it examines the genes for horse gonadotrophins and growth hormones by creating complementary DNA libraries from horse pituitary gland and endometrial cup tissues.

Approach and Methodology

• The study team constructed cDNA libraries from horse pituitary gland and endometrial cup tissues. cDNA libraries are created by using a process called reverse transcription to convert the RNA messages found in a cell into stable, double-stranded DNA molecules.
• These cDNA libraries were created with the goal of isolating the genes responsible for the hormones FSH, LH, CG, and GH in horses. FSH, LH, and CG are types of gonadotrophins – hormones that regulate the function of the gonads, or sexual organs. GH is a growth hormone that stimulates growth, cell reproduction, and cell regeneration.
• The team applies Southern (DNA) and Northern (RNA) blotting techniques. These techniques are used to identify specific sequences of DNA and RNA, respectively, within a complex mixture.
• They successfully applied heterologous (human and ovine) cDNA probes to isolate the horse hormones’ genes. These probes work like a kind of genetic ‘lock and key’, binding to specific sequences of DNA that match their own.

Findings and Conclusions

• The researchers successfully used a human cDNA probe to isolate the horse gonadotrophin alpha-subunit cDNA from the pituitary and endometrial cup libraries. This alpha-subunit is a key component of the larger gonadotrophin hormones.
• The research found that the nucleotide sequences from both tissue sources were identical, confirming that there is only a single gene responsible for the alpha-subunit in horses.
• Given this discovery, the scientists intend to begin expressing the beta-subunit sequences (another integral part of the gonadotrophin hormones) alongside the alpha-subunit to generate biologically active hormones.
• The team was also able to successfully isolate horse GH cDNA using a sheep cDNA probe. Furthermore, they found that mRNA for a GH-like protein was present in horse endometrial cup tissues but not in the allantochorion (part of the placenta) at the same stage of pregnancy. The presence of this mRNA suggests that either the GH gene is being expressed at a low level, or that genes for a different but related protein (like prolactin or placental lactogen) are being expressed.