Nucleotide sequences from pbaseolln cDNA clones: the major storage proteins fromPhaseolus vulgarisare encoded by two unique gene families

Abstract

The nucleotide sequences of eight partial and five full-length phaseolin cDNA clones show that phaseolin polypeptides are encoded by two distinct gene families which differ in their coding regions by the presence or absence of two different size direct repeats. The alpha-type phaseolin polypeptides are encoded by genes containing direct repeats which encode 14 additional amino acids. Aside from these differences, the alpha-and beta-type phaseolin genes show a high degree of homology (98%) which is consistent with these genes being derived from a common ancestral gene. Much of the heterogeneity found in the phaseolin polypeptides appears to be due to post-translational processing. Nucleotide sequence analysis demonstrates that the alpha-type genes contain only a few amino acid replacement substitutions and that the beta-type genes appear to contain no amino acid replacement substitutions. S1 nuclease mapping shows a complex pattern for transcriptional initiation of phaseolin mRNA. Hydropathy analysis shows that phaseolin polypeptides are predominately hydrophilic, and that the two N-glycosyl recognition sites are located in different hydropathic environments.

Sequence and Spacing of TATA Box Elements Are Critical for Accurate Initiation from the β-Phaseolin Promoter

“…After prolonged exposure, minor products could also be detected which initiated at Ϫ9, ϩ4, and ϩ16/ϩ17. These findings are consistent with previous mapping studies in bean cotyledons which identified a complex pattern of phas messages predicted to originate between Ϫ12 and ϩ5, with the predominant forms localizing around ϩ1 and Ϫ11 (21). Substitution of hTBP with purified recombinant wheat TBP (a gift of S. Ackerman, University of Massachusetts) (32) had no effect on the location of transcription initiation, although the preference for ϩ1 was slightly enhanced.…”

“…In vitro and in vivo analysis of phas promoter activity demonstrated that the TATA box elements are required for basal transcription. Mapping of the primary and secondary phas transcription start sites confirmed that the HeLa in vitro transcription system accurately reconstituted the in vivo pattern of phas transcription initiation (21). The reconstitution of phas basal transcription regulation on naked DNA templates indicates that the observed requirement for core promoter organization is chromatin-independent.…”

“…3A, compare lanes 8 and 9) suggests that the transcription products are RNA polymerase II-specific (31). One major product (ϳ105 bp) was detected, the expected size of a transcript originating from the ϩ1 site previously identified as the primary transcription start site in vivo (21). This site conforms to the consensus for a mammalian Inr element (5Ј-PyPyCAPyPyPyPy-3Ј) with transcription initiating at the A (17), except that the phas ϩ1 sequence contains a puridine in place of the penultimate underlined pyrimidine residue.…”

“…The detection of multiple phas mRNAs with variable 5Ј ends in vivo (21) suggested the existence of several functional transcription initiation sites. We show here that both the sequence and spacing of the TATA elements is critical for high levels of accurately initiated transcription.…”

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system

“…Both enzymes combine in an unusual pathway, not known to exist in higher plants, to produce indole-3-acetic acid whereby iaaM is the rate-limiting enzyme (2-4). Seed-specific expression is mediated by the embryo-specific phaseolin promoter from French bean, Phaseolus vulgaris (5). Repression of the SL construct is conferred by the R element tetR that acts on Fig.…”