Nek1 é uma serina/treonina quinase humana envolvida na ciliogênese, na resposta a danos ao DNA e na regulação do ciclo celular. Devido ao possível envolvimento dessa proteína em patologias como a Doença Policística do Rim (PKD), a Síndrome de Costelas Curtas e Polidáctilia tipo Majewski e no desenvolvimento de tumores, muitos estudos tem sido feitos buscando entender a via de atuação dessa quinase nas células. Em vista disso, o objetivo desse trabalho foi avaliar o papel da Nek1 em resposta a danos ao DNA, principalmente gerados por agentes indutores de Crosslinks (ICLs), como cisplatina, buscando localizar a Nek1 nas vias de sinalização conhecidas e identificar proteínas que interagem com essa quinase. Para isto, foi utilizado um modelo de silenciamento estável de Nek1 por shRNAi na linhagem celular humana Hek293t. Essas células, quando comparadas à linhagem selvagem expressando a proteína, apresentaram um reparo deficiente de lesões induzidas por cisplatina e ausência de quebras de dupla fita através do ensaio cometa, confirmado pelos baixos níveis de fosforilação da histona H2AX. Entretanto, análises com outro agente indutor de ICLs, Nimustina (ACNU), demonstrou que a Nek1 parece atuar nos mecanismos envolvidos no reparo de lesões induzidas por cisplatina. Isso sugere que essa proteína possa estar agindo no reconhecimento da lesão do DNA e até mesmo regulando vias checagem de ciclo celular, uma vez que a linhagem silenciada apresentou modificações no ciclo celular e na sinalização de Chk1 e Chk2, após a exposição com cisplatina. Além disso, essas células também apresentaram alterações na sinalização das proteínas envolvidas no reparo de ICLs, BRCA1 e FANCD2. O estudo de interação proteica por espectrometria de massas destacou algumas proteínas de reparo, como proteínas da via de Fanconi, e principalmente moléculas relacionadas com o processo de ubiquitinação, sugerindo que este pode ser o mecanismo pelo qual essa molécula está atuando e integrando diferentes sistemas. Esse conjunto de dados indica o papel da Nek1 como uma proteína sensora de danos que atua no início das vias de reparo, regulando diferentes moléculas e interagindo em distintos processos celulares como controle do ciclo celular, reparo e morte. / Nek1 is a human serine/threonine protein kinase involved in ciliogenesis, DNA damage response and cell cycle regulation. Because of its possible involvement in human diseases such as Polycystic Kidney Disease (PKD), Short-Rib Polydactyly Syndrome Type Majewski and development of tumors, many studies have been done to understand how this kinase acts in cells. Therefore, the objective of this study was to evaluate a role for Nek1 in response to DNA damage, mainly generated by agents that induce crosslinks (ICLs), such as cisplatin, identifying pathways in which participate and how proteins interact with this kinase. To accomplish that, a stable silencing of Nek1 by shRNAi in human cell line Hek293t was employed. These cells, when compared to the wild type, showed a deficient repair of lesions induced by cisplatin and absence of double strand breaks, when analysed by comet assay and, confirmed by low levels of histone H2AX phosphorylation. However, analysis with another inducing ICLs agent, Nimustine (ACNU) demonstrated that Nek1 seems to act on mechanisms involved in repair of lesions induced by cisplatin. It suggesting that this protein might be acting on the recognition of DNA damage and even regulating checkpoint pathways, since the silenced strain showed changes in cell cycle and in signaling of Chk1 and Chk2 after exposure to cisplatin. Moreover, these cells also showed changes in signaling of proteins involved in the repair of ICLs FANCD2 and BRCA1. The study of protein interaction by mass spectrometry highlighted some repair proteins, like Fanconi pathway proteins, and protein involved in the ubiquitin-related process, suggesting a possible mechanism of action and integration with different systems. This dataset indicates the role of Nek1 as a DNA damage sensor protein that acts at the beginning of the repair pathways regulating and interacting with distinct molecules in different cellular processes such as cell cycle control, repair and death.

