Browsing by Author "Esen, Asim"

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    Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 beta-glucosidase
    Opassiri, Rodjana; Pomthong, Busarakum; Onkoksoong, Tassanee; Akiyama, Takashi; Esen, Asim; Ketudat Cairns, James R. (2006-12-29)
    Background Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice. Results Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3-6 glucose residues and laminaribiose. Conclusion Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.
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    cDNA Cloning and Gene Characterization of Large and Small Subunits of Ribonucleotide Reductase in Soybean
    Xiong, Xinsheng (Virginia Tech, 1999-09-16)
    Ribonucleotide reductase (RNR) reduces four ribonucleoside diphosphates to corresponding deoxyribonucleoside diphosphates, which are transformed into deoxyribonucleoside triphosphates, substrates for DNA polymerase. By controlling the supply and balance of deoxyribonucleoside diphosphates, RNR regulates DNA synthesis. RNR in E. coli and in animals consists of two identical large and two identical small subunits. Until recently, little was known about RNR in plants. For cloning RNR cDNA in plants, soybean (Glycine max) cDNAs were amplified with highly degenerate primers and the Rapid Amplification of cDNA Ends techniques. The cDNAs encoding two complete large subunits, one partial large subunit and one complete small subunit of RNR in soybean were cloned and sequenced. The RNR large subunits in soybean contain a motif with 20 amino acids, which appears to be specific for the RNR large subunits in plants. Southern hybridization results imply that a gene family encodes at least three different large subunits of RNR in soybean, and that a single gene encodes the small subunit. The presence of three different large subunits of RNR in soybean suggests that RNR complex in some plants may have a non-homodimer structure; alternatively, some plants may have different RNR isozymes. Northern hybridization results show that RNR large and small subunit genes in soybean are expressed both in dark-grown and light-grown seedlings, and that light does not increase RNR mRNA levels. Multiple poly(A) sites and different lengths of the 3â untranslated regions were found in cDNAs encoding some subunits of RNR in soybean. The same cis-acting elements may imprecisely locate some multiple poly(A) sites in plants.
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    The Cell Wall Integrity-Associated Map Kinase Homolog, AbSlt2 in the Necrotrophic fungus Alternaria brassicicola is Required for Pathogenicity of Brassicas
    Scott, Derrick Cornelius (Virginia Tech, 2009-01-22)
    Using the genome database of the phytopathogenic fungus, Alternaria brassicicola, we identified a gene with high homology to the cell wall integrity-associated mitogen-activated protein (MAP) kinase, Slt2 in the yeast, Saccharomyces cerevisiae. This MAP kinase consists of a predicted 1,251-bp open reading frame, and encodes a 416-amino-acid protein weighing 47501 Da. This homolog was designated AbSlt2 (A. brassicicola Slt2) and gene disruption knockout (KO) mutants were generated in an A. brassicicola wild type background. Several altered phenotypes were found in the mutants compared to the wild type. During growth in various liquid and solid media, the abslt2 mutants displayed slightly aberrant hyphal growth and were unable to develop at the same rate as wild type. Furthermore, scanning electron microscopy (SEM) analysis revealed the abslt2 mutants showed decreased penetration ability, underdeveloped appresoria, and altered morphology on the leaf surface of the host plant, Brassica oleracea (cabbage) when compared to wild type. Abslt2 mutant hyphae exhibited slower growth in planta ultimately resulting in highly reduced virulence. Complementation of the disruption mutant with the wild type gene fully restored pathogenicity. Therefore, AbSlt2 is a new pathogenicity and developmental factor in A. brassicicola.
