Construction and use of a cloned cDNA library to messenger RNAs from pollen

Corn pollen at maturity contains a store of messenger RNAs (N. T. Mascarenhas et al. 1984, Theoret. Appl. Genet. 68:323-326). In order to study the regulation and function of these mRNAs, we have constructed a library of complementary DNA (cDNA) clones made to poly(A) RNAs isolated from mature pollen of maize, hybrid "Gold Cup" (Harris Seeds, Rochester, New York). The cDNAs were cloned in pBD1, a plasmid vector-primer system modified from Okayama, H. P. and Berg, P. (1982 Mol. Cell. Biol. 2:161-170; D. C. Alexander, B. G. Williams, D. McKnight, manuscript in preparation). The vector was kindly provided by Drs. Danny C. Alexander and Bill G. Williams. The plasmids containing the double stranded inserts were introduced into E. coli (HB 101) by transformation. Colonies containing sequences complementary to pollen messenger RNAs (mRNAs) were identified by colony hybridization to32P-cDNA made to pollen poly(A) RNA.

We have selected 100 clones from the library for further study. Several of the clones are pollen-specific. This has been determined by Northern blot hybridizations using RNA isolated from pollen and several vegetative tissues. The majority of the clones, however, are expressed in both pollen and vegetative tissues. The cloned inserts range in size from about 50 nucleotide pairs to 1268. The mRNAs to which the clones are complementary range from about 600 to 2500 nucleotides, as determined by Northern hybridizations. Based on Southern hybridizations to restriction-enzyme-digested genomic DNA, the pollen-specific clones thus far tested seem to be represented by single genes or a very few genes.

RNA was isolated from pollen of different stages of development and analyzed by Northern hybridizations with one of the pollen-specific clones (pZmc30), which hybridizes to a mRNA, approximately 2000 nucleotides in size. The results indicate that transcription of the mRNA is initiated during the interphase following microspore mitosis but prior to generative cell division, and the mRNA continues to accumulate reaching a maximum in the mature pollen grain. Similar analyses are being carried out with several other pollen-specific clones and clones that are expressed both in pollen and vegetative tissues.

We are currently using several of the cDNA clones to isolate genomic clones of the inbred line W22, with the aim of characterizing the pollenexpressed genes in greater detail.

Arthur Eisenberg, R. Paul Willing, Jeffery R. Stinson, M. Enrico Pe and Joseph P. Mascarenhas

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