Academic literature on the topic 'INTRONE'

AbstractRetinoic X receptor alpha (RXRA), a member of nuclear receptor superfamily, plays a key role in development, metabolism, glucose homeostasis, and intestinal cholesterol balance. The aim of this study was to examine an association of RXR alpha introne 5 A(39526)AA polymorphism and ABO blood groups with chronic heart failure (CHF) in the Czech population. A total of 238 patients with chronic heart failure and a control group of 246 subjects were included in the study. The RXR alpha gene polymorphism and ABO blood groups were detected by PCR and RFLP methods. Significant differences in distributions of RXRA A(39526)AA alleles and genotypes between CHF patients and controls were observed (Pg=0.03, Pa=0.02). The RXRA gene polymorphism differences of within blood group A between CHF patients and controls were highly significant in genotype distributions (Pg=0.002) and in allele frequency comparisons (Pa=0.0001). The prevalence of AA allele in CHF patients with A blood group was four-fold lower than in controls with the same blood group (OR=0.24; Pcorr=0.0001). A highly significant association of RXRA introne single-nucleotide insertion polymorphism and A blood group in chronic heart failure was observed. Our results suggest close linkage between RXRA A(39526)AA polymorphism and ABO blood groups.

Actin is one of the reference genes that is often used as an internal control in gene expression analysis. This study aimed to isolate actin cDNA from Pandanus sp originated from Riau. Fresh leaves Pandanus sp. Lake Kajuik, Langgam District, Pelalawan Regency, Riau Province. Isolation of RNA, synthesis of total cDNA, amplification of actin genes used McDowell's designed degenerate primer (PlAc46S-20/PlAc245N-20), electrophoresis, sequencing, and data analysis. Actin cDNA fragments obtained were 353 pb in size, registered at GenBank and encoded 117 amino acids. Actin cDNA fragment consists of two exons and one introne. Specific actin primers from Riau Pandanus sp. can be designed based on sequences obtained for the purpose of analyzing certain gene expressions.

AbstractIn the present study, a genomic DNA of MIH (GenBank: #EU869539) was cloned from the swimming crab, Portunus trituberculatus (Miers, 1876). The genome DNA, consisting of 2865 bp, is comprised of three exons interrupted by two introns. Multiple sequence alignments revealed that in the 5 upstream region of MIH, sequences with high similarity to arthropod initiator, TATA box, CREB (cyclic AMP response element binding) protein were the common structure. The signal peptide in the genomic DNA was encoded by exon1 and exon2, which was interrupted by 242 bp-intron (intron1), located between gln12 and arg13. The mature peptide was encoded by exon2 and exon3, which was interrupted by 313 bp-intron (intron2), at the position between 2nd and 3rd nucleotides of the codon encoding arg41. Pot-MIH was expressed only in the eyestalk ganglia, ovaries, testes, posterior spermatic duct, bristle in ejaculatory duct, cranial ganglia, and thoracic ganglia, as determined in various tissues by semi-quantitative RT-PCR. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using the Escherichia coli (Migula, 1895) expression system. Two constructs were designed to yield either a mature MIH fusion protein with and without histidine (His) tag at the carboxyl terminus. The rMIH protein was detected by SDS-PAGE and Western blot analysis, indicating that the antibody prepared by two rMIH proteins has high specificity.

Abstract Group II introns are self-splicing ribozymes and mobile genetic elements. Splicing is required for both expression of the interrupted host gene and intron retromobility. For the pRS01 plasmid-encoded Lactococcus lactis group II intron, Ll.LtrB, splicing enables expression of the intron's host relaxase protein. Relaxase, in turn, initiates horizontal transfer of the conjugative pRS01 plasmid and stimulates retrotransposition of the intron. Little is known about how splicing of bacterial group II introns is influenced by environmental conditions. Here, we show that low temperatures can inhibit Ll.LtrB intron splicing. Whereas autocatalysis is abolished in the cold, splicing is partially restored by the intron-encoded protein (IEP). Structure profiling reveals cold-induced disruptions of key tertiary interactions, suggesting that a kinetic trap prevents the intron RNA from assuming its native state. Interestingly, while reduced levels of transcription and splicing lead to a paucity of excised intron in the cold, levels of relaxase mRNA are maintained, partially due to diminished intron-mediated mRNA targeting, allowing intron spread by conjugal transfer. Taken together, this study demonstrates not only the intrinsic cold sensitivity of group II intron splicing and the role of the IEP for cold-stress adaptation, but also maintenance of horizontal plasmid and intron transfer under cold-shock.

ABSTRACT A group I intron has been found to interrupt the anticodon loop of the tRNALeu(UAA) gene in a bacterium belonging to the γ-subdivision of Proteobacteria and isolated from a deep subsurface environment. The subsurface isolate SMCC D0715 was identified as belonging to the genus Pseudomonas. The group I intron from this isolate is the first to be reported for γ-proteobacteria, and the first instance of a tRNALeu(UAA) group I intron to be found in a group of bacteria other than cyanobacteria. The 231-nucleotide (nt) intron's sequence has group I conserved elements and folds into a bona fide group I secondary structure with canonical base-paired segments P1 to P9 and a paired region, P10. The D0715 intron possesses the 11-nt motif CCUACG … UAUGG in its P8 region, a feature not common in bacterial introns. To date, phylogenetic analysis has shown that bacterial introns form two distinct families, and their complex distribution suggests that both lateral transfer and common ancestry have taken part in the evolutionary history of these elements.