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    Characterization and localization of adenylate cyclase during development of Dictyostelium discoideum
    Merkle, Roberta Gayle Kurpit (Virginia Polytechnic Institute and State University, 1982)
    Cyclic AMP functions as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cyclic AMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. Biochemical characterization of adenylate cyclase, the cyclic AMP synthetic enzyme, was accomplished using a sensitive radioimmunoassay. The enzyme was found to be pellet-bound. The non-ionic detergents, Triton X-100 and Lubrol PX, were not effective for solubilizing this activity. Magnesium or manganese could serve as the required divalent cation, with the Mn-supported activity over 4-fold greater than the Mg-supported activity. Typical mammalian adenylate cyclase modulators such as guanyl nucleotides, fluoride, and cholera toxin did not activate the Dictyostelium enzyme. Calcium, in conjunction with its protein receptor calmodulin, did not appear to regulate the enzyme. An endogenous extracellular, heat-stable substance was found to inhibit Dictyostelium adenylate cyclase. By use of ultramicrotechniques adenylate cyclase activity was localized in the pre-spore cells of the culminating individual with no activity detected in the pre-stalk region. Lack of detectable activity in the pre-stalk cells may be due to a masking by the endogenous inhibitor. An increasing gradient of activity was found in the pre-spore mass with activity increasing from the uppermost area to the base. No striking localization was seen prior to the pre-culmination stage of development. Two peaks in cyclic AMP levels, as measured in individuals were found during development. One coincided with aggregation, the other occurred at the culmination stage. A gradient of cyclic AMP within the culminating individual paralleled the gradient of adenylate cyclase activity. The tip of the individual had greater levels of cyclic AMP than the middle pre-spore region, and the upper stalks had higher levels than the lower stalks. These data indicate an enzymatic potential for establishing a gradient of cyclic AMP. At the culmination stage of development this molecule could act to direct the chemotactic movements of the pre-stalk cells as well as provide positional information for the terminal differentiation of the pre-spore cells into mature spores.
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    Characterization of a Beta-glucosidase Aggregating Factor Responsible for the Null Beta-glucosidase Phenotype in Maize (Zea mays L.)
    Blanchard, David Joseph (Virginia Tech, 2000-04-25)
    β-Glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of aryl and alkyl β-D-glucosides as well as glucosides with a carbohydrate moiety such as cellobiose and other beta-linked oligosaccharides. In maize (Zea mays L.), β-glucosidase exists as 120 kD homodimers, but also forms high-molecular-weight (HMW) aggregates in certain maize inbreds (nulls). In this study we show that the null β-glucosidase phenotype is caused by the formation of HMW enzyme aggregates (>1.5 X 10⁶ Daltons), caused by a β-glucosidase aggregating factor (BGAF). BGAF is a 32 kD protein that binds specifically to β-glucosidase and renders it insoluble during extraction. The data unequivocally demonstrate that BGAF is solely responsible for β-glucosidase aggregation and insolubility, and thus, the apparent null phenotype. Additionally, I have isolated the cDNA encoding BGAF and have identified BGAF as a member of the small heat-shock protein (sHsp) family. Interestingly, BGAF binds to both maize β-glucosidase isozymes (Glu1 and Glu2), but does not bind to their sorghum homolog Dhurrinase-1 (Dhr1; Sorghum beta-glucosidase), that shares 70% sequence identity with Glu1 and Glu2. Therefore, these proteins provide an excellent system to study functional differences at nonconserved residues and elucidate the mechanism of enzyme aggregation and insolubility. By examining the behavior of β-glucosidase chimeras in binding assays, I demonstrate that BGAF binding is conformation dependent, highly specific, and reminiscent of antigen-antibody interactions. Additionally, I have identified two disparate polypeptide segments in the primary structure of the maize beta-glucosidase isozyme Glu1 that form a BGAF binding site in the tertiary structure of the enzyme.
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    The chemotaxonomy of the fungal genus Endothia fr.
    Micales, Jessie Ann Daubert (Virginia Polytechnic Institute and State University, 1985)
    The taxonomic status of the fungal genus Endothia was recently changed in a controversial monograph by Barr (Barr, M. E. 1978. The Diaporthales of North America. Mycol. Mem. 7. J. C. Cramer. 232 p.), who divided the genus into two separate genera, Endothia and Cryphonectria, based on differences in ascospore shape and septation, stromatic configuration and distribution of stromatic tissues. This group of fungi traditionally contains some important plant pathogens; its taxonomic position needs to be resolved. The morphological criteria used by Barr were reinvestigated. Polyacrylamide gel electrophoresis and fungicide sensitivity assays were also used to examine biochemical relationships among the organisms and to establish additional means of distinguishing among the closely related taxa. The morphological features of 12 species of Endothia were examined. Those species with 2-celled, ovoid ascospores produced valsoid stromata, while organisms associated with nonseptate, allantoid ascospores formed diatrypoid stromata. Pseudoparenchymatous tissue was observed along the edge of the stroma, while prosenchyma was confined to the stromatic center. The major criteria used by Barr were confirmed. Polyacrylamide gel electrophoresis was used to separate the buffer-soluble proteins of 78 isolates, representing 13 species of Endothia and Cryphonectria cubensis. Intraspecific variation of banding patterns was less than interspecific differences; the species were separated by this technique. The banding patterns of E. eugeniae isolates closely resembled those of C. cubensis: these organisms may be conspecific. Hypovirulent isolates of E. parasitica could not be distinguished from wild isolates. The banding patterns of specific isozymes were species specific; the use of isozyme analysis has great potential for future taxonomic and genetic studies. The sensitivities of E. parasitica and E. gyrosa were determined for 23 different fungitoxicants. The two species were differentially sensitive to cycloheximide, with ED₅₀ values of 0.01-0.03μg/ml and 1.0-2.0 μg/ml for E. gyros and E. parasitica respectively. Differential sensitivities were not exhibited toward the remaining fungitoxicants; these fungi probably share many biochemical processes and response mechanisms. Barr’s classification system is technically correct and it seems to organize relationships within the entire order in a uniform manner. Its adoption is recommended with some hesitation since the influence of host on stromal development is not fully understood.
