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A recently isolated human alphoid DNA (in plasmid pHH550) has been sequenced and found to have an exceptionally high degree of similarity to the human alphoid consensus sequence, while its component monomers are unusually heterogeneous in sequence. In contrast to other alphoid DNAs, this DNA is found in all primates tested. Thus this may be an evolutionarily old sequence similar to the one from which other human alphoid DNAs diverged. The pHH550 sequences are found on a number of human chromosomes, including 21 and 22. On chromosome 21 most members of this new sequence group are located distal to other alphoid DNAs.

With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function. This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis. More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses. <b>Keywords:</b>functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.

<p>With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function.</p><p>This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis.</p><p>More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses.</p><p><b>Keywords:</b>functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and occurs in ~1 of every 700 live births. Individuals with DS present craniofacial abnormalities, specifically an undersized, dysmorphic mandible which may lead to difficulty with eating, breathing, and speech. Using the Ts65Dn DS mouse model, which mirrors these phenotypes and contains three copies of ~50% Hsa21 homologues, our lab has traced the mandibular deficit to a neural crest cell (NCC) deficiency in the first pharyngeal arch (PA1 or mandibular precursor) at embryonic day 9.5 (E9.5). At E9.5, the PA1 is reduced in size and contains fewer cells due to fewer NCC populating the PA1 from the neural tube (NT) as well as reduced cellular proliferation in the PA1. We hypothesize that both the deficits in NCC migration and proliferation may cause the reduction in size of the PA1. To identify potential genetic mechanisms responsible for trisomic PA1 deficits, we generated RNA-sequence (RNA-seq) data from euploid and trisomic E9.25 NT and E9.5 PA1 (time points occurring before and after observed deficits) using a next-generation sequencing platform. Analysis of RNA-seq data revealed differential trisomic expression of 53 genes from E9.25 NT and 364 genes from E9.5 PA1, five of which are present in three copies in Ts65Dn. We also further analyzed the data to find that fewer alternative splicing events occur in trisomic tissues compared to euploid tissues and in PA1 tissue compared to NT tissue. In a subsequent study, to test gene-specific treatments to rescue PA1 deficits, we targeted Dyrk1A, an overexpressed DS candidate gene implicated in many DS phenotypes and predicted to cause the NCC and PA1 deficiencies. We hypothesize that treatment of pregnant Ts65Dn mothers with Epigallocatechin gallate (EGCG), a known Dyrk1A inhibitor, will correct NCC deficits and rescue the undersized PA1 in trisomic E9.5 embryos. To test our hypothesis, we treated pregnant Ts65Dn mothers with EGCG from either gestational day 7 (G7) to G8 or G0 to G9.5. Our study found an increase in PA1 volume and NCC number in trisomic E9.5 embryos after treatment on G7 and G8, but observed no significant improvements in NCC deficits following G0-G9.5 treatment. We also observed a developmental delay of embryos from trisomic mothers treated with EGCG from G0-G9.5. Together, these data show that timing and sufficient dosage of EGCG treatment is most effective during the developmental window the few days before NCC deficits arise, during G7 and G8, and may be ineffective or harmful when administered at earlier developmental time points. Together, the findings of both studies offer a better understanding of potential mechanisms altered by trisomy as well as preclinical evidence for EGCG as a potential prenatal therapy for craniofacial disorders linked to DS.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the result of trisomy of human chromosome 21 (Hsa 21) and occurs in approximately 1/700 live births. Mouse models of DS have been crucial in understanding the gene-phenotype relationships that underlie many DS anomalies. The Ts65Dn mouse model, trisomic for half of the Hsa 21 orthologs replicates many DS phenotypes including craniofacial alterations such as a small, dysmorphic mandible, midface, and maxilla. Other mouse models, such as the Ts1Rhr which contains a triplication of 33 Hsa 21 orthologs, have been used to better understand the genes responsible for craniofacial alterations. Our laboratory has demonstrated that the postnatal mandibular phenotype found in Ts65Dn mice can be traced back to an original neural crest cell (NCc) deficit in the developing first pharyngeal arch (PA1) at embryonic day 9.5 (E9.5). Furthermore, evidence suggested that both a proliferation deficit in the PA1 and a migration deficit in the NCC from the neural tube (NT) could be the mechanism behind this