Errata slip inserted at back / Bibliography: leaves 159-192 / vi, 142, [75] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Primary object ... was to construct a pulsed field gel electrophoresis (PFGE) derived long range restriction map of this region by physically linking adjacent DNA probes to common high molecular weight genomic DNA fragments / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1994

The human genome contains ~1.5% coding sequence, with the remaining 98.5% being non-coding. The functional potential of the majority of this non-coding sequence remains unknown. Much of this non-coding sequence is derived from transposable element (TE) sequences. These TE sequences contain their own regulatory information, e.g. promoter and transcription factor binding sites. Given the large number of these sequences, over 4 million in the human genome, it would be expected that the regulatory information that they contain would affect the expression of nearby genes. This dissertation describes research that characterizes that alternation of and contribution to the human transcriptome by non-coding elements, including TE sequences.

Seven years into the postgenomic era, we sit atop a mountain of data whose generation was enabled by gene sequencing. The creation, integration, and analysis of these large scale data sets allow us to move forward toward the complementary goals of determining the individual roles of the thousands of uncharacterized mammalian genes and understanding how they work together to produce a healthy human being -- or, perhaps more importantly, how their malfunction results in disease. Collapsing the results of large-scale assays into gene networks provides a useful framework from which we can glean information that advances both of these goals. However, the utility of networks is limited by the quality of the data that goes into them. This study offers seeks to shed some light on the quality and breadth of protein interaction networks, describes a new experimental technique for functional genetic assays in mammalian cell lines, and ultimately suggests a strategy for how to improve the overall utility of the output generated by the systems biology community. / text