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    Chromosome aberrations in field strains of Blattella germanica
    Crouch, Joelle H. (Virginia Tech, 1993-07-05)
    Resistant and susceptible field strains of the German cockroach were compared for possible chromosome aberrations. Resistant strain females produced significantly higher numbers of aberrant oothecae ( >5 unhatched eggs ) than the susceptible strains. Chromosome aberrations found in the susceptible strains were attachments (autosome-autosome, autosome-x) and fragments that did not reappear in outcrosses. Attachments (autosome-autosome, autosome-x), fragments, three translocation configurations that did not reappear in outcrosses and two reciprocal translocation heterozygotes occurred in the resistant strains. These two translocations have been tentatively identified as T(12:8)/12:8 from the Bowl strain and T(11:6)/11:6 from the K851 strain. T(12:8)/12:8 exhibits random disjunction at metaphase I. There were no differences related to susceptible vs. resistant strains in the frequency of chromosome aberrations from the aberrant oothecae. There was no evidence, except in the K851 strain, to support a relationship between egg arrest and chromosome aberrations, or the hypothesis that chromosome aberrations result from the selective pressure of insecticides. It is suggested by this study that translocations are the most common type of “floating” polymorphism in the German cockroach. The first occurrence of three known phenotypic mutants, bent bristle, yellow body, and pallid eye, and one new phenotypic mutant, colorless eye, in field strains are reported by this study.
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    Chromosome banding of German cockroach spermatocytes
    Keil, Clifford B. (Virginia Tech, 1982-06-05)
    A chromosome banding protocol for Blattella germanica speramtocytes was developed to reveal locations of constitutive heterochromatin (C-bands). Normality of the acid treatment used to denature basic (histone) proteins, temperature of the salt solution used to extract DNA from unhanded regions, and the components of the Romanowsky-type stain proved to be critical components in the C-banding process. The thiazine component of a standard Gieasa stain had an absorbance maximum altered from that of methylene blue, the putative major thiazine in this stain and was ineffective in producing C-bands. Two samples of Leishman's stain were examined chromatographically, spectrophotometrically, and for their C-bandinq ability as they aged. Banding failure was accompanied by a rounding of the thiazine spectral peak and the appearance of unknowns with cbromatographic mobility intermediate to azures A and B, and an apparent increase in azure C content. Experimental thermal and photo-degradation of Leishman's stain shoved similar alterations of the thiazine components. c-banded prop base II and diplotene kacyotypes revealed a hiqhly heteroaorphic pattern of C-band distribution. Blocks of constitutive heterochromatin were not soley associated with centroaeres bat occurred interstitially and terminally also. One chromosome was C-band negative, number 8, while two others. 9 and the X, were almost completely heterochromatic. Differential rates of condensation from prophase I to prophase II for euchromatic and heterochromatic regions were documented. The karyotype of B. germanica contained many gray bands that may indicate euchroaatin interspersed with heterochromatin. Translocation heterozygote stocks were used to correlate the banded karyotype with linkage groups. Translocation multivalents frequently contained C-bands Dot resolved in wild type chromosomes. A C-banded prophase II karyotype of a closely related species, Blattella vaga, was prepared to assess the variability of heterochromatin distribution. The basic banding pattern was preserved in four of the twelve chromosomes although the bands were larger in this species. Tvo chromosomes, 11 and 8, had a single additional C-band in each. The basic banding pattern was preserved in four of the twelve chromosomes although the bands larger in this species. Two chromosomes, 11 and 89, had a single additional C-band in each. The B. vaga X chromosome was about twice as large as that in B. germanica. The mid-sized chromosomes were extensively repatterned. Overall, B. vaga chromosomes were longer than those of B. germanica. Increased heterochromatin content appeared to be the cause of the greater length.
