Biochemistry of Nectar Proteins

박상규,Robert W. Thornburg 한국식물학회 2009 Journal of Plant Biology Vol.52 No.1

Thymine-Starvation Up-Regulates a Region of the Arabidopsis thaliana Chromosome Ⅲ Containing Both the UMP Synthase Gene and a Proximal Gene Encoding a Novel F-Box Protein

Kafer, Chris,Thornburg, Robert 한국식물학회 2002 Journal of Plant Biology Vol.45 No.3

We have cloned the Arabidopsis thaliana uridine 5'-monophosphate synthase (UMP synthase) locus and characterized transcript levels of this gene as well as the transcript encoding a novel F-box protein found upstream of UMP synthase, Skp1-interacting partner 5 (SKIP5). The 3' end of the SKIP5 gene is 405 bp from the 5' end of the UMP synthase gene. To determine whether these proximate genes are coordinately or independently regulated, we have used an RT-PCR method to quantitate the transcript levels of each gene relative to 18S ribosomal RNA. Previous work has demonstrated an up-regulation of the UMP synthase gene in tobacco callus in the presence of compounds such as 5-fluoro-orotate and aminopterin that induce thymine starvation. Here, we present results showing that both the UMP synthase and SKIP5 genes are coordinately up-regulated in the presence of fluoroorotic acid.

Germin-Like Proteins : Structure, Phylogeny, and Function

Carter, Clay,Thornburg, Robert W. 한국식물학회 1999 Journal of Plant Biology Vol.42 No.2

Sequence Analysis of Rice UMP Synthase cDNAs and Comparison with Other Genes

Sanggyu Park,Robert W. Thornburg 한국토양비료학회 2001 한국토양비료학회 학술발표회 초록집 Vol.2001 No.5

Arabidopsis thaliana Cytidine Deaminase 1 Shows More Similarity to Prokaryotic Enzymes Than to Eukaryotic Enzymes

Kafer, Chris,Thornburg, Robert W. 한국식물학회 2000 Journal of Plant Biology Vol.43 No.3

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to the Arabidopsis thaliana gene, cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with the Escherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the E coli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the E. coli enzyme. In this model, the active site zinc is coordinated by His^73, Cys^103, Cys^107, and an active site hydroxyl. Additional residues that participate in catalysis, Asn^64, Glu^66, Ala^78, Glu^79, and Pro^102, are conserved between the Arabidopsis and E. coli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the E. coli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to the A thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His_6 tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K_m= 226μM) and 2'-deoxycytidine (K_m=49μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP.

T7 RNA polymerase 유전자의 담배식물에서의 발현

Miguel A . Caviedes,박상규,Robert W . Thornburg ( Sanggyu Park ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.4

We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase.

감자의 단백질 분해효소 억제제 Ⅱ 유전자의 특별한 염기서열의 자연적 제거로 인한 상처 유발성 발현의 소실

박상규 ( Sang Gyu Park ),Robert W . Thornburg 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.2

We have isolated several proteinase inhibitor Ⅱ genes pin2 from a Russet Burbank potato DNA library. One of these, pin2T was subcloned and a 1.8 kb XbaI/NsiI insert was sequenced. This fragment contained the complete Inhibitor Ⅱ gene including 965 bp of flanking DNA upstream from the gene and 200 bp of flanking DNA downstream from the gene. The open reading frame encodes a protein that is similar to other reported proteinase Inhibitor II proteins. The DNA sequence of the 5` flanking region of pin2T from -714 to +1 is highly homologous (91% identity) with that of the previously isolated wound-inducible pin2K. There are, however, four small deletions in the pin2T promoter which are located at -221 to -200, -263 to -254, -523 to -426 and -759 to -708 relative to the transcription start site of the wound-inducible pin2K. Three of these deletions map to a portion of the promoter that controls the wound-inducibility of the proteinase inhibitor genes. Chimeric genes containing the promoter of the pin2T gene linked with the both CAT and GUS were constructed and transfered into tobacco plants. Analysis of these plants indicated that pin2T is not a wound-inducible gene but is expressed at low levels. Thus, wound-inducibility is lost with the concomitant natural deletion of three small regions of the promoter. Comparison of the sequences deleted in pin2T relative to the pin2K with Genebank sequences indicates that the deleted sequences contain a motif (consensus 5`AGTAAA-3`) that is found in many other wound-inducible genes but not easily found in the published promoter sequences of other plant genes. Nuclear proteins from unwounded and wounded potato leaves were bound to the proximal promoter region, downstream of the 5`-AGTAAA-3`, of pin2T. The comparison of the pin2T gene with the pin2K gene indicates that the natural internal promoter deletions are likely responsible for loss of the wound-inducible phenotype in the pin2T gene.

