Etwa ein Drittel der Weltbevölkerung ist mit Mycobacterium tuberculosis (Mtb), der Erreger der Tuberkulose (TB), infiziert. Daher ist es unbedingt notwendig vorhandenen Behandlungsstrategien weiter zu verbessern. Diese Study beschäftigt sich mit dem Mtb Protein Ag85C, einer Mycolyltransferase, als ein neues Ziel für die medikamentöse Behandlung der TB. Ag85C ist eines von drei verwandten Proteinen, Ag85A, B und C, welche zusammen an der Biogenese der Zellwand von Mtb beteiligt sind. Eine Gruppe von chemischen Molekülen mit den Namen Ag85C-1 bis -4 wurde als Inhibitoren von Ag85C getestet. Alle Verbindungen waren in der Lage das Wachstum von Mtb in Flüssigkulturen zu inhibieren, aber nur Ag85C-3 hatte ebenfalls einen Effekt auf intrazelluläre Bakterien, welches in einem Makrophagen-Infektions-System getestet wurde. Hervorzuheben ist, dass Ag85C-3 darüber hinaus auch das in vitro Überleben eines MDR Stammes inhibierte. Dies macht dieses Molekül zu einem interessanten Kandidaten für neue anti-mycobakterielle Therapieansätze. Desweiteren wurden detaillierte, funktionelle Charakterisierungen der Effekte von Ag85C-3 auf Mtb durchgeführt. Die Verbindung modifiziert die Lipide der mykolischen Säuren in der Zellwand durch die Blockierung der Ag85 Funktionen. Dieser Effekt führt dann zu einer Veränderung in der Durchlässigkeit der Außenhülle von Mtb. Mit Hilfe der microarray Analyse wurden die Regulierungen der Signalwege durch Ag85C-3 umfassend untersucht. Es konnte gezeigt werden, dass lebensnotwendige Siderophore durch das Molekül modifiziert werden, was auf mehrere Wirkungsmechanismen schließen lässt. Diese Erkenntnisse machen Ag85C, als Ziel, und Ag85C-3, als Inhibitor, zu vielversprechende Kandidaten für zukünftige Medikamentenforschung auf dem Gebiet der TB-Therapien. Diese Studie hebt zudem die zielbasierte Identifizierung von chemischen Inhibitoren als wichtigen und wertvollen Ansatz für die Medikamentenentwicklung hervor. / Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) infects about one-third of the world’s population. Therefore there is an urgent need to improve existing intervention strategies. This study evaluated the Mtb Ag85C protein, a mycolyl transferase, as a novel target for drug mediated intervention. Ag85C belongs to a family of three cognate proteins, Ag85A, B and C. They are involved in the final steps of Mtb cell envelope biogenesis. A panel of chemical molecules, Ag85C-1-4, which bind to Ag85C were utilized as inhibitors of Ag85C. All compounds inhibited growth of Mtb in vitro in liquid medium cultures but only Ag85C-3 had an effect on intracellular bacteria in macrophage infection system. Importantly, Ag85C-3 can inhibit in vitro survival of a MDR strain of Mtb making it a relevant molecule in the search for novel classes of anti-mycobacterial compounds. Furthermore a detailed functional characterization of Ag85C-3 effect on Mtb was performed. It modified the cell wall mycolic acid containing lipid amounts by blocking Ag85 function that led to changes in permeability of Mtb envelope. A comprehensive analysis of Mtb signalling pathways regulated by Ag85C-3 was investigated through microarray analysis. It showed modification of vital siderophore biosynthesis indicating multiple mechanisms of action. Thus the target, Ag85C and the inhibitor, Ag85C-3 are promising candidates for future TB drug research aimed at combating broad spectrum resistance development. This study also reinforces target based identification of chemical inhibitors as a valid and valuable approach in drug development.

