Application of Synthetic Oligonucleotides in the Isolation of Murine Transplantation Antigen cDNA Clones

Citation

Reyes, Antonio Arevalo (1982) Application of Synthetic Oligonucleotides in the Isolation of Murine Transplantation Antigen cDNA Clones. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/hmha-5v32. https://resolver.caltech.edu/CaltechTHESIS:05162018-154934919

Abstract

Cytoplasmic poly(A)⁺ RNA was extracted from the murine thymoma cell line EL4 (b haplotype) and used to construct a library of cloned cDNA in pBR322. Initially, 30,000 colonies were screened with a mixture of eight hexadecanucleotides representing all possible coding sequences for residues 51-56 of H-2K^b. The only clone isolated, pH2K01, contains the coding sequence for residues 50 through 91 of H-2K^b, followed by a Glu codon and a termination codon. It is speculated that the mRNA from which pH2K01 was derived and H-2K^b mRNA have a common precursor but differ in the manner of post-transcriptional splicing.

A 133-nucleotide probe containing most of the coding sequence of pH2K01 was constructed and used to screen the remainder of the library. Two clones were isolated. pH202 encodes H-2K^b from residue 66 through the carboxy-terminus and includes 386 nucleotides of 3'-untranslated sequence. pH203 codes for H-2D^b, from residue 82 through the carboxy-terminus, together with 476 nucleotides of 3'-untranslated sequence.

H-2K^b and H-2D^b share sequence homologies of 83% and 91% at the protein and nucleotide levels, respectively. The cytoplasmic region of the molecule proximal to the membrane is identical in both antigens. The next most conserved region is the third external domain.

The H-2K^b molecule is 10 amino acid residues longer than the H-2D^b molecule. Analysis of 3'-end coding sequences of pH202, pH203 and other H-2 clones reported in the literature suggests that the difference in length could be the result of different splicing patterns of the mRNAs.

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