Biochemistry, Molecular Biology : Sequence Analysis of a Futative Membrane Transporter cDNA of Arabidopsis thaliana

Sang Gyu Park,Robert W. Thornburg 한국응용생명화학회 2002 Journal of Applied Biological Chemistry (J. Appl. Vol.45 No.4

Six different clones of Arabidopsis thaliana expressed sequence tags (ESTs) were sequenced, among which ATTS1488 clone appeared to code a putative membrane transporter of purines. Comparison of the nucleotide sequence of ATTS1488 with that of a genomic put

Selection of Wound Minus Mutants in Arabidopsis thaliana Containing pin2-codA Transgene

Park, Sang-Gyu,Thornburg, Robert 대구대학교 생명과학연구소 2002 생명과학연구 Vol.1 No.2

Based on the cytosine deaminase assay in the presence of inducing materials, wounding, sucrose, abscisic acid (ABA), and methyl jasmonate, Tr349#3 was selected. Seeds of Tr349#3 were treated with mutagen and 66,000 plants were plated on selective media containing both fluorocytosine and methyl jasmonate to get wound minus mutant(wnd^(-)) plants. 18 plants survived the second round of screening. Each of these plants was scored for the expression of the wound-inducible cytosine deaminase activity. Four of these plants had lost the wound-inducible pin2-codA expression and they were termed as wnd1, wnd4, wnd6, and wnd7. None of the selected wnd plants could be induced by wounding, methyl jasmonate, sucrose, and ABA. Because exogeneously added methyl jasmonate does not induce the pin2-codA construct, we conclude that the defect in each of the four mutations must occur downstream from methyl jasmonate production. These acquired wound minus mutant plants will be used to find out some factor(s) affecting wound minus expression of pin2-codA in Arabidopsis.

Loss of specific sequences in a natural variant of potato proteinase inhibitor Ⅱ gene may cause the loss of the inducibility

Park, Sang Gyu,Thornburg, Robert W 한국농화학회 1992 Applied Biological Chemistry (Appl Biol Chem) Vol.35 No.4

A potato proteinase inhibitor II gene (pin2T) was isolated and characterized both at molecular and functional levels. The open reading frame as well as the 5′ and 3′ flanking regions were sequenced. The 5′ flanking region of the pin2T is highly homologous (91% identity) with that of pin2K from -767 to +29 relative to the transcription start site of the wound-inducible pin2K. The 5′ flanking region was linked to the reporter genes (chloramphenicol acetyl transferase, CAT; or β-glucuronidase, GUS) coding sequences in constructions that contained the terminator from the wound-inducible pin2K gene. The chimeric genes were transferred to tobacco plants using Asrobacterium tumefaciens. The presence of the constructions in the transgenic plants was confirmed by polymerase chain reaction (PCR). Expression of CAT or GUS activities in transgenic tobacco plants driven by pin2T promoter indicated that pin2T is not a wound-inducible gene. Comparison of pin2T promoter sequence with that of the wound-inducible pin2K indicates that there are four small deletion which are located at -221 to 200, -263 to -254, -523 to -462, and -759 to 708 relative to the transcription start site of pin2K. When these deleted sequences are searched through Genebank, we identified one sequence that is found three times in pin2K and is completely deleted from pin2T. This sequence, 5′-AGTAAA-3′, is found in a wide variety of other wound-inducible genes but is not frequently found in the published promoter sequences of the other plant genes. A third deletion located at -523 to -462 relative to the transcription initiation site of pin2K, was moderately homologous (72% identity) with a putative sucross-responsive element of sucrose-inducible genes. Deletion of this sequence was correlated with a loss of sucross inducibility in the pin2K promoter.