Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF.

BACKGROUND: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies. RESULTS: The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections. CONCLUSION: E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.

Whole genome analysis of Fusobacterium nucleatum, Rickettsia typhi and Francisella tularensis

The genomes of Fusobacterium nucleatum subspecies polymorphum strain ATCC 10953, Rickettsia typhi strain Wilmington, and Francisella tularensis subspecies holarctica strain OSU18 were sequenced, annotated, and analyzed. Each genome was then compared to the sequenced genomes of closely related bacteria. The genome of F. nucleatum ATCC 10953 was compared to two additional F. nucleatum subspecies, subspecies nucleatum and subspecies vincentii. This analysis revealed substantial evidence of horizontal gene transfer along with considerable genetic diversity within the species of F. nucleatum. R. typhi was compared to R. prowazekii and R. conorii. This analysis uncovered a hotspot for chromosomal rearrangements in the Spotted Fever Group but not the Typhus Group Rickettsia and revealed the close genetic relationship between the Typhus Group rickettsial species. F. tularensis OSU18 was compared to two additional F. tularensis strains. These comparisons uncovered significant chromosomal rearrangements between F. tularensis subspecies due to recombination between insertion sequence elements. ^

Human TNF receptor p75/80 gene structure and promoter characterization

Tumor necrosis factor receptor p75/80 ((TNF-R p75/80) is a 75 kDa type 1 transmembrane protein expressed predominately on cells of hematopoietic lineage. TNF-R p75/80 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report, we have characterized, for the first time, the complete gene structure for human TNF-R p75/80 which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 bp to 2.5 kbp) and 9 introns (343 bp to 19 kbp). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5$\sp\prime$ flanking region including TCF-1, Ikaros, AP-1, CK-2, IL-6RE, ISRE, GAS, NF-$\kappa$B and SP1, as well as an unusually high GC content and CpG frequency that appears characteristic of some TNF-R family members. The unusual (GATA)$\sb{\rm n}$ and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. The human TNF-R p75/80 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and non-random translocations in hematopoietic malignancies. The region 1.8 kb 5$\sp\prime$ of the ATG was able to drive luciferase expression when transfected into cell lines expressing TNF-R p75/80. Further characterization of the 5$\sp\prime$-regulatory region will aid in determining factors and signal transduction pathways involved in regulating TNF-R p75/80 expression. ^

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Genome-wide gene-gene interaction analysis for cardiovascular disease

Numerous studies have been carried out to try to better understand the genetic predisposition for cardiovascular disease. Although it is widely believed that multifactorial diseases such as cardiovascular disease is the result from effects of many genes which working alone or interact with other genes, most genetic studies have been focused on identifying of cardiovascular disease susceptibility genes and usually ignore the effects of gene-gene interactions in the analysis. The current study applies a novel linkage disequilibrium based statistic for testing interactions between two linked loci using data from a genome-wide study of cardiovascular disease. A total of 53,394 single nucleotide polymorphisms (SNPs) are tested for pair-wise interactions, and 8,644 interactions are found to be significant with p-values less than 3.5×10-11. Results indicate that known cardiovascular disease susceptibility genes tend not to have many significantly interactions. One SNP in the CACNG1 (calcium channel, voltage-dependent, gamma subunit 1) gene and one SNP in the IL3RA (interleukin 3 receptor, alpha) gene are found to have the most significant pair-wise interactions. Findings from the current study should be replicated in other independent cohort to eliminate potential false positive results.^

Pathway analysis of genome-wide association study data in primary biliary cirrhosisa