O carrapato Boophilus microplus é um dos mais importantes ectoparasitas dos rebanhos bovinos, estando em todas as áreas tropicais e subtropicais entre o paralelo 32 °N e 32 °S, abrangendo regiões que se dedicam à pecuária na América, África, Ásia e Oceania. O controle do carrapato B. microplus é realizado principalmente com o uso de acaricidas, entretanto devido a crescente preocupação com os problemas criados pela poluição química do ambiente, ao alto custo e toxicidade das drogas e ao aparecimento de carrapatos resistentes aos acaricidas, alternativas para o controle do B. microplus devem ser encontradas. A sobrevivência dos carrapatos depende grandemente da sua capacidade de evadir o sistema imunológico dos hospedeiros, portanto antígenos da glândula salivar poderiam ser alvos para intervenção imunoprofilática. Neste trabalho foram isolados cDNAs não previamente descritos correspondentes a antígenos presentes na glândula salivar. cDNAs que codificam para proteínas similares a paramiosina e calreticulina foram seqüenciados, expressos em Escherichia coli e as proteínas recombinantes purificadas, tendo sido produzidos soros policlonais contra as proteínas recombinantes em coelhos. As seqüências foram caracterizadas e alinhamentos múltiplos com outras seqüências foram determinados. O gene da calreticulina mostrou ser expresso em todos os estágios e tecidos testados, tanto em experimentos de RT-PCR quanto de Western blot, tendo sido demonstrado também a sua secreção pela saliva. Análise filogenética indica o agrupamento do gene de B. microplus com seqüências de outros artrópodos. Soros de bovinos infestados não reconhecem a calreticulina recombinante, apesar dela ser reconhecida pelo soro de cães infestados pelo carrapato Rhippicephalus sanguineus. A paramiosina mostrou-se, em ensaios de Western blot, também estar presente em todos os estágios testados, porém não na saliva. A paramiosina recombinante foi capaz de ligar colágeno e IgGs. Ambas as proteínas correspondem a proteínas multi-funcionais possivelmente envolvidas na imunomodulação do hospedeiro, tendo sido sugeridas como imunógenos protetores contra outros parasitas.

by Brian C. Freeman. / Typescript. / Thesis (M.S.)--Florida State University, 1991. / Includes bibliographical references.

Nek1 é uma serina/treonina quinase humana envolvida na ciliogênese, na resposta a danos ao DNA e na regulação do ciclo celular. Devido ao possível envolvimento dessa proteína em patologias como a Doença Policística do Rim (PKD), a Síndrome de Costelas Curtas e Polidáctilia tipo Majewski e no desenvolvimento de tumores, muitos estudos tem sido feitos buscando entender a via de atuação dessa quinase nas células. Em vista disso, o objetivo desse trabalho foi avaliar o papel da Nek1 em resposta a danos ao DNA, principalmente gerados por agentes indutores de Crosslinks (ICLs), como cisplatina, buscando localizar a Nek1 nas vias de sinalização conhecidas e identificar proteínas que interagem com essa quinase. Para isto, foi utilizado um modelo de silenciamento estável de Nek1 por shRNAi na linhagem celular humana Hek293t. Essas células, quando comparadas à linhagem selvagem expressando a proteína, apresentaram um reparo deficiente de lesões induzidas por cisplatina e ausência de quebras de dupla fita através do ensaio cometa, confirmado pelos baixos níveis de fosforilação da histona H2AX. Entretanto, análises com outro agente indutor de ICLs, Nimustina (ACNU), demonstrou que a Nek1 parece atuar nos mecanismos envolvidos no reparo de lesões induzidas por cisplatina. Isso sugere que essa proteína possa estar agindo no reconhecimento da lesão do DNA e até mesmo regulando vias checagem de ciclo celular, uma vez que a linhagem silenciada apresentou modificações no ciclo celular e na sinalização de Chk1 e Chk2, após a exposição com cisplatina. Além disso, essas células também apresentaram alterações na sinalização das proteínas envolvidas no reparo de ICLs, BRCA1 e FANCD2. O estudo de interação proteica por espectrometria de massas destacou algumas proteínas de reparo, como proteínas da via de Fanconi, e principalmente moléculas relacionadas com o processo de ubiquitinação, sugerindo que este pode ser o mecanismo pelo qual essa molécula está atuando e integrando diferentes sistemas. Esse conjunto de dados indica o papel da Nek1 como uma proteína sensora de danos que atua no início das vias