Carbohydrate molecules are essential parts of living cells. They are used as energy storage and signal substances, and they can be found incorporated in the cell membranes as attachments to glycoproteins and glycolipids, but also as free molecules. In this thesis the effect of carbohydrate molecules on phospholipid model membranes have been investigated by the means of Molecular Dynamics (MD) computer simulations. The most abundant glycolipid in nature is the non-bilayer forming monogalactosyldiacylglycerol (MGDG). It is known to be important for the membrane stacking typical for the thylakoid membranes in plants, and has also been found essential for processes related to photosynthesis. In Paper I, MD simulations were used to characterize structural and dynamical changes in a lipid bilayer when MGDG is present. The simulations were validated by direct comparisons between dipolar couplings calculated from the MD trajectories, and those determined from NMR experiments on similar systems. We could show that most structural changes of the bilayer were a consequence of lipid packing and the molecular shape of MGDG. In certain plants and organisms, the enrichment of small sugars such as sucrose and trehalose close to the membrane interfaces, are known to be one of the strategies to survive freezing and dehydration. The cryoprotecting abilities of these sugar molecules are long known, but the mechanisms at the molecular level are still debated. In Papers II–IV, the interactions of trehalose with a lipid bilayer were investigated. Calculations of structural and dynamical properties, together with free energy calculations, were used to characterize the effect of trehalose on bilayer properties. We could show that the binding of trehalose to the lipid bilayer follows a simple two state binding model, in agreement with recent experimental investigations, and confirm some of the proposed hypotheses for membrane–sugar interactions. The simulations were validated by dipolar couplings from our NMR investigations of TRH in a dilute liquid crystal (bicelles). Furthermore, the assumption about molecular structure being equal in the ordered and isotropic phases was tested and verified. This assumption is central for the interpretation of experimentally determined dipolar couplings in weakly ordered systems. In addition, a coarse grain model was used to tackle some of the problems with slow dynamics that were encountered for trehalose in interaction with the bilayer. It was found that further developments of the interaction models are needed to properly describe the membrane–sugar interactions. Lastly, from investigations of trehalose curvature sensing, we concluded that it preferably interacts in bilayer regions with high negative curvature. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

O gene AGT1 do locus mal1g, do sistema de transporte de maltose de S. cerevisiae, codifica uma proteína de 67 kDa, que tem 75% de similaridade e 58% de identidade com o transportador de maltose do locus MAL1. Sua expressão é ativada por genes reguladores constitutivos do sistema MAL, e é reprimida pela glicose. A cepa AP68-7A carrega o gene AGT1, e possivelmente o gene transportador MAL31, e transporta trealose ao final da primeira fase de crescimento anaeróbico. SDS-PAGE comparando proteínas de membranas de células reprimidas pela glicose (taxa de transporte &#60;0,5 U/mg), com aquelas de células induzidas por &#945;-metilglicosídeo (taxa de transporte de ~35 U/mg), verificou-se 2 bandas (PMs 57 e 66 kDa) exclusivas em membranas de células induzidas. As 2 bandas foram isoladas por três métodos diferentes (cromatografia de troca iônica, lavagens da membrana com tampão de alta força iônica e cromatografia de afinidade) e testadas para ligação de 14C-trealose. A ligação foi enriquecida ~3 X após a cromatografia de troca iônica. O transporte de trealose na cepa AP77-4C (que não tem nenhum dos genes transportadores dos loci MAL) foi recuperado após sua transformação com plasmídeo YEp366-AGT1. De membranas plasmáticas destas células (transporte de trealose ~25 U/mg) foram isoladas por cromatografia de afinidade, 2 bandas cujos PMs em SDS-PAGE são idênticos aos das proteínas isoladas das membranas da cepa AP68-7A. Estes resultados permitem concluir que o transportador de trealose de leveduras é codificado pelo gene AGT1. / The AGT1 gene presente in the mal1g locus from S. cerevisiae maltose transport system encodes a 67 