Early nodulins in root nodule development

The symbiotic interaction between bacteria of the genus Rhizobium and leguminous plants leads to the formation of root nodules, which are specific nitrogen-fixing organs on the roots of plants. Bacteria enter the root by infection threads, and concomitantly cell divisons are induced in the root cortex, which lead to the formation of a meristern. From this meristern the different tissues of the root nodule originate. In the nodule bacteria are released in plant cells and then differentiate into the endosymbiotic bacteroids. These bacteroids are capable of nitrogen fixation.The formation of root nodules involves expression of both bacterial and plant genes. Rhizobium genes involved in nodule formation are the nodulation ( nod ) genes. Nodulespecific plant genes are termed nodulin genes. According to their timing of expression they can be divided into early and late nodulin genes. Early nodulin genes are expressed well before the onset of nitrogen fixation, at the time that the nodule tissue is formed and the roots become infected by bacteria, while expression of late nodulin genes starts shortly before the onset of nitrogen fixation, when the nodule structure has been formed. Therefore only early nodulins can be involved in the infection process and in nodule development. Early nodulin genes expressed during the pea ( Pisum sativum L.) - Rhizobium leguminosarum bv. viciae interaction are the subject of this thesis. Several cDNA clones representing pea early nodulin genes have been isolated and they have been used to study root nodule development and the communication between bacteria and host plant.In chapter 2 we review general aspects of plant development. Recent progresses in understanding the molecular mechanisms underlying animal development are listed, and the possible significance of such mechanisms for plant development is discussed. The features of the root nodule formation system that make it suitable to study particular questions on the molecular basis of plant development are put forward.In chapter 3 the pea early nodulin cDNA clone pPsENOD2 is characterized. The nature of the encoded polypeptide is compared with that of the soybean early nodulin described before. ENOD2 transcripts are localized both in pea and soybean root nodules throughout successive stages of development by in situ hybridization. Data on the primary structure of the ENOD2 protein and localization data are then combined to hypothesise that the function of this early nodulin is to create an oxygen barrier in the root nodule.In chapter 4 the early nodulin ENOD12 is described. The spatial distribution of the corresponding transcript throughout root nodule development is depicted to demonstrate the involvement of ENOD12 in the infection process. We describe the primary structure of the ENOD12 protein and we examine whether ENOD12 gene expression is related to a defense respons. Using a sensitive detection method based on the polymerase chain reaction (PCR) we demonstrate that ENOD12 gene expression is induced by excreted Rhizobium factors and that bacterial nod genes are involved. ENOD12 transcripts found in flower and stem tissue are compared to the ENOD12 mRNAs in nodules using, among other techniques, a novel adaptation of RNase mapping to determine whether the same genes are expressed in these different tissues or not.In chapter 5 it is demonstrated that the accumulation pattern of the transcripts corresponding to the pPsENOD5, pPsENOD3 and pPsENOD14 cDNA clones differs from that of ENOD2 and ENOD12 mRNA. The distribution of the former three transcripts is compared with the distribution of ENOD12 mRNA and the late nodulin leghemoglobin transcript. It is shown that the different transcripts are present at successive stages of development of the infected cell type. The primary structure of the ENOD5, ENOD3 and ENOD14 early nodulins is determined and these data are combined with the localization data of the transcripts to speculate on functions of these proteins, The involvement of different factors to induce expression of different early and late nodulin genes is discussed.In chapter 6 the results described in the previous three chapters are summarized and some additional data on early nodulins are presented. The significance of the availability of early nodulin gene probes to elucidate the mechanisms of communication between rhizobia and legumes, which underly the process of root nodule formation, is discussed. Finally, in chapter 7, the value of the obtained information on early nodulins for studying both specific and general aspects of root nodule development is discussed

The role of the aryl hydrocarbon receptor interacting protein (AIP) in pituitary tumorigenesis: A proteomic approach for explaining the clinical behaviour of AIP mutation-associated pituitary adenomas