de reparo, regulando diferentes moléculas e interagindo em distintos processos celulares como controle do ciclo celular, reparo e morte. / Nek1 is a human serine/threonine protein kinase involved in ciliogenesis, DNA damage response and cell cycle regulation. Because of its possible involvement in human diseases such as Polycystic Kidney Disease (PKD), Short-Rib Polydactyly Syndrome Type Majewski and development of tumors, many studies have been done to understand how this kinase acts in cells. Therefore, the objective of this study was to evaluate a role for Nek1 in response to DNA damage, mainly generated by agents that induce crosslinks (ICLs), such as cisplatin, identifying pathways in which participate and how proteins interact with this kinase. To accomplish that, a stable silencing of Nek1 by shRNAi in human cell line Hek293t was employed. These cells, when compared to the wild type, showed a deficient repair of lesions induced by cisplatin and absence of double strand breaks, when analysed by comet assay and, confirmed by low levels of histone H2AX phosphorylation. However, analysis with another inducing ICLs agent, Nimustine (ACNU) demonstrated that Nek1 seems to act on mechanisms involved in repair of lesions induced by cisplatin. It suggesting that this protein might be acting on the recognition of DNA damage and even regulating checkpoint pathways, since the silenced strain showed changes in cell cycle and in signaling of Chk1 and Chk2 after exposure to cisplatin. Moreover, these cells also showed changes in signaling of proteins involved in the repair of ICLs FANCD2 and BRCA1. The study of protein interaction by mass spectrometry highlighted some repair proteins, like Fanconi pathway proteins, and protein involved in the ubiquitin-related process, suggesting a possible mechanism of action and integration with different systems. This dataset indicates the role of Nek1 as a DNA damage sensor protein that acts at the beginning of the repair pathways regulating and interacting with distinct molecules in different cellular processes such as cell cycle control, repair and death.

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:36:16Z (GMT). No. of bitstreams: 1 Sampaio_ClaudioLuisMarques_M.pdf: 2335220 bytes, checksum: 0b5237faca34d31d6e2743587c4b32fd (MD5) Previous issue date: 2010 / Resumo: Um problema recorrente em Filogenética é saber de antemão que melhores tamanhos de genes ou proteínas se deve ter para a construção de dendrogramas mais precisos. Neste trabalho, examinamos quais os efeitos de variados tamanhos de um alinhamento conhecido na qualidade da inferência de filogenia, em particular a filogenia dos fungos, utilizando 198 táxons fungais e 16 de grupo externo. Adicionalmente, calculamos a entropia de Shannon de cada ponto do alinhamento e fizemos iterações semelhantes por seus limiares. Para isto construímos um programa open-source baseado no toolkit bioperl que calcula estes dados. Concluímos que tanto para as iterações por tamanho quanto os para entropia, os limiares ideais são aquém do tamanho total do gene, podendo justificar uso de drafts de seqüenciamentos em inferências filogenéticas usando um pequeno número de regras / Abstract: A recurring issue in phylogenetics is knowing beforehand which best sizes for genes or proteins one should have for building more accurate cladograms. Herein we examine the effects or varying sizes of a known aligment on the quality of its inferred phylogeny, specifically considering the fungi phylogeny by using 198 fungal taxa plus 16 outgroup taxa. Additionally, we calculate the Shannon entropy of each point of the alignment and iterate similarly by its thresholds. To that end, we developed an open-source software based on the bioperl toolkit to calculate this data. Finally, we concluded that either for the size iterations or for the entropy iterations, the ideal thresholds are below the gene full size, justifying the use of sequencing drafts in phylogenetic inferrences using a handful of rules / Mestrado / Bioinformatica / Mestre em Genética e Biologia Molecular

