An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts) prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.

The Consequences of Phenotypic Plasticity in Cyclically Varying Environments: A Genetic Algorithm Study

By “phenotypic plasticity” we refer to the capacity of a genotype to exhibit different phenotypes, whether in the same or in different environments. We have previously demonstrated that phenotypic plasticity can improve the degree of adaptation achieved via natural selection (Behera & Nanjundiah, 1995). That result was obtained from a genetic algorithm model of haploid genotypes (idealized as one-dimensional strings of genes) evolving in a fixed environment. Here, the dynamics of evolution is examined under conditions of a cyclically varying environment. We find that the rate of evolution, as well as the extent of adaptation (as measured by mean population fitness) is lowered because of environmental cycling. The decrease is adaptation caused by a varying environment can, however, be partly or wholly compensated by an increase in the degree of plasticity that a genotype is capable of. Also, the reduction of population fitness caused by a variable environment can be partially offset by decreasing the total number of genetic loci. We conjecture that an increase in genome size may have been among the factors responsible for the evolution of phenotypic plasticity.

Secondary metabolites in Gerbera hybrida

Plants produce a diversity of secondary metabolites, i.e., low-molecular-weight compounds that have primarily ecological functions in plants. The flavonoid pathway is one of the most studied biosynthetic pathways in plants. In order to understand biosynthetic pathways fully, it is necessary to isolate and purify the enzymes of the pathways to study individual steps and to study the regulatory genes of the pathways. Chalcone synthases are key enzymes in the formation of several groups of flavonoids, including anthocyanins. In this study, a new chalcone synthase enzyme (GCHS4), which may be one of the main contributors to flower colour, was characterised from the ornamental plant Gerbera hybrida. In addition, four chalcone synthase-like genes and enzymes (GCHS17, GCHS17b, GCHS26 and GCHS26b) were studied. Spatial expression of the polyketide synthase gene family in gerbera was also analysed with quantitative RT-PCR from 12 tissues, including several developmental stages and flower types. A previously identified MYB transcription factor from gerbera, GMYB10, which regulates the anthocyanin pathway, was transferred to gerbera and the phenotypes were analysed. Total anthocyanin content and anthocyanidin profiles of control and transgenic samples were compared spectrophotometrically and with HPLC. The overexpression of GMYB10 alone was able to change anthocyanin pigmentation: cyanidin pigmentation was induced and pelargonidin pigmentation was increased. The gerbera 9K cDNA microarray was used to compare the gene expression profiles of transgenic tissues against the corresponding control tissues to reveal putative target genes for GMYB10. GMYB10 overexpression affected the expression of both early and late biosynthetic genes in anthocyanin-accumulating transgenic tissues, including the newly isolated gene GCHS4. Two new MYB domain factors, named as GMYB11 and GMYB12, were also upregulated. Gene transfer is not only a powerful tool for basic research, but also for plant breeding. However, crop improvement by genetic modification (GM) remains controversial, at least in Europe. Many of the concerns relating to both human health and to ecological impacts relate to changes in the secondary metabolites of GM crops. In the second part of this study, qualitative and quantitative differences in cytotoxicity and metabolic fingerprints between 225 genetically modified Gerbera hybrida lines and 42 non-GM Gerbera varieties were compared. There was no evidence for any major qualitative and quantitative changes between the GM lines and non-GM varieties. The developed cell viability assays offer also a model scheme for cell-based cytotoxicity screening of a large variety of GM plants in standardized conditions.

Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae

Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level. In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function. The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose. The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.

Regulation of GABAergic neuron identity and diversity in the developing midbrain

Gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the vertebrate brain. In the midbrain, GABAergic neurons contribute to the regulation of locomotion, nociception, defensive behaviours, fear and anxiety, as well as sensing reward and addiction. Despite the clinical relevance of this group of neurons, the mechanisms regulating their development are largely unknown. In addition, their migration and connectivity patterns are poorly characterized. This study focuses on the molecular mechanisms specifying the GABAergic fate, and the developmental origins of midbrain GABAergic neurons. First, we have characterized the function of a zink-finger transcription factor Gata2. Using a tissue-specific mutagenesis in mouse midbrain and anteror hindbrain, we showed that Gata2 is a crucial determinant of the GABAergic fate in midbrain. In the absence of Gata2, no GABAergic neurons are produced from the otherwise competent midbrain neuroepithelium. Instead, the Gata2-mutant cells acquire a glutamatergic neuron phenotype. Ectopic expression of Gata2 was also sufficient to induce GABAergic in chicken midbrain. Second, we have analyzed the midbrain phenotype of mice mutant for a proneural gene Ascl1, and described the variable and region-dependent requirements for Ascl1 in the midbrain GABAergic neurogenesis. These studies also have implications on the origin of distinct anatomical and functional GABAergic subpopulations in midbrain. Third, we have identified unique developmental properties of GABAergic neurons that are associated with the midbrain dopaminergic nuclei, the substantia nigra pars reticulata (SNpr) and ventral tegmental area (VTA). Namely, the genetic regulation of GABAergic fate in these cells is distinct from the rest of midbrain. In accordance to this phenomenon, our detailed fate-mapping analyses indicated that the SNpr-VTA GABAergic neurons are generated outside midbrain, in the neuroepithelium of anterior hindbrain.