Abstract The occurrence of group II introns in plant mitochondrial genomes is strikingly different between the six major land plant clades, contrasting their highly conserved counterparts in chloroplast DNA. Their present distribution likely reflects numerous ancient intron gains and losses during early plant evolution before the emergence of seed plants. As a novelty for plant organelles, we here report on five cases of twintrons, introns-within-introns, in the mitogenomes of lycophytes and hornworts. An internal group II intron interrupts an intron-borne maturase of an atp9 intron in Lycopodiaceae, whose splicing precedes splicing of the external intron. An invasive, hypermobile group II intron in cox1, has conquered nine further locations including a previously overlooked sdh3 intron and, most surprisingly, also itself. In those cases, splicing of the external introns does not depend on splicing of the internal introns. Similar cases are identified in the mtDNAs of hornworts. Although disrupting a group I intron-encoded protein in one case, we could not detect splicing of the internal group II intron in this ‘mixed’ group I/II twintron. We suggest the name ‘zombie’ twintrons (half-dead, half-alive) for such cases where splicing of external introns does not depend any more on prior splicing of fossilized internal introns.

AbstractIntron sizes show an asymmetrical distribution in a number of organisms, with a large number of “short” introns clustered around a minimal intron length and a much broader distribution of longer introns. In Drosophila melanogaster, the short intron class is centered around 61 bp. The narrow length distribution suggests that natural selection may play a role in maintaining intron size. A comparison of 15 orthologous introns among species of the D. melanogaster subgroup indicates that, in general, short introns are not under greater DNA sequence or length constraints than long introns. There is a bias toward deletions in all introns (deletion/insertion ratio is 1.66), and the vast majority of indels are of short length (<10 bp). Indels occurring on the internal branches of the phylogenetic tree are significantly longer than those occurring on the terminal branches. These results are consistent with a compensatory model of intron length evolution in which slightly deleterious short deletions are frequently fixed within species by genetic drift, and relatively rare larger insertions that restore intron length are fixed by positive selection. A comparison of paralogous introns shared among duplicated genes suggests that length constraints differ between introns within the same gene. The janusA, janusB, and ocnus genes share two short introns derived from a common ancestor. The first of these introns shows significantly fewer indels than the second intron, although the two introns show a comparable number of substitutions. This indicates that intron-specific selective constraints have been maintained following gene duplication, which preceded the divergence of the D. melanogaster species subgroup.

