<p>Genes encoding homeodomain leucine zipper, HDZip, transcription factors constitute a large gene family in <i>Arabidopsis thaliana</i>. In this thesis the isolation and characterization of four HDZip genes (<i>ATHB3</i>, -<i>13</i>, -<i>20</i> 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 <i>pointed cotyledon</i> HDZip genes. </p><p>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 <i>ATHB23</i> is expressed in the entire leaf and cotyledon from early stages of development while <i>ATHB20</i> is predominantly expressed in the root cortex. <i>ATHB13</i> is expressed in basal parts of mature leaves and floral organs and <i>ATHB3</i> 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. <i>ATHB3</i> specifically inhibits primary root development without affecting the development of secondary roots when constitutively expressed. </p><p>Reduced expression of <i>ATHB3</i> by antisense suppression results in increased expression of <i>ATHB13</i>, indicating that ATHB3 acts as a repressor of <i>ATHB13</i> expression in the wild type.</p><p>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.</p>

<p>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. </p><p>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. </p><p>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. </p><p>Finally, detailed <i>in vivo</i> and <i>in vitro</i> 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. </p><p>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.</p>

<p>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 (<i>Picea abies</i>) and the club moss (<i>Lycopodium annotinum</i>).</p><p>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, <i>DAL2</i>, is an ortholog to</p><p>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 <i>DAL2</i> 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 <i>DAL2</i> in the angiosperm Arabidopsis resulted in homeotic conversions very similar to those resulting from constiutive expression of the Arabidopsis C-class gene.</p><p>Angiosperm B-class MADS-box genes determine petal and stamen identity. The isolated Norway spruce B-class orthologs: <i>DAL11, DAL12</i>, and <i>DAL13</i> 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.</p><p>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.</p><p>The club moss MADS-box genes, <i>LAMB2, LAMB4</i> and <i>LAMB6</i>, conform structurally to plant type MADS-box genes, whereas <i>LAMB1</i> is divergent in details. The genes <i>LAMB3</i> and <i>LAMB5 </i>encode shorter proteins.</p><p><i>LAMB1</i> expression is restricted to reproductive structures, whereas <i>LAMB2</i>, <i>LAMB4, LAMB5</i> and <i>LAMB6</i> are broadly expressed. The implications from these expression patterns on the ancestral function of plant type MADS-box genes are discussed. </p>

<p>The molecular mechanisms of the immune system in shrimp, <i>Penaeus monodon</i>, 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, <i>Pacifastacus leniusculus</i>, than to insect proPOs. </p><p>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. </p><p>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 <i>Vibrio harveyi</i> did not show any significant changes in relevant mRNA levels. </p><p>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.</p>

<p>Cyanobacteria belonging to the genus <i>Nostoc</i> have the capacity to form symbiotic associations with a wide range of organisms. Diversity, specificity and cellular modifications of the symbiosis between <i>Nostoc</i> and fungi in the formation of lichens were investigated in this thesis.</p><p>The use of the tRNA^LeuUAA intron as a genetic marker for the subgeneric identification of Nostoc in complex field material was developed. Lichens belonging to the genera <i>Peltigera</i> and <i>Nephroma</i> show limited variability in their <i>Nostoc</i> symbionts. The <i>in situ</i> symbiont consists of a single strainn rather than a community of different <i>Nostocs</i>, 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 <i>Nostoc</i> symbiont. Examination of a <i>P. aphthosa</i> photosymbiodeme revealed that one <i>Nostoc</i> has the capacity to perform the physiological roles found in both bipartite and tripartite lichens. The symbiotic association between bryophytes and <i>Nostoc</i> on the other hand exhibited a much greater variation of <i>Nostoc</i> symbionts.</p><p>Evolutionary patterns in the tRNA^LeuUAA 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.</p><p>Protein profiles of symbiotic and free living <i>Nostoc</i> 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 <i>N. punctiforme</i> was homologous to symbiotically induced genes found in different symbiotic systems. </p><p>This thesis gives new perspectives on lichens and pr for further exaovides a platform for further examiniations using tools provided by modern biology.</p>

