In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database... more
In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the E...
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Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen... more
Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; AT and DT genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Be...
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Soybean [Glycine max (L.) Merr.] genetic diversity is limited because domesticated soybean has undergone multiple genetic bottlenecks. Its progenitor, the wild soybean [Glycine soja Siebold & Zucc], has not undergone the same intense... more
Soybean [Glycine max (L.) Merr.] genetic diversity is limited because domesticated soybean has undergone multiple genetic bottlenecks. Its progenitor, the wild soybean [Glycine soja Siebold & Zucc], has not undergone the same intense selection and is much more genetically diverse than domesticated soybean. However, the agronomic importance of diversity in wild soybean is unclear, and its weedy nature makes assessment difficult. To address this issue, we chose for study a highly selected, adapted F4-derived progeny of wild soybean, NMS4-44-329. This breeding line is derived from the hybridization between G. max cultivar N7103 and G. soja PI 366122. Agronomic comparisons were made among N7103, NMS4-44-329 and their F1 and F2 progeny in replicated yield trials at two North Carolina locations. Significant F1 mid-parent heterosis was observed at each location for seed yield (189 and 223 kgha-1, P<0.05 and P<0.10, respectively), seed protein content (1.1g/100g, P<0.01) and protei...
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ADP-glucose pyrophosphorylase (ADPGp, EC 2.7.7.27) is a tetrameric protein composed of two small and two large subunits that catalyzes the biosynthesis of ADP-glucose from glucose-phosphate which is used to provide the glucose subunits... more
ADP-glucose pyrophosphorylase (ADPGp, EC 2.7.7.27) is a tetrameric protein composed of two small and two large subunits that catalyzes the biosynthesis of ADP-glucose from glucose-phosphate which is used to provide the glucose subunits for starch biosynthesis. A second cotton gene encoding an ADPGp small subunit has been cloned and characterized. The gene contains eight introns similar to previously reported potato and cotton ADPGp small subunit genes. The deduced translation of the gene contained a poorly conserved transit peptide and well conserved catalytic and regulatory elements typical of other plant ADPGps. The 5&amp;amp;#39; end of the mRNA was cloned and sequenced to identify the transcriptional start site (TSS). The promoter region upstream of the TSS did not contain the core promoter sequence in the typical positions indicating this gene may not use a standard core promoter. Other sequence motifs associated with tissue specific expression and phytohormone response were present. Reverse transcription (RT)-PCR with gene specific primers identified the sites of expression of this gene. Expression was most abundant in the meristem region, and immature stem and relatively lower in starch accumulating roots demonstrating that this gene has a different pattern of expression than the previously reported cotton ADPGp small subunit gene. Additionally this gene was differentially expressed in cotton fibers. The presence of starch was confirmed in developing cotton fibers suggesting that starch metabolism plays a role in cotton fiber development.
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Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in... more
Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR.
Research Interests: Plant Biology, Gene expression, Free Radical, Plant Physiology, DBA, and 15 moreSoil sciences, Enzyme, Plant Physiology and Biochemistry, Molecular cloning, Isoenzymes, Molecular Mass, Amino Acid Sequence, Base Sequence, Heterologous Expression, Polyacrylamide Gel Electrophoresis, Benzoquinones, Full Length Movies, Molecular Sequence Data, restriction enzyme, and Gossypium
Broadening the genetic base of upland cotton (Gossypium hirsutum L.) is essential for continu- ous genetic improvement of yield and fiber quality through breeding. The objectives of this study were to evaluate a species polycross (SP)... more
Broadening the genetic base of upland cotton (Gossypium hirsutum L.) is essential for continu- ous genetic improvement of yield and fiber quality through breeding. The objectives of this study were to evaluate a species polycross (SP) popula- tion for phenotypic and genotypic variations in yield and fiber quality, investigate morphological variations among the SP lines, and analyze the interrelationships among
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The pedicel (basal maternal tissue) of maize (Zea mays L.) kernels contains a physically and kinetically unique form of glutamine synthetase (GSp1) that is involved in the conversion of transport forms of nitrogen into glutamine for... more
The pedicel (basal maternal tissue) of maize (Zea mays L.) kernels contains a physically and kinetically unique form of glutamine synthetase (GSp1) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch [1989] Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific GS that does not cross-react either with a second GS isozyme found in the pedicel or with the GS isozymes from the embryo, roots, or leaves. When used as a probe for tissue printing, the antibody labeled the pedicel tissue uniformly and also labeled some of the pericarp surrounding the lower endosperm. Silver-enhanced immunogold staining of whole-kernel paraffin sections revealed the presence of GSp1 in both the vascular tissue that terminates in the pedicel and the pedicel parenchyma cells, which are located between the vascular tissue and the basal endosperm transfer cells. Light staining of the subaleur...