Simple repetitive sequences in the genome: structure and functional significance

The current explosion of DNA sequence information has generated increasing evidence for the claim that noncoding repetitive DNA sequences present within and around different genes could play an important role in genetic control processes, although the precise role and mechanism by which these sequences function are poorly understood. Several of the simple repetitive sequences which occur in a large number of loci throughout the human and other eukaryotic genomes satisfy the sequence criteria for forming non-B DNA structures in vitro. We have summarized some of the features of three different types of simple repeats that highlight the importance of repetitive DNA in the control of gene expression and chromatin organization. (i) (TG/CA)n repeats are widespread and conserved in many loci. These sequences are associated with nucleosomes of varying linker length and may play a role in chromatin organization. These Z-potential sequences can help absorb superhelical stress during transcription and aid in recombination. (ii) Human telomeric repeat (TTAGGG)n adopts a novel quadruplex structure and exhibits unusual chromatin organization. This unusual structural motif could explain chromosome pairing and stability. (iii) Intragenic amplification of (CTG)n/(CAG)n trinucleotide repeat, which is now known to be associated with several genetic disorders, could down-regulate gene expression in vivo. The overall implications of these findings vis-à-vis repetitive sequences in the genome are summarized.

Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.

Risk factors for open angle glaucoma; a clinical and molecular genetic study

Glaucoma, optic neuropathy with excavation in the optic nerve head and corresponding visual field defect, is one of the leading causes for blindness worldwide. However, visual disability can often be avoided or delayed if the disease is diagnosed at an early stage. Therefore, recognising the risk factors for development and progression of glaucoma may prevent further damage. The purpose of the present study was to evaluate factors associated with visual disability caused by glaucoma and the genetic features of two risk factors, exfoliation syndrome (ES) and a positive family history of glaucoma. The present study material consisted of three study groups 1) deceased glaucoma patients from the Ekenäs practice 2) glaucoma families from the Ekenäs region and 3) population based families with and without exfoliation syndrome from Kökar Island. For the retrospective study, 106 patients with open angle glaucoma (OAG) were identified. At the last visit, 17 patients were visually impaired. Blindness induced by glaucoma was found in one or both eyes in 16 patients and in both eyes in six patients. The cumulative incidence of glaucoma caused blindness for one eye was 6% at 5 years, 9% at 10 years, and 15% at 15 years from initialising the treatment. The factors associated with blindness caused by glaucoma were an advanced stage of glaucoma at diagnosis, fluctuation in intraocular pressure during treatment, the presence of exfoliation syndrome, and poor patient compliance. A cross-sectional population based study performed in 1960-1962 on Kökar Island and the same population was followed until 2002. In total 965 subjects (530 over 50 years) have been examined at least once. The prevalence of exfoliation syndrome (ES) was 18% among subjects older than 50 years. Seventy-five of all 78 ES-positives belonged to the same extended pedigree. According to the segregation and family analysis, exfoliation syndrome seemed to be inherited as an autosomal dominant trait with reduced penetrance. The penetrance was more reduced for males, but the risk for glaucoma was higher in males than in females. To find the gene or genes associated with exfoliation syndrome, a genome wide scan was performed for 64 members (28 ES affected and 36 controls) of the Kökar pedigree. A promising result was found: the highest two-point LOD score of 3.45 (θ=0.04) in chromosome18q12.1-21.33. The presence of mutations in glaucoma genes TIGR/MYOC (myocilin) and OPTN (optineurin) was analysed in eight glaucoma families from the Ekenäs region. An inheritance pattern resembling autosomal dominant mode was detected in all these families. Primary open angle glaucoma or exfoliation glaucoma was found in 35% of 136 family members and 28% were suspected to have glaucoma. No mutations were detected in these families.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

'Mutiny on the Bounty': The genetic history of Norfolk Island reveals extreme gender-biased admixture

Background The Pacific Oceania region was one of the last regions of the world to be settled via human migration. Here we outline a settlement of this region that has given rise to a uniquely admixed population. The current Norfolk Island population has arisen from a small number of founders with mixed Caucasian and Polynesian ancestry, descendants of a famous historical event. The ‘Mutiny on the Bounty’ has been told in history books, songs and the big screen, but recently this story can be portrayed through comprehensive molecular genetics. Written history details betrayal and murder leading to the founding of Pitcairn Island by European mutineers and the Polynesian women who left Tahiti with them. Investigation of detailed genealogical records supports historical accounts. Findings Using genetics, we show distinct maternal Polynesian mitochondrial lineages in the present day population, as well as a European centric Y-chromosome phylogeny. These results comprehensively characterise the unique gender-biased admixture of this genetic isolate and further support the historical records relating to Norfolk Island. Conclusions Our results significantly refine previous population genetic studies investigating Polynesian versus Caucasian diversity in the Norfolk Island population and add information that is beneficial to future disease and gene mapping studies.

Genetic and epigenetic variants in the MTHFR gene are not associated with non-Hodgkin lymphoma

The methylenetetrahydrofolate reductase (MTHFR) gene codes for the MTHFR enzyme which plays a key role in the pathway of folate and methionine metabolism. Polymorphisms of genes in this pathway affect its regulation and have been linked to lymphoma. In this study we examined whether we could detect an association between two common non-synonomous MTHFR polymorphisms, 677C>T (rs1801133) and 1298A>C (rs1801131), and susceptibility to non-Hodgkin lymphoma (NHL) in an Australian case-control cohort. We found no significant differences between genotype or allele frequencies for either polymorphisms between lymphoma cases and controls. We also explored whether epigenetic modification of MTHFR, specifically DNA methylation of a CpG island in the MTHFR promoter region, is associated with NHL using blood samples from patients. No difference in methylation levels was detected between the case and control samples suggesting that although hypermethylation of MTHFR has been reported in tumour tissues, particularly in the diffuse large B-cell lymphoma subtype of NHL, methylation of this MTHFR promoter CpG island is not a suitable epigenetic biomarker for NHL diagnosis or prognosis in peripheral blood samples. Further studies into epigenetic variants could focus on genes that are robustly associated with NHL susceptibility.