INTRODUCTION For many years, introns have been considered only an additional cost to replication and transcription; earliest studies described them as ‘junk DNA’ (Wong, 2000). Subsequent observations revealed that some intronic sequences show regulative control on their own genes. Nevertheless, the enormous space occupied by introns in the cell genome cannot be justified only by these data. Some remarks about intron presence can be done considering the literature. Roy and Gilbert (2006) showed that introns density and organism complexity share a relation of proportionality. Introns can also increase transcription efficiency, as proved by some studies on transgenic mice (Le Hir et al., 2003). mRNA stability is improved by intron presence that protects the transcript from degradation. Moreover, despite introns being not as conserved as associated exons, many studies demonstrated unexpected conservation patterns between orthologue introns (Mattick, 1994). These and other data suggest that introns play a pivotal role in the regulatory network of the cell. AIM OF THE THESIS In the present study, intronic sequences of estrogen receptor alpha (ERalpha) gene in mammals and teleosts have been analyzed. A preliminary bioinformatic investigation has been utilized to choose a conserved intronic sequence in order test the potential binding to protein factors. The examination of these sequences concerns the structure, the location of important sites for transcription and splicing, the research of conserved regions, and the recognition of repetitive elements and potential binding sites for transcription factors. After that, by means of gelshift experiment, the binding of DNA and RNA probes to elements of nuclear protein extracts has been examined. MATERIALS AND METHODS Bioinformatic analysis was led on intronic and exonic sequences of ERalpha gene available at Ensembl database (Homo sapiens, Macaca mulatta, Canis familiaris, Mus musculus, Bos taurus, Monodelphis domestica, Oryctolagus cuniculus, Echinops telfairi and Ochotona princeps). Also one intron of some teleosts was sequenced and analyzed (Atherina boyeri, Barbus plebejus, Chondrostoma genei, Rutilus aula, Rutilus rutilus, Phoxinus phoxinus, Rhodeus amarus, Gasterosteus aculeatus, Psetta maxima, Scorpaena porcus, Polyprion americanus and Sparus aurata). The same intron for Danio rerio, Takifugu rubripes and Oryzia latipes was obtained from Ensembl database. Alignment: peculiar features of intronic sequences are their considerable lengths that are hardly comparable, low conservation rates and presence of insertions, deletions and inversions. MLAGAN alignment software (Brudno et al., 2003) has been a useful tool to align sequences that show these cha-racteristics. The software also gives back fragments of sequences, defined as Conserved Non-Coding Sequences (CNS), that show more than 70% of identity. Dotplot: The software dotplot was used to find exact conserved words between two sequences. Transcription factors: the presence of putative binding sites for tran-scription factors was identified by means of TFSEARCH version 1.3. The potential binding sites were analyzed in and out of CNS. Repetitions: the search of tandem repetitions inside introns was done with mreps software (Kolpakov et al., 2003). After that , the repetitions inside CNS were compared to the non-conserved repetitions. MicroRNAs: the presence of putative precursors of microRNAs was ex-amined with the softwares RNA22, proMirII (Nam et al., 2006) and mipred (Jiang et al., 2007). Gelshift: binding experiments were performed with DNA and RNA probes of intron I117 from D. rerio and nuclear protein extracts from tissues expressing ERalpha in female D. rerio. RESULTS AND DISCUSSION Splicing: the presence of consensus sequences necessary for the branch-ing of introns and the 5’ and 3’ splice sites (Zhuang et al., 1989) was investigated. The consensus sequence for the formation of the lariat isn’t present in some of the introns considered, while in others it exists in many copies. Conversely, 5’ and 3’ termini are highly conserved and always present. Alignments: conserved regions were studied calculating the average length and percentage of average identity of the conserved regions, the number of conserved regions and the number of nucleotides in the conserved regions, the ratio between the number of nucleotides inside the conserved regions and the length of the same intron sequence. These data show a great number of regions sharing more than 70% identity. Some pairwise alignments reveal that more than half of the intron’s length may show conservation (for example the same intron in H. sapiens and C. familiaris). Transcription factors: this analysis disclosed a great number of putative binding sites for transcription factors, mostly for CdxA (caudal type homeo-box transcription factor 1) and SRY (sex determining factor). The massive presence of binding sites for SRY is probably related to ERα function. Consi-dering the percentages of transcription factor binding sites nucleotides inside and outside CNS, a common pattern was not found. Repetitions: nearly all the repeated words found in the intronic se-quences are microsatellites. In teleosts, repeated words are present only in some introns and always outside conserved regions. In mammalian introns, repetitions are more frequently placed in non-conserved region. MicroRNAs: in mammalian introns, RNA22, ProMirII and mipred softwares found stem-loop structures that are potential precursor of micro-RNA. These structure have been found in introns of C. familiaris, H. sapiens, M. domestica and M. musculus. In teleosts, no precursors were identified. Gelshift: considering the result of bioinformatic studies, a conserved re-gion of intron I117 from D. rerio was chosen for the binding experiments. This intron is placed between the DNA binding domain region and the D region of the ERalpha gene. EMSA experiments reveal binding of both DNA and RNA probes. With DNA probes, the best result was obtained with liver nuclear protein extract. For RNA probes, a binding reaction was revealed probably involving many factors. These results suggest a putative regulative role of I117 in D. rerio.
INTRODUZIONE Per molti anni gli introni sono stati considerati un semplice costo aggiuntivo alla replicazione e alla trascrizione; nei primi studi compiuti sugli introni, questi ultimi sono stati descritti essenzialmente come “Junk DNA” (Wong, 2000). Successive osservazioni hanno rivelato che alcune delle sequenze introniche mostrano un controllo di tipo regolativo nei confronti dei geni in cui sono collocati. Nonostante ciò, l’enorme spazio occupato dagli introni nella cellula non può essere giustificato solo da questi dati. In base alla letteratura, sono state tratte alcune considerazioni riguardo alla presenza degli introni. Roy e Gilbert (2006) mostrano che densità intronica e complessità condividono una relazione di proporzionalità diretta. Gli introni possono inoltre aumentare l’efficienza di trascrizione, come dimostrato da alcuni studi compiuti su topi transgenici (Le Hir et al., 2003). La stabilità degli mRNA è migliorata dalla presenza degli introni che agiscono proteggendo il trascritto dalla degradazione. Inoltre, nonostante gli introni non siano conservati quanto gli esoni a loro associati, molti studi dimostrano un inatteso pattern di conservazione tra introni ortologhi (Mattick, 1994). Questi e altri dati suggeriscono che gli introni abbiano un ruolo cruciale nel network regolatorio della cellula. SCOPO DELLA TESI Nel presente studio, sono state analizzate le sequenze introniche del gene del recettore degli estrogeni forma alfa (ERalfa) in mammiferi e teleostei. Un’indagine preliminare di tipo bioinformatico è stata utile per scegliere una sequenza intronica conservata al fine di testare il possibile binding a fattori proteici. L’esame di queste sequenze ha riguardato la struttura, la posizione di siti importanti per la trascrizione e lo splicing, la ricerca di regioni conservate, l’individuazione di elementi ripetitivi e di siti di binding potenziali per fattori di trascrizione. In seguito a questo, per mezzo di esperimenti di gelshift, è stato testato il legame di sonde a RNA e DNA a elementi di estratto nucleare proteico. MATERIALI E METODI Indagini bioinformatiche sono state condotte sulle sequenze introniche ed esoniche del gene ERalfa disponibili nella banca dati Ensembl (Homo sapiens, Macaca mulatta, Canis familiaris, Mus musculus, Bos taurus, Monodelphis domestica, Oryctolagus cuniculus, Echinops telfairi e Ochotona princeps). Anche un introne del medesimo gene è stato sequenziato ed analizzato (Atherina boyeri, Barbus plebejus, Chondrostoma genei, Rutilus aula, Rutilus rutilus, Phoxinus phoxinus, Rhodeus amarus, Gasterosteus aculeatus, Psetta maxima, Scorpaena porcus, Polyprion americanus and Sparus aurata). Lo stesso introne è stato ricavato dal database Ensembl per le specie Danio rerio, Takifugu rubripes e Oryzia latipes. Allineamento: caratteristiche tipiche delle sequenze introniche sono una lunghezza considerevole e difficilmente comparabile con altre sequenze, basso tasso di conservazione e presenza di inserzioni, delezioni e inversioni. Il software MLAGAN (Brudno et al., 2003) è stato un valido strumento per allineare sequenze che condividono queste caratteristiche. Il programma restituisce anche porzioni di sequenza, definite Conserved Non-Coding Sequences o CSN, che mostrano più del 70% di identità. Dotplot: il software Dotplot è stato sfruttato per ritrovare parole esatte conservate tra due sequenze. Fattori di trascrizione: la presenza di potenziali siti di binding per fattori di trascrizione all’interno e all’esterno delle zone di conservazione è stata individuata tramite in programma TFSEARCH versione 1.3. Ripetizioni: l’analisi sulle ripetizioni tandem all’interno degli introni è stata effettuata col programma mreps (Kolpakov et al., 2003). Dopo ciò, è stato eseguito un confronto tra le ripetizioni conservate e non. microRNA: è stata infine ricercata la presenza di precursori di microRNA con i software RNA22, proMirII (Nam et al., 2006) e mipred (Jiang et al., 2007). Gelshift: esperimenti di binding sono stati effettuati con sonde a DNA e RNA dell’introne I117 di D. rerio ed estratti nucleari proteici da tessuti e-sprimenti ERalfa nelle femmine di D. rerio. RISULTATI E DISCUSSIONE Splicing: è stata ricercata la presenza di sequenze consenso necessarie per il branching degli introni e di splice sites conservati. La sequenza consenso necessaria per la formazione del laccio non è stata riscontrata in tutti gli introni, in altri invece era presente in copie multiple. Gli splice sites al terminale 5’ e 3’ sono risultati altamente conservati. Allineamento: le regioni conservate sono state studiate considerando la lunghezza media e la percentuale di identità media delle regioni conservate, il numero di regioni conservate, il numero di nucleotidi conservati nella sequenza, il rapporto tra il numero di nucleotidi all’interno delle CNS e la lunghezza totale della sequenza. Questi dati mostrano una grande quantità di regioni che condividono più del 70% di identità. Alcuni allineamenti pairwise rivelano che più di metà della lunghezza dell’introne può essere conservata (questo avviene ad esempio tra alcuni introni di C. familiaris e H. sapiens). Fattori di trascrizione: questa analisi ha rivelato un gran numero di potenziali siti di binding per fattori di trascrizione. Si può inoltre precisare che i fattori di trascrizione più rappresentati sono CdxA (caudal type homeobox transcription factor 1) e SRY (sex determining factor). La considerevole presenza di copie del sito di binding per SRY è probabilmente correlabile alla funzione di ERalfa. Tuttavia considerando la percentuale di siti di binding all’interno e all’esterno delle CNS, non si riscontra un pattern comune per tutte le sequenze. Ripetizioni: quasi tutte le sequenze ripetute trovate nelle sequenze introniche sono microsatelliti. Nei teleostei le ripetizioni sono presenti solo in alcuni introni e comunque sempre al di fuori delle zone conservate. Anche nei mammiferi, le ripetizioni sono state riscontrate più frequentemente nelle regioni non conservate. MicroRNA: negli introni dei mammiferi sono state trovate strutture a stem-loop potenziali precursori di microRNA. Sono stati riscontrati negli introni C. familiaris, H. sapiens, M. domestica e M. musculus. Nei teleostei non sono stati ritrovati precursori. Gelshift: considerando i risultati degli studi bioinformatici, per gli esperimenti di binding è stato scelto l’introne I117 di D. rerio. Questo introne è collocato tra il DNA binding domain e la regione cerniera del gene dell’ERalfa. Gli esperimenti di gel retardation hanno rivelato la presenza di binding sia per la sonda a RNA che per la sonda a DNA. Per le sonde a DNA sono stati ottenuti i risultati migliori con l’estratto nucleare proteico di fegato. Per le sonde a RNA è stato individuato il binding ma, probabilmente a causa del legame con più fattori, i risultati sono al momento di complessa interpretazione. Ad ogni modo, questi risultati suggeriscono un potenziale ruolo regolativo per l’introne I117 di D. rerio.