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

With the advent of large scale genome sequencing efforts along with more sophisticated methods of genetic mapping, a number of loci have been identified that are associated with human diseases. Intriguingly, many genes identified in these loci remain uncharacterized. Although current annotation can provide a prediction of putative function of some of these proteins at a biochemical level, understanding their cellular roles require analysis at a single gene level. Bioinformatic analysis carried out in the laboratory during studies on cyclic nucleotide metabolism in mycobacteria identified putative Class III cyclic nucleotide phosphodiesterases (Class III cNMP PDEs) from the non-redundant database of proteins. One of the proteins identified was the Rv0805 gene product from Mycobacterium tuberculosis. Detailed biochemical characterization of this protein revealed that Rv0805 is indeed a phosphodiesterase (PDE) and could hydrolyze 3’, 5’-cyclic adenosine monophosphate (cAMP) as well as 3’, 5’-cyclic guanosine monophosphate (cGMP). Structural analysis of Rv0805 revealed a metallophosphoesterase (MPE) like fold and presence of two metal atoms at the binuclear metal centre of the protein. Moreover, overexpression of Rv0805 in E. coli and M. smegmatis reduced intracellular cAMP levels indicating that it possesses cAMP PDE activity in vivo. The majority of proteins identified in this bioinformatic analysis were of bacterial or archaebacterial in origin but it was interesting to find some mammalian proteins, since, till date, no Class III cNMP PDE has been found in higher eukaryotes. Interestingly, two genes were identified in the human genome. These genes, 239FB and 239AB, are expressed in the fetal brain and adult brain, respectively and have been annotated as metallophosphoesterases but there has been no biochemical or functional characterization of these proteins. The 239FB gene is present between the FSHB and PAX6 genes on chromosome 11. This gene locus is present within a deletion interval (11p13-14) that is associated with the mental retardation phenotype of WAGR syndrome (Wilms’ tumor, aniridia, genitourinary anomalies, mental retardation). Inspection of available sequenced mammalian genomes indicated a shared synteny of the genes in the WAGR locus, highlighting it’s evolutionary conservation. Most interestingly, nucleotide sequences within the WAGR locus (which include the 5 genes WT1, PAX6, RCN1, ELP4 and 239FB) are amongst the 481 ultra conserved regions of the human genome. Moreover, 239FB is one of only 24 instances where an ortholog of an ultra-conserved element could be partially traced back by sequence similarity in lower eukaryotes such as Ciona intestinalis, Drosophila melanogaster, or Caenorhabditis elegans. Although the function of the 239FB protein is unknown so far, the distinctive expression of the gene in the fetal brain and the presence of an “ancient conserved region” in this gene suggest that this gene may be vital for the development of the nervous system. The work carried out in this thesis has attempted to understand the physiological functions of the 239FB/AB gene family. Amino acid sequence comparison revealed two amino acids changes between the human and rat proteins indicating the extra-ordinary sequence conservation of these proteins. Therefore, to characterize the biochemical properties of 239FB and 239AB proteins, rat proteins were used as model enzymes. Reverse transcription-PCR analysis of RNA prepared from the fetal and adult rat brains as well as Western blot analysis on cytosolic fractions of rat brains from various developmental stages indicated that 239FB is predominantly expressed in fetal brain. Detailed biochemical analyses of the rat 239FB and 239AB proteins were performed which showed that they possess metallophosphodiesterase activity. 239FB showed activity only in the presence of Mn2+ and Co2+ as the added metal cofactors. Surprisingly, the Km for Mn2+ of 239FB was found to be 1.5 mM, which is nearly 60-fold higher than that of its mycobacterial ortholog, Rv0805. A systematic mutational analysis was performed to characterize the residues that are involved in binding either one or both the metals found in the catalytic site of 239FB. Although 239FB shares some of the residues that have been shown to be essential for metal binding and catalytic activity with other MPEs including Rv0805, there are some differences as well. One histidine residue that has been conserved in other MPEs and has been shown to be important for metal binding is replaced by glycine (Gly-252) in 239FB. To study the consequence of replacing the glycine with a histidine in 239FB, a 239FBGly252His mutant protein was generated and characterized. Interestingly, the single mutation of Gly-252 to a histidine residue not only increased the affinity of the protein for metals but increased catalytic activity as well with various phosphodiesters. Moreover, 239FBGly252His mutant protein showed significant activity with cAMP and cGMP which were not hydrolysed by wild type 239FB. Interestingly, in the 239AB protein, histidine 284 is present at a position equivalent to Gly-252 in the 239FB protein. Biochemical characterization of 239AB showed 2’, 3’-cAMP hydrolyzing activity similar to 239FBGly252His mutant protein. A rat 239FB protein with a mutation (His67Arg) corresponding to a single nucleotide polymorphism seen in human 239FB, led to complete inactivation of the protein. The occurrence of this SNP at a very low frequency and only as a heterozygous condition suggests that a complete loss-of-function mutation of 239FB in human populations cannot be tolerated. To gain insights into the function of 239FB in its physiological milieu, yeast two-hybrid screening was performed with 239FB using human fetal brain cDNA library. Dipeptidyl peptidase III, a zinc dependent metallopeptidase, was found as an interacting partner of 239FB in this analysis and the functional consequences of this interaction would be an interesting area of study in future. While a number of metallophosphoesterases have been characterized biochemically and structurally, their biological role(s) and in vivo substrate(s) remain elusive. In order to elucidate the physiological role of 239FB/AB family, the ortholog of 239FB/AB in D. melanogaster was characterized. Sequence comparison of Drosophila ortholog with both the mammalian proteins indicated that it may be an ortholog of 239AB and hence, it was named as d239AB. Enhancer-promoter analysis with a putative promoter region of the d239AB indicated the expression of d239AB in the mushroom bodies in brain and in enterocytes in mid gut. Characterization of a Drosophila line, BS#16242, with a piggybac element inserted in the intron of d239AB showed disruption of d239AB expression. This suggested that BS#16242 line can serve as a d239AB knockout line and hence, was selected for further phenotypic characterization to unravel the physiological roles of d239AB. Though, BS#16242 flies did not show any developmental defects, a severe reduction in the fecundity of these files was observed. Further analysis revealed defective ovulation as a probable reason for reduced fecundity of these flies. In addition to compromised fecundity, BS#16242 flies showed a significant reduction in the life span of male as well as female flies. Moreover, these flies showed less resistance to thermal stress and desiccation. Most interestingly, all these phenotypes were rescued upon neuronal expression of the d239AB transgene in BS#16242 flies indicating that neuronal function of d239AB is important for diverse physiological processes. The phenotypes observed in BS#16242 flies mimic the physiological state under increased insulin signaling, such as decrease in life span, and susceptibility to various stress conditions suggesting that d239AB could play a role in the insulin signaling pathway. Interestingly, overexpression of d239AB transgene in neurons reduced cAMP levels in the brains of Drosophila, indicating that the protein may have cAMP phosphodiesterase activity in vivo. This is the first analysis of the presence of a Class III phosphodiesterase in eukaryotes. Thus, d239AB mediated regulation of cAMP levels in a particular subsets of cells, such as neurons, could also be one of the molecular mechanisms responsible for reduced fecundity and longevity of BS#16242 flies. Interacting partners of d239AB were inspected in the Drosophila interactome (built on protein-protein interactions identified using a yeast two-hybrid approach). Strikingly, most of the d239AB interacting proteins were involved either in transcriptional or translational regulation indicating that d239AB could be involved in the regulation of expression of genes involved in diverse physiological processes. This could explain why disruption of d239AB led to various physiological defects such as reduced fecundity, decreased life span and compromised fitness. In summary, studies described in this thesis suggest that 239FB and 239AB proteins are the first Class III cyclic nucleotide phosphodiesterases reported in eukaryotes. Results shown here suggest the critical role of their ortholog in the physiology of Drosophila. Further genetic manipulation in D. melanogaster and other organisms which harbor orthologs of the 239FB/AB gene could throw light on the diverse biological roles of these enzymes in humans.