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... Analyses of Two Cell Wall Invertase Genes in Maize 33 22 EARL W TALIERCro\ JAE-YEAN KIM, ALINE MAHE, SAVITA SHANKER, JAE CH0I, WAN ... in a humid chamber at 56 °c for 20 h. After hybridization the slides were incubated for 4 h at 56... more
... Analyses of Two Cell Wall Invertase Genes in Maize 33 22 EARL W TALIERCro\ JAE-YEAN KIM, ALINE MAHE, SAVITA SHANKER, JAE CH0I, WAN ... in a humid chamber at 56 °c for 20 h. After hybridization the slides were incubated for 4 h at 56 °c in wash buffer composed of 50 ...
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Vapour pressure deficit (VPD) is considered an important environmental factor that might affect leaf expansion and transpiration rate (TR) in plants. Two slow-wilting soybean (Glycine max (L.) Merr.) genotypes PI 416937 and PI 471938... more
Vapour pressure deficit (VPD) is considered an important environmental factor that might affect leaf expansion and transpiration rate (TR) in plants. Two slow-wilting soybean (Glycine max (L.) Merr.) genotypes PI 416937 and PI 471938 along with commercial cultivar Hutcheson were subjected to low (1.2-1.6 kPa) and high VPD (2.8-3 kPa) environments to study their leaf expansion and TR over five days. Among the three genotypes, PI 416937 had the lowest increase in its TR (34%) at high VPD compared with low VPD and the greatest decrease in leaf area (31%). In contrast, Hutcheson had the highest increase in TR (87%) under high VPD and the lowest decrease in leaf expansion rate (18%). Expansin and extensin genes were isolated in PI 416937 to determine if changes in leaf expansion were associated with changes at the molecular level. The four studied genes were all suppressed after five days in the high VPD environment.
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lhe pedicel (basal maternal tissue) of maize (Zea mays 1.) kernels contains a physically and kinetically unique form of glutamine synthetase (GS,,) that is involved in the conversion of transport forms of nitrogen into glutamine for... more
lhe pedicel (basal maternal tissue) of maize (Zea mays 1.) kernels contains a physically and kinetically unique form of glutamine synthetase (GS,,) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch (1989) Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific CS that does
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Roots and stems of cotton (Gossypium hir- sutum L.) plants store photoassimilate as starch. Partitioning of fixed carbon between starch in vegetative storage tissues and seed is likely to impact cotton development and yield. The enzyme... more
Roots and stems of cotton (Gossypium hir- sutum L.) plants store photoassimilate as starch. Partitioning of fixed carbon between starch in vegetative storage tissues and seed is likely to impact cotton development and yield. The enzyme ADP-glucose pyrophosphorylase (ADPGp) plays a rate-limiting role in starch production, and its temporal and spatial expression plays a critical role in determining patterns of starch deposi- tion in plants. The objective of this study was to identify, sequence, and analyze a cotton ADPGp (small subunit) gene involved in starch produc- tion in stems and roots. A genomic sequence with extensive similarity to an mRNA encoding the small subunit of ADPGp that is expressed in starchy stems was identified and sequenced. The gene was composed of nine exons and eight introns. The introns were bound by typical splice sites. The open reading frame (ORF) encoded a peptide of 518 amino acids with many catalytic and regulatory features common to plant ADPGp. This g...
