Karen Talmadge

Structure, Expression, and Evolution of the Genes for the Human Glycoprotein Hormones

Publisher Summary The recombinant DNA technology is used to understand the tissue-specific, developmentally regulated expression of the four different glycoprotein hormones: chorionic gonadotropin (CG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The isolation of a full-length cDNA encoding the common α subunit demonstrates that there is a single human gene for this protein, expressed in the placenta for production of hCG and in the pituitary for the production of hLH, hFSH, and hTSH. From this, it is concluded that the control for the expression of glycoprotein hormones probably resides in the β subunit genes. The isolation of a full-length cDNA encoding the β subunit of hCG allows to isolate the full human complement of seven hCG β subunit genes or pseudogenes and, by cross-hybridization, the single β hLH gene. The β hLH gene is linked to at least three of the β hCG genes, and together, they show a complex organization of inverted and tandem pairs. In the analysis of independently isolated β hCG cDNA clones and from transfection of six of the seven β hCG genes into mammalian tissue culture cells, it was found that only three of the seven hCG genes are expressed. From a comparison of the nucleotide sequence of two of the β hCG genes and of the single β hLH gene, β hCG is shown to arise from β hLH by a series of selected changes with very little neutral drift.

Construction of plasmid vectors with unique PstI cloning sites in a signal sequence coding region

We have constructed a series of plasmids with unique PstI restriction sites within or near the pre-penicillinase signal sequence for protein secretion. To do this, we devised a rapid, simple method to eliminate undesirable unique restriction sites within plasmids while maintaining antibiotic resistance. We thus obtained a plasmid with a conveniently located, unique HincII site in the penicillinase gene of plasmid pBR322 which was used to generate, with BAL 31 exonuclease, deletions extending into the region encoding the signal sequence. DNA inserted into these plasmids can be translated in all three reading frames both including signal sequence, or starting immediately beyond it.

Proinsulin from Bacteria

One problem we face in the cloning and expression of a small hormone like insulin, is that the normal hormone is made in the pancreas through a series of precursors. Preproinsulin is a molecule some 100 amino acids long that has on its amino terminal end a hydrophobic presequence of 24 amino acids which is cleaved off as that molecule is passed through the cell membrane (1, 2). The resulting fragment, proinsulin, folds up; disulphide bonds form, and then a portion of the peptide chain, the C peptide, is cleaved out between two pairs of basic amino acid residues to produce the final molecule, insulin itself. When we make insulin in bacteria, we can do the final maturation ourselves with a mixture of trypsin and carboxypeptidase B. However, how can we arrange that the amino terminus will be the correct one for insulin rather than bearing some other amino acid or the presequence?