Human Globin Messenger RNA: Importance of Cloning for Structural Analysis

Abstract

The sequence of most of the human beta globin messenger RNA and large sections of the alpha globin messenger RNA has been determined. Partly because of genetic polymorphism, it was necessary to clone globin complementary DNA in order to extend the analysis. Purified human fetal globin messenger RNA was isolated and used as a template by reverse transcriptase to produce duplex complementary DNA molecules. These molecules were linked in vitro to plasmid DNA by use of T4 ligase in the presence of Escherichia coli Pol 1. Several colonies transformed by these molecules have been shown to hybridize with labeled human globin complementary RNA.

Detection of proteins like human gamma and beta globins in Escherichia coli carrying recombinant DNA plasmids.

“…Bacterial cultures were grown at 37°C to midlogarithmic phase. Preparation of plasmid DNA has been described (10,11). All growth experiments were performed in a P3 physical containment facility in compliance with National Institues of Health guidelines for recombinant DNA research (13).…”

“…Procedures for restriction endonuclease digestion and gel electrophoresis have been described (10,11 site (10,11). This site is flanked by two Hha I cleavage sites (21), one approximately 900 bases to the left of the EcoRI site and the other approximately 400 bases to the right.…”

“…Also, Mercereau-Puijalon et al (9) have reported the synthesis of chicken ovalbumin-like protein in E. coli K-12 harboring a recombinant DNA plasmid in vector pBR322. More than 2 years ago, we produced cDNA clones of human a, 1, and 'y, globin mRNA (10,11) inserted into bacterial plasmid pMB9. Here we report the expression of proteins from human cDNA in E. coli, by demonstrating that these proteins contain antigenic determinant sites specific for human y chain and proteins that react with antibodies to human 1 chain.…”

Insertion of synthetic copies of human globin genes into bacterial plasmids

“…Extensive structural information is already available for the human a globin chain mRNA and partial information is available for the human a chain mRNA (2)(3)(4)(5)(6). There are limitations in perforrfing extensive structural analyses of isolated mRNAs both because of possible heterogeneity (polymorphism) between multiple gene loci for the same globin chain (7), and because of the difficulty in obtaining large quantities of pure mRNA. To facilitate the nucleotide sequence determination of the a and y globin chain structural genes and to provide pure molecular probes for the human globin genes which can be used in mapping experiments of normal and mutant (thalassemic) globin genomic DNA, we have prepared cloned bacterial plasmids (Luria broth) containing, per liter: lOg of trypone; 5g of yeast extract (Difco); 5g of NaCl; lg of glucose; 1 ml of 1N NaOH, and 25 mg of tetracycline (for pMB9) or 10 mg of kanamycin (for pCRl).…”

“…With single stranded globin cDNA as template, second strand synthesis was accomplished in a self primed reaction (20,21) using either E. coli DNA polymerase I (20) or reverse transcriptase (7). The resulting double stranded "hairpin" cDNA molecules were digested with S1 nuclease as described (7,20) (9).…”