deficit. However, the molecular mechanisms behind these deficits remain to be elucidated. Due to the involvement of the Hsa 21 genes DYRK1A and RCAN1 in regulation of signaling pathways including NFATc (NFAT2), a transcription factor known to influence cellular proliferation and, later, bone development, we hypothesized that dysregulation of these genes could underlie the cellular deficit in the PA1. Furthermore, we hypothesized that targeting Dyrk1a by decreasing activity or available protein could ameliorate the established deficits. Through the use of RNA isolation techniques and cell culture systems of cell from the PA1 and NT of E9.5 Ts65Dn, Ts1Rhr, and control embryos, we established that trisomic genes Dyrk1a and Rcan1 ara dysregulated in both structures and that these two genes may interact. Furthermore, we established that a proliferation deficit in the Ts65Dn PA1 and a migration deficit in the Ts65Dn PA1 and NT exists at E9.5 and can be rescued to euploid levels in vitro with the addition of the Dyrk1a inhibitor, EGCG, a green tea polyphenol. We also confirmed that harmine, a more highly studied and specific Dyrk1a inhibitor, is capable of similar effects on proliferation of PA1 cell from E9.5 Ts65Dn embryos. Furthermore, when Ts65Dn pregnant mothers were treated with EGCG in vivo, the cellular deficit found in the developing E9.5 embryonic PA1 was rescued to near euploid volume and NCC number. Treatment with EGCG did not adversely impact litter size or embryonic development. Interestingly, euploid embryonic volume increased with EGCG treatment. Expression analysis of the E9.5 PA1 of EGCG treated Ts65Dn and control embryos revealed dysregulation of several genes involved in craniofacial and developmental pathways including Dyrk1a, Rcan1, Ets2 and members of the sonic hedgehog pathways. Our novel results provide a foundation for better understanding the molecular mechanisms of craniofacial development and may provide evidence-based therapeutic options to improve the quality of life for individuals with DS.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in an array of phenotypes including intellectual disability. Ts65Dn mice, the most extensively studied DS model, have three copies of ~50% of the genes on Hsa21 and display many phenotypes associated with DS, including cognitive deficits. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including CNS development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have shown that a three-week EGCG treatment (~10mg/kg/day) during adolescence normalizes skeletal abnormalities in Ts65Dn mice, yet the same dose did not rescue deficits in the Morris water maze spatial learning task (MWM) or novel object recognition (NOR). Others have reported that An EGCG dose of 2-3 mg per day (90mg/ml) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated deficits in a radial arm maze pattern separation task in Ts65Dn mice. Pattern separation requires differentiation between similar memories acquired during learning episodes; distinguishing between these similar memories is thought to depend on distinctive encoding in the hippocampus. Pattern separation has been linked to functional activity of newly generated granule cells in the dentate gyrus. Recent studies in Ts65Dn mice have reported significant reductions in adult hippocampal neurogenesis, and after EGCG treatment, enhanced hippocampal neurogenesis. Thus, it was hypothesized that Ts65Dn mice would be impaired in the pattern separation task, and that EGCG would alleviate the pattern separation deficits seen in trisomic mice, in association with increased adult hippocampal neurogenesis. At weaning, Ts65Dn mice and euploid littermates were randomly assigned to the water control, or EGCG [0.4 mg/mL], with both treatments yielding average daily intakes of ~50 mg/kg/day. Beginning on postnatal day 75, all mice were trained on a radial arm maze-delayed non-matching-to-place pattern separation task. Euploid mice performed significantly better over training than Ts65Dn mice, including better performance at each of the three separations. EGCG did not significantly alleviate the pattern separation deficits in Ts65Dn mice. After the behavioral testing commenced, animals were given ad libitum food access for five days, received a 100mg/kg injection of BrdU, and were perfused two hours later. Coronal sections through the dorsal hippocampus were processed for BrdU labeling, and cells were manually counted throughout the subgranular zone of the dentate gyrus. The euploid controls had significantly more BrdU labeled cells than Ts65Dn mice, however, EGCG does not appear to increase proliferation of the hippocampal neuroprogenitor cells. This is the first report of deficits in Ts65Dn mice on a pattern separation task. To the extent that pattern separation depends on the functional involvement of newly generated neurons in an adult dentate gyrus, this approach in Ts65Dn mice may help identify more targeted pharmacotherapies for cognitive deficits in individuals with DS.