kDa protein which shares 75% similarity and 58% identity with the maltose transporter protein encoded in MAL6 locus. The expression of this gene is regulatory genes from MAL system and is repressed by glucose. The strain AP68-7A which harbors the AGT1 gene and probably the MAL31 transporter gene, expresses trehalose transport activity at the end of first anaerobic growth. The comparison from the SDS-PAGE of membrane proteins from glucose repressed cells (trehalose transport activity of &#60;0.5 U/mg), and &#945;-methylglucoside induced cells (trehalose transport activity of ~35 U/mg), revealed 2 bands (Mr 57 and 66 kDa) present only in the induced cells membranes. Those bands were isolated by 3 different methods (ionic exchange chromatography, high strength ionic washes and affinity chromatography, and tested for 14C-trehalose binding. Both bands bind trehalose and this activity was enriched about 3 times after the ionic exchange chromatography. The trehalose transport activity was recovered by strain AP77-4C, (which does not harbor any MAL transporter gene) after its transformation with a plasmid containing the AGT1 gene. From the membranes of these cells (trehalose transport activity of ~25 U/mg) 2 bands were isolated by affinity chromatography with similar Mrs to those isolated from AP68-7A strain. The results permit to conclude that the AGT1 gene encodes the yeast trehalose transporter.

It is of great importance to be able to unambiguously interpret deoxyribonucleic acid (DNA) profiles, especially with Low Template (LT) DNA and mixture DNA that may contain major and minor contributors. Reducing stochastic effects, such as heterozygote peak imbalance, dropouts, and stutter artifacts have been studied by scientists in order to improve the evaluation of low quality DNA profile. There has been much research on a compatible solute, trehalose, in its effectiveness in enhancing the polymerase chain reaction (PCR), especially with GC-rich templates of DNA, and thermal stabilizing Thermus Aquaticus (Taq) DNA polymerases. Based on previous research, the effect of trehalose on peak heights, peak height ratios, and stutter ratios (n-1) from 15 short tandem repeats (STR) loci of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit was evaluated with 0.025ng, 0.05ng, 0.1ng, and 1ng of DNA, through the addition of 0M (control), 0.2M, and 0.4M of trehalose for each quantity of DNA. Although there was an observation regarding changes in average peak heights at 1ng of DNA with the addition of 0.2M, and 0.4M of trehalose, no conclusions could be made with the average peak heights for 0.025ng, 0.05ng, 0.1ng, and 1ng of DNA. The reason is that the propagation of pipetting error during the preparation of each batch could have contributed to the difference in the amount of DNA between each conditions which can be directly reflected in peak heights. Furthermore, unexpected discrepancy between the average peak heights for 0.1ng of DNA from the first and the second experiments rendered 0.1ng of DNA incompatible for comparison. With regards to average peak height ratios for 0.025ng, 0.05ng, 0.1ng, and 1ng of DNA, and average reverse stutter ratios for 0.1ng, and 1ng of DNA, there were no evidence to suggest that 0.2M or 0.4M of trehalose had any effects. Consistent trends for 0.1ng (Exp. 1 and 2) and 1ng of DNA from a statistical analysis through one-way ANOVA of individual loci, suggested that trehalose may have varying effects on certain loci. However, this observation must be approached with caution as it is uncertain whether unique trends across each data sets for certain loci were observed by chance due to small sample sizes or due to mechanisms of stutters and trehalose that are currently unknown. Future studies regarding the effect of trehalose on peak heights should be done with more precision through minimizing pipetting error, which can be accomplished by preparing one batch from which aliquots are taken. The result of the research does not show enough evidence to prove the usefulness of trehalose since the addition of trehalose does not yield consistently higher average peak heights and peak height ratios, and lower average reverse stutter ratios across 15 STR loci. Therefore, our results do not support that 0.2M and 0.4M of trehalose are useful within the parameter of forensic DNA analysis as they do not enhance the polymerase chain reaction (PCR) and improve stochastic effects for DNA profiles.

