Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Starch-containing agricultural crops are widely available as feedstocks for the production of fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructose corn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, are usually used for the production of whisky. One of the first steps in the production of whisky is to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step is performed to disrupt and solubilise the starch granules to make them more accessible for enzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase and then saccharified by glucoamylase and a debranching enzyme. Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effective a-amylases and glucoamylases, making them two of the most efficient raw-starchdegrading yeasts known. However, L. kononenkoae and S. fibuligera cannot be used in existing industrial fermentations because of their low ethanol tolerance, slow growth rate, catabolite repression, poorly characterised genetics and lack of GRAS (Generally Regarded As Safe) status. This study is divided into two sections. The aim of the first section was to clone a gene (LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) from L. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid, YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) and terminator (PGKh) expression cassette. This recombinant plasmid was designated pJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquid assays revealed that the recombinant yeast secreted active a-amylase into the medium. The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C. The aim of the second part of the study was to construct recombinant strains of S. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were cloned into a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expression cassette. Two indigenous yeasts were selected on the basis of their ability to utilise raw starch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing the L. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase (SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmids expressing the individual coding sequences under the control of the PGK1 p-PGK1.,- expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively; two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of the PGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective native promoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and two single-cassette plasmids expressing SFA 1 and SFG1 with their native promoters and terminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs were transformed into a laboratory strain of S. cere visiae , L1278b. By homologous recombination, each plasmid was integrated into the yeast genome at the ura3 locus. S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 under the control of the PGK1 rrPGK1,expression cassette resulted in the highest levels of a-amylase activity when assayed for amylolytic activity in a liquid medium. This recombinant strain resulted in the most efficient starch utilisation in batch fermentations, consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation. The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassette gave the highest levels of glucoamylase activity.' These results confirmed that co-expression of a-amylase and/or glucoamylase synergistically enhance starch degradation. This study paves the way for the development of efficient starch-degrading strains of S. cerevisiae for the production of whisky, beer and biofuel ethanol. / AFRIKAANSE OPSOMMING: Styselbevattende landbougewasse kom wydverspreid voor as die substraat vir die produksie van brandstofetanol, drinkbare spiritualië of bier, enkelselproteïen en hoëfruktose graanstroop. Styselbevattende gewasse, soos mielies, rog, gars en koring, word gewoonlik vir die produksie van whisky gebruik. Die eerste stap in die produksie van whisky is om die stysel by temperature bo 1DOOG te kook. Hierdie jelatinisasie stap word uitgevoer om die styselkorrels te versteur en vloeibaar te maak sodat hulle meer toeganklik vir ensimatiese hidrolise is. Na dié kookproses word die stysel deur o-arnilases vervloei en dan deur glukoamilases en 'n vertakkingsensiem versuiker. Lipomyces kononenkoae en Saccharomycopsis filuligera skei hoogs effektiewe a-amilases en glukoamilases