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    Cis and trans signals for the replication of bovine parvovirus
    Metcalf, John Brockway (Virginia Tech, 1990)
    The cis and trans signals important in BPV replication were identified using a transient replication assay, the mobility shift assay, and a comparison between the BPV and LPV genomes. Replication of deleted BPV genomic clones, which contain the natural left (3’ OH end of the viral minus strand) and right (5’ PO, end of the viral minus strand) BPV termini, defined the minimum size of the BPV origin of replication (ori) to be the terminal 171 nucleotides of each terminus. Clones containing duplicate termini or altered left ends were also shown to replicate. The BPV ori was determined to have two domains identified by a computer analysis of homologus regions between these termini. Three proteins were identified that bind to the left terminal 171 nucleotides in the hairpin conformation. Inhibition of the formation of the DNA-protein complexes with competitor DNA localized two potential binding sites that correspond to the domains mentioned above. Two of the DNA-protein complexes were formed by BPV-coded proteins as determined by inhibition of the complex by anti-BPV antibodies. The third complex resulted from binding of a host cell S-phase protein that is a likely candidate for the S-phase factor required for autonomous parvovirus replication. The BPV ori thus appears to function by binding both cellular and viral proteins for the initiation of DNA synthesis from the hairpinned termini. The comparison of the BPV and LPV genome sequence suggest that the genomic organization of LPV may be more like BPV than that of the rodent parvovirus minute virus of mice; and therefore, LPV may contain similar cis signals.
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    A comparison of determinate and indeterminate soybean lines for double cropping in Virginia
    Pyle, Marjorie Elizabeth (Virginia Polytechnic Institute and State University, 1982)
    Forty lines, 20 determinate and 20 indeterminate, from four soybean (Glycine max L. Merrill) crosses were evaluated under full-season and double cropping conditions to compare the performance of the two plant types under both cropping systems. The parents and selected lines were in the range of Maturity Groups IV and V. In 1980, the lines were planted 1 July in a replicated test at Warsaw, VA while in 1981 these lines were planted in three replicated tests on 11 June and 8 July at Warsaw and on 12 June at Orange, VA. Both June plantings were considered full-season. Standard cultivars in 1980 included 'Essex', 'Williams', and 'Crawford'. 'Bay' and 'Will' were added in 1981. Yields were similar in 1980 for both plant types with the determinates yielding 10.1 q/ha and the indeterminates yielding 9.9 q/ha. In 1981, the yields were similar for both types at Orange with the determinates and indeterminates yielding 26.4 and 26.5 respectively. The types were significantly different in the 11 June planting at Warsaw, with yields of 28. 6 and 28. 0 q/ha for the determinate and indeterminate lines, respectively. The opposite was observed for the 8 July planting in which the indeterminates yielded 21.2 q/ha and the determinates yielded 20.8 q/ha. A comparison of the two determinate and indeterminate lines with the highest yields in both 1981 Warsaw plantings showed that indeterminates were more adaptable to double cropping, though high yielding lines of both types were present. The high yielding indeterminates of the 8 July planting had a tendency to be taller and more erect than the determinates, an attribute desirable for double cropping. Lines that were highest yielding in the 11 June planting were ranked lower in the 8 July planting and vice versa. Spearman' s rank correlation for yield in the two Warsaw plantings had a coefficient of 0. 23, indicating a high degree of specific adaptation to the two environments. Selection of better adapted lines for double cropping appears feasible. The indeterminate trait appears to make some contributions to this adaptation.
