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  • phaseolin  (4)
  • kafirin  (2)
  • Bin 19  (1)
  • 1
    Electronic Resource
    Electronic Resource
    Expression, glycosylation and secretion of phaseolin in a baculovirus system (1988)
    Springer
    Plant molecular biology 10 (1988), S. 475-488 
    Details
    ISSN: 1573-5028
    Keywords: baculovirus ; eukaryotic expression vectors ; phaseolin ; plant glycoproteins ; protein targeting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this report, we describe the efficient expression and glycosylation, in insect cells, of β-phaseolin polypeptides (M r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/106 cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033603
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  • 2
    Electronic Resource
    Electronic Resource
    A 68 bp element of the β-phaseolin promoter functions as a seed-specific enhancer (1996)
    Springer
    Plant molecular biology 32 (1996), S. 579-588 
    Details
    ISSN: 1573-5028
    Keywords: embryo ; endosperm ; phaseolin ; spatial regulation ; tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In beans, expression of the β-phaseolin gene (phas), encoding the major seed storage protein of bean (Phaseolus vulgaris) is confined to the cotyledons of developing embryos. Phaseolin has not been detected in the endosperm, which remains liquid and is lost early in development. However, fusion constructs between the phas promoter and the gus-coding region yield expression in both embryo and endosperm of developing seeds from transgenic tobacco (Nicotiana tabacum) plants. Although elements extending 1470 bp upstream of the transcription start site are known to modulate phas expression, the proximal 295 bp (p295) are sufficient to drive high levels of seed-specific GUS activity. This region was dissected into three elements: a 68 bp element (seed specific enhancer, SSE: −295 to −227), a middle region ( −227 to −109) and a basal phas promoter ( −109 to +20: p109). Different promoter constructs containing the SSE or middle region upstream of p 109 or a CaMV 35S basal promoter ( −64 to +6) were fused to gus. Each construct was expressed in seed, but not in vegetative tissues. Use of the various phas promoter regions yielded notable differences in relative GUS activity in embryo or endosperm. Addition of both the SSE and middle region resulted in higher activity than the sum of adding either element alone to p 109, indicating synergistic interaction between these elements. Seeds from plants transformed with the proximal 227 bp of promoter (p227) showed embryo-specific GUS activity. In contrast, constructs containing two copies of the SSE element were preferentially expressed in the endosperm. These results illustrate the modular nature of the proximal phas promoter, where distinct elements contribute to high levels of expression in different parts of the seed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020199
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  • 3
    Electronic Resource
    Electronic Resource
    Analysis of kafirin promoter activity in transgenic tobacco seeds (1996)
    Springer
    Plant molecular biology 32 (1996), S. 1029-1035 
    Details
    ISSN: 1573-5028
    Keywords: α-prolamin ; kafirin ; monocot gene expression ; transgenic tobacco ; zein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sequences corresponding to 855 bp of 5′ promoter region and the transit peptide from ⋋GK.1, a genomic clone encoding a 22 kDa α-kafirin seed protein from sorghum, were translationally fused to a cloned β-glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants. However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10–15 days after flowering. Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument. These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00041386
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  • 4
    Electronic Resource
    Electronic Resource
    Characterization of the kafirin gene family from sorghum reveals extensive homology with zein from maize (1989)
    Springer
    Plant molecular biology 12 (1989), S. 245-256 
    Details
    ISSN: 1573-5028
    Keywords: kafirin ; prolamin ; seed storage protein ; sorghum genomic library ; zein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Electrophoretic analysis of translation products of polyadenylated RNA isolated from mid-maturation sorghum seed in the presence of [35S]met, [3H]leu, or [3H]val revealed two major proteins of kDa and 21 kDa. These products were not detected when [3H]lys was supplied as the radioactive substrate. Under similar electrophoretic conditions, kafirin (a major seed storage prolamin of sorghum), migrated as two bands of 22 kDa and 19 kDa. Sequence analysis of two cDNA clones (pSK8 and pSKR2) from sorghum seed mRNA revealed them to be highly homologous with each other and to the 22 kDa zeins from maize, suggesting that they represented kafirin cDNAs. Compared with pSKR2, pSK8 had an insertion of 24 nucleotides and a deletion of 24 nucleotides, so that the coding regions were nearly identical in length. The deduced amino acid sequence for these cDNA clones reveals that kafirin, like zein, is rich in glutamine and nonpolar amino acids, but contains no lysine. Both kafirin and zein have a 21 amino acid signal peptide exhibiting 80% homology and eight copies of a repetitive amino acid block in the C-terminal domain with the consensus: infI supP LL finP supA LN infQ supP LALANPAAYLQQQQ. The kafirin cDNAs were used as probes to screen a sorghum genomic library; one genomic clone (λGK.1) was sequenced and found to be very similar (97.8%) to the pSK8 cDNA clone. Clone λGK.1 contains features typical for a functional gene in that the intronless open reading frame encoding 268 amino acids is flanked at the 5′ end by sequences corresponding to the CAAT and TATA promoter boxes (positioned at about −60 and −30 bp, respectively, from the transcriptional initiation site), and at the 3′ end by a consensus polyadenylation signal. In common with zein genomic clones, kafirin clones contain a 15 basepair consensus sequence centered at postion −320 relative to the transcriptional initiation site. Under similar hybridization conditions, genomic reconstruction analysis using an oligonucleotide probe indicated the presence of less than 20 copies of kafirin per haploid sorghum genome compared with approximatley 140 copies of zein per haploid maize genome.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00043202
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  • 5
    Electronic Resource
    Electronic Resource
    Differential accumulation of four phaseolin glycoforms in transgenic tobacco (1991)
    Springer
    Plant molecular biology 16 (1991), S. 381-395 
    Details
    ISSN: 1573-5028
    Keywords: French bean (Phaseolus vulgaris) ; N-glycosylation ; phaseolin ; plant introns ; protein stability ; seed storage proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical β-phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (βwti−) and mutant phaseolin glycoforms (βdgly 1, βdgly 2 and βdgly 1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the βdgly 1 and βdgly 2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin βdgly 1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023990
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  • 6
    Electronic Resource
    Electronic Resource
    Complete sequence of the binary vector Bin 19 (1995)
    Springer
    Plant molecular biology 27 (1995), S. 405-409 
    Details
    ISSN: 1573-5028
    Keywords: Bin 19 ; binary vector ; t-DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11 777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size. Precise knowledge of restriction sites and removal of unnecessary sequences will facilitate plasmid manipulations and plant transformation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020193
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  • 7
    Electronic Resource
    Electronic Resource
    The β-phaseolin 5′ matrix attachment region acts as an enhancer facilitator (1997)
    Springer
    Plant molecular biology 33 (1997), S. 553-557 
    Details
    ISSN: 1573-5028
    Keywords: boundary element ; chromatin domain ; enhancer blocking assay ; matrix attachment region ; phaseolin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract MARs found flanking the β-phaseolin gene (phas) were tested for insulating activity in an enhancer blocking assay. True insulators should block enhancer dependent expression of a reporter gene when placed between the enhancer and a promoter. Insertion of phas 3′ MAR or coding sequences lowered CaMV 35S enhancer driven GUS expression from the phas basal promoter, indicating a distance dependence of the 35S enhancer. 5′ MAR or 5′ MAR core fragments could not act as independent enhancers when fused to the phas basal promoter, and did not lower expression when inserted in the enhancer blocking assay construct, indicating that they facilitated 35S enhancer expression at a distance when located between the enhancer and the promoter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005765525436
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