Nous avons montré la présence d'un intron de type intron de gènes nucléaires de protéines, dans les gènes codant pour le snrna u3 de s. Cerevisiae. Le pre-snrna u3 et épisse dans un spliceosome. S. Cerevisiae contient deux gènes a et b codant pour le snrna u3. Dans chacun l'intron est situé à la même position, mais à une taille et une séquence différente. Le gène b est présent chez les saccharomyces, mais pas chez les kluyveromyces et les pichia. La séquence des introns de type a est très conservée chez les saccharomyces, kluyveromyces et pichia. La détermination de la structure secondaire du snrna u3 de levure, en solution montre qu'il est constitué: d'un domaine 3 fortement structuré qui fixe les protéines de la snrnp u3 et d'un domaine 5, de structure plus lâche, qui interagit avec le pre-arn ribosomique. Ce domaine a une structure secondaire différente chez les eucaryotes inferieurs et chez les eucaryotes supérieurs. Nous avons aussi déterminé la structure secondaire du pre-snrna u3. La majorité des éléments de structure de l'arn mature se retrouvent dans le pre-arn. L'intron présente une longue structure tige-boucle centrale. Les sites fonctionnels sont regroupés dans l'espace. Ce qui favorise, sans doute, l'assemblage du spliceosome. L'épissage in vitro de différents pre-arn u3 mutes et chimériques montre que la conformation et la séquence du site de branchement sont des paramètres essentiels pour l'efficacité d'épissage et que l'addition d'un segment très structuré en 5 du premier exon augmente le taux d'épissage in vitro, lorsque la boite de branchement est gactaac