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    The construction and testing of maize transcriptional fusions in yeast (Saccharomyces cerevisiae)
    Bennett, Selester (Virginia Tech, 1993-12-06)
    The specific goal of this study was to construct and test transcriptional fusions of zein promoters and a yeast reporter gene that will serve as part of a two plasmid system that will allow for the identification of maize transcriptional regulators of zein genes. Zein genes are expressed coordinately and tempO~ly during endosperm development and are controlled at the transcriptional level (Pedersen et aL, 1980; Kodryzcki et al. t 1989). The accumulation of zein proteins in the endosperm presents an ideal model system to study plant gene regulation. These proteins are synthesized only in the endosperm tissue, and their concentration in the endosperm determine the nutritional quality of the seed. Because of the coordinate and temporal regulation of zein gene transcription, there is a strong likelihood that there exists positive regulatory elements of zein gene expression during early endosperm development. We know that the control of storage protein gene expression is mediated by regulatory elements in the endosperm of maize seeds. It has been shown that the recessive mutation opaque-2 (02) specifically reduces the 22,000 zein polypeptide .. Schmidt et at (1990) and Aukennan et at (1991) show that the wild-type 02 encodes a protein containing a basic leucine zipper domain
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    Determination of the Binding Site and the Key Amino Acids on Maize β-Glucosidase Isozyme Glu1 Involved in Binding to β-Glucosidase Aggregating Factor (BGAF)
    Yu, Hyun Young (Virginia Tech, 2009-04-10)
    β-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called β-glucosidase aggregating factor (BGAF) is responsible for the absence of β-glucosidase activity bands on zymograms. BGAF specifically binds to maize β-glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu⁵⁰-Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum β-glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize β-glucosidases, and an examination of the 3-D structure of Glu1 suggested that the BGAF binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²-Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹-Thr²⁹, together with C-terminal region Phe⁴⁶⁶-Ala⁵¹², affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (Kd) of chimeric β-glucosidase-BGAF interactions. The results demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²-Thr⁸² on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val⁷²-Glu⁸² region was replaced with the Ile⁷²-Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²-Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding. We also constructed mutant β-glucosidases to identify and define the contribution of individual amino acids in the above three regions to BGAF binding. In the N-terminal region (Ile⁷²-Thr⁸²), critical region for BGAF binding, Glu1 mutants K81E and T82Y failed to bind BGAF in the gel-shift assay and their frontal affinity chromatography (FAC) profiles were essentially similar to that of sorghum β-glucosidase (dhurrinase 2, Dhr2), a non-binder, indicating that these two amino acids within Ile⁷²-Thr⁸² region are essential for BGAF binding. In the extreme N-terminal (Ser¹-Thr²⁹) and C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions, N481E [substitution of asparagine-481 with glutamic acid (as in Dhr)] showed lower affinity for BGAF, whereas none of the single amino acid substitutions in the Ser¹-Thr²⁹ region showed any effect on BGAF binding indicating that these regions play a minor role. To further confirm the importance of lysine-81 and threonine-82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four unique amino acids (isoleucine-72, asparagine-75, lysine-81, and threonine-82) of Glu1 in the peptide span Ile⁷²-Thr⁸² are required to impart BGAF binding ability to Dhr2. The sequence comparison among plant β-glucosidases supports the hypothesis that BGAF binding is specific to maize β-glucosidases because only maize β-glucosidases have threonine at position 82.
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    Development of genetic transformation systems in creeping bentgrass (Agrostis palustris Huds.)
    Xiao, Lian (Virginia Tech, 1994)
    As a first step toward improving creeping bentgrass (Agrostis palustris Huds.) via genetic engineering, this study was conducted to develop genetic transformation systems in creeping bentgrass. Establishment of embryogenic cell cultures is a prerequisite for crop improvement via genetic engineering. A protocol for initiating and maintaining embryogenic callus and suspension cultures in creeping bentgrass was developed by substantially modifying and combining a few existing protocols. A high frequency of plant regeneration was obtained following this protocol. Several factors affecting electroporation efficiency were studied using transient expression assay of the reporter uuid gene encoding B-glucuronidase (GUS). Increases in plasmid DNA resulted in increases in GUS activity. Maximal GUS activity was observed at field strength of 950 V/cm, protoplast density of 2 x 10⁶/ml, and KCl concentration of 125 mM in the electroporation buffer. Information obtained from this study facilitated optimization of electroporation conditions. To identify a 5’ regulatory sequence conferring a high level of transgene expression in creeping bentgrass, the effect of six different 5’ regulatory sequences on transient gene expression was studied in electroporated creeping bentgrass protoplasts. The cauliflower mosaic virus (CaMV) 35S promoter was least active; whereas the rice actin 1 gene 5’ sequence was most active among the six sequences tested. Ranked in order of activity (high to low), the other four 5’ sequences were: 1) the CaMV 35S promoter plus the maize alcohol dehydrogenase 1 gene (Adh1) intron 6; 2) the 5’ sequence of the maize ubiquitin gene (Ubi-1), 3) the maize Adh1 promoter and its intron 1, and 4) the 35S promoter plus the Adh1 intron 1. Stable transformation of creeping bentgrass was conducted via particle bombardment and electroporation using a plasmid, pZO1052, containing the reporter B-glucuronidase (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene under the control of CaMV 35S promoter plus the maize Adh1 intron 6. Putative transformants were selected by culturing cells on medium containing hygromycin. Transgenic plants and calli were obtained following particle bombardment. The frequency of putative transformants was 4.6 hygromycin-resistant colonies per bombardment. Integration of the transgenes was confirmed by Southern blot hybridization. A high frequency of escapes, however, occurred in the transformant selection following electroporation, which resulted in inefficient transformant recovery. In this study, efficient genetic transformation systems using particle bombardment were established. Use of these systems will facilitate the improvement of creeping bentgrass.