uit, wat dit twee van die effektiefste rou-stysel-afbrekende giste bekend, maak. L. kononenkoae en S. fibuligera kan egter nie in reeds bestaande industriële fermentasies gebruik word nie, as gevolg van hulle lae etanoltoleransie, stadige groeitempo, katabolietonderdrukking, swak gekarakteriseerde genetika en gebrek aan ABAV (Algemeen Beskou As Veilig) status. Hierdie tesis is in twee afdelings verdeel. Die doel van die eerste deel was om 'n geen (LKA2) wat vir 'n nuwe, unieke styselafbrekende ensiem kodeer, te kloneer, 'n tweede a-amilase (Lka2p) van L. kononenkoae. LKA2 is in 'n multikopie plasmied, die gis episomale plasmied, YEp352, onder beheer van die fosfogliseraatkinasepromotor- en termineerder-kasset (PGK1 p-PGK1 r), gekloneer. Hierdie rekornbinante plasmied is pJUL3 genoem en in 'n laboratoriumras van Saccharomyces cerevisiae, L:1278b, getransformeer. Plaat- en vloeibare-ensiem toetse het getoon dat die rekombinante gis aktiewe a-amilase in die medium uitskei. Die optimum pH vir Lka2p is 3.5, is en die optimum temperatuur 60oG. Die doel van die tweede deel van die studie was om rekombinante rasse van S. cerevisiae te konstrueer wat a-amilases en/of glukoamilases uitskei. Die individuele gene is toe in 'n gis-integreringsplasmied, Ylp5, onder beheer van die PGK1p-PGK1,ekspressiekasset, gekloneer. Twee inheemse giste is op grond van hulle vermoë om stysel te benut geselekteer, L. kononenkoae en S. filuIigera, as geen donors. Agt konstrukte bevattende die L. kononenkoae se a-amilasegene, LKA 1 en LKA2, en S. filuligera se a-amilasegeen (SFA 1) en glukoamilasegeen (SFG1), moes gekonstrueer word: vier _enkel-kasset plasmiede wat die individuele koderende sekwense onder beheer van die PGK1 p-PGK1, ekspressiekasset uitdruk, wat onderskeidelik plPLKA 1, pIPLKA2, plPSFA 1 en plPSFG1 lewer; twee dubbel-kasset plasmiede (wat beide LKA 1 en LKA2 onder beheer van die PGK1 p-PGK1,ekspressiekasset, en SFA 1 en SFG1 met hulle onderskeie inheemse promotors en termineerders) uitdruk, wat onderskeidelik pIPLKA1/2 en plPSFAG lewer, en twee enkel-kasset plasmiede wat SFA 1 and SFG1 met hulonderskeie inheemse promotors en termineerders, en wat onderskeidelik pSFA 1 en pSFG1 lewer. Die onderskeie konstrukte is in 'n laboratoriumras van S. cerevisiae, L1278b, getransformeer. Deur middel van homoloë rekombinasie, is die onderskeie plasmiede in die ura3-lokus van die gisgenoom geïntegreer. S. cerevisiae L1278b, getransformeer met plPLKA 1/2, LKA 1 en LKA2 onder die beheer van die PGK1 ~PGK1 ïekspressiekasset, het die hoogste vlakke van a-amilase aktiwiteit gelewer toe dit vir amilolitiese aktiwiteit in vloeibare medium getoets is. Hierdie rekombinante ras het stysel die effektiefste benut, nl. 80% van die stysel en 'n opbrengs van 6 gIL etanol na 156 ure in lotfermentasies. Die ras wat SFG1 onder beheer van die PGK1~PGK1ïekspressiekasset uitdruk, het die hoogste vlakke van glukoamilase-aktiwiteit gelewer. Hierdie resultate bevestig dat die gesamentlike uitdrukking van a-amilase- en/of glukoamilase-ensieme styselafbreking sinergisties . bevorder. Hierdie studie baan die weg vir die ontwikkeling van 'n effektiewe styselfermenterende ras van S. cerevisiae wat moontlik gebruik kan word vir die produksie van whisky en biobrandstofalkohol.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with tartaric acid, are natural constituents of grape must and wine, and can be released as free acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms present in wine. These volatile phenols contribute to the aroma of the wine. The Bretfanomyces yeasts are well known for their ability to form volatile phenols in wine. However, these species are associated with the more unpleasant and odorous formation of the ethylphenols and the formation of high concentrations of volatile phenols. Other organisms, including some bacterial species, are responsible for the formation of volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the organoleptic properties of the wine. The enzymes responsible for the decarboxylation of phenolic acids are called phenolic acid decarboxylases; and several bacteria and fungi have been found to contain the genes encoding these enzymes. The following genes have been characterised: PAD1 from Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified. S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast. Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not show any higher enzyme activity than the control strain. Both bacterial genes, under the control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the SMR1 gene as selective marker. The cloning and transformation of pdc and padc into industrial wine yeast strains can therefore be used to detect the effect of phenolic acid decarboxylase genes in the winemaking process for the possible improvement of wine aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1 disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile phenols in the wine. The manipulated strains also produced enzymes capable of producing large amounts of favourable monoterpenes in the wine. This study paves the way for the development of wine yeast starter culture strains for the production of optimal levels of volatile phenols, thereby improving the sensorial quality of wine. / AFRIKAANSE OPSOMMING: Die fenoliese sure (p-kumaarsuur en ferolsuur), wat as natuurlike komponente in mos en wyn voorkom, word gewoonlik as esterverbindings in wynsteensuur gevind. Seker esterase-aktiwiteite kan die fenoliese sure as vrye sure vrystel gedurende die wynmaakproses. Hierdie vrye fenoliese sure kan dan weer deur verskillende mikroorganismes na 4-viniel en 4-etiel derivate omgesit word. Hierdie derivate staan as vlugtige fenole bekend en kan tot die aroma van wyn bydra. Die Brettanomyces giste is baie bekend vir hulle vermoeë om vlugtige fenole in wyn te vorm, maar dit is gewoonlik die formasie van hoë konsentrasies van vlugtige fenole, veral die 4-etiel derivate, wat met af geure geassosieer word. Ander organismes besit egter die vermoeë om vlugtige fenole teen lae konsentrasies te vorm, veral die 4-viniel derivate, wat 'n aanvullende effek op die wyn aroma kan hê. . Die ensieme wat verantwoordelik is vir die dekarboksilasie van fenoliese sure staan as fenolsuurdekarboksilases bekend. Verskeie bakterieë en fungi bevat gene wat vir hiedie ensieme kodeer. Die volgende gene is reeds gekarakteriseer: PAD1 van Saccharomyces cerevisiae, fdc van Bacillus pumilus, pdc van Lactobacillus plantarum en padc van Bacillus subtilis. PadA van Pediococcus pentosaceus is ook reeds geïdentifiseer. S. cerevisiae bevat die PAD1- (fenielakrielsuurdekarboksilase) geen, wat teen 'n vaste tempo in gis getranskribeer word. Die aktiwiteit van hierdie ensiem is egter laag. Fenolsuurdekarboksilase van B. subtilis, sowel as p-kumaarsuurdekarboksilase van L. plantarum, vertoon "n substraat-induseerbare dekarboksilerende aktiwiteit met fenoliese sure. Beide die p-kumaarsuur dekarboksilase en die fenolsuurdekarboksilase gene is in die PGK1PT ekspressie kasset gekloneer. Episomale en gisintegreringsplasmiede is vir die PAD1-geen onder beheer van die PGK1 PT ekspressiekasset gekonstrueer vir die ooruitdrukking van hierdie geen in gis. Die PGK1 PT ekspressiekasset het bestaan uit die promotor- (PGK1 p) en termineerdersekwense (PGK1 T) van die gisfosfogliseraatkinasegeen (PGK1). Industriële gisrasse is ontwikkel waarin die PAD1-geen onderbreek is. Ooruitdrukking van p-kumaarsuurdekarboksilase (Pdc) en fenolsuurdekarboksilase (pade) in S. cerevisiae toon hoë ensiemaktiwiteit in laboratoriumgisrasse. Die ooruitdrukking van die PAD1-geen het nie hoër aktiwiteit as die kontroleras gewys nie. Albei die bakteriële gene, onder die beheer van die PGK1 PT ekspressiekasset, is ook in "n gisintegreringsplasmied met die SMR1-geen as selektiewe merker geplaas. Die klonering en transformasie van pdc en padc in industriële wyngiste kan dus gebruik word vir die bepaling van die effek van fenolsuur dekarboksilases in die wynmaakproses en die moontlike verbetering van wynaroma. Wyn is met al drie die industriële rasse (die ooruitgedrukte bakteriële gene en die ontwrigte PAD1- geen) gemaak. Die effek van die teenwoordigheid van hierdie gene in die wynmaakproses is deur gaschromatografie bepaal. Die resultate het aangedui dat die bakteriële gene op In effektiewe wyse vlugtige fenole in die wyn kan produseer. Sekere monoterpene is ook in In verhoogde mate gedurende hierdie proses gevorm. Hierdie studie baan die weg vir die ontwikkeling van reingisinentingskulture vir die produksie van optimale vlakke van vlugtige fenole om sodoende die sensoriese gehalte van die wyn te verbeter.