Pathway optimization is, in general, a very demanding task due to the complex, nonlinear and largely unknown interactions of enzymes, regulators and metabolites. While in vitro reconstruction and pathway analysis is a viable alternative, a major limitation of this approach is the availability of the pathway enzymes for reliable pathway reconstruction. Here, we report the application of RNA display methods for the construction of fusion (chimeric) molecules, comprising mRNA and the protein they express, that can be used for the above purpose. The chimeric molecule is immobilized via hybridization of its mRNA end with homologous capture DNA spotted on a substrate surface. We show that the protein (enzyme) end of the fusion molecule retains its function under immobilized conditions and that the enzymatic activity is proportional to the amount of capture DNA spotted on the surface of a microarray or 96-well microplate. The relative amounts of all pathway enzymes can thus be changed at will by changing the amount of the corresponding capture DNA. Hence, entire pathways can be reconstructed and optimized in vitro from genomic information alone by generating chimeric molecules for all pathway enzymes in a single in vitro translation step and hybridizing on 96-well microplates where each well contains a different combination of capture DNA. We provide validation of this concept with the sequential reactions catalyzed by luciferase and nucleoside diphosphate kinase and further illustrate this method with the optimization of the five-step pathway for trehalose synthesis. Multi-enzyme pathways leading to the synthesis of specialty molecules can thus be optimized from genomic information about the pathway enzymes, provided the latter retain their activity under the in vitro immobilized conditions. / Singapore-MIT Alliance (SMA)

Background: Biological systems adapt to changing environments by reorganizing their cellula r and physiological program with metabolites representing one important response level. Different stresses lead to both conserved and specific responses on the metabolite level which should be reflected in the underl ying metabolic network. Methodology/Principal Findings: Starting from experimental data obtained by a GC-MS based high-throughput metabolic profiling technology we here develop an approach that: (1) extracts network representations from metabolic conditiondependent data by using pairwise correlations, (2) determines the sets of stable and condition-dependent correlations based on a combination of statistical significance and homogeneity tests, and (3) can identify metabolites related to the stress response, which goes beyond simple ob servation s about the changes of metabolic concentrations. The approach was tested with Escherichia colias a model organism observed under four different environmental stress conditions (cold stress, heat stress, oxidative stress, lactose diau xie) and control unperturbed conditions. By constructing the stable network component, which displays a scale free topology and small-world characteristics, we demonstrated that: (1) metabolite hubs in this reconstructed correlation networks are significantly enriched for those contained in biochemical networks such as EcoCyc, (2) particular components of the stable network are enriched for functionally related biochemical path ways, and (3) ind ependently of the response scale, based on their importance in the reorganization of the cor relation network a set of metabolites can be identified which represent hypothetical candidates for adjusting to a stress-specific response. Conclusions/Significance: Network-based tools allowed the identification of stress-dependent and general metabolic correlation networks. This correlation-network-ba sed approach does not rely on major changes in concentration to identify metabolites important for st ress adaptation, but rather on the changes in network properties with respect to metabolites. This should represent a useful complementary technique in addition to more classical approaches.