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    A diallel study of flowering and of ear components of yield in Corn Belt maize and their interactions with population density
    McClane, John Michael (Virginia Polytechnic Institute and State University, 1985)
    A diallel study of American Corn Belt maize (Zea mays L.) was conducted at Holland, Virginia in 1981 and 1982. All possible crosses of twelve inbred parents (A619, A632, B73, H60, H93, H96, Mo17, Oh7B, Pa91, Val7, Va.79:419, Va85) were planted in three replications with population density treatments of 39,536, 49,420, 59,304, and 69,188 pl/ha in strips across hybrid treatments. Analyses of variance and combining ability analyses were performed on traits measuring the timing of anthesis (pollen shed) and silk emergence, on ear components of yield, and on components of kernel size. Density effects were highly significant for all traits, except for that of pollen shed duration, in the analyses combined over years. Hybrid-by-year interactions were highly significant for all traits. Correlations between GCA effects of grain yield and GCA effects of silking delay (anthesis-to-silking interval), kernels per row on the ear, ear kernel number, and kernel depth[(ear diameter - cob diameter)/2] were -0.79, 0.64, 0.66, and 0.80 in 1981, and 0.24, 0.81, 0.71, and 0.26 in 1982, respectively. Moisture stress sufficient to cause wilting occurred before and during silking in 1981. Apparently, short silking delay was associated with high moisture stress tolerance for grain yield in 1981 and was associated with long ear shoot length in 1982. Deep kernel depth apparently was associated with drought stress tolerance for yield as well. The heritabilities of ear traits were higher the earlier they became established in the sequence of development. Heritabilities of silking delay and most ear components of yield were increased by increasing planting density. However, the correlations among flowering and ear traits largely were unaffected by density, perhaps because densities were not high enough to make barrenness a substantial factor in grain yield. The most important traits related to yield were silking delay, kernels per row, kernel depth, and kernel row number. GCA to SCA variance component ratios were increased by combining data over years and by the more optimum season for yield.
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    DNA systematic studies in the genus Bromus L. (Poaceae)
    Pillay, Michael (Virginia Tech, 1991)
    Chloroplast and ribosomal DNA restriction endonuclease analyses were used to assess phylogenetic relationships between different subgenera of Bromus, a genus of over 100 diploid and polyploid species. Variation in chloroplast DNA (cpDNA) fragment pattern was examined, initially, in 15 species of subgenera Festucaria and Ceratochloa. Subsequently, cpDNA restriction site variation was examined in 38 species, using 10 enzymes and filter hybridization experiments. Variation in the ribosomal DNA (rDNA) repeat units was examined in 56 species, using four restriction endonucleases. Generally, higher polyploid species of Bromus showed very little or no interspecific variation in chloroplast DNA sequences. For example, nine species of Ceratochloa were identical in cpDNA structure. In contrast, diploid species showed various degrees of nucleotide sequence divergence. Cladistic analysis of cpDNA restriction site variation indicated two major lines of evolution within Bromus. One clade includes species of Festucaria, Neobromus and Ceratochloa and the other comprises species of Stenobromus and Bromus. cpDNA trees indicate greater genetic relationships among the subgenera of Bromus when compared to results from morphology and cytogenetics. The restriction site data suggests that Festucaria, Ceratochloa and Neobromus are closely related, while Stenobromus and Bromus are genetically isolated from the other subgenera and appear to be evolutionarily advanced. However, the cpDNA results cannot differentiate species of subgenera Stenobromus and Bromus since species of both subgenera were interwoven in the same clade. The cpDNA results suggest that additional characters are needed to provide further information on the taxonomy and evolution of Bromus. Restriction analysis of rDNA produced a wide variety of patterns. Digestion with BamHI produced extensive length heterogeneity within and among species. EcoRI digests were not useful for phylogenetic analysis since the enzyme generally cleaves only once per repeat unit. EcoRI and Kpni endonuclease fragment patterns were used to identify the number of repeat unit length variants per species. The BstEIIand Kpnl patterns were useful in determining relationships at the subgenus level. The unique 2.1 kb BstEII fragment of the coding region in subgenera Festucaria, Ceratochloa, Neobromus and Stenobromus suggests close genetic relationship among these subgenera. A similar fragment of 1.1 kb was present in subgenus Bromus suggesting genetic isolation from the other subgenera of Bromus.
