Characterization and complete sequence of a cDNA encoding for ubiquitin from W22

--Keqin Chen, John Hunsperger and Irwin Rubenstein

Ubiquitin is a small (76 amino acid residues) protein found in eukaryotic cells. Its amino acid sequence is highly conserved. The ubiquitin protein forms a covalent attachment to cytosolic proteins destined for degradation. It also is found conjugated to certain histones in chromatin and is involved in ribosomal RNA processing and cell surface receptor modification. The structure of genes coding for ubiquitin has been studied in yeast, chicken, Xenopus, pig, humans, Drosophila, barley, and Arabidopsis.

We have determined the DNA sequence of a cDNA clone coding the ubiquitin protein from W22 inbred. The clone UB14 was found in the cDNA library made from endosperm protein body. The UB14 sequence contains seven perfect ubiquitin (76 amino acid) repeats. The amino acid sequence deduced from the cDNA sequence is identical to the sequence of ubiquitin from barley, oats, and Arabidopsis. Plant ubiquitin differs from animal and yeast ubiquitin by only three amino acid substitutions. In addition, there is an additional Gln (Q) at the C-terminal end of the last 76 amino acid maize ubiquitin repeat (Fig. 1). The 3' untranslated region is about 170 nucleotides long. This cDNA clone includes a 5' untranslated region 65 nucleotides long.

(MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG)6

-MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGGQ

Fig. 1. Amino acid sequence of maize polyubiquitin.

Total RNAs were isolated from different tissues of 4-day-old seedlings: coleoptile, scutellum, radicle, and endosperm. Northern blots of these RNAs indicated several ubiquitin transcripts of different lengths: 600 to >2000 nucleotides. The sizes of the transcripts found were specific to a given tissue. Some high molecular weight ubiquitin-related RNA sequences found in coleoptile and radicle total RNA are absent from scutellum and endosperm. Total RNAs were isolated from 4 day-old seedlings which had undergone a heat shock (1 hr, 42 C). Northern blots of this RNA indicated that the amount of ubiquitin RNAs is dramatically increased in all four tissues.

Southern blots of W22 genomic DNA show two intensive ubiquitin-specific hybridization bands and several faint bands. We estimate that there are two or more ubiquitin genes in W22 inbred.

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