Orientador: Eduardo Daruge Júnior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T20:17:11Z (GMT). No. of bitstreams: 1 Santos_JulianaMendoncaFerreirados_M.pdf: 905410 bytes, checksum: 2000e7b5451e5f8d58ef540bdeedbd5a (MD5) Previous issue date: 2012 / Resumo: O processo de identificação humana envolve diferentes etapas, contexto no qual se faz necessário o aprimoramento das técnicas tradicionais e o desenvolvimento de novas metodologias aplicáveis na rotina da prática pericial. É através da investigação do gênero biológico, precedida da determinação da espécie, que se dá o passo inicial no delineamento do perfil de vítimas não-identificadas. Ao encontro dos exames antropológicos, as técnicas de DNA consolidaram-se como adjuvantes e certificadoras; porém, trazem consigo limitações atinentes ao custo, agilidade e aplicabilidade. O presente estudo buscou investigar o gênero biológico, através do isolamento do gene amelogenina. Para tanto, foram utilizadas 52 polpas dentais obtidas de dentes extraídos de corpos em decomposição, as quais foram submetidas à extração do material genético por meio da resina quelante Chelex 100® (BioRad) e à amplificação pela reação em cadeia da polimerase (PCR). Após eletroforese com seqüenciador automático (Applied Biosystems 3130 Genetic Analyzer®), os picos foram obtidos, e os resultados interpretados como AMEL X para indivíduo feminino (106 pb), e AMEL Y para indivíduo masculino (112 pb). Não foi possível determinar o gênero biológico em 15,4% (n=8) dos dentes e não houve influência do sexo na viabilidade do material coletado para a determinação do gênero (p = 0, 072). Quanto aos grupos dentais, divididos em molares, pré-molares, caninos e incisivos, os mesmos não exerceram influência na obtenção dos resultados (p = 0,892). Houve concordância de 95,5% (n=42) entre a determinação do gênero biológico e o gênero real dos indivíduos que compuseram a amostra (Kappa = 0, 909). Dentre aqueles do gênero masculino houve discordância de 5,6% (n=2) entre o sexo real e o sexo obtido pela amplificação, revelando resultado de falso-feminino, ou seja, indivíduos masculinos com genótipo XX (AMEL X). Concluiu-se que, apesar da amostra ser composta por material degradado, a amplificação da amelogenina apresentou resultados satisfatórios na investigação do gênero biológico, mostrando-se como uma metodologia de ótimo custo-benefício capaz de complementar estudos antropológicos de identificação humana / Abstract: The process of human identification involves differents stages, in wich context it is necessary to the improvement of tradicional techniques and the development of new methods applicable in forensic practice routine. It is througth the investigation of biological gender, preceded by the determination of species, wich takes the initial step in designing the profile of unidentified victms. To meet the anthropological examination, DNA techniques established themselves as adjuvants and certifiers, but bring with them limitations pertaining to cost, flexibility and applicability. This study aimed to investigate the biological gender by isolating the amelogenin gene. For this purpose, we used dental pulp obtained from 52 teeth extracted from decomposing bodies, wich were genetic material extracted throught the chelating resin Chelex 100® (Bio Rad) and amplification by polymerase chain reaction (PCR). After electrophoresis on automatic sequencer (Applied Biosystems 3130 Genetic Analyser®), the peaks were obtained and the results interpreted as AMEL X for females individuals (106 pb) and AMEL Y for male individuals (112 pb). Could not be determined the biological gender of 15,4% (n = 08) of the teeth and there was no influence of gender on the viability of the material collected for the determination of sex (p = 0,072). As dental groups, divided in into molars, premolars, canines and incisors, they were not influential in obtaining the results (p = 0,892). There was concordance of 95,5% (n = 42) between the determination of the actual biological gender and the gender of sampled individuals (Kappa = 0,909). Among those of males, there was disagreement of 5,6% (n = 02) between real sex and sex obtained by amplification, revealing results of false-female, or males subjects with XX genotype (AMEL X). We concluded that, althought the samples were composed of degraded material, the amplification of amelogenin presented satisfactory results in the investigation of biological gender, showing himself as a cost-effective methodology, capable of complement anthropological studies in human identification / Mestrado / Odontologia Legal e Deontologia / Mestre em Biologia Buco-Dental

No description available.