M.Sc. / A common feature of multi gene families is that their members are expressed in different ways in response to environmental and developmental signals. In the present study the expression of a CHS multi gene family in bean (Phase°lus vulgari.), was studied. using a RT-PCR technique that focuses on the 3' divergent regions of the isogenes. Tissue-specific expression in roots, stems, leaves and the flowers of Phaseolus vulgaris, as well as in callus tissue, were investigated. Patterns and levels of gene expression were investigated after treatment with different elicitors as well as light. In most cases time and concentration studies were performed. The four CHS transcripts (CHS4. CHS1. CHS17 and CHS14) showed tissue-specific expression. The four CHS transcripts were differently expressed in the seven organs investigated: and different levels of activity were observed. The highest level of transcript expression for CHS14 and CHS4 could be observed in the roots, whereas relatively low levels were obtained in the leaves. stems and flowers of the green as well as etiolated seedlings. Higher levels of CHS were found in flower buds. High levels of all four transcripts were also found in callus. Elicitor treatment with structurally diverse abiotic agents showed induction of all four CHS mRNA transcripts. Concentration studies revealed high levels of CHS transcript levels. Elicitation with different concentrations of the elicitors: glutathione. mercuric chloride and sodium salicylate showed high levels of the CHS transcripts after exposure of 6 h to the different elicitation agents. The transcript levels increased significantly to levels above those observed in untreated (control) plants. The CHS transcripts showed higher levels of induction after elicitation with mercuric chloride (1 mM) relative to treatment with sodium salicvlate (10 rnM), suggesting differential regulation at the transcriptional level. The expression patterns observed with glutathione were very similarly to those induced by mercuric chloride. The kinetics of induction of all CHS transcripts. except for CHS1 were low at 2 and at 8 h postelicitation and maximal levels of transcript. although transiently induced, could be observed at 4 - 6 h. The use of 4 mM mercuric chloride did not give any induction. most probably because it was a lethal concentration. Etiolated and green bean seedlings, exposed to UV light. showed expression of all four CHS transcripts. In green leaves no significant differences in the induction kinetics between the different chs genes were observed. Three of the transcripts (CHS4. CHSI7. CHS14) accumulated rapidly (within ca. 3h). reaching a maximum after 6 h of irradiation. followed by a decline. CHS4 revealed a 18.2 fold induction. CHSI7 showed a 4.8 fold increase and CHS14 a 4 fold increase after 6 h of illumination in green leaves. In contrast. CHS1 showed a delay ed response which was still observable after 15 h. It was also demonstrated that CHS transcripts accumulated rapidly but transiently. Following illumination of etiolated leaves with white light. except for CHS I. CHS17 and CHS4 showed similar expression levels and patterns. with maximal induction at 1,5 h after white light exposure, whereas maximal induction for CHS14 was at 2 h. At 2.5 Ii the levels for all three transcripts dropped to preinduction levels. It is therefore evident that CHS is a key metabolic control point in the phenylpropanoid pathway leading specifically to isollavonoid biosynthesis. The results strongly suggest that the activation of plant defence genes are regulated in a tissue-specific manner and that induction by different elicitor-active agents. may be regulated by different. But convergent signal transduction regulatory networks.

L’érythritol est un polyol aux propriétés édulcorantes utilisé comme substitut de sucre par l’industrie agroalimentaire. Le but principal du projet est l’amélioration du procédé de production d’érythritol par génie métabolique. L’idée est de construire des souches surexprimant les gènes liés à la voie de synthèse de l’érythritol. L’objectif principal est donc d’identifier les gènes clés permettant d’augmenter la synthèse d’érythritol et d’évaluer cette dernière en bioréacteur.Parallèlement à cela, un autre objectif est d’identifier les gènes liés au catabolisme de l’érythritol. En effet, Y. lipolytica est capable de produire de l’érythritol, mais aussi de le reconsommer en cas d’absence d’autre source de carbone. L’objectif est donc d’identifier les gènes liés au catabolisme de l’érythritol afin de les déléter, et ainsi obtenir une souche capable de produire de grandes quantités d’érythritol sans le reconsommer en fin de culture.Les résultats obtenus ont permis d’identifier les étapes clés de la voie de synthèse de l’érythritol et d’obtenir des souches à haut rendement et productivité par génie génétique. Par ailleurs, deux gènes de la voie de dégradation de l’érythritol ont pu être identifiés pour la première fois chez une levure. En combinant la surexpression de gènes liés à la synthèse de l’érythritol et la délétion de gènes liés à sa dégradation, une souche présentant une productivité 74% plus importante que la souche sauvage a pu être créée. Par ailleurs, une souche capable de convertir l’érythritol en érythrulose, un autre composé d’intérêt, a également pu être construite. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.