The chloroplast genes of Euglena gracilis contain more than 100 introns. A comparison of intron content and position among plastid and prokaryote genes has led to the hypothesis that introns have been inserted into chloroplast genes during evolution. Several Euglena loci contain unusual introns. These introns have been characterized by direct primer extension cDNA sequencing, cDNA cloning and sequencing, and northern hybridization. The psbF locus has a 1042 nt intron that appears to be one group II intron inserted into domain V of another group II intron. It was determined that a 618 nt internal intron is first excised from the 1042 nt intron, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. The term "twintron" was used to define this new genetic element. Splicing of the internal and external introns occurs via lariat intermediates. The splicing of the 409 nt intron of the rps3 gene was also examined. This intron is a "mixed" twintron, composed of a 311 nt group II intron internal to a 98 nt group III intron. The splicing of four additional introns with mean lengths twice typical group III introns, three within the rpoC1 gene and one within the rpl16 gene, was analyzed. The 1604 nt intron in the psbC gene, which encodes orf458, was also examined. These introns are group III twintrons. Orf458 is encoded within the internal group III intron of the psbC twintron. Splicing of internal introns in three of the five group III twintrons involves multiple 5'- and/or 3'-splice sites. Excised group III introns accumulate as lariat RNAs. Twintrons represent evidence for intron insertion during gene evolution. One possible mechanism for twintron formation is by intron transposition. The disruption of functional domains by internal introns may necessitate a sequential in vivo splicing pathway, requiring excision of internal introns prior to excision of external introns. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.

A partir da descoberta dos íntrons, muitas questões sobre sua origem vêm sendo discutidas como: porque eles existem em eucariotos e não são encontrados em procariotos, quando e como eles se originaram. Basicamente duas hipóteses existem para explicar a origem dos íntrons: \"introns-early\" e \"introns-late\". A primeira hipótese sugere que íntrons e éxons já existiam nos primeiros genes e os íntrons foram perdidos posteriormente na linhagem de bactérias. A hipótese oponente, introns-late, assume que os íntrons foram adicionados posteriormente durante a evolução, somente em eucariotos. Introns de genes diferentes podem sofrer recombinação durante a divisão celular e assim formar novos genes. Este processo permite e aumenta a freqüência de troca completa de éxons e, conseqüentemente, aumenta a probabilidade de novos genes funcionais serem formados. Este fenômeno é chamado de \"exon-shuffling\" e é um mecanismo importante em relação a origem de muitas proteínas novas em eucariotos. Entretanto, o papel do exon-shuffling na criação das proteínas no ancestral comum dos procariotos e eucariotos é o ponto de discordância entre as hipóteses introns-early e introns-late porque este mecanismo depende da presença dos introns no progenoto. Excesso de éxons simétricos é considerado uma evidência de exon-shuffling, já que a troca de éxons flanqueados por íntrons da mesma fase não muda o quadro de leitura do gene receptor. Nesta tese, apresentamos dois estudos relacionados ao papel do fenômeno de exon-shuffling na evolução das proteínas. No primeiro estudo, observamos que existe uma correlação significativa entre unidades simétricas de shuffling e a idade de domínios protéicos. Domínios antigos, presentes em procariotos e eucariotos, são mais freqüentemente flanqueados por íntrons de fase zero e são preferencialmente localizados nas partes centrais das proteínas. Domínios modernos são mais freqüentemente flanqueados por íntrons de fase um e estão presentes predominantemente nas extremidades das proteínas. Propomos um modelo no qual o shuffling de domínios antigos flanqueados por íntrons de fase zero deve ter sido importante durante a criação das partes centrais das proteínas no ancestral comum de eucariotos e procariotos. Shuffling de domínios modernos, predominantemente flanqueados por íntrons de fase um, deve ter sido importante para a origem das extremidades das proteínas durante a evolução de eucariotos. O segundo estudo trata do possível papel do exon-shuffling na evolução de peptídeos sinal em proteínas humanas. Recentemente, foi mostrado que existe uma predominância de íntrons de fase um próximo ao sítio de clivagem de peptídeos sinal em genes humanos [Tordai, H., Patthy, L. (2004) FEBS lett. 575:109-111]. Os autores sugeriram que tal distribuição é causada pela inserção de íntrons em sítios de inserção preferencial AGG. Apresentamos evidências de que o sinal observado não é tão forte como inicialmente mostrado e que não existe excesso desproporcional de AGG que daria suporte a inserção em sítios preferenciais. Como estas proteínas evoluíram através de exon-shuffling, levantamos a possibilidade de que este fenômeno possa também ter sido amplamente responsável pelo excesso de íntrons de fase um. Acreditamos que os dados presentes nestes dois estudos representam uma contribuição importante para o campo de estudo de evolução de íntrons e do fenômeno de exon-shuffling porque estes apresentam dados importantes e originais acerca do papel do \"exon-shuffling\" antigo e moderno durante a evolução das proteínas.
Since the discovery of introns, many questions about their origin have been raised such as: why they exist in eukaryotic organisms and not in prokaryotes, when and how did they originate. Mainly, there are two hypotheses explaining the origin of introns: ?introns-early? and ?introns-late?. The first hypothesis suggests that introns and exons already existed in the first genes and were lost later in the bacteria lineage. The opposing hypothesis, introns-late, assumes that introns were inserted late in evolution, in eukaryotic organisms only. Introns from different genes may suffer recombination during cell division and this way form new genes. This process allows and increases the frequency of exchanging complete exons and, consequently, increases the probability of forming new functional genes. This phenomenon is called ?exon-shuffling? and is an important mechanism accounting for the origin of many new proteins in eukaryotes. However, the role of exon-shuffling in the creation of proteins in the ancestor of prokaryotes and eukaryotes is the point of disagreement between the hypotheses \"introns-early\" and \"introns-late\" because this mechanism depends of the presence of introns in the progenote. Excess of symmetric exons is thought to represent evidence for exon-shuffling since the exchange of exons flanked by introns of the same phase does not disrupt the reading frame of the host gene. In this thesis, we present two studies concerning the role of the exon-shuffling phenomenon in protein evolution. In the first study, we found that there is a significant correlation between symmetric units of shuffling and the age of protein domains. Ancient domains, present in both prokaryotes and eukaryotes, are more frequently bounded by phase zero introns and their distribution is biased towards the central part of proteins. Modern domains are more frequently bounded by phase one introns and are present predominantly at the ends of proteins. We propose a model in which shuffling of ancient domains mainly flanked by phase zero introns would have been important during the creation of the central part of proteins in the ancestor of eukaryotes and prokaryotes. Shuffling of modern domains, predominantly flanked by phase one introns, would have accounted for the origin of the extremities of proteins during eukaryotic evolution. The second study accounts the possible role of exon-shuffling in the acquisition of signal peptides in human proteins. It was recently shown that there is a predominance of phase one introns near the cleavage site of signal peptides of human genes [Tordai, H., Patthy, L. (2004) FEBS lett. 575:109-111]. The authors suggested that it was due to intron insertion at AGG proto-splice sites. We present evidence that the signal observed is not as strong as initially shown and that there is no disproportional excess of AGG that would support insertion at proto-splice sites. As these proteins evolved by exon-shuffling, we raise the possibility that this phenomenon might also be largely responsible for such excess of phase one introns. We believe the data present in these two studies represent an important contribution to the field of introns and exon-shufling evolution due to their important and original data concerning the role of ancient and modern exon-shuffling during the evolution of the proteins.