The binding of different proteins involved in processes such as transcription, replication and chromatin compaction to regions of the genome is regulated by the structure of DNA. Thus, DNA structure acts as the crucial link modulating evolutionary selection of the DNA sequence based on its own function, and the function of the proteins it encodes. The aim of this work is to examine the role of intrinsic, sequence-dependent structural variations vis-a -vis the protein-induced variations, in allowing DNA to assume geometries necessary for binding by proteins. For this purpose, we carried out analyses of datasets of X-ray crystal structures of free and protein-bound DNA, and molecular dynamics simulation studies of few free DNA structures and a protein-DNA complex. Each of the projects described below will appear as a separate chapter in the thesis. Analysis of X-ray crystal structure datasets Dataset of high-resolution X-ray crystal structures of free and protein-bound DNA This project was initiated with the aim of investigating the variation in A-and B-forms of DNA and the role they play in the binding of proteins. However, a survey of the existing literature indicated that the terms ‘A-DNA’ and ‘B-DNA’ were being used rather loosely and several different parameters at the local structural level were being used by various investigators to characterise these structures. Hence a systematic study was taken up to analyse all high-resolution free DNA structures comprising of sufficient number of contiguous Watson-Crick basepairs, irrespective of how they were classified by the existing databases. We also carried out a study of double-helical, Watson-Crick basepaired, free RNA structures for comparison. The structures in the RNA dataset were observed to rigidly assume the A-form and hence the average values of different parameters for that dataset were used to characterise the A-form. The analysis of free DNA and RNA structures was accompanied by an analysis of protein-bound DNA crystal structures. DNA structures bound to the helix-turn-helix motif in proteins were also analysed separately. The analysis of free DNA and RNA structures allowed us to pinpoint the parameters suitable for discriminating A-and B-forms of DNA at the local structural level. The results illustrated that the free DNA molecule, even in the crystalline state, samples a large amount of conformational space, encompassing both the A-and the B-forms. Most protein-bound DNA structures, including those with large, smooth curvature, were observed to assume the B-form. The A-form was observed to be limited to a small number of dinucleotide steps in DNA structures bound to the proteins belonging to a few specific families. Thus our study highlighted the structural versatility of B-form DNA, which allowed it to take up a range of global geometries to accommodate most DNA-binding protein motifs. Dataset of X-ray crystal structures of the nucleosome The study of high-resolution structures of free and protein-bound DNA was followed by an analysis of a dataset of X-ray crystal structures of the nucleosome, which is the fundamental repeating unit of the eukaryotic chromosome, and has been shown to play an important role in transcription regulation. Our results indicated that there is an ensemble of dinucleotide and trinucleotide level parameters that can give rise to similar global nucleosome structures. We therefore raise doubts about the use of the best resolved nucleosome structure as the template to calculate the energy required by putative nucleosome-forming sequences for adopting the nucleosome structure. Based on our results, we have proposed that the local and global level structural variability of DNA may act as a significant factor influencing the formation of nucleosomes in the vicinity of high-plasticity genes, and in determining the probability of binding by regulatory proteins. Molecular dynamics simulation studies of free and protein-bound DNA structures The analysis of crystal structure databases was complemented by molecular dynamics (MD) studies to investigate the dynamic evolution of the DNA structure in its free and protein-bound states. The following three simulation studies were carried out: Study to examine the biological relevance of the presence of 