The unusual 5? splicing border GC is used in myrosinase genes of the Brassicaceae

Xue, Jiaping; Rask, Lars

doi:10.1007/bf00019128

Cited by 33 publications

(22 citation statements)

References 21 publications

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“…Whereas the exon-intron junction sequences of the first and third introns agree with the GT-AG rule for RNA splicing (Mount, 1982), the second intron is flanked by the dinucleotides GC and AG. A similar junction sequence was reported for several other plant genes, including seven myrosinase genes (Xue and Rask, 1995).…”

Section: Ldentification Of the Exon-lntron Organization Of The Md13 Cenesupporting

confidence: 74%

Sequencing, Genomic Organization, and Preliminary Promoter Analysis of a Black Cherry (R)-(+)-Mandelonitrile Lyase Gene

Poulton

1997

Plant Physiology

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Section: Ldentification Of the Exon-lntron Organization Of The Md13 Cenesupporting

confidence: 74%

Sequencing, Genomic Organization, and Preliminary Promoter Analysis of a Black Cherry (R)-(+)-Mandelonitrile Lyase Gene

Poulton

1997

Plant Physiology

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“…The 5Ј splicing border of five introns was GT and the 3Ј border of all six was AG. Only the fourth intron i4 contained the unusual 5Ј splicing border GC, which has been found in genes of several plant species (Xue and Rask, 1995). The reliability of this intron sequence was confirmed by sequencing two other PCR-amplified clones over this region.…”

Section: Structure Of the Genementioning

confidence: 68%

Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens

et al. 1998

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SummaryThe moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel ∆5-and ∆6-desaturases are required. To isolate one of these, PCR-based cloning was used, and resulted in the isolation of a full-length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N-terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b 5 -related region and a C-terminal sequence with low similarity (27% identity) to acyl-lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ-linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a ∆6-desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC-MS. Only the transformed yeast cells were able to introduce a further double bond into the ∆6-position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.

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“…Selmar et al, 1987), most plant ␤-glucosidases display high aglycone specificities (Hö sel and Conn, 1982;Conn, 1993). Prime examples include ␤-glucosidases exhibiting pronounced specificities toward such endogenous substrates as cyanoglucosides (Hö sel and Nahrstedt, 1975;Hö sel et al, 1987;Poulton, 1993) Rask, 1995). This high degree of aglycone specificity raises the question as to which amino acid residues confer such selectivity.…”

Section: Identification Of Amino Acid Residues That May Confer Aglycomentioning

confidence: 99%

“…All exon-intron junction sequences of the black cherry ␤-glucosidase genes conform to the GT-AG rule for RNA splicing with the exceptions of intron 1 of Ah1 and Ph3 and intron 8 of Ah1, which have GC-AG junction sequences (Brown, 1986). Interestingly, a GC donor splice site was noted for intron 1 of the Arabidopsis tgg1 and tgg2, and probably also tgg3, myrosinase genes (Xue and Rask, 1995).…”

Section: Exon-intron Organization Of Ph and Ah Genesmentioning

confidence: 99%

Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases

et al. 2002

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In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity.O-Glycoside hydrolases (EC 3.2.1.x) constitute a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. These enzymes have been classified into distinct families based on amino acid sequence similarities (Henrissat, 1991; Henrissat and Bairoch, 1996). One of these, family 1, includes many ␤-glucosidases (EC 3.2.1.21) that play diverse and important roles in prokaryotes and eukaryotes. In bacteria and fungi, the cellulolytic ␤-glucosidases are key enzymes in biomass conversion (Béguin, 1990; Fowler, 1993), whereas in animals, lysosomal glucocerebrosidase, for example, is critical to glycosphingolipid metabolism (Grabowski et al., 1993). In higher plants, ␤-glucosidases have been implicated in such fundamental processes as chemical defense against herbivores and pathogens (Conn, 1979), lignification (Dharmawardhana et al., 1995), and regulation of the biological activity of phytohormones by hydrolysis of their inactive hormone-glucoside conjugates (Falk and Rask, 1995).In recent years, our laboratory has been investigating the nature and regulation of ␤-glucosidases involved in large-scale cyanogenesis (HCN product...

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The unusual 5? splicing border GC is used in myrosinase genes of the Brassicaceae

Cited by 33 publications

References 21 publications

Sequencing, Genomic Organization, and Preliminary Promoter Analysis of a Black Cherry (R)-(+)-Mandelonitrile Lyase Gene

Sequencing, Genomic Organization, and Preliminary Promoter Analysis of a Black Cherry (R)-(+)-Mandelonitrile Lyase Gene

Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens

Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases

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