X-disease, caused by phytoplasmas, is one of the destructive diseases in stone fruit trees, causing yield loss and poor fruit quality. So far no effective methods are available to control X-disease. X-disease resistance has been first discovered in chokecherry (Prunus virginiana, 2n=4x=32), which is a native woody species of North America. To identify molecular markers linked to X-disease resistance, simple sequence repeat (SSR) markers were used to construct genetic linkage maps for chokecherry and to identify markers associated with X-disease resistance in chokecherry. In this research, three segregating populations of chokecherry were developed by crossing one X-disease resistant (CL) with three susceptible chokecherry lines (a, c, and d), of which the progenies were 101, 177, and 82, respectively. In order to construct a genetic map for chokecherry, 108 pairs of SSR primers were employed from other Prunus species. Additionally, a set of 246 SSRs were developed from chokecherry sequencing by Roche 454 sequencing technology. A total of 354 pairs of SSR primers were used to screen individuals of all three populations. Two software programs, TetraploidMap and JoinMap, were used to construct linkage map based on single-dose restriction fragments (SDRFs) and two parental linkage maps were generated for each population from both software programs. Bulked segregant analysis (BSA) was applied for identification of X-disease resistance markers. As a result, one SSR marker was found to be linked to the X-disease resistance. The set of 246 chokecherry SSRs was later used to test transferability among another 11 rosaceous species (sour cherry, sweet cherry, wild cherry, peach, apricot, plum, apple, crabapple, pear, june berry, and raspberry). As a result, chokecherry SSR primers can be transferable in Prunus species or other rosaceous species. An average of 63.2% and 58.7% of amplifiable chokecherry primers amplified DNA from cherry and other Prunus species, respectively, while 47.2% of amplifiable chokecherry primers can be transferable to other rosaceous species. The genetic information, including genetic map, disease linked marker, chokecherry sequence, and confirmed transferability of the identified chokecherry SSRs to other species, will benefit the genetic research in Prunus and other rosaceous species.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / There are many mechanisms for the creation of new genes. In this study, the newborn genes i.e. de novo genes are the genes that are created from scratch. These are created by two mechanisms, polymerization (de novo genes produced from non-coding regions) and overprinting (de novo genes produced from overlapping frames). Rancurel et al has found that de novo genes in overlapping coding regions tend to be more disordered than their ancestral counterparts. It was suggested that it is natural for the newborn genes to be disordered, as it must be very difficult for newborn genes to obtain order at such an early stage, so that the structure is only developed after the evolutionary development. The two hypotheses tested in this study state (1) that genes generated de novo will have a tendency to be disordered, and (2) this tendency is due to a natural inclination of these genes to be disordered at birth. The origin and evolution of some de novo coding regions have been studied in detail. We analyzed genes reported in literature that have been produced de novo; either by overprinting or by polymerization, and their tendency for disorder was evaluated using the VSL2 disorder predictor. The de novo coding regions produced by both ways indeed shows a tendency towards disorder, which supports hypothesis 1. For hypothesis 2 to be tested on a larger dataset the exonic and intronic materials of two human chromosomes were studied and the tendency for disorder was assessed for any new peptide sequence arising from the translation of non-coding sequences arising from introns and exons (overlapping frames). It was shown that the tendency of disorder for protein products of newborn genes arising from introns were not inclined towards being ordered or disordered, but they can become disordered by evolution. The new exonic material created from the existing exons tends to be more disordered when translated, and this tendency does not seem to be dependent upon the disorder content of the original exons. This difference could be a consequence of the fact that the overlapping frames of coding sequences have indirectly been subjected to evolutionary pressure along with the original exon, whereas intronic sequences do not seem to have this constraint, but the exact nature of this discrepancy needs further study to be explained. The tendency of disorder in the existing new exons seems to be higher than the artificial exons (generated in this study). We conclude that the intrinsic disorder in the protein products of de novo genes is selected by the evolution rather than an initial condition. Thus, the newborn genes were not born disordered. / indefinitely

Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.