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The nucleosome structure of the nuclear rRNA genes was investigated in a variety of tumorous and nontumorous Nicotiana tabacum cell lines, and in a genetic tumor produced by crossing Nicotiana langsdorffii with Nicotiana glauca. The rRNA... more
The nucleosome structure of the nuclear rRNA genes was investigated in a variety of tumorous and nontumorous Nicotiana tabacum cell lines, and in a genetic tumor produced by crossing Nicotiana langsdorffii with Nicotiana glauca. The rRNA genes from two unorganized octopine type crown gall tumors were found in an altered nucleosome conformation compared to those of the other cell lines and N. tabacum leaves. The altered nucleosome structure of the rRNA genes in the octopine type crown gall lines was not due to the tumorous state of the tissue, nor was it related directly to the morphology of the tumor. These two lines did have, however, a greatly reduced rRNA gene copy number. Several Eco R1 fragments homologous to the rRNA gene probe were preferentially lost from one of these tumor lines. The alteration of the nucleosome structure of the remaining rRNA genes in the octopine type crown gall tumors may result from rapid transcription necessitated by their reduced copy number.
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Differential display has been widely and successfully used to identify differentially expressed genes based on physiological treatments or genetic variation. We used differential display to identify genes based on their site of... more
Differential display has been widely and successfully used to identify differentially expressed genes based on physiological treatments or genetic variation. We used differential display to identify genes based on their site of translation. Identification was made based on the fractionation of RNA from soybean leaves into total RNA, free polyribosomal RNA, and membrane-bound (MB) polyribosomal RNA. Sequences were identified representing
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A strategy was developed to isolate a complete gypsy-element from Gossypium hirsutum L. based on the sequence of a RAPD that was polymorphic in near isoganic lines of cotton that varied in leaf shape. A 5998 nt clone was isolated and its... more
A strategy was developed to isolate a complete gypsy-element from Gossypium hirsutum L. based on the sequence of a RAPD that was polymorphic in near isoganic lines of cotton that varied in leaf shape. A 5998 nt clone was isolated and its gene order and sequence confirmed it was a gypsy-type retroelement. Sequences homologous to the gag portion of this clone were also found in Gossypium herbaceum L. and Gossypium raimondii L. A portion of the open reading frame of the integrase gene was amplified from three Gossypium species under investigation. PCR products of the expected size were amplified from all three Gossypium species. G. raimondii sequences were statistically more degenerate than sequences from either G. hirsutum or G. herbaceum. Finally, analysis of the open reading frames of selected integrase clones from the three Gossypium species revealed a second gypsy element based on similarity of the deduced amino acid sequence.
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A genomic clone representing a putative retinoblastoma binding (RBB) protein was isolated from a Gossypium hirsutum BAC library. Alignment of the gene sequence with the cDNA sequence indicated the gene consists of six exons that have... more
A genomic clone representing a putative retinoblastoma binding (RBB) protein was isolated from a Gossypium hirsutum BAC library. Alignment of the gene sequence with the cDNA sequence indicated the gene consists of six exons that have standard eukaryotic splice junctions. The conceptual spliced transcript was 98% identical to TC37171 in the TIGR gene index, however it encoded an ORF 107 amino acids longer than best deduced protein from TC37171. The conceptual translation of the genomic clone was 56% identical to a tomato gene experimentally demonstrated to be a RBB protein and able to complement the yeast growth mutant IRA. The mRNA encoded by the genomic clone was abundantly expressed in meristems and expression levels increased as the cotton fiber matured. We propose that this gene may regulate growth and/or cell division in cotton based on homology of the clone with a protein of known function and sites of expression.