Genome-wide association studies (GWAS) have successfully identified several genetic loci associated with inherited predisposition to primary biliary cirrhosis (PBC), the most common autoimmune disease of the liver. Pathway-based tests constitute a novel paradigm for GWAS analysis. By evaluating genetic variation across a biological pathway (gene set), these tests have the potential to determine the collective impact of variants with subtle effects that are individually too weak to be detected in traditional single variant GWAS analysis. To identify biological pathways associated with the risk of development of PBC, GWAS of PBC from Italy (449 cases and 940 controls) and Canada (530 cases and 398 controls) were independently analyzed. The linear combination test (LCT), a recently developed pathway-level statistical method was used for this analysis. For additional validation, pathways that were replicated at the P <0.05 level of significance in both GWAS on LCT analysis were also tested for association with PBC in each dataset using two complementary GWAS pathway approaches. The complementary approaches included a modification of the gene set enrichment analysis algorithm (i-GSEA4GWAS) and Fisher's exact test for pathway enrichment ratios. Twenty-five pathways were associated with PBC risk on LCT analysis in the Italian dataset at P<0.05, of which eight had an FDR<0.25. The top pathway in the Italian dataset was the TNF/stress related signaling pathway (p=7.38×10 -4, FDR=0.18). Twenty-six pathways were associated with PBC at the P<0.05 level using the LCT in the Canadian dataset with the regulation and function of ChREBP in liver pathway (p=5.68×10-4, FDR=0.285) emerging as the most significant pathway. Two pathways, phosphatidylinositol signaling system (Italian: p=0.016, FDR=0.436; Canadian: p=0.034, FDR=0.693) and hedgehog signaling (Italian: p=0.044, FDR=0.636; Canadian: p=0.041, FDR=0.693), were replicated at LCT P<0.05 in both datasets. Statistically significant association of both pathways with PBC genetic susceptibility was confirmed in the Italian dataset on i-GSEA4GWAS. Results for the phosphatidylinositol signaling system were also significant in both datasets on applying Fisher's exact test for pathway enrichment ratios. This study identified a combination of known and novel pathway-level associations with PBC risk. If functionally validated, the findings may yield fresh insights into the etiology of this complex autoimmune disease with possible preventive and therapeutic application.^

Gene-gene interaction and gene-pathway analysis of genome-wide association for schizophrenia

Schizophrenia (SZ) is a complex disorder with high heritability and variable phenotypes that has limited success in finding causal genes associated with the disease development. Pathway-based analysis is an effective approach in investigating the molecular mechanism of susceptible genes associated with complex diseases. The etiology of complex diseases could be a network of genetic factors and within the genes, interaction may occur. In this work we argue that some genes might be of small effect that by itself are neither sufficient nor necessary to cause the disease however, their effect may induce slight changes to the gene expression or affect the protein function, therefore, analyzing the gene-gene interaction mechanism within the disease pathway would play crucial role in dissecting the genetic architecture of complex diseases, making the pathway-based analysis a complementary approach to GWAS technique. ^ In this study, we implemented three novel linkage disequilibrium based statistics, the linear combination, the quadratic, and the decorrelation test statistics, to investigate the interaction between linked and unlinked genes in two independent case-control GWAS datasets for SZ including participants of European (EA) and African (AA) ancestries. The EA population included 1,173 cases and 1,378 controls with 729,454 genotyped SNPs, while the AA population included 219 cases and 288 controls with 845,814 genotyped SNPs. We identified 17,186 interacting gene-sets at significant level in EA dataset, and 12,691 gene-sets in AA dataset using the gene-gene interaction method. We also identified 18,846 genes in EA dataset and 19,431 genes in AA dataset that were in the disease pathways. However, few genes were reported of significant association to SZ. ^ Our research determined the pathways characteristics for schizophrenia through the gene-gene interaction and gene-pathway based approaches. Our findings suggest insightful inferences of our methods in studying the molecular mechanisms of common complex diseases.^

Nonlinear dimension reduction in pathway-based genome-wide association analysis

Pathway based genome wide association study evolves from pathway analysis for microarray gene expression and is under rapid development as a complementary for single-SNP based genome wide association study. However, it faces new challenges, such as the summarization of SNP statistics to pathway statistics. The current study applies the ridge regularized Kernel Sliced Inverse Regression (KSIR) to achieve dimension reduction and compared this method to the other two widely used methods, the minimal-p-value (minP) approach of assigning the best test statistics of all SNPs in each pathway as the statistics of the pathway and the principal component analysis (PCA) method of utilizing PCA to calculate the principal components of each pathway. Comparison of the three methods using simulated datasets consisting of 500 cases, 500 controls and100 SNPs demonstrated that KSIR method outperformed the other two methods in terms of causal pathway ranking and the statistical power. PCA method showed similar performance as the minP method. KSIR method also showed a better performance over the other two methods in analyzing a real dataset, the WTCCC Ulcerative Colitis dataset consisting of 1762 cases, 3773 controls as the discovery cohort and 591 cases, 1639 controls as the replication cohort. Several immune and non-immune pathways relevant to ulcerative colitis were identified by these methods. Results from the current study provided a reference for further methodology development and identified novel pathways that may be of importance to the development of ulcerative colitis.^