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    Ecology of algal mats and their role in the formation of stromatolites in Antarctic dry valley lakes
    Wharton, Robert A. (Virginia Polytechnic Institute and State University, 1982)
    Algal mats comprised primarily of Phormidium frigidum Fritsch, Lyngbya martensiana Menegh., and several species of pennate diatoms are found in the below-ice benthic regions of Lakes Bonney, Chad, Hoare, Fryxell, and Vanda, southern Victoria Land, Antarctica. Mats are also found in the littoral moats and ice-covers of several lakes, and in cryoconite holes on Canada Glacier. Variations in temperature, light, oxygen, salinity, and nutrient levels between lakes and different habitats in the same lake result in differences in species composition, morphology, biomass, and photosynthetic pigment content of the mats. Algal mats are trapping and binding sediment, and precipitating minerals, particularly calcite. Mats are removing organic and inorganic matter from the arheic lakes via transfer through the icecovers or by incorporation into the sediments. Some of the algal mats are laminated, organosedimentary structures and can be considered stromatolitic. Depending upon ambient and subsequent environmental conditions non-columnar, columnar, and pinnacle-shaped stromatolites are forming, some of which are partially lithified. If environmental variables (i.e. low light intensity, lack of burrowers or browsers, and relative lack of turbulence) associated with these stromatolites do not vary significantly, it is probable that they may result in a lacustrine carbonate sedimentary deposit.
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    Efforts to Manage Disputes in the Construction Industry: A Comparison of the New Engineering Contract and the Dispute Review Board
    Thompson, Roxene Marie II (Virginia Tech, 1998-04-20)
    The construction industry has been plagued with an increasing number of claims and high litigation costs. How do we reduce conflict and litigation in the construction process? On one hand, leaders of the construction industry in the United States (US) focused their efforts on improving alternative dispute resolution mechanisms. For instance, the American Society of Civil Engineers has introduced the Dispute Review Board (DRB) as a complementary provision to standard US construction practices. The establishment of the DRB to solve construction disputes on the job, avoid claims, and reduce project costs has proven considerable success. On the other hand, construction industry leaders in the United Kingdom (UK) have focused some of their efforts on improving general contract conditions. The Council of the Institution of Civil Engineers of the UK has introduced the New Engineering Contract (NEC) to the construction industry as an alternative to presently used contracts. The NEC proposes to be an innovative, non-adversarial mechanism to resolve disputes on the job, avoid and reduce claims, and to assuage rising litigation costs in the construction industry. It too has proven considerable success in its efforts. This research concentrates on the DRB and the NEC as attempts by construction leaders to modernize and improve construction practices. In summary, the research compares the success stories of the DRB and the NEC as approaches to combating the adversarial nature, increasing number of disputes and rising litigation costs in the construction industry. The main conclusions ascertained in this research are as follows. Despite coming from similar business environments, construction industry leaders in the US and the UK embarked on different methods to address the issues plaguing the industry and to improve construction practices. Both in the US and the UK, construction leaders were mostly influenced to proactively seek and implement change in construction practices by experts from within the engineering and construction industry vanguard. The undertaking of these changes have shown similar success stories and the results have produced substantial impacts on the construction process. In conclusion, the efforts of construction leaders to implement the DRB and the NEC have provided effective mechanisms in improving communication and relations, and managing disputes in a timely fashion at the job site level.
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    Footprint Analysis of the Transcriptional Control of Glycogen Phosphorylase 2 in Dictyostelium Discoideum
    Col, Bekir (Virginia Tech, 1997-12-12)
    Glycogen phosphorylase 2 (gp-2) is a key enzyme during the development of Dictyostelium discoideum. The gp-2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. I present in this thesis the identification of several putative cis-acting elements of gp-2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an endlabeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), purified replication protein A and several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for both protein sources. However, using a more sensitive footprinting strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It can be concluded from the data obtained that the TAG and C boxes are very likely cis-acting elements involved in the regulation of gp-2 expression.
