University of Florida, Gainesville, Florida 3261 1. The 14-3-3 proteins represent a family of acidic proteins with a molecular mass of approximately 30 kD (Moore ...
Plant Physiol. (1994) 105: 1457-1458
Plant Gene Register
Two Tomato Fruit Homologs of 14-3-3 Mammalian Br in Proteins’ Beth Laughner, Susan D. Lawrence’, and Robert J.Ferl* Department of Horticultural Sciences and Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, Florida 3261 1
The 14-3-3 proteins represent a family of acidic proteins with a molecular mass of approximately 30 kD (Moore and Perez, 1967). Originally classified as mammalian brain proteins, homologs have been identified through deduced amino acid sequences of cDNAs from diverse eukaryotes including Drosophila, yeast, barley, Arabidopsis, com, and Oenothera (Aitken et al., 1992; De Vetten et al., 1992; Lu et al., 1992). Although the first reported functional role as an activator protein of Tyr and Trp hydroxylase suggested that these proteins were limited to nervous tissue and the physiological realm of neurotransmission (Ichimura et al., 1987), subsequent studies have shown that they possess much broader tissue specificity as well as additional potential physiological roles. For example, one family member demonstrates involvement with protein kinase C in calcium-dependent exocytosis (Morgan and Burgoyne, 1992). Another 14-3-3 homolog identified from barley leaves was shown to be encoded by a pathogen-induced gene (Brandt et al., 1992). Furthermore, a 14-3-3 homolog from pea leaves demonstrates inhibition of protein kinase C activity (Hirsch et al., 1992). A consistent theme among the 14-3-3 homologs is their purported role in signal transduction through their association with protein kinases. However, there is no apparent correlation between evolutionary relationship and purported function that might guide prediction of the range of activities possessed by any one homolog (Ferl et al., 1994). Two cDNA clones were selected by immunoscreening a tomato (Lycopersicon esculentum) fruit cDNA library with antisera to GF14, monoclonal antisera that had previously identified a 14-3-3 homolog from an Arabidopsis library (Lu et al., 1992). These two tomato clones, designated pBLT3 and pBLT4, share 71% identity at the nucleotide level and 84% identity at the predicted amino acid level (Table I). Furthermore, both clones share approximately 84% identity at the deduced amino acid level with both an Arabidopsis and a com cDNA identified with the same antisera (De Vetten et al., 1992). Such high deduced amino acid identities might
Table 1. Characteristics of two tomato cDNAs encoding 14-3-3 homoloes
Organ ism : Lycopersicon esculentum var Alisa Craig. Source: A ripening tomato fruit cDNA library commercially prepared by Clontech. Techniques: Library screened with monoclonal anti-CF14 sera, which had previously identified 14-3-3 homologs in an Arabidopsis cDNA library. Positive plaques were purified and their inserts amplified from phage eluates via PCR. Upon cloning into pUC 18, both strands were sequenced by automated dideoxy methods on an Applied Biosystems (Foster City, CA) ABI 373 system. Features of cDNAs: pBLT3 is a partia1 cDNA 859 bp in length truncated at both the 5’ and 3’ ends. pBLT4 is a 1101-bp clone containing 49 nucleotides upstream of the first ATC and a 273-nucleotide 3’ nontranslated region with a 7-nucleotide poly(A)tail. Potential adenylation signals begin at either nucleotide 945 or 1061. The 5’ terminus of the transcript has not been determined. Structural Features of the Peptides: The open reading frame of pBLT3 is 230 amino acids with a predicted molecular m a s of 26,066 D. The open reading frame of pBLT4 is 260 amino acids with a predicted molecular mass of 29,302 D. A comparison of pBLT3 and pBLT4 shows 71% identity at the nucleotide level and 84% identity at the amino acid level.
suggest that plant 14-3-3 homologs evolved similar potential functional roles prior to the divergence of monocots and dicots. Furthermore, the highly conserved amino acid primary structure among a11 eukaryotic 14-3-3 homologs may reflect a strong selective pressure to ensure presently undefined general regulatory roles that in tum affect multiple physiological functions.
This work was supported by National Institutes of Health grant R01 GM40061 and U.S. Department of Agriculture/National Research Initiative grant 93-3704-9608 to R.J.F. This is manuscript number R-03703 of the Florida Agricultura1 Experiment Station. Present address: School of Forest Resources and Conservation: Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611. * Corresponding author; fax 1-904-392-4072.
ACKNOWLEDCMENT
The authors gratefully acknowledge Dr. Emie Almira for automated dideoxy sequencing through the University of Florida’s Interdisdplinary Center for Biotechnology Research.
Received March 4, 1994; accepted April 4, 1994. 1457
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Copyright Clearance Center: 0032-0889/94/105/1457/02. The GenBank accession numbers for the tomato clones pBLT3 and pBLT4 reported in this article are L29151 and L 29150, respectively. LITERATURE ClTED
Aitken A, Collinge DB, van Heusden BPH, Isobe T, Roseboom PH, Rosenfield G, Soll J (1992) 14-3-3 proteins: a highly conserved widespread family of eukaryotic proteins. Trends Biochem Sci 17: 498-501 Brandt J, Thordal-Christensen H, Vad K, Gregersen P, Collinge DB (1992) A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator. Plant J 2 815-820 De Vetten NC, Lu G, Ferl RJ (1992) A maize protein associated with the G-box binding complex has homology to brain regulatory proteins. Plant Cell4 1295-1307 Ferl RJ, Lu G, Bowen B (1994) Evolutionary implications of the family of 14-3-3 brain protein homologs in Arabidopsis thaliana.
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