5-methyl group in thymine nucleotides An investigation of the biological relevance of the 5-methyl group in thymine nucleotides was carried out. For this purpose, comparison of molecular dynamics studies on structures with sequences d(CGCAAAUUUGCG)2and d(CGCAAATTTGCG)2was carried out. Our results showed that the presence of the thymine 5-methyl group was necessary for the A-tract to assume characteristic properties such as a narrow minor groove. It was also shown to modulate local level structural parameters and consequently, the curvature of the longer DNA fragment in which the A-tract was embedded. The analysis also provided possible explanation for the experimentally observed interaction of A-tracts with drugs and DNase-I in the presence and the absence of the thymine 5-methyl group. This project was the first of a series of MD studies, and hence several protocols were tested before finalising the correct protocol. Simulations were carried out using the Berendsen temperature equilibration scheme as well as the Langevin temperature equilibration scheme on both the structures. The Langevin temperature equilibration scheme was found to be unsuitable for nucleic acid simulations, as it caused long-term and possibly permanent disruption of the double-helical structure at the terminal and the neighbouring two positions in the sequence. The Berendsen temperature equilibration scheme was not observed to cause such disruptions. Simulations were also carried out on both structures, with or without initialising the initial ion positions. The position of minimum electrostatic potential, where AMBER8 placed the first counterion, was observed to act as a minimum energy trap from which the counterion could not escape even during the course of several nanoseconds of simulation. Hence, the actual simulations were carried out using the Berendsen temperature equilibration scheme, and after randomisation of initial ion positions. The results of protocol testing have been reported in an appendix. Study of DNA bending and curvature An analysis of DNA bending and curvature was carried out, by MD simulation on structures of three, ∼thirty basepair long sequences, namely, d(G-3(CA4T4G)-C)2, d(G-3(CT4A4G)-C)2and d(T-GACTA5T-GACTA6T-GACTA5T-G). For each molecule, snapshots belonging to a particular global geometry (linear, curved, bent in a particular direction etc.) were grouped together, and the average values of the dinucleotide step parameters for different groups were compared. It was observed that for all the three molecules, the average values for groups corresponding to different global geometries were within 1of each other, indicating that ensemble average values of dinucleotide level parameters are incapable of predicting the global geometry of a DNA molecule. Study of the TraR-Trabox complex The study on DNA bending and curvature was followed by simulations of a protein-DNA complex comprising of the bacterial quorum sensing transcription factor TraR with its promoter region known as Trabox. Simulations of a protein-free wild-type Trabox and a Trabox with two mutations in the spacer region were also carried out. Grouping of DNA snapshots in all the three simulations based on average values of dinucleotide parameters in the spacer region shows how selection of the ‘right’ DNA geometry by proteins works at several levels. The number of snapshots of free mutated Trabox assuming a geometry favourable for protein-binding in terms of average twist alone are less than one-fourth of the corresponding number for free wild-type Trabox. When one applies further selection criteria in terms of other parameters such as roll and slide, the number of mutated Trabox snapshots with a geometry favourable for protein-binding drops to less than 0.5%ofthe total number of MD snapshots. Thus our results highlight how sequence-dependent changes in the structrure of DNA regions, adjacent to those that directly hydrogen-bond to proteins, can also critically influence processes such as transcription. General Conclusion Overall, our results indicate that intrinsic, sequence-dependent structural variations in free B-DNA allow it to sample a large volume of the double-helical conformational space, and assume global geometries that can accomodate most DNA-binding proteins.