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    Genetic analysis and phenotypic characterization of Lon mutants of Escherichia coli K-12
    Torres-Cabassa, Angel S. (Virginia Polytechnic Institute and State University, 1982)
    A systematic study of a collection of Lon⁻ mutants has been made in order to determine whether their pleiotropic phenotype is due to mutations affecting one or more genes. A fine structure map of the lon locus was constructed by Pl mediated generalized transduction. The lon⁻ mutations were found to map in two "clusters" within the region. Phenotypic characterization of a set of isogenic Lon⁻ strains derived from these experiments indicated that all Lon-associated phenotypes (e.g. sensitivity to UV irradiation, decreased ability to inherit plasmid and prophage, abnormal polypeptide degradation and regulation of capsular polysaccharide biosynthesis) are differentially expressed in Lon⁻ strains. A direct correlation exists between the intracistronic ordering of the lon⁻ alleles and the degree of expression the Lon⁻ phenotypes in each strain. All isogenic Lon⁻ strains exhibit conditional lethality upon a nutritional shift-up. However, some filamenting Lon⁻ mutants are not able to overcome this defect when exposed to growth conditions known to promote cell division in Lon⁻ strains. Evidence was obtained that suggest a role for nucleotide pools in the control of cell division and capsular polysaccharide production. Reversion studies indicated that all lon⁻ mutations studied are point mutations. The failure to generate deletions of the lon region in χ573, an F' strain carrying the lac to minE region on the plasmid, and the inability to cure F' strains carrying a lon⁻ mutation on the plasmid suggest that the lon gene product may be indispensable for the cell's survival. From transductional crosses, two intermediate phenotypic classes: UV-resistant, mucoid (UVRMuc), (Class A) and UV-sensitive, nonmucoid (UVSRou) (Class B), were obtained that did not segregate colonies of the opposite morphology. Genetic analysis of these strains by back-transduction into a proC⁻ lon⁺ background, indicated that complete genetic separation of all Lon-associated phenotypes tested was not achieved, although differences in the expression of some of these persisted. Data obtained from complementation analysis ruled out the presence of two genes at the lon locus. The patterns of complementation observed were compatible with the existence of one lon gene, having at least two distinct domains, and whose product is a multifunctional polypeptide.
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    Genetic analysis of androgenetic competence and plants regenerated from callus culture of diploid potato species
    Singsit, Chongkhohao (Virginia Polytechnic Institute and State University, 1988)
    Inheritance of androgenetic competence was studied in 10 diploid potato species hybrids and 16 backcross progeny. Ten hybrid families including three reciprocals were generated between competent clones of S. phureja and incompetent clones of S. berthaultii, S. microdontum and S. phureja. The F₁ hybrid families segregated for androgenetic competence with some highly competent and some incompetent genotypes in all families. The expression of androgenetic competence was modified by parents lacking competence. The cytoplasm of species lacking competence exerted a greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. The segregation data of 16 backcross progeny between a highly competent hybrid and its incompetent parent suggested that competence may be under control of a single dominant gene. Androgenetic competence can be transferred among sexually compatible potato species. The transfer of desirable traits to a monoploid background can be expected using an- drogenetically competent selections in hybrid combination with germplasm expressing the desired attributes. In an attempt to determine genetic changes of regenerated potato plants following anther and callus cultures, 20 callilines of two S. phureja clones were examined. Four of 20 callilines selected for fertility and diploidy were morphologically indistinguishable among themselves and from the parental clone that had not undergone a tissue culture cycle. Even though morphologically indistinct from the parental clone, all four callilines exhibited higher seed set as pollen parents in 4x-2x crosses and two of the four exhibited higher recombination frequency between the centromere and the y gene. The estimated increase in map distance of the y locus ranged from 3.4 to 10.0 units. Progeny analysis revealed no significant morphological differences among 4x-2x hybrid families under field conditions, and only a single difference among 2x-2x hybrid families under screenhouse conditions. Hence, variation induced in tissue culture may have occurred without detectable morphological change. Assuming no adverse tissue culture effects, breakage of undesirable linkage groups may be an advantage of caulogenesis before backcrossing.