<p>Species of the genus <i>Rickettsia</i> 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.</p><p>This thesis presents a comparative study of evolutionary processes such as gene rearrangements, deletions and duplications in a variety of <i>Rickettsia</i> 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 <i>Rickettsia</i>. Likewise, it was found that a repetitive element, the so-called <i>Rickettsia</i> Palindromic Element (RPE) flourished prior to species divergence in <i>Rickettsia</i>. 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 <i>Rickettsia</i>. Taken together, this suggests that <i>Rickettsia</i> have been intracellular parasites for an extensive period of time.</p><p>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 <i>Rickettsia</i> species reflect species-specific differences in rates of deterioration rather than variation in rates of intra-genomic sequence proliferation. </p>

<p>Two new genes involved in important processes of plant development were identified in the model plant <i>Arabidopsis thaliana</i>. The genes were isolated from mutants generated through insertional mutagenesis based on a transposon tagging approach.</p><p>The first gene, ALB3, was isolated through the identification of the mutant <i>albino3 (alb3),</i> 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.</p><p>The second gene, SHI, was identified through the <i>short internodes</i> (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.</p><p>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.</p>

<p>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 <i>in vitro</i> 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 <i>in vitro</i> 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. </p><p>High-level expression of the HDZip gene <i>ATHB5</i> in transgenic plants results in</p><p>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 <i>Arabidopsis</i> ABA response mutants <i>abi3</i> and <i>abi5</i>. 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. </p><p>Enhanced levels of the HDZip gene ATHB13 in transgenic <i>Arabidopsis</i> 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. </p><p>Loss-of-function mutations in <i>ATHB5</i> and <i>ATHB13</i> 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. </p>

<p>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.</p><p>Pedigree analysis of large numbers of barn swallows revealed extremely high mutation frequencies for the tetranucleotide <i>HrU6</i> and pentanucleotide <i>HrU10</i> repeat loci. A detailed analysis showed that both the rate and the pattern of mutation differed significantly between the two loci.</p><p>Further analysis of <i>HrU6</i> and <i>HrU10</i> mutations, as well as mutation data for another hypermutable locus (<i>HrU9</i>) 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.</p><p>Single molecule genotyping of DNA prepared from human sperm cells was used to detect mutations at the tetranucleotide repeat <i>D21S1245.</i> 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. </p><p>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.</p><p>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.</p>

<p>The coordinated regulation of gene expression must be tightly controlled for normal development to occur. In mammals, this issue is further complicated by the requirement of both the maternal and paternal genomes for normal development, reflecting the fact that a subset of genes are monoallelically expressed depending on parental inheritance, a phenomenon known as genomic imprinting. </p><p>The imprinted <i>H19</i> and <i>Igf2 </i>genes are often considered as paradigms of genomic imprinting, since their monoallelic expression patterns are coordinated <i>via</i> a short stretch of sequence upstream of <i>H19</i>, known as the imprinting control region (ICR). This region is differentially methylated, with specific CpG methylation on the paternal allele. It is shown here that the ICR harbours several maternal-specific hypersensitive sites, located in linker regions between positioned nucleosomes. Furthermore, this region functions as an orientation-dependent insulator, that binds the chromatin insulator factor CTCF. The hypothesis that the methylation status of the ICR dictates the activity of the <i>Igf2</i> gene 90 kb further upstream was confirmed by the demonstration that the insulator function is lost when the ICR is CpG methylated. </p><p>The ICR has previously been shown to act as a silencer when positioned in a promotor-proximal position. The cause of this silencing was shown to be distance-dependent, suggesting that the silencing features of the ICR depend on a chromatin conformation that renders adjacent sequences inaccessible to the RNA polymerase. These data issue a cautionary note with respect to the interpretation of silencer functions.</p><p>In several forms of cancer, the normally silent maternal <i>IGF2</i> gene is expressed, possibly as a result of loss of insulator function at the ICR. The utilisation of CTCF target-sites was analysed in different tumours, and was shown to be highly variable. Methylation analysis showed that potential loss of insulator function and gain of methylation at the maternal ICR did not always correlate with biallelic expression of <i>IGF2</i>. Further investigations uncovered a novel mechanism, in which the activation of the <i>IGF2</i> promoter was independent of insulator function in some cancers.</p><p>This thesis shows that the regulation of the imprinted state of <i>Igf2</i> depends on the formation of an epigenetically regulated chromatin insulator, and that the loss of <i>IGF2</i> imprinting in human cancer can be attributed to several mechanisms, including a novel mechanism that neutralises chromatin insulator function.</p>