Characteristics of maize leaf cDNA clone Zm7gg --G. Gowri, Björn Ingemarsson and Wilbur H. Campbell In the process of screening a maize leaf cDNA library in lambda gt11 with the nitrate reductase antibody, we isolated and purified 4 positive clones. All these clones show very strong reaction with the antibody when the inserts are expressed as lacZ fusion proteins. All 4 clones were subcloned into pUC12 for further characterization. Two clones had identical restriction maps and one of them (Zmnr1) was sequenced and shown to be a partial clone for the NADH:nitrate reductase, from which the polypeptide had been partially sequenced. The third clone (Zm6) was sequenced and found to be a near full-length clone for chloroplastic glyceraldehyde-3-phosphate dehydrogenase by comparison to the sequence published for another cDNA clone of this enzyme. We have described these results (Gowri & Campbell, Plant Physiol. 90:792, 1989). However, the fourth clone (Zm7gg) has not been described.

Zm7gg contains an insert DNA of 0.9 kb. Since our nitrate reductase clone encoded only about two-thirds of the enzyme's sequence as compared to the full-length polypeptide deduced from clones of tobacco and Arabidopsis, we sequenced Zm7gg to determine if it encoded the rest of the maize enzyme. Unfortunately, the amino acid sequence deduced from the nucleotide sequence of Zm7gg does not match the amino acid sequence expected for the N-terminal region of nitrate reductase and does not match strongly to any sequence in the databases. However, the sequence of Zm7gg does have a polyA tail and appears to represent a mRNA expressed in maize leaves. A Northern blot of total RNA and polyA+RNA from nitrate-induced maize leaves was probed with Zm7gg and several bands were found to hybridize, which appeared at first to suggest that the probe was non-specifically hybridizing to rRNA despite the stringent conditions used for developing the blot. However, a Northern blot of nitrate-induced maize scutella RNA showed no hybridization to Zm7gg, which ruled out non-specific binding to rRNA as an explanation for the clone's binding to leaf RNA. In addition, a Northern blot of nitrate-induced root RNA probed with Zm7gg had a single RNA of 3 kb which weakly hybridized.

Since the deduced amino acid sequence of Zm7gg is very rich in proline residues, we compared it to the sequence of the proline-rich domain of CTF, a eukaryotic DNA-binding protein with affinity for the CCAAT-box (Santoro et al. Nature 334:218, 1988). We found a significant degree of homology between these sequences. Another DNA-binding protein found in HeLa cells and rat liver, NF1, also has a protein-rich domain with homology to the deduced amino acid sequence of Zm7gg. A comparison of the proline-rich regions of these three sequences is shown in Figure 1. Zm7gg, CTF and NF1 have 18, 19 and 23 proline residues in these proline-rich sequences, which yields from 16 to 24% proline. The consensus sequence derived from these three sequences, which is shown in Figure 1, indicates that 13 proline residues are shared. However, the degree of homology over the entire sequence of Zm7gg and the C-terminal half of CTF/NF1 is only about 18%, which indicates that Zm7gg is probably not similar in function to these DNA-binding proteins. But it does indicate that Zm7gg probably falls into the class of proline-rich domains commonly found in DNA-binding proteins. Most interestingly, the Northern blots of RNA from rat liver and other tissues resemble those we have found for Zm7gg, which have multiple transcripts hybridizing to the probe (Paonessa et al., EMBO J. 7:3115, 1988). The sizes of the transcripts found for corn leaf are: 3.5, 2.0 and 1.8 kb, with the middle band being the strongest.

Figure 1. A comparison of the proline-rich regions of Zm7gg and two mammalian DNA-binding proteins. Residues 82 to 195 are shown of the open-reading frame of Zm7gg. The proline-rich domains of CTF and NF1 are in the C-terminal half, while the DNA-binding domain, which is a highly positively charged alpha-helix region, is in the N-terminal half of CTF and NF1 (Mitchell and Tjian, Sci. 245:371, 1989).

More recently, we isolated a cDNA clone with some sequence homology to Zm7gg when using the same anti-nitrate reductase antibodies to screen a lambda gt11 library of corn roots, which had been induced with nitrate. However, this root clone, which is 1.5 kb in length, hybridizes to mRNAs of 4.5 and 3 kb in a Northern blot of polyA+RNA from nitrate-induced corn roots, but not from uninduced root RNA. Slot blots indicate that the transcripts, which hybridize to the new cDNA clone from maize roots, are increased in level by at least 3-fold after 1.5 hours of nitrate treatment of the roots. We are unable at this time to explain why antibodies for nitrate reductase pick up these clones when their inserts are expressed as lacZ fusion proteins in lambda gt11, nor do we have any clearer idea what this protein does in maize. We are doing more detailed analysis of the nucleotide sequence of the maize root clone, which we hope will assist in unraveling more about its function and perhaps shed light on its relationship to Zm7gg. If other investigators are interested in utilizing Zm7gg, we would be glad to share it.

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