The genus Fusarium constitutes fungi with diverse biological behaviours. This study focused on four plant pathogenic species. These were F. verticillioides which infects maize, F. oxysporum which infects tomato, F. graminearum, a pathogen of wheat and F. circinatum, which is pathogenic to pine. The genomes of F. verticillioides, F. oxysporum, F. graminearum and F. circinatum have been sequenced. These genomes were annotated using different gene prediction software. To study the architecture and distribution of Spliceosomal introns in these a set of Housekeeping (HK) genes common to all eukaryotes were used. These analyses revealed discrepancies in the annotations of these genomes, which most commonly included intron position incongruences, misidentified introns and sequencing errors. Spliceosomal introns have four cis-elements which include the 5ꞌ and 3ꞌ splice sites, the branch site and the polypyrimidine tract. Analysis of the first three elements of Spliceosomal introns in the four Fusarium species and comparisons to those in other fungi showed significant differences in the consensus sequences of these elements. Two additional branch site motifs were also found for Fusarium, while the polypyrimidine tract of these species was found to be very diverse. The results also indicated that the first introns of the HK genes of the Fusarium species significantly longer, which is consistent with what have been found for genes of other eukaryotic. Also consistent with what is known for other eukaryotes, the analysed Fusarium genes had much lower intron densities than those observed in higher eukaryotes. An average of 2.53 introns per gene was observed in Fusarium and most of these introns were located closer to the 5ꞌ end of the HK genes. This average is low compared A. thaliana and Homo sapiens which have averages of 4.3 and 8.82 introns per gene, respectively. With the aid of EST and genome data, F. circinatum was shown to harbour putative alternative introns at a frequency of approximately 0.3%. Homologues of a number of these genes from F. verticillioides, F. oxysporum and F. graminearum also harboured alternative splicing signals. Certain alternatively spliced transcripts harbored premature stop codons. These transcripts are targeted by non-sense mediated mRNA decay (NMD) system. The high rate at which these transcripts included premature stop codons suggested that such a quality control system is indeed needed for these fungi. Overall, however, it remains to be investigated whether these alternative transcripts are functional as is the case with some of them in humans, plants and insects. As more fungal genomes are being sequenced the need for accurate gene prediction methods is soaring. The incorporation of the findings of the architecture and distribution of Spliceosomal introns in Fusarium into gene prediction methods will thus increase the accuracy of such methods for Fusarium species, especially those related to F. circinatum. The identification of genes that are potentially regulated through alternative intron splicing also provide valuable targets for future studies on important biological processes such as pathogenicity and virulence. Note: The disc provided contains a spreadsheet with all the data included in this dissertation.
Dissertation (MSc)--University of Pretoria, 2012.
Microbiology and Plant Pathology
MSc
Unrestricted

Saccharomyces cerevisiae, the best-known representative of the Saccharomycotina subphylum, is an intron-poor organism with introns in only 5 % of its protein-coding genes. The most popular model of intron evolution suggests that intron-poor eukaryotes, such as S. cerevisiae, have undergone extensive intron loss throughout their evolutionary history. Against this background of intron loss, the retention of specific introns in the S. cerevisiae genome might be attributable to an evolutionary advantage that they provide. Introns have been shown to exhibit ‘function’ in various ways: through recognition of their sequence by RNA binding proteins, the adoption of secondary structures after transcription, the mechanism of splicing itself, and noncoding RNA genes embedded within them. In order to understand how RNA structures contribute to intron function, we first performed a computational screen using 306 alignments of S. cerevisiae intron orthologs. We identified conserved RNA structures in 19 introns that act either in trans as independent intron-encoded ncRNA genes or in cis within the pre-mRNA. Our results showed that introns with conserved secondary structures are conserved in yeast and experimental validation revealed they are frequently maintained in the cells after splicing. Our results suggest that the intron in GLC7 contains a novel ncRNA that regulates expression of its host transcript under stress conditions. Secondly, we focused on the HAC1 intron, which is known to be spliced upon the unfolded protein response by an endoribonuclease IRE1. We showed that the conservation of known intron-defining RNA hairpins in HAC1 extends to Fungi and Metazoa. Concurrently, we identified with high confidence those species that have lost the mechanism of this unconventional splicing. Thirdly, we investigated rates and mechanisms of intron loss within the whole Saccharomycetaceae family in order to develop our findings on the conservation of introns with RNA structures within the context of yeast evolution on both the species and clade level. Computational intron prediction supplemented by RNAseq data from four yeast species demonstrated that both intron loss and conservation of intronic ncRNAs were prevalent in yeast species, and that these patterns have been shaped by whole genome duplication. Lastly, we hypothesise that intron loss in recent yeast evolutionary history has been promoted by double strand break repair machinery.