A subset of familial and sporadic pituitary adenomas is due to germline mutations in the aryl hydrocarbon receptor interacting protein gene (AIP). A systematic follow-up of cases and families with AIP mutation (AIPmut)-associated pituitary adenomas is lacking. The product of this novel tumour suppressor gene is a ubiquitously expressed co-chaperone of the heat shock proteins HSPA8 and HSP90, but besides of pituitary adenomas, there is no clear association of AIPmuts to other neoplasms. The molecular processes leading to pituitary tumorigenesis in the presence of AIPmuts and the mechanism for tissue-specific tumour suppressor function are unclear. This research work describes the clinical features of AIPmut positive familial and sporadic pituitary adenomas in a large international cohort of patients, aiming to increase the knowledge about this condition and focusing on the screening-led detection of pituitary adenomas. To define the repertoire of interactions of AIP in the pituitary gland and to determine which interactions are lost by AIP mutants, a proteomic screening for molecular partners of AIP in a pituitary cell line was conducted. The stability of a panel of missense AIP mutant proteins and the mechanism of protein degradation were evaluated in half-life studies, and the relationship between protein stability and phenotype was analysed. A number of novel features of AIPmut positive pituitary disease were identified, drawing attention to the high percentage of positive clinical screening of the apparently unaffected AIPmut carriers. The AIP tumour suppressor function is apparently mediated by its interaction with molecular chaperones, perhaps modifying their affinity for specific client proteins. AIP could exert an additional anti-tumorigenic action by regulating cytoskeletal organisation. AIP is processed via ubiquitination and proteasomal degradation, probably mediated by the FBXO3- containing SKP1-CUL1-F-BOX protein complex E3 ubiquitin-ligase. Enhanced proteasomal degradation conferred shorter half-life to most of the AIP mutants tested, with implications for the clinical presentation.National Council of Science and Technology (CONACYT) and the Secretariat of Public Education (SEP) Mexico, Barts and The London Charity

Functional and evolutionary analysis of the mouse Muc-1 gene

The mouse homologue of the human tumour-associated mucin, MUC1, was cloned and full-length sequence was determined. This mucin (previously called polymorphic epithelial mucin) is expressed by the majority of simple secretory epithelial cells in both the mouse and human and is also overexpressed in a large percentage of carcinomas. The mouse gene, Muc-1, encodes an integral membrane protein with 44% of its coding capacity made up of serine, threonine and proline, a composition typical of a highly O-glycosylated protein. The Muc-1 core protein consists of an amino-terminal signal sequence, a repetitive domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing membrane-spanning and cytoplasmic domains. Although overall homology with the human MUC1 protein is only 53%, the transmembrane and cytoplasmic domains exhibit homologies of 90% and 87%, respectively. This level of sequence conservation would suggest that these regions may be functionally important. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat (VNTR) polymorphism. However, this type of polymorphism was found to be present in all other mammalian groups analysed. Data is presented, including sequence obtained for the Muc-1 gene from a large number of species, to suggest how this gene has evolved and to explain possible reasons why the mouse Muc-1 gene does not exhibit minisatellite characteristics. Numerous functions have been suggested for this molecule, yet it still remains unclear what role this protein plays in the tissues and tumours in which it is expressed. In an effort to learn more of the function of the mouse Muc-1 gene, the gene was specifically mutated in embryonic stem (ES) cells. Targeting vectors derived through genomic clones from two strains of mice were utilised and their relative targeting efficiencies are discussed. Several mouse cell lines were created carrying a disruption in the Muc-1 gene. These cell lines were injected into nude mice to create tumours and also injected into blastocysts, in order to generate mice carrying the Muc-1 mutation. These mouse lines will provide a crucial tool in the analysis of the function of this molecule in vivo

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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Laboratory directed research and development. Annual report, fiscal year 1995

This document is a compilation of the several research and development programs having been performed at the Pacific Northwest National Laboratory for the fiscal year 1995