[Truncated abstract] Polymorphic Alu insertions (POALINs) are known to contribute to the variation and genetic diversity of the human genome. In this report specific POALINs of the Major Histocompatibility Complex (MHC) were studied. Previous population studies on the MHC POALINs have focused on individuals of African, European and Asian descent. In this study, we expand the research by studying a new and previously uncharacterised population, focusing on the Bedouin from the Middle East. Specifically we report on the individual insertion frequencies of four POALINs within the MHC class I region of this population. POALINs are members of a young Alu subfamily that have only recently been inserted into the human genome. POALINs are either present or absent at particular sites. Individuals that share the inserted (or deleted) polymorphism inherited the insertion (or deletion) from a common ancestor, making Alu alleles identical by decent. In population genetics a comparison of the resulting products from each population can then be done by comparing the lengths of the PCR products in a series of unrelated individuals and may also detect polymorphisms with regard to the presence or absence of the Alu repeats. As a direct result of their abundance and sequence identity, they promote genetic recombination events that are responsible for large-scale deletions, duplication and translocations. The deletions occur mostly in the A-T rich regions and have found to be unlikely to have been created independently of the insertions of the Alu elements (Callinan et al, 2005) The easy genotyping of the POALINs has proven to be very valuable as lineage markers for the study of human population genetics, pedigree and forensics as well as genomic diversity and evolution. POALINs have been used in a range of applications, primarily focusing on anthropological analysis of human populations. As a result of its ease of use and its utility as a marker in human evolutions studies, combining the POALINs along with other markers used in forensics could lead to improved identity testing in forensic science. More specifically, in combination with more traditional markers, race specific genotypes and haplotypes could be used for profiling crime scene samples. ... This is supported by previously reported molecular data using various types of genetic markers. In a study using six separate Alu genes, Antunez-de-Mayolo et al were able to generate a phylogenetic tree, in which the biogeographical groups followed a pattern. The biogeographical groups started with African populations that were found to relate closely to the hypothetical ancestral African population. The African populations were then followed in order by Southwest Asian populations, European populations which include Middle Eastern groups (Antunez-de-Mayolo et al, 2002). This study shows the similarities and differences between the frequencies of the Middle Eastern Bedouin and the rest of the compared populations. Though no clear results were determined, the information from the POALINs along with information provided from other genetic markers can lead to further research on the Bedouin population and the improvement of the forensic population database in order to accurately test individual ethnic background of samples to be analysed.

Thesis (M. A.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 118-130). Also available via the World Wide Web

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Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer's disease (AD) is the most common form of dementia defined by loss in memory and cognitive abilities severe enough to interfere significantly with daily life activities. Amnestic mild cognitive impairment (MCI) is a clinical condition in which an individual has memory deficits not normal for the individual's age, but not severe enough to interfere significantly with daily functioning. Every year, approximately 10-15% of individuals with MCI will progress to dementia. Currently, there is no treatment to slow or halt AD progression, but research studies are being conducted to identify causes that can lead to its earlier diagnosis and treatment. Genetic variation plays a key role in the development of AD, but not all genetic factors associated with the disease have been identified. Copy number variants (CNVs), a form of genetic variation, are DNA regions that have added genetic material (duplications) or loss of genetic material (deletions). The regions may overlap one or more genes possibly affecting their function. CNVs have been shown to play a role in certain diseases. At the start of this work, only one published study had examined CNVs in late-onset AD and none had examined MCI. In order to determine the possible involvement of CNVs in AD and MCI susceptibility, genome-wide CNV analyses were performed in participants from three cohorts: the ADNI cohort, the NIA-LOAD/NCRAD Family Study cohort, and a unique cohort of clinically characterized and neuropathologically verified individuals. Only participants with DNA samples extracted from blood/brain tissue were included in the analyses. CNV calls were generated using genome-wide array data available on these samples. After detailed quality review, case (AD and/or MCI)/control association analyses including candidate gene and genome-wide approaches were performed. Although no excess CNV burden was observed in cases compared to controls in the three cohorts, gene-based association analyses identified a number of genes including the AD candidate genes CHRFAM7A, RELN and DOPEY2. Thus, the present work highlights the possible role of CNVs in AD and MCI susceptibility warranting further investigation. Future work will include replication of the findings in independent samples and confirmation by molecular validation experiments.