The endothelium plays a crucial role in the regulation of the fibrinolytic process, since it synthesizes and secretes tissue-type plasminogen activator (t-PA) as well as the fast-acting plasminogen activator inhibitor (PAI-1). Molecular cloning of full-length PAI-1 cDNA, employing a human endothelial cDNA expression library, and a subsequent determination of the complete nucleotide sequence, allowed a prediction of the amino-acid sequence of the PAI-1 glycoprotein. It was observed that the amino-acid sequence is significantly homologous to those of members of the serine protease inhibitor ("Serpin") family, e.g. αl-antitrypsin and antithrombin III. Serpins are regulators of various processes, such as coagulation, inflammatory reactions, complement activation and share a common functional principle and a similar structure, indicative for a common primordial gene. The intron-exon arrangement of Serpin genes may provide a record for the structure of a primordial gene. A comparison of the location of introns among members of the Serpin family reveals that some introns are indeed present at identical or almost identical positions, however in many other cases there is no correspondence between the intron positions among different Serpin genes.Obviously, more data on the chromosomal gene structure of members of this family are required to formulate a scheme for the evolutionary creation of the Serpins. To that end, we have established the number and the precise location of the introns in the PAI-1 gene and have compared these data with those reported on other Serpin genes. For that purpose a human genomic cosmid DNA library of about 340.000 independent colonies was screened with radiolabelled full-length PAI-1 cDNA as probe. Two clones were found which contain the entire PAI-1 gene. Restriction site mapping, electron microscopic inspection of heteroduplexes and nucleotide sequence analysis demonstrate that the PAI-1 gene comprises about 12.2kilo basepairs and consists of nine exons and eight introns. Intron-exon boundaries are all in accord with the "GT-AG" rule, including a cryptic acceptor splice site found in intron 7. Furthermore, it is observed that intron 3 of the PAI-1 gene occupies an identical position as intron E of chicken ovalbumin and intron E of the ovalbumin-related gene Y. The location of the other seven introns is unrelated to the known location of introns in the genes encoding the Serpins, rat angiotensin, chicken ovalbumin (and gene Y), human antithrombin III and human al-antitrypsin. The 3' untranslated region of the PAI-1 gene is devoid of introns, indicating that the two mRNA species detected in cultured endothelial cells which share an identical 5' untranslated segment and codogenic region, but differ in the length of the 3' untranslated region, arise by alternative polyadenylation. An extrapolation of the position of the introns to the amino-acid sequence of PAI-1, and adaption of the view that the subdomain structure of the Serpins is analogous, shows that the introns of PAI-1 are non-randomly distributed. Except for intron 7, the position of the other seven introns corresponds with randon-coil regions of the protein or with the borders of β-sheets and a-helices. Extrapolation of the position of introns in the genes of other Serpins to their respective amino-acid sequences and subdomain structures also reveals a preference for random-coil regions and borders of subdomains. These observations are reminiscent of an evolutionary model, called "intron sliding", that accounts for variations in surface loops of the same protein in different species by aberrant splicing (Craik et al., Science 220 (1983) 1125). The preferential presence of introns in gene segments, encoding these variable regions, and absence in regions determining the general folding of these proteins would explain conservation of the structure during the evolution of those genes.

Surface activation of the plasma systems involved with coagulation, fibrinolysis, renin formation and kinin generation involves factor XII (Hageman factor). This protein is a 76,000 dalton glycoprotein which circulates in plasma as an inactive form of a serine protease. A human liver cDNA coding for factor XII was used to screen a human genomic phage library. Two overlapping clones were isolated, XHXII27 and XHXII76, and contain the entire gene for human factor XII. The gene is 13.5 Kbp in length and consists of 14 exons and 13 introns. The transcriptional start site of the mRNA was determined using S1 mapping and primer extension analysis. The results indicate that the 5′ untranslated end of the mRNA has a leader sequence of 47 bp and is not interrupted by an intron in the gene. DNA sequence analysis of the region upstream of the transcriptional start site does not contain TATA or CAAT sequences, which are often found in other genes transcribed by RNA polymerase II. The positions of the introns in the coding sequence separate the protein into domains which are homologous to similar regions found in fibronectin and tissue-type plasminogen activator. Furthermore, wherever protein homologies are found, the positions of the introns in the triplet codon occur in the same reading frame as in the tissue-type plasminogen activator, urokinase plasminogen activator and factor XII genes. The intron/exon organization of the factor XII gene is different to the organization of other coagulation genes such as prothrombin, factor IX and factor X. Therefore, factor XII appears to have evolved as a member of the plasminogen activator family of genes rather than as a member of the clotting factor gene family.