Network-based analysis of genome-wide scans of natural selection

The genomic era brought by recent advances in the next-generation sequencing technology makes the genome-wide scans of natural selection a reality. Currently, almost all the statistical tests and analytical methods for identifying genes under selection was performed on the individual gene basis. Although these methods have the power of identifying gene subject to strong selection, they have limited power in discovering genes targeted by moderate or weak selection forces, which are crucial for understanding the molecular mechanisms of complex phenotypes and diseases. Recent availability and rapid completeness of many gene network and protein-protein interaction databases accompanying the genomic era open the avenues of exploring the possibility of enhancing the power of discovering genes under natural selection. The aim of the thesis is to explore and develop normal mixture model based methods for leveraging gene network information to enhance the power of natural selection target gene discovery. The results show that the developed statistical method, which combines the posterior log odds of the standard normal mixture model and the Guilt-By-Association score of the gene network in a naïve Bayes framework, has the power to discover moderate/weak selection gene which bridges the genes under strong selection and it helps our understanding the biology under complex diseases and related natural selection phenotypes.^

GENETIC ANALYSIS OF THE FUNCTION OF THE DROSOPHILA DOUBLESEX-RELATED FACTOR dmrt93B

DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be expressed sex-specifically in the developing somatic gonad and to affect testis morphogenesis in RNAi knockdowns. In order to disrupt this gene, the GAL4 yeast transcriptional activator followed by a polyadenylation signal was inserted after the dmrt93B start codon and introduced into the genome by homologous recombination. Analysis of the knock-in mutation as well as a small deletion removing all dmrt93B sequence demonstrate that loss of function causes partial lethality at the late pupal stage. Surprisingly, these mutations have no significant effect on gonad formation or male fertility. Analysis of GAL4-driven GFP reporter expression indicates that the dmrt93B promoter activity is highly specific to neurons in the suboesophageal and proventricular ganglion in larva and adult of both sexes suggesting a possible role in digestive tract function. Using the Capillary Feeder (CAFÉ) assay to measure daily food intake we find that reduction in this gene’s function leads to an increase in food consumption. These results suggest dmrt93 plays an important role in the formation or maintenance of neurons that affect feeding and support the idea that dmrt genes may not be restricted to roles in sexual differentiation.

HLA-DPB1 and DPB2 are genetic loci for systemic sclerosis: a genome-wide association study in Koreans with replication in North Americans.

OBJECTIVE: To identify systemic sclerosis (SSc) susceptibility loci via a genome-wide association study. METHODS: A genome-wide association study was performed in 137 patients with SSc and 564 controls from Korea using the Affymetrix Human SNP Array 5.0. After fine-mapping studies, the results were replicated in 1,107 SSc patients and 2,747 controls from a US Caucasian population. RESULTS: The single-nucleotide polymorphisms (SNPs) (rs3128930, rs7763822, rs7764491, rs3117230, and rs3128965) of HLA-DPB1 and DPB2 on chromosome 6 formed a distinctive peak with log P values for association with SSc susceptibility (P=8.16x10(-13)). Subtyping analysis of HLA-DPB1 showed that DPB1*1301 (P=7.61x10(-8)) and DPB1*0901 (P=2.55x10(-5)) were the subtypes most susceptible to SSc in Korean subjects. In US Caucasians, 2 pairs of SNPs, rs7763822/rs7764491 and rs3117230/rs3128965, showed strong association with SSc patients who had either circulating anti-DNA topoisomerase I (P=7.58x10(-17)/4.84x10(-16)) or anticentromere autoantibodies (P=1.12x10(-3)/3.2x10(-5)), respectively. CONCLUSION: The results of our genome-wide association study in Korean subjects indicate that the region of HLA-DPB1 and DPB2 contains the loci most susceptible to SSc in a Korean population. The confirmatory studies in US Caucasians indicate that specific SNPs of HLA-DPB1 and/or DPB2 are strongly associated with US Caucasian patients with SSc who are positive for anti-DNA topoisomerase I or anticentromere autoantibodies.

