Septinas são proteínas que ligam GTP e interagem entre si formando heterocomplexos, os quais se organizam em filamentos e estruturas de maior nível de organização. Em humanos são encontrados 13 genes que codificam septinas, as quais se dividem em 4 grupos com base na similaridade de sua organização estrutural. Análises filogenéticas identificaram quatro septinas ortólogas no deuterostômio Ciona intestinalis, as quais apresentam, cada uma, identidade com um dos quatro grupos de septinas de mamíferos. Tais proteínas foram nomeadas CiSEPT2, CiSEPT6, CiSEPT7 e CiSEPT9 devido a identidade com as septinas humanas. Sabe-se que septinas de diferentes grupos interagem entre si formando heterocomplexos e, assim, a possibilidade de formar um oligômero único em C. intestinalis permite um modelo mais simples para o estudo da organização dos filamentos. Análises de bioinformática identificaram nessas proteínas motivos estruturais típicos de septinas, incluindo resíduos conservados para a ligação e hidrólise do GTP. As proteínas foram produzidas de forma heteróloga e a capacidade hidrolítica tanto de CiSEPT2 como de CiSEPT7 foiram confirmadas diretamente. Em contrapartida, CiSEPT6 não apresentou atividade catalítica. Um sistema de coexpressão foi construído no qual as quatro septinas de C. intestinalis foram co-expressas e co-purificadas em diferentes arranjos, resultando na formação de heterocomplexos parciais além daquele contendo as quatro subunidades. A avaliação do estado oligomérico dos heterocomplexos parciais mostrou que estes correspondem majoritariamente a tetrâmeros e hexâmeros quando formados, respectivamente, por duas ou três subunidades. Estes oligômeros foram analisados por microscopia eletrônica de transmissão (MET) em que se observou que os mesmos se dispõem na forma de um bastão. Ensaios ainda mostraram que a polimerização desses oligômeros é dependente de CiSEPT2 sugerindo que esta subunidade esteja possivelmente posicionada na extremidade do heterocomplexo. Adicionalmente, ensaios de polimerização realizados com o complexo CiSEPT2/6/7/9 revelaram que este é capaz de interagir com filamentos adjacentes, possivelmente via coiled-coil, formando estruturas de mais alta ordem, similar aos já observados em S. cerevisiae. Finalmente, experimentos utilizando termoforese em microescala (MST) mostraram uma maior afinidade de interação entre as subunidades quando oligômeros estão envolvidos, indicando que estes podem atuar como fator de nucleação para a formação dos heterocomplexos. Esse trabalho apresenta os primeiros estudos de caracterização das septinas de C. intestinalis mostrando a formação do heterocomplexo e representa um modelo simplificado e viável para estudos estruturais relativos a formação de filamentos de septinas. Assim, através de uma abordagem comparativa com complexos oriundos de outros organismos, este novo complexo poderá contribuir para a compreensão do mecanismo de montagem e controle da polimerização de septinas. / Septins are GTP-binding proteins that interact with each other forming heterocomplexes, filaments and higher order structures. The human genome encodes 13 septins, which are divided into 4 subgroups based on sequence similarity. Phylogenetic analysis demonstrated that only a single representative of each of these subgroups is present in the non-vertebrate chordate Ciona intestinalis. These proteins are named CiSEPT2, CiSEPT6, CiSEPT7 and CiSEPT9 due to their great similarity with human septins. Once septins from different subgroups are able to interact among themselves to form hetero-oligomeric complex, C. intestinalis representatives can be used as a simpler model for studies of septin complex assembly. Bioinformatics analysis identified typical structural motifs of septins in these proteins, including conserved residues for binding and hydrolysis of GTP. These septins were expressed and characterized biophysically and biochemically revealing that CiSEPT2 and CiSEPT7 are capable of hydrolyzing GTP. In contrast, CiSEPT6 showed no catalytic activity. A coexpression system were designed in which the four septins of C. intestinalis were co-expressed and co-purified in different arrangements, resulting the formation of partial heterocomplexes in addition to that containing the four subunits. The oligomeric state of the partial heterocomplexes were confirmed corresponding to tetramer and hexamers when formed, respectively, by two or three subunits. Transmission electron microscopy analysis revealed that these oligomers assemblies are rod-like structures. The polymerization of these oligomers is dependent on CiSEPT2 suggesting that this subunit occupies possibly the terminal positions of the heterocomplex. Additionally, polymerization assays performed with the CiSEPT2/6/7/9 complex revealed that it is capable of interacting with adjacent filaments, possibly via coiled-coil, forming higher order structures similar to those already reported in S. cerevisiae. Finally, the interaction strenght among C. intestinalis septins were determined using microscale thermoforesis (MST), revealing a higher binding affinity when titration involves small oligomers instead of monomers, indicating that these may act as a nucleating factor for heterocomplex assembly. This work constitutes the first characterization of C. intestinalis septins presenting the heterocomplex assembly and represents a simplified and potential model for structural studies regarding the filaments assembly of septins. Thus, comparative studies involving complexes from other organisms may contribute to the understanding of the assembly mechanism and polymerization control of septins.