Factor VII is a member of a family of vitamin K-dependent, gamma-carboxylated plasma protein which includes factor IX, factor X, protein C, protein S and prothrombin. Activated factor VII (factor Vila) is a plasma serine protease which participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylated at multiple sites but contains only one AAUAAA poly-A signal sequence. The mRNA can undergo alternative splicing forming one transcript containing eight segments as exons and another with an additional exon which encodes a larger pre-pro leader sequence. The portion of the pre-pro leader coded for by the additional exon has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family, including a pre-pro leader (the essential exon la and alternative exon lb), a gamma-carboxylated domain (exons 2 and 3) a growth factor domain (exons 4 and 5) an activation region (exon 6) and a serine protease (exon 8). The corresponding introns in these genes are dissimilar with respect to size and sequence, with the exception of the third intron in factor VII and protein C. Four introns and a portion of exon 8 in factor VII contain regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats. This type of structure is responsible for polymorphisms due to allelic variation in repeat copy number in other areas of the human genome. Tandem repeats can evolve as a result of random crossover in DNA whose sequence is not maintained by selection. This suggests that much of the sequence information present in the introns and untranslated portion of the message is dispensable.

The task of identifying intron and exon regions in genes is a verycomplex task, and it is necessary to identify certain nucleotidepatterns in the gene sequence. This task can be done manually orthrough software that most often uses genetic alignment techniques, which is not a very effective way for this purpose. In this oppor-tunity for collaboration between biology and computer science using machine learning techniques, the objective was to predictthe intron and exon regions in filamentous fungi genes as well totranslate the identified regions intro proteic codons. In this paper,the problem was modeled as a supervised learning problem, basedon training a set of genes obtained from GenBank that alreadyhave the intron and exon regions identified. The machine learningmodel used in this work was the Condicional Random Fields (CRF).Through the values resulting from the metrics applied to the model,it can be seen that it is possible to achieve a good precision in thetask of identifying the intron and exon regions as well the proteiccodons. Thus, although there is a need for a greater diversity ofdatabase characteristics to support the effectiveness of identifyingthe splicing sites, this paper gives evidence that it is possible topredict these splicing sites with a good accuracy.

An exploratory survey of business professionals from a broad range of organizations indicates that potential intranet benefits are both difficult to achieve and highly interrelated. Although many organizations implement intranets in the hope of generating cost savings or improving knowledge sharing across organizational units, this study indicates that the only likely benefit of implementing an intranet is faster access to information. The study also suggests that the most difficult benefit to achieve is generating a more cooperative work environment, and that developing an intranet that improves the quality and relevance of information is foundational to achieving most other intranet benefits.

Taking a critical theory approach and the pluralist view of technology, this paper examines the problems in organizational communication that arose due to the implementation of a limited intranet electronic mail system as the main channel of communication between a rural state-owned organization and its city-based Head Office, installed at the sole discretion of the latter. The intranet was provided only to the administration division and managers of some units due to financial constraints. This required others to receive information carried via the intranet through a gatekeeper who due to information and work overload, failed to disseminate the information effectively and efficiently. Using a combination of qualitative data collection methods, this study found that the intranet had marginalized those without access to it and reinforced the privileged position of those already with higher status within the organization, contrary to the utopian predictions of new technologies as leading to social equality.

Mitochondria are the site of respiration and numerous other metabolic processes required for plant growth and development. Increased demands for metabolic energy are observed during different stages in the plants life cycle, but are particularly ample during germination and reproductive organ development. These activities are dependent upon the tight regulation of the expression and accumulation of various organellar proteins. Plant mitochondria contain their own genomes (mtDNA), which encode for a small number of genes required in organellar genome expression and respiration. Yet, the vast majority of the organellar proteins are encoded by nuclear genes, thus necessitating complex mechanisms to coordinate the expression and accumulation of proteins encoded by the two remote genomes. Many organellar genes are interrupted by intervening sequences (introns), which are removed from the primary presequences via splicing. According to conserved features of their sequences these introns are all classified as “group-II”. Their splicing is necessary for organellar activity and is dependent upon nuclear-encoded RNA-binding cofactors. However, to-date, only a tiny fraction of the proteins expected to be involved in these activities have been identified. Accordingly, this project aimed to identify nuclear-encoded proteins required for mitochondrial RNA splicing in plants, and to analyze their specific roles in the splicing of group-II intron RNAs. In non-plant systems, group-II intron splicing is mediated by proteins encoded within the introns themselves, known as maturases, which act specifically in the splicing of the introns in which they are encoded. Only one mitochondrial intron in plants has retained its maturaseORF (matR), but its roles in organellar intron splicing are unknown. Clues to other proteins required for organellar intron splicing are scarce, but these are likely encoded in the nucleus as there are no other obvious candidates among the remaining ORFs within the mtDNA. Through genetic screens in maize, the Barkan lab identified numerous nuclear genes that are required for the splicing of many of the introns within the plastid genome. Several of these genes are related to one another (i.e. crs1, caf1, caf2, and cfm2) in that they share a previously uncharacterized domain of archaeal origin, the CRM domain. The Arabidopsis genome contains 16 CRM-related genes, which contain between one and four repeats of the domain. Several of these are predicted to the mitochondria and are thus postulated to act in the splicing of group-II introns in the organelle(s) to which they are localized. In addition, plant genomes also harbor several genes that are closely related to group-II intron-encoded maturases (nMats), which exist in the nucleus as 'self-standing' ORFs, out of the context of their cognate "host" group-II introns and are predicted to reside within the mitochondria. The similarity with known group-II intron splicing factors identified in other systems and their predicted localization to mitochondria in plants suggest that nuclear-encoded CRM and nMat related proteins may function in the splicing of mitochondrial-encoded introns. In this proposal we proposed to (i) establish the intracellular locations of several CRM and nMat proteins; (ii) to test whether mutations in their genes impairs the splicing of mitochondrial introns; and to (iii) determine whether these proteins are bound to the mitochondrial introns in vivo.