Rhodococcus is an important industrial organism that possesses diverse metabolic capabilities, it also has a unique cell envelope, composed of an outer layer of mycolic acids and glycolipids (free or bound lipids generally linked to the sugar trehalose). Rhodococcus is able to transform nitriles to the corresponding amide by the enzyme Nitrile Hydratase (NHase), therefore rhodococcal cells can be utilized as biocatalysts in the detoxification of nitrile waste water or in the production of industrially important amides such as acrylamide. However, the NHase within the native cells must be stable with high activity. This research examined how NHase activity and stability can be increased in native cells by changing growth media composition, the impact on the rhodococcal cell envelope was also studied. Growth media composition was altered by supplementing different sugars such as fructose, maltose or maltodextrin to replace glucose in rich solid media containing cobalt and urea for induction of NHase. The supplementation of maltose or maltodextrin resulted in significantly higher NHase activities and greater NHase stability at 55„aC. The supplementation of these different sugars was shown to alter cellular and lipid bound trehalose levels, a sugar known to stabilize proteins and a component of the rhodococcal cell envelope. Cells that had higher levels of cellular trehalose had significantly greater NHase stability at 55„aC. The effect of the different sugar supplements and inducers of NHase, such as cobalt, on cell envelope components such as mycolic acids and glycolipids were examined by High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). The results showed that changes in mycolic acids and glycolipids occurred when the cells were grown in the presence of different sugar supplements and when the cells were induced for NHase. Susceptibility of Rhodococcus sp DAP 96253 to different antibiotics was examined to indicate if changes were occurring in the cell envelope. Differences in antibiotic susceptibility were observed when the cells were grown on media with different sugar supplements and when the cells were induced for NHase. In the presence of cobalt Rhodococcus sp DAP 96253 showed a significant increase in sensitivity to antibiotics. Changes in growth media composition influences the cell envelope of Rhodococcus sp DAP 96253 and also affects NHase activity and stability. Therefore, achieving increased enzyme activity and stability is not entirely dependent on the actual enzyme, but is related to other aspects of the cell, such as the cell envelope and metabolites of the cell.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 3 uppsatser.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 191-216). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family. Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production. Sugar yields from sugarcane have been improved in the past by either creating transgenic sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane is still of interest and new cisgenic strategies are being considered to alleviate consumer concerns over transgenic plants. This thesis consists of two parts. The first was aimed at understanding the relation between trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed that four out of the nine otsA lines had significantly decreased and one line significantly increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose levels in sugarcane, but further research of TPS activity is required before a conclusion can be reached. Further analysis of mature cane material in regard to relevant enzyme levels, carbohydrate levels and gene expression should contribute to more conclusive results. Three novel sugarcane TPS encoding sequences were isolated and proven to be functional through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by successful application of 5‟ RACE alongside standard PCR using primers based on other monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N terminal truncation, which removes one of the thirteen amino acids found within the active site of the TPS domain. Future characterization of the encoded enzymes will determine the effects of these modifications on TPS activity. The second part of this thesis describes initial efforts made in attempting to develop a cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco (NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO, used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid organelle, so as to ensure translocation of the enzyme to that subcellular location. Also, random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE herbicide resistance, however this work is currently on hold until a heme deficient E. coli can be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was successful in revealing additional nucleotide sequence adding 1006 bp to the already known partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence. / AFRIKAANSE OPSOMMING: Suikerriet is 'n tropiese meerjarige gras spesie wat deel is van die Poaceae (ware grasse) familie. Volwasse suikerriet bestaan hoofsaaklik uit suikerrietstamme, wat hoë hoeveelhede sukrose akkumuleer, 'n feit wat gelei het tot die wye verbouing van suikerriet vir sukrose produksie. In die verlede is suikeropbrengste vanuit suikerriet verbeter deur die skep van transgeniese suikerriet óf die gebruik van tradisionele teelmetodes. Toenemende suiker opbrengste in suikerriet is steeds van belang en nuwe cisgeniese strategieë word oorweeg om verbruikerskommer oor transgeniese plante te akkommodeer. Hierdie tesis bestaan uit twee dele. Die eerste deel is daarop gemik om die begrip van die verhouding tussen trehalose-6-fosfaat (T6P) sintese en sukrose ophoping in suikerriet te verstaan. In hierdie studie is die E. coli gene wat betrokke is in trehalose sintese, otsA en otsB, ooruitgedruk in suikerriet ten einde die uitwerking daarvan in die opgaar van oplosbare suiker te bestudeer. Nege otsA en twee otsB verhoogte uitdrukkings lyne is geskep, bevestig deur gDNA bygevoegde PKR, sq-RT-PKR en immuno opsporing van geïnkripteerde ensieme. Voorlopige metings van oplosbare suikers toon dat vier van die nege otsA lyne ʼn beduidende afname in sukrose vlakke en een lyn „n beduidende toegeneem in sukrose vlakke getoon het. Korrelerende sq-RT-PKR resultate met oplosbare suikermetings dui daarop dat trehalose-6- fosfaat sintese (TPS) geenuitdrukking sukrose vlakke sal affekteer, maar verdere navorsing van TPS aktiwiteit is nodig voordat 'n gevolgtrekking gemaak kan word. Verdere ontleding van volwasse riet materiaal met betrekking tot relevante ensiem vlakke, koolhidrate vlakke en geenuitdrukking, behoort by te dra tot meer volledige resultate. In hierdie studie is drie nuwe suikerriet TPS gene geïsoleer en dit is bewys as funksioneel deur die komplimentering van die groeidefek van tps1Δ gis, gegroei op glukose as 'n koolstof bron. Suikerriet TPS isoforme, genoem SoTPSa, SoTPSb en SoTPSc, is geïsoleer deur die suksesvolle toepassing van 5 'RACE, in kombinasie met standaard PKR, deur van spesiaal ontwerpte primers, gebaseer op ander eensaadlobbige TPS gene, gebruik te maak. Die gekodeerde SoTPSa bevat 'n 25 aminosuur invoeging binne-in die gedeeltelike TPP domein. Die gekodeerde SoTPSc bevat 'n 126 aminosuur lange N terminaal afkapping, wat een van die dertien aminosure binne die aktiewe terrein van die TPS domein verwyder. Toekomstige karakterisering van hierdie geïnkripteerde ensiemes sal die effek van hierdie veranderinge op TPS aktiwiteit bepaal. Die tweede deel van hierdie tesis beskryf die aanvanklike probeerslae wat gemaak is in 'n poging om „n cisgeniese in vitro selekteerbare merker vir suikerriet, S. officinarum kallus te ontwikkel. Hierin word gebruik gemaak van 'n difenylether tipe (DPE) onkruiddoder as 'n seleksie agent, en 'n suikerriet protoporphyrinogen oksidase (PPO) geen as 'n seleksie merker. In 'n poging om dit te bewerkstellig is daar eerstens plastied geteikende PPO van tabak (NtPPO-1) geïsoleer en geteikende mutagenese suksesvol daarop uitgevoer. Mutasies wat geinduseer is, is gegrond op die dubbele gemuteerde Arabidopsis PPO, wat gebruik was in mielies deur Li et al., (2003). Alhoewel die suikerriet kallus getransformeer is met die dubbele gemuteerde NtPPO-1 konstruk en geselekteer is op media wat fomesafen onkruiddoder bevat, was die kallus nie in staat om te regenereer nie. In toekomstige pogings sal probeer word om 'n N-terminale volgorde, geteiken op „n plastied organel, te benut sodat translokasie van die ensiem aan die plastied organel verseker kan word. So ook is toevallige mutasies veroorsaak in die NtPPO-1 gene om te soek vir nuwe mutasies wat DPE onkruiddoderweerstand verleen, maar hierdie werk is tans gestop totdat 'n heem gebrekkige E. coli mutant verkry kan word. Tweedens, is pogings aangewend om 'n vermeende suikerriet plastied geteikende PPO gene te isoleer, om uiteindelik te gebruik in die ontwikkeling van 'n cisgeniese strategie in suikeriet. 5 'RACE was suksesvol in die onthulling van bykomende nukleotiede volgorde deur 1006 bp by te voeg by die reeds bekende gedeeltelike suikerriet PPO fragment. Nie teenstaande is die fragment wat nuut geïsoleer is, steeds slegs 'n gedeeltelike volgorde volgens vergelykings met ander bekende plant PPO gene.