Habilitationsschrift--Jena. / "Litteratur": p. 75-79.

Alternative splicing from a single gene or transcription from distinct genes are the two most important genetic mechanisms used to generate protein isoforms. This study concerns the ascidian Ciona intestinalis and the genetic mechanism used in this primitive chordate organism to produce differentially expressed isoforms of the muscle protein troponin I (TnI). / Through cDNA cloning and reverse-transcription PCR approaches, the mRNA sequences encoding two distinct Ciona ThI isoforms were determined. These mRNAs were found to be tissue-specifically expressed; one in the heart and the other in body-wall muscle. They encode long (229-residue) and short (182-residue) TnI protein isoforms homologous to vertebrate heart and skeletal muscle TnI isoforms, respectively. By sequence comparison, allelic polymorphism analysis, and gene structure determination, the two mRNAs were shown to be produced from a single gene by inclusion/exclusion of two serial internal exons. This tissue-specific alternative splicing mechanism contrasts with the vertebrate strategy of TnI isoform. production by differential transcription of a multigene family. This is the first reported case in which homologous tissue-specific protein isoforms are produced by non-homologous molecular mechanisms in related organismal lineages, implying that the molecular mechanism of isoform generation was entirely reworked in one of these lineages. An evolutionary scenario is presented that can account for such a reworking through a series of reasonable steps.

Sequence analysis of 5.4 kilobases (kb) of DNA upstream of the ATG initiation codon of the troponin I (TnI) gene of the ascidian Ciona intestinalis failed to identify a sequence corresponding to the first 16 nucleotides (nt) at the 5' end of the mature TnI mRNA. Database searching revealed that three additional and unrelated Ciona mRNAs contained the same, or almost identical, 16 nt sequence at the 5' -end. Reverse-transcriptase/polymerase chain reaction (RT-PCR) experiments proved that at least three additional mRNAs contain the same 5' -end sequence. These results suggested the possibility that the 5 ' end of the TnI, and other, Ciona mRNAs is derived by spliced leader (SL) trans-splicing. Also consistent with SL trans-splicing were Ciona embryo transfect ion studies with a TnI/beta-galactosidase reporter construct that demonstrated the existence of a functional TnI promoter within 1.5 kb of ATG-upstream DNA Moreover, RT-PCR experiments showed that TnI/beta-galactosidase transcripts contained the 16 nt mRNA common 5' end sequence despite the absence of this sequence from the transcriptional template construct. / This data is a demonstration of SL trans-splicing and represents the first reported case of SL trans-splicing among the deuterostomes. It suggests that SL trans-splicing may represent an ancestral metazoan feature. Moreover, because vertebrates probably arose from ascidian-like ancestors, it may be that many vertebrate genes, none of which are thought to undergo SL trans-splicing, are descended from trans-spliced ancestral genes. Such a history could explain the common occurrence in vertebrate genes of 5' untranslated region enhancer-containing introns.

A long-range research interest of this laboratory is the evolution of the transcriptional control mechanisms of the vertebrate troponin I (TnI) gene family. It is likely that the vertebrate TnI gene family arose from a single TnI gene present in early chordate ancestors. Analysis of transcriptional control mechanisms of the TnI gene of a primitive chordate, such as the ascidian Ciona intestinalis, may therefore provide insight into the regulatory mechanisms of the vertebrate ancestral TnI gene. As an initial step in such a study, I localized transcriptional control regions within 1.5 kb of 5'-flanking DNA of the Ciona TnI gene. I prepared a series of deletion constructs in which Ciona TnI 5'-flanking DNA segments were fused to a nuclear-targeted beta-galactosidase reporter gene. Constructs were introduced into fertilized Ciona eggs by electroporation, and following development up to the mid tailbud stage (12 h), reporter gene expression was assessed by whole-mount beta-galactosidase histochemistry. (Abstract shortened by UMI.)

My research goal was to functionally dissect the regulatory region of the gene encoding the muscle protein troponin I (TnI) in the simple chordate Ciona intestinalis. Based on evolutionary sequence conservation and computational prediction of cis-elements in the regulatory region, I designed mutants and assayed their effects in a recombinant DNA construct reporter system. I electroporated TnI DNA-linked reporter constructs into Ciona zygotes and assayed reporter activity following 12-14 hr of development. These studies showed that a conserved E-box is essential for high-level embryonic tail muscle-specific expression whereas conserved MEF-2 and overlapping CREB/TEF-1 sites were not. I carried out functional promoter identification experiments and 5' RACE studies and localized the core-promoter/transcription start-site in the Ciona TnI gene. My results point out the similarities/differences between Ciona and vertebrate Tnl gene regulation, which presumably reflect the conservation/modification of TnI gene regulatory mechanisms during chordate/vertebrate evolution.

Thesis (doctoral)--Stockholm University, 1992. / Recueil d'articles. Réf. bibliogr.

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