A cDNA library was constructed in l Zap (Stratagene) from polyribosome-associated poly(A)+ RNAs isolated from the radicles of heat-shocked five-day-old maize seedlings. The library was screened with a fragment from the BMS HSP18 gene and two clones, cMHS18-3 and cMHS18-9, were isolated from it. RNA transcripts, generated from cMHS18-3 and cMHS18-9, were translated in a cell-free system and their protein products were separated on a two-dimensional gel (IEF/SDS-PAGE) electrophoretic system. The proteins synthesized by these RNAs show the same electrophoretic characteristics as those displayed by the LMW HSPs numbered 6 and 3, and are immunoreactive with anti-HSP18 antibodies generated against the 6 LMW HSPs of maize (Oh43).
Sequence analyses revealed that the cDNA cMHS18-3 is 982 nucleotides (nt) in length, and contains a putative open reading frame of 495 nt which is capable of encoding a polypeptide consisting of 164 amino acid residues (Mr = 17.8 kDa and pI = 5.1). The cDNA cMHS18-9 is 843 nucleotides in length, and contains a putative open reading frame of 486 nt which is capable of encoding a polypeptide consisting of 161 amino acid residues (Mr = 17.5 kDa and pI = 5.5). The two cDNA sequences share 21% and 32% homology in their 5'- and 3'- untranslated regions, respectively, and are 92% homologous in their putative coding region. The apparent conservation of the protein encoding nucleotide sequences and the marked differences in the untranslated regions suggest that the LMW HSPs numbered 3 and 6 are the products of a gene duplication event that has since undergone divergence. In fact, many of the apparent insertions evident in cMHS18-3 and cMHS18-9 are at sites of imperfect repeats, possibly created by the imperfect excision of a transposable element. Interestingly, the sequence of the first 194 nucleotides of the 5'-untranslated region of the clone cMHS18-3 is 97% complementary to the 3' region of RNA found in rice 25 S ribosomal RNA (rRNA) and shows equally high complementarity (74-95%) to the 25 S or 28 S rRNA of other organisms. The relevance of these observations is being explored.
Finally, comparisons of the amino acid
sequences deduced from these clones reveal that some, albeit limited, homology
exists with the class VI LMW HSPs of soybean (particularly in the carboxy
terminus; 46% homology) while considerably less homology is evident among
the LMW HSPs of other organisms. Despite these amino acid differences,
inspection of Hopp-Woods hydrophilicity plots suggests that the LMW HSPs
of maize and soybean contain highly conserved structural regions in their
carboxy termini. Therefore, it appears that some of the LMW HSPs of maize
(cv. Oh43), specifically those designated as number 3 and number 6, are
the products of different but related genes and that these proteins appear
to share some structural similarities with the LMW HSPs of other organisms.
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