52-kD @entity6540 : genomic structure and identification of an alternatively spliced transcript encoding a novel XXXX zipper-minus autoantigen expressed in fetal and adult heart.

@entity1 @entity1522 @entity368 @entity6540 @entity219 @entity8314
The 52-kD @entity6540 protein is one of the antigenic targets strongly associated with the autoimmune response in mothers whose @entity1 have manifestations of @entity8314 . In addition to the cDNA clone we previously reported for the full-length 52-kD @entity6540 protein, an interesting MOLT-4 cDNA clone, p52-2, was found to have an internal deletion of 231 nucleotides including the domain encoding the @entity368 zipper motif. To further investigate the nature of this deletion, genomic DNA clones were isolated from a lambda FIXII library. The complete gene for the full-length 52-kD protein (alpha form, 52 alpha) spans 10 kb of DNA and is composed of seven exons. Exon 1 contains only the 5' untranslated sequence, while the translation initiation codon is located 3 kb downstream in exon 2, which also encodes the three @entity219 finger motifs. Exon 4 encodes @entity1522 168-245, including the coiled coil/ @entity368 zipper domain. Exon 7 is the longest and encodes the rfp-like domain and the 3' untranslated region. The cDNA p52-2 can now be accounted for as a product of alternative messenger RNA (mRNA) derived from the splicing of exon 3 to exon 5, skipping exon 4, which results in a smaller protein (52 beta) with a predicted molecular weight of 45,000. An initial approach to identifying this alternatively spliced form in the @entity1 heart used a ribonuclease protection assay. Using an RNA probe corresponding to bases 674-964 of the full-length cDNA, two protected mRNA fragments were identified, a 290-bp fragment corresponding to expression of 52 alpha and a smaller fragment of 144 bp, the predicted size of 52 beta. Using reverse transcription followed by polymerase chain reaction, cDNAs from a 16-wk fetal heart, 24-wk heart, and adult heart were amplified with primers flanking exon 4. Two polymerase chain reaction products were observed in each tissue, one 1.0 kb likely representing 52 alpha and a second 0.78 kb, consistent with 52 beta. The 0.78-kb fragment identified in the 16-wk heart was cloned, and DNA sequencing confirmed the 52 beta type. Immunoprecipitation of in vitro-translated 35S-labeled 52 beta form was performed to evaluate the antigenicity of this novel form of 52-kD @entity6540 . 26 (87%) of 30 sera tested from mothers whose @entity1 were known to have @entity8314 immunoprecipitated the 52 beta form.(ABSTRACT TRUNCATED AT 400 WORDS)
