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D K Fisher

Publications and source records attributed to D K Fisher.

8 recordsLinked to original sources
PubMed1998

Genomic organization and promoter activity of the maize starch branching enzyme I gene.

Starch branching enzymes (SBE) which catalyse the formation of alpha-1,6-glucan linkages are of crucial importance for the quantity and quality of starch synthesized in plants. In maize (Zea mays L.), three SBE isoforms (SBEI, IIa and IIb) have been identified and shown to exhibit differential expression patterns. As a first step toward understanding the regulatory mechanisms controlling their expression, we isolated and sequenced a maize genomic DNA (-2190 to +5929) which contains the entire coding region of SBEI (Sbe1) as well as 5'-and 3'-flanking sequences. Using this clone, we established a complete genomic organization of the maize Sbe1 gene. The transcribed region consists of 14 exons and 13 introns, distributed over 5.7kb. A consensus TATA-box and a G-box containing a perfect palindromic sequence, CCACGTGG, were found in the 5'-flanking region. Genomic Southern blot analysis indicated that two Sbe1 genes with divergent 5'-flanking sequences exist in the maize genome, suggesting the possibility that they are differentially regulated. A chimeric construct containing the 5'-flanking region of Sbe1 (-2190 to +27) fused to the beta-glucuronidase gene (pKG101) showed promoter activity after it was introduced into maize endosperm suspension cells by particle bombardment. 1998 Elsevier Science B.V.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Molecular cloning and characterization of the Amylose-Extender gene encoding starch branching enzyme IIB in maize.

The amylose-extender (Ae) gene encoding starch-branching enzyme IIb (SBEIIb) in maize is predominantly expressed in endosperm and embryos during kernel development. A maize genomic DNA fragment (-2964 to +20,485) containing the Ae gene was isolated and sequenced. The maize Ae mRNA is derived from 22 exons distributed over 16,914 bp. Twenty-one introns, differing in length from 76 bp to 4020 bp, all have conserved junction sequences (GT..AG). Sequence analysis of the 5'- and 3'-flanking regions revealed a consensus TATA-box sequence located 28 bp upstream of the transcription initiation site as determined by primer extension analysis, and a putative polyadenylation signal observed 29 bp upstream of the polyadenylation site based on cDNA sequence. Genomic Southern blot analysis suggests that a single Ae gene is present in the maize genome. Promoter activity was confirmed by testing a transcriptional fusion of the Ae 5'-flanking region between -2964 and +100 to a luciferase reporter gene in a transient expression assay using maize endosperm suspension cultured cells. 5' deletion analysis revealed that the 111 bp region from -160 to -50 is essential for high-level promoter activity.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1997

Independent genetic control of maize starch-branching enzymes IIa and IIb. Isolation and characterization of a Sbe2a cDNA.

In maize (Zea mays L.) three isoforms of starch-branching enzyme (SBEI, SBEIIa, and SBEIIb) are involved in the synthesis of amylopectin, the branched component of starch. To isolate a cDNA encoding SBEIIa, degenerate oligonucleotides based on domains highly conserved in Sbe2 family members were used to amplify Sbe2-family cDNA from tissues lacking SBEIIb activity. The predicted amino acid sequence of Sbe2a cDNA matches the N-terminal sequence of SBEIIa protein purified from maize endosperm. The size of the mature protein deduced from the cDNA also matches that of SBEIIa. Features of the predicted protein are most similar to members of the SBEII family; however, it differs from maize SBEIIb in having a 49-amino acid N-terminal extension and a region of substantial sequence divergence. Sbe2a mRNA levels are 10-fold higher in embryonic than in endosperm tissue, and are much lower than Sbe2b in both tissues. Unlike Sbe2b, Sbe2a-hybridizing mRNA accumulates in leaf and other vegetative tissues, consistent with the known distribution of SBEIIa and SBEIIb activities.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1996

Two closely related cDNAs encoding starch branching enzyme from Arabidopsis thaliana.

Two starch branching enzyme (SBE) cDNAs were identified in an Arabidopsis seedling hypocotyl library using maize Sbe1 and Sbe2 cDNAs as probes. The two cDNAs have diverged 5' and 3' ends, but encode proteins which share 90% identity over an extensive region with 70% identity to maize SBE IIb [12]. Genomic Southern blots suggest that the two cDNAs are the products of single, independent genes, and that additional, more distantly related SBE genes may exist in the Arabidopsis genome. The two cDNAs hybridize to transcripts which show similar expression patterns in Arabidopsis vegetative and reproductive tissues, including seedlings, inflorescence rachis, mature leaves, and flowers. This is the first report of the identification of cDNAs encoding two closely related starch branching enzymes from the same species.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1996

Evolutionary conservation and expression patterns of maize starch branching enzyme I and IIb genes suggests isoform specialization.

Expression of the maize (Zea mays L.) starch branching enzyme (SBE) genes Sbe1 and Sbe2 were characterized during kernel development and in vegetative tissues. The onset of Sbe1 and Sbe2 expression during endosperm development was similar to that of other genes involved in starch biosynthesis (Wx, Sh2 and Bt2). However, the expression of Sbe2 peaked earlier than that of Sbe1 in developing endosperm and embryos resulting in a shift in the ratio of Sbe1 to Sbe2 relative message levels during kernel and embryo development. Transcripts hybridizing to the Sbe2 probe were not detectable in leaves or roots which nonetheless have SBEII enzymatic activity, suggesting that there may be another divergent SBEII-like gene(s) in maize. A similar expression pattern is shared between the maize genes and related genes in pea, which together with their evolutionary conservation, suggests that the SBE isoforms may play unique roles in starch biosynthesis during plant development.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1994

A sensitive, high-volume, colorimetric assay for protein phosphatases.

Protein phosphatases are intimately involved in a variety of cellular processes, many of which are of interest to the pharmaceutical industry. Phosphatase assays generally employ radioisotopes, making them tedious to perform, costly, and hazardous, while other procedures require antibodies and/or are unsuitable for mass screening efforts. To facilitate screening for inhibitors of the CD45 protein tyrosine phosphatase (PTPase), we have developed a sensitive colorimetric assay, using small volumes in 96-well microtiter plates and read on a standard ELISA plate reader. The assay was sensitive down to 0.5 nmol of released phosphate and can be easily run by robotics to assay thousands of compounds in a day. The assay is sparing of reagents and has been successfully used with all classes of phosphatases. The reagents are nonradioactive, readily obtainable, and minimal in cost. This assay should facilitate the search for specific inhibitors of phosphatases.

Amino Acid Sequence↗