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Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of basic and acidic portions joined by disulfide bridges. There are five glycinin subunit... more
Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of basic and acidic portions joined by disulfide bridges. There are five glycinin subunit isoforms designated Gy1-Gy5. The purpose of this study is to identify epitopes from selected glycinin subunits that are antigenic in pigs. Twenty-seven out of 30 pigs had antibodies against glycinin in their sera. Ten of these sera had immunoglobulin G (IgG) against the Gy4 (A5A4B3) or Gy1 (A1aBx) subunit. Three sera recognised overlapping regions between the two subunits tested, though no serum stained both A5A4B3 and A1aBx. Two sera stained a highly conserved region between A5A4B3 and A1aBx, though again neither serum stained both peptides. The basic part of the A1aBx subunit was not recognised by any of the sera tested even though immunoblot data indicated that the basic and acidic subunits of glycinin are nearly equally antigenic. Two antigenic regions of A5A4B3 and A1aBx were identified that bound antibodies in half of the sera that reacted with these two proteins. Half of the sera reacted with unique regions of A5A4B3 and A1aBx. The failure of the basic portion of A1aBx to bind pig antibodies may indicate that it is less antigenic than the basic portion of A5A4B3 and other glycinin subunits.
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β-Conglycinin (conglycinin) is one of the major seed storage proteins of soybean. Conglycinin is a 7S trimer composed of different combinations of β, α and... more
β-Conglycinin (conglycinin) is one of the major seed storage proteins of soybean. Conglycinin is a 7S trimer composed of different combinations of β, α and α&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; subunits. All subunits of conglycinin have been reported to be allergenic in humans. The goal of this research is to identify epitopes of the β subunit of conglycinin that are antigenic in multiple animal species. Sera from pigs, dogs, rabbits and hybrid striped bass that had antibodies against soybean conglycinin were identified by ELISA. Most of these sera recognized peptides that represent the β subunit of conglycinin. One antigenic region of the β subunit of conglycinin had considerable overlap among all species tested. One region that was similar to a peanut allergenic epitope in humans overlapped with a region that binds IgE from dogs. One region was antigenic in multiple rabbits and pigs, suggesting it may play a role in the response of pigs to soybean in the diet. One region of the β subunit of conglycinin is an important antigen across species and abuts a region similar to the peanut allergen ARA h 1. A second region is particularly antigenic in pigs and rabbits. Variants of these antigenic regions of the β subunit of conglycinin may be useful in determining the role these regions play in the health of animals fed soybean. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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ABSTRACT Soybean meal is commonly added to a variety of animal feeds to supplement protein sources and to optimise growth. While soybean protein is a valuable food supplement it has been recognised as an important food allergen. The... more
ABSTRACT Soybean meal is commonly added to a variety of animal feeds to supplement protein sources and to optimise growth. While soybean protein is a valuable food supplement it has been recognised as an important food allergen. The soybean seed storage protein, glycinin, has been identified as an allergen. A tiled peptide array of the A1aBx and A5A4B3 subunits of glycinin was screened to identify the epitopes that bind antibodies from multiple species. We have identified four regions in these two glycinin subunits that are antigenic in most or all of the species tested. One region is implicated in an allergic response in dogs by the dog&#39;s ability to bind IgE. Three regions overlap or abut regions that are similar to allergenic epitopes in peanut. It will be critical to identify immunogenic regions able to cause allergies to soy in order to prioritise them for mitigation.