An attempt has been made to demonstrate an association between the carcinogenic activity of a chemical compound and its capacity to induce DNA damage and chromosome aberrations which may result in mutations and/or neoplastic transformation. Twenty-five 4-nitroquinoline 1-oxide (4NQO) derivatives and five related compounds of 4-nitropyridine 1-oxide (4NPO) of varying carcinogenicity were examined. [Formulae omitted] The induction of DNA damage, chromosome aberrations and clone forming capacity were used as end points. Monolayer cultures of embryonal Syrian-hamster cells and an established line of baby hamster kidney cells (BHK-21) were employed in this study. DNA damage, as measured by the unscheduled incorporation of tritiated thymidine (³H-TdR), was assayed by the autoradiographic procedure. To distinguish DNA repair synthesis from DNA replication synthesis at S-phase, cultured embryonal hamster cells were arrested at G₁ by growing them in an arginine deficient medium (ADM) prior to the application of the various carcinogens. The unscheduled uptake of radioisotope was estimated by counting the number of grains per diploid nucleus of carcinogen treated cells. The highly oncogenic derivatives of 4NQO and 4NPO elicited an elevated level of unscheduled ³H-TdR incorporation in treated cells, while the weakly oncogenic compounds induced only a smaller amount of DNA repair synthesis. The non-oncogenic N-oxides failed to provoke any detectable ³H-TdR uptake. Chromosome aberrations were studied in ADM-arrested cells which were exposed to the various compounds and then triggered into division by transferring them into the regular growth medium. A direct proportionality was observed between the degree of carcinogenicity of a compound and the frequency of induced chromosome aberrations. The clone forming ability of treated cells was employed as a means to compare the cytotoxicity of the 4NQO and 4NPO derivatives. Potent carcinogens were highly cytotoxic; weakly carcinogenic compounds showed only a slight lethal effect and non-oncogenic derivatives did not affect cell survival. This study demonstrated the capacity of carcinogens to induce alterations at the chromosome and DNA level. The possible role of DNA repair and chromosome aberrations in neoplastic transformation was discussed. The use of DNA repair synthes as an economic and relevant tool for identifying mutagens and/or carcinogens has been suggested. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Cell duplication in Escherichia coli involves complex events, coordinated with chromosome replication. Because of the importance of chromosomes in perpetuating the normal cell cycle the initiation of their replication must be coordinated with cellular division. Following initiation, the cell must replicate and segregate its chromosomes, create a site necessary for septation and divide. These events could be coordinated by either; (1) biochemical reactions involving diffusible enzymes, or (2) multienzyme complexes which are localized at the site of DNA replication and cell division. In the latter case, the cyclic events of replication, segregation and cell division may be coordinated by physical-chemica1 or biochemical means. In any case, physical association implies pleiotropic effects. To test this hypothesis, cell division of the initiator mutant of E. coli , isolated by Kohiyama (1968) was studied. The temperature-sensitive initiator mutant E. coli CR 34T83 (ts DnaA) grew normally at 30 C, and at the restrictive temperature (42 C). The DNA replication as measured by radioactive precursor uptake, stopped after approximately 40 minutes and was equivalent to completion of rounds of replication started. Measurement of ribo- and deoxyribonucleotide triphosphate pools by thin-layer chromatography at 30 C and 42 C indicated residual DNA synthesis was not due to a limitation in the DNA precursors. Using a combination of density and differential radioactive labelling for the starts and ends of chromosomes, a preferred place for reinitiation of new replication cycles was shown. It was shown that DNA replication at 42 C terminated at a fixed region of the chromosome, and was identical to the 150 μg/ml chloramphenicol sensitive step involved in the process of initiation of chromosome replication in E. coli. A cessation of cellular division was noted by measurement of cell growth by Coulter Counter, at a shift from 30 C to 42 C, resulting in filamentous growth. Upon a return to 30 C, the cells resume division after approximately 15 - 20 min. The pleiotropic behaviour, that is, the cessation of cell division and initiation of DNA replication was a result of a point mutation in the gene DnaA, coding for a membrane bound protein involved in initiation. This mutation was mapped by transduction and was located at the isoleucine-valine region of the E. coli map. When this gene was transduced to different strains of E. coli K(12) the same pleiotropy was observed. This pleiotropy could be uncoupled, however, at 30 C by inhibitors of DNA synthesis or initiation. During recovery at 30 C from growth under 42 C, expression of cell division was proportional to cell equivalents generated at the restrictive temperature. RNA and protein synthesis, for 10 minutes during the recovery period, was obligatory for initiation of new rounds of replication, but not for the expression of cell division. A cell division "potential" protein was present under the restrictive growth condition. This "potential" was made at a derepressed rate and underwent a rapid degradation if kept at 42 C. At any given time, when returning from 42 C to 30 C, this "potential" allowed expression cell division based on DNA/mass or normal cell equivalents generated at 42 C. The half-life for decay of the division "potential" was estimated to be 1.4 minutes. The results were interpreted, in terms of an enzyme complex, which is common to the initiation of DNA replication and cellular division. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate