This work is focused on the molecular genetic basis for morphological change in evolution. Genes belonging to the MADS-box gene family, which includes members, that determine angiosperm floral organ identity, were isolated and characterised from two non-angiosperm plants; Norway spruce (Picea abies) and the club moss (Lycopodium annotinum). The exon/intron organisation of the isolated genes was determined, and its significance as an independent test of the position of a gene within the gene family tree evaluatad. identity genes were identified. One Norway spruce gene, DAL2, is an ortholog to Norway spruce genes that are closely related to the angiosperm floral organ angiosperm C-class MADS-box genes that specify stamen and carpel identity. The expression of DAL2 in male and female cones suggests that orthologous genes in conifers and argiosperms determine the identities of pollen- and seed-bearing structures. Constitutive expression of DAL2 in the angiosperm Arabidopsis resulted in homeotic conversions very similar to those resulting from constiutive expression of the Arabidopsis C-class gene. Angiosperm B-class MADS-box genes determine petal and stamen identity. The isolated Norway spruce B-class orthologs: DAL11, DAL12, and DAL13 are expressed in the developing male cones exclusively, suggesting a conserved function of B-class related genes in the determination of pollen forming organs among seed plants. No orthologs to the floral organ identity genes couId be isolated from the club moss, suggesting that the origin of these gene classes may be coupled to the origin of the pollen and the seed. The club moss MADS-box genes, LAMB2, LAMB4 and LAMB6, conform structurally to plant type MADS-box genes, whereas LAMB1 is divergent in details. The genes LAMB3 and LAMB5 encode shorter proteins. LAMB1 expression is restricted to reproductive structures, whereas LAMB2, LAMB4, LAMB5 and LAMB6 are broadly expressed. The implications from these expression patterns on the ancestral function of plant type MADS-box genes are discussed.

Two new genes involved in important processes of plant development were identified in the model plant Arabidopsis thaliana. The genes were isolated from mutants generated through insertional mutagenesis based on a transposon tagging approach. The first gene, ALB3, was isolated through the identification of the mutant albino3 (alb3), displaying severe defects in pigmentation and chloroplast biogenesis. The ALB3 protein shows sequence similarity to a yeast protein, OXA1, which is required in the mitochondria for proper assembly of the cytochrome oxidase complex. As ALB3 is localised in thylakoid membranes, we suggest that the ALB3 protein acts in the assembly of thylakoid membrane protein complexes and thereby is crucial for proper chloroplast development and function. The second gene, SHI, was identified through the short internodes (shi) mutation, a dwarfing mutation conferring a phenotype similar to mutants defective in the biosynthesis of the plant hormone gibberellin (GA). However, the shi mutant is unable to elongate following treatment with exogenous GA, which indicates that shi is defective in the response to GA. The level of active GA is elevated in the shi mutant, which is the expected result of reduced feedback control of GA biosynthesis. As the shi mutant phenotype is the result of overexpression of the SHI gene, we suggest that the SHI protein is a component of the GA signalling pathway, possibly acting as a repressor of GA-induced cell elongation. Sequence similarity database searches revealed that the SHI gene belongs to a new Arabidopsis gene family comprising at least eight members (SHI, LRP1, and SRS1 to SRS6). These genes encode regulatory proteins containing a putative zinc-binding RING finger-like domain. We have cloned SRS1 and SRS2, and have shown by overexpression of these genes in transgenic Arabidopsis that their gene products might function in similar processes as SHI.

Homeodomain-leucine zipper (HDZip) proteins constitute a large family of transcription factors apparently unique to plants. To elucidate the function of these factors, the biochemical properties in vitro as well as the effects on transgenic plants when expressed at high levels were studied. The conclusion is that HDZip proteins are very similar with respect to DNA-binding specificity in vitro but appear to be active in different aspects of plant development. Thus, functional specificity of HDZip proteins is most likely determined by other aspects of proteins function, e.g. their capacity to interact with other proteins. High-level expression of the HDZip gene ATHB5 in transgenic plants results in hypersensitivity to the inhibitory effect of the plant hormone abscisic acid (ABA) on seed germination and seedling root growth. Furthermore, the expression of ATHB5 in germinating seedlings is downregulated in the Arabidopsis ABA response mutants abi3 and abi5. Together, these data suggest that ATHB5 acts as a regulator of seed germination and postgerminative growth downstream of ABI3 and ABI5 in an ABA response-signaling pathway. Enhanced levels of the HDZip gene ATHB13 in transgenic Arabidopsis confer a sugar-dependent reduction of cotyledon width. In addition, a subset of known sugar-dependent genes was hyperinduced by sucrose in Arabidopsis seedlings overexpressing ATHB13. These data suggest that ATHB13 affects both cotyledon morphology and gene regulation as a component of a sucrose-signaling pathway. Loss-of-function mutations in ATHB5 and ATHB13 did not result in any discernable mutant phenotypes, suggesting that these genes are only required under specific physiological conditions or that they act in a redundant fashion in the plant.

Genes encoding homeodomain leucine zipper, HDZip, transcription factors constitute a large gene family in Arabidopsis thaliana. In this thesis the isolation and characterization of four HDZip genes (ATHB3, -13, -20 and -23) is described. These genes are similar in sequence and form a distinct subclass within the HDZip gene family. Since the genes cause similar alterations in cotyledon shape when expressed constitutively, we refer to the members of this subclass as pointed cotyledon HDZip genes. To determine the biological functions of the genes, the phenotypes of plants constitutively expressing the genes have been analysed. Each of the genes specifically inhibits lateral cell expansion in cotyledons and leaves and thereby causes them to be abnormally narrow. Detailed expression analysis shows that only ATHB23 is expressed in the entire leaf and cotyledon from early stages of development while ATHB20 is predominantly expressed in the root cortex. ATHB13 is expressed in basal parts of mature leaves and floral organs and ATHB3 in root and stem cortex. The ATHB13 protein acts within a signalling pathway that mediates a response to sucrose that specifically regulates the expression of specific sugar-regulated genes. ATHB3 specifically inhibits primary root development without affecting the development of secondary roots when constitutively expressed. Reduced expression of ATHB3 by antisense suppression results in increased expression of ATHB13, indicating that ATHB3 acts as a repressor of ATHB13 expression in the wild type. This thesis also reports the isolation of seven new genes of HDZip class I and reviews available functional information on the genes in this class. One conclusion is that HDZip I proteins that are closely related phylogenetically are also functionally related, in most cases. Seven different mutations in HDZip I genes were also identified. The lack of phenotypic deviations from wild type of these mutants suggests that these HDZip proteins act in a redundant fashion in the plant.

The molecular mechanisms of the immune system in shrimp, Penaeus monodon, are completely unknown, despite its economic importance as an aquaculture species, especially in Asia and Latin America. The genes and their gene products involved in the prophenoloxidase activating system, which is considered to be a non-self recognition and defence system in many invertebrates, have been isolated and characterised in shrimp. These include a zymogen of this cascade, prophenoloxidase (proPO); a cell adhesion protein, peroxinectin and a pattern recognition protein, β-1,3-glucan binding protein (GBP). All proteins are synthesised in shrimp hemocytes, not in the hepatopancreas. The shrimp proPO cDNA clone has 3,002 bp and contains an open reading frame of 2,121 bp encoding a putative polypeptide of 688 amino acids, with a molecular mass of 78.7 kDa. Comparison of amino acids sequences showed that this shrimp proPO was more closely to that of another crustacean, the freshwater crayfish, Pacifastacus leniusculus, than to insect proPOs. Upon activation of the proPO system in shrimp, a cell adhesion activity in the hemolymph is generated. Inhibition of adhesion by an antiserum against the crayfish cell adhesion protein, peroxinectin, revealed that the cell adhesion activity detected in shrimp hemolymph might result from a peroxinectin in shrimp. Indeed, a cDNA clone which encoded shrimp peroxinectin was isolated with an open reading frame of 2,337 bp encoding a putative protein of 778 amino acids including a signal peptide. Two putative integrin-binding motifs (RGD and KGD) are present suggesting that integrin is involved in the adhesion activity. The peroxinectin transcript was slightly reduced in shrimp injected with a β-1,3-glucan or laminarin. Also found in shrimp hemolymph was a 31 kDa-GBP that could bind to β-1,3-glucan polymers such as curdlan and zymosan, but not to LPS. The cDNA sequence of shrimp GBP showed high similarity to that of crayfish LGBP, other insect recognition proteins as well as bacterial and sea urchin glucanases. Shrimp injected with an insoluble β-1,3-glucan, curdlan or heat-killled Vibrio harveyi did not show any significant changes in relevant mRNA levels. An attempt to knock out the LGBP expression by its exogeneous dsRNA was done in a proliferating blood cell culture from the hematopoietic tissue of crayfish. We found that the expression of endogeneous LGBP mRNA could be substantially inhibited by incubation of dsRNA-LGBP in the cell culture. The effect is quick, specific, and also affects the cell behaviours.

Cyanobacteria belonging to the genus Nostoc have the capacity to form symbiotic associations with a wide range of organisms. Diversity, specificity and cellular modifications of the symbiosis between Nostoc and fungi in the formation of lichens were investigated in this thesis. The use of the tRNALeuUAA intron as a genetic marker for the subgeneric identification of Nostoc in complex field material was developed. Lichens belonging to the genera Peltigera and Nephroma show limited variability in their Nostoc symbionts. The in situ symbiont consists of a single strainn rather than a community of different Nostocs, and single thalli consistently contained the same symbiont. Patterns in symbiont identity were found in geographically remote populations and the lichen species, rather than growth locality, was shown to be important for the identity of the Nostoc symbiont. Examination of a P. aphthosa photosymbiodeme revealed that one Nostoc has the capacity to perform the physiological roles found in both bipartite and tripartite lichens. The symbiotic association between bryophytes and Nostoc on the other hand exhibited a much greater variation of Nostoc symbionts. Evolutionary patterns in the tRNALeuUAA intron were analyzed and it was shown that sequence variation was caused by several processes other than random mutations. Such evolutionary processes in genetic markers are crucial to consider, especially if phylogenetic reconstructions are attempted. Protein profiles of symbiotic and free living Nostoc were analyzed using 2-dimensional gel electrophoresis. One of the major proteins in the extracts from freshly isolated symbionts was partially sequenced and shown to contain a fasciclin domain. The corresponding ORF in N. punctiforme was homologous to symbiotically induced genes found in different symbiotic systems. This thesis gives new perspectives on lichens and pr for further exaovides a platform for further examiniations using tools provided by modern biology.

Species of the genus Rickettsia are obligate intracellular parasites of the a-proteobacterial subdivision. It has been suggested that obligate intracellular bacteria have evolved from free-living bacteria with much larger genome sizes. Transitions to intracellular growth habitats are normally associated with radical genomic alterations, particularly genome rearrangements and gene losses. This thesis presents a comparative study of evolutionary processes such as gene rearrangements, deletions and duplications in a variety of Rickettsia species. The results show that early intrachromosomal recombination events mediated by duplicated genes and short repeats have resulted in deletions as well as rearrangements. For example, an exceptional organization of the elongation factor genes was found in all species examined, suggesting that this rearrangement event occurred at the early stage of the evolution of Rickettsia. Likewise, it was found that a repetitive element, the so-called Rickettsia Palindromic Element (RPE) flourished prior to species divergence in Rickettsia. Finally, a phylogenetic analysis shows that the duplication events that gave rise to the five genes encoding ATP/ADP transporters occurred long before the divergence of the two major groups of Rickettsia. Taken together, this suggests that Rickettsia have been intracellular parasites for an extensive period of time. A detailed analysis of the patterns of nucleotide changes in genes and intergenic regions among the different species provides evidence for a gradual accumulation of short deletions. This suggests that different distributions of genes and repeated sequences in modern Rickettsia species reflect species-specific differences in rates of deterioration rather than variation in rates of intra-genomic sequence proliferation.

Sequence comparisons of orthologous microsatellite loci in cattle and sheep revealed that the substitution rate in microsatellite flanking sequences does not differ from the rate in presumably neutrally evolving intron sequences. This suggests that microsatellites are generally located in regions that are not subjected to selection. Interestingly, a propensity for substitutions to occur in the border region between flanking and repeat sequence was found. Pedigree analysis of large numbers of barn swallows revealed extremely high mutation frequencies for the tetranucleotide HrU6 and pentanucleotide HrU10 repeat loci. A detailed analysis showed that both the rate and the pattern of mutation differed significantly between the two loci. Further analysis of HrU6 and HrU10 mutations, as well as mutation data for another hypermutable locus (HrU9) in barn swallows, revealed that mutations were more likely to arise in some families than others. This was partly, but probably not only, due to an effect of allele length on mutation rate. The mutation rate was found to vary between colonies of breeding birds, but, overall, not between two different populations. Single molecule genotyping of DNA prepared from human sperm cells was used to detect mutations at the tetranucleotide repeat D21S1245. A tenfold difference in mutation rate between alleles was found. Three phylogenetically distinct allele lineages could be defined, which differed significantly in mutation rate. Unexpectedly, the mutation rate was not found to increase with male age. Microsatellites are commonly applied in a wide range of genetic contexts including linkage mapping, forensic science and population genetics. Obtaining a detailed picture of the evolution of these tandem repeats is important in order to fully understand how to interpret microsatellite data. In addition, studies of the mechanisms underlying microsatellite mutation will provide insights in the processes that shape the eukaryotic genome. This thesis demonstrates that microsatellite evolution is a highly heterogeneous process that is dependent on more factors than was previously thought. As the rate and pattern may vary between loci, caution must therefore be taken when building models to handle microsatellite data.

Genetic information is encoded by the linear sequence of the DNA double helix, while epigenetic information is overlayed as the packaging of DNA and associated proteins into the chromatin structure. Variations in chromatin structure play a vital role in establishing and maintaining patterns of gene expression during differentiation and development of higher eukaryotes, and disruption of this epigenetic gene regulation can lead to cancer. Mammals display an epigenetic phenomenon known as genomic imprinting, which provides an ideal model system for the study of epigenetics. Genes subject to genomic imprinting are differentially expressed within a single cell depending on the parental origin of the chromosome. Imprinting of the maternally expressed H19 gene and the adjacent paternally expressed Igf2 gene is regulated by the chromatin insulator protein CTCF. The studies presented in this thesis aim to investigate the functional mechanisms of CTCF-dependent gene regulation at the H19/Igf2 locus and at numerous other target sites throughout the genome. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. We have developed novel microarray based methods to identify and characterize numerous new CTCF target sites throughout the mouse genome. We have shown that CTCF acts as part of the RNA polymerase II complex. We have identified the post-translational modification by addition of ADP-ribose polymers to CTCF, and demonstrated that this modification regulates its insulating ability. The results of these studies of CTCF-dependent epigenetic gene regulation are discussed in light of the evolution of genomic imprinting and chromatin insulators, and a novel role for poly ADP-ribosylation of CTCF in the progression of cancer is proposed.

Strict control of gene expression is essential during development and in response to physiological stimuli. This thesis describes the functional characterisation of the gene regulatory mechanisms controlling the expression of the potent human growth factor Platelet Derived Growth Factor B gene, in a cell type specific context and in response to low oxygen tension. In addition, analysis of hypoxia in neuroblastoma indicates a role during tumour differentiation. Initally, a promoter-specific enhancer system controlling the expression of PDGF-B was characterised in placentally derived choriocarcinoma cells. The specificity of this enhancer promoter interaction was shown to be dependent on specific sequence elements identified in both the promoter and enhancer regions. It was then shown that the activity of the PDGF-B promoter is controlled via modulation of histone acetylation status in a cell type specific manner and furthermore, that one role of its enhancer could be to regulate transcription via alterations in acetylation status at the promoter. PDGF-B expression was then shown to be controlled by hypoxia in a biphasic manner in bladder carcinoma cells. An initial induction was followed by repression of transcription following chronic hypoxia. The biphasic response was shown to be dependent on glucose levels and uniquely amongst hypoxia regulated genes studied so far, PDGF-B expression was shown to be repressed by low glucose. Finally, detailed in vivo and in vitro analysis revealed that the major form of differentiation in childhood neuroblastoma is towards chromaffin-like rather than ganglionic lineages. This type of differentiation did not correlate with disease progression but was suggested to be dependent on tumour hypoxia, since chromaffin differentiation markers co-localised with markers of tumour hypoxia in both clinical samples and xenogenic tumours. In conclusion, the work presented in this thesis has identified several novel, highly specific gene regulatory mechanisms that are involved in development, the response to physiological stimuli and in disease progression.