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


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.


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-2Kb. The only clone isolated, pH2K01, contains the coding sequence for residues 50 through 91 of H-2Kb, followed by a Glu codon and a termination codon. It is speculated that the mRNA from which pH2K01 was derived and H-2Kb 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-2Kb from residue 66 through the carboxy-terminus and includes 386 nucleotides of 3'-untranslated sequence. pH203 codes for H-2Db, from residue 82 through the carboxy-terminus, together with 476 nucleotides of 3'-untranslated sequence.

H-2Kb and H-2Db 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-2Kb molecule is 10 amino acid residues longer than the H-2Db 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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology
Degree Grantor:California Institute of Technology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Mitchell, Herschel K.
Thesis Committee:
  • Attardi, Giuseppe (chair)
  • Hood, Leroy E.
  • Mitchell, Herschel K.
  • Owen, Ray David
  • Wallace, R. Bruce
Defense Date:2 April 1982
Funding AgencyGrant Number
Record Number:CaltechTHESIS:05162018-154934919
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter II. adapted for Chapter III. adapted for Chapter IV. adapted for Appendix II.
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10903
Deposited By: Mel Ray
Deposited On:29 Jun 2018 18:17
Last Modified:16 Apr 2021 22:12

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