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ABSTRACT Soybean meal in the post-weaning diet of pigs results in a growth lag correlated with Immunoglobulin G (IgG) production against soybean proteins, including the seed storage protein β-conglycinin. We screened sera from 30 pigs for... more
ABSTRACT Soybean meal in the post-weaning diet of pigs results in a growth lag correlated with Immunoglobulin G (IgG) production against soybean proteins, including the seed storage protein β-conglycinin. We screened sera from 30 pigs for IgG directed against β-conglycinin. Analysis of the same sera on protein blots identified sera that had IgG against the α-subunit of β-conglycinin. A tiled peptide array (16 amino acids with an 11 amino acid overlap) of the α-subunit of β-conglycinin was screened to identify the epitopes of the α-subunit that bind IgG. One major IgG binding epitope previously identified was confirmed. A second major IgG binding epitope was identified that bound IgG from half of the animals tested. Minor epitopes that bound IgG from one or two other sera were also identified. Binding of IgG to β-conglycinin in an enzyme-linked immunosorbent assay (ELISA) was inhibited by a synthetic peptide representing one of the minor epitopes.
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We have used antibodies directed against the two sucrose synthase (SS) isozymes, and the cDNA clones corresponding to the two nonallelic genes in maize to describe sorghum (Sorghum bicolor) SS genes and their expressions at protein andRNA... more
We have used antibodies directed against the two sucrose synthase (SS) isozymes, and the cDNA clones corresponding to the two nonallelic genes in maize to describe sorghum (Sorghum bicolor) SS genes and their expressions at protein andRNA levels. ...
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We have investigated the chromatin structure of the integrated T-DNA in two N. tabacum crown gall tumor lines, and compared the results to those obtained in a previous study of the methylation patterns of these same integrated DNA... more
We have investigated the chromatin structure of the integrated T-DNA in two N. tabacum crown gall tumor lines, and compared the results to those obtained in a previous study of the methylation patterns of these same integrated DNA sequences (Gelvin et al., Nucleic Acids Res. 11:159-174, 1983). The E9 octopine-type tumor contains a single copy of TL, whose transcription is essential for tumor maintenance, and 15-30 copies of TR, a non-essential region. The HT37#15 nopaline type teratoma contains a single copy of the nopaline T-DNA. All these integrated sequences can be found associated with nucleosomes, although the diffuse nature of the nucleosome bands on Southern transfers implies an &#39;open&#39; chromatin conformation. In addition, all the sequences are more sensitive to digestion with deoxyribonuclease I than the bulk of the chromatin. We present evidence suggesting that, despite the previously published data that the majority of copies of the TR-DNA are highly methylated at the sequence CCGG whereas the TL-DNA is not, the majority of copies of the TR-DNA in the E9 tumor line are in the same chromatin conformation as TL. These data therefore suggest that most of the copies of TR-DNA are likely to be transcriptionally competent.
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ADP-glucose pyrophosphorylase (ADPGp, EC 2.7.7.27) is a tetrameric protein composed of two small and two large subunits that catalyzes the biosynthesis of ADP-glucose from glucose-phosphate which is used to provide the glucose subunits... more
ADP-glucose pyrophosphorylase (ADPGp, EC 2.7.7.27) is a tetrameric protein composed of two small and two large subunits that catalyzes the biosynthesis of ADP-glucose from glucose-phosphate which is used to provide the glucose subunits for starch biosynthesis. A second cotton gene encoding an ADPGp small subunit has been cloned and characterized. The gene contains eight introns similar to previously reported potato and cotton ADPGp small subunit genes. The deduced translation of the gene contained a poorly conserved transit peptide and well conserved catalytic and regulatory elements typical of other plant ADPGps. The 5&amp;amp;#39; end of the mRNA was cloned and sequenced to identify the transcriptional start site (TSS). The promoter region upstream of the TSS did not contain the core promoter sequence in the typical positions indicating this gene may not use a standard core promoter. Other sequence motifs associated with tissue specific expression and phytohormone response were present. Reverse transcription (RT)-PCR with gene specific primers identified the sites of expression of this gene. Expression was most abundant in the meristem region, and immature stem and relatively lower in starch accumulating roots demonstrating that this gene has a different pattern of expression than the previously reported cotton ADPGp small subunit gene. Additionally this gene was differentially expressed in cotton fibers. The presence of starch was confirmed in developing cotton fibers suggesting that starch metabolism plays a role in cotton fiber development.