Phenotypic and genotypic analysis of mouse hepatitis virus (JHM) complementation group A temperature-sensitive mutants

Temperature sensitive (ts) mutant viruses have helped elucidate replication processes in many viral systems. Several panels of replication-defective ts mutants in which viral RNA synthesis is abolished at the nonpermissive temperature (RNA$\sp{-})$ have been isolated for Mouse Hepatitis Virus, MHV (Robb et al., 1979; Koolen et al., 1983; Martin et al., 1988; Schaad et al., 1990). However, no one had investigated genetic or phenotypic relationships between these different mutant panels. In order to determine how the panel of MHV-JHM RNA$\sp{-}$ ts mutants (Robb et al., 1979) were genetically related to other described MHV RNA$\sp{-}$ ts mutants, the MHV-JHM mutants were tested for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). The three ts mutant panels together were found to comprise eight genetically distinct complementation groups. Of these eight complementation groups, three complementation classes are unique to their particular mutant panel; genetically equivalent mutants were not observed within the other two mutant panels. Two complementation groups were common to all three mutant panels. The three remaining complementation groups overlapped two of the three mutant sets. Mutants MHV-JHM tsA204 and MHV-A59 ts261 were shown to be within one of these overlapping complementation groups. The phenotype of the MHV-JHM mutants within this complementation class has been previously characterized (Leibowitz et al., 1982; Leibowitz et al, 1990). When these mutants were grown at the permissive temperature, then shifted up to the nonpermissive temperature at the start of RNA synthesis, genome-length RNA and leader RNA fragments accumulated, but no subgenomic mRNA was synthesized. MHV-A59 ts261 produced leader RNA fragments identical to those observed with MHV-JHM tsA204. Thus, these two MHV RNA$\sp{-}$ ts mutants that were genetically equivalent by complementation testing were phenotypically similar as well. Recombination frequencies obtained from crosses of MHV-A59 ts261 with several of the gene 1 MHV-A59 mutants indicated that the causal mutation(s) of MHV-A59 ts261 was located near the overlapping junction of ORF1a and ORF1b, in the 3$\sp\prime$ end of ORF1a, or the 5$\sp\prime$ end of ORF1b. Sequence analysis of this junction and 1400 nucleotides into the 5$\sp\prime$ end of ORF1b of MHV-A59 ts261 revealed one nucleotide change from the wildtype MHV-A59. This substitution at nucleotide 13,598 (A to G) was a silent mutation in the ORF1a reading frame, but resulted in an amino acid change in ORF1b gene product (I to V). This amino acid change would be expressed only in the readthrough translation product produced upon successful ribosome frameshifting. A revertant of MHV-A59 ts261 (R2) also retained this guanidine residue, but had a second substitution at nucleotide 14,475 in ORF1b. This mutation results in the substitution of valine for an isoleucine.^ The data presented here suggest that the mutation in MHV-A59 ts261 (nucleotide 13,598) would be responsible for the MHV-JHM complementation group A phenotype. A second-site reversion at nucleotide 14,475 may correct this defect in the revertant. Sequencing of gene 1 immediately upstream of nucleotide 13,296 and downstream of nucleotide 15,010 must be conducted to test this hypothesis. ^

Cost and power analysis of linkage disequilibrium methods for locating genes influencing quantitative traits in humans

Linkage disequilibrium methods can be used to find genes influencing quantitative trait variation in humans. Linkage disequilibrium methods can require smaller sample sizes than linkage equilibrium methods, such as the variance component approach to find loci with a specific effect size. The increase in power is at the expense of requiring more markers to be typed to scan the entire genome. This thesis compares different linkage disequilibrium methods to determine which factors influence the power to detect disequilibrium. The costs of disequilibrium and equilibrium tests were compared to determine whether the savings in phenotyping costs when using disequilibrium methods outweigh the additional genotyping costs.^ Nine linkage disequilibrium tests were examined by simulation. Five tests involve selecting isolated unrelated individuals while four involved the selection of parent child trios (TDT). All nine tests were found to be able to identify disequilibrium with the correct significance level in Hardy-Weinberg populations. Increasing linked genetic variance and trait allele frequency were found to increase the power to detect disequilibrium, while increasing the number of generations and distance between marker and trait loci decreased the power to detect disequilibrium. Discordant sampling was used for several of the tests. It was found that the more stringent the sampling, the greater the power to detect disequilibrium in a sample of given size. The power to detect disequilibrium was not affected by the presence of polygenic effects.^ When the trait locus had more than two trait alleles, the power of the tests maximized to less than one. For the simulation methods used here, when there were more than two-trait alleles there was a probability equal to 1-heterozygosity of the marker locus that both trait alleles were in disequilibrium with the same marker allele, resulting in the marker being uninformative for disequilibrium.^ The five tests using isolated unrelated individuals were found to have excess error rates when there was disequilibrium due to population admixture. Increased error rates also resulted from increased unlinked major gene effects, discordant trait allele frequency, and increased disequilibrium. Polygenic effects did not affect the error rates. The TDT, Transmission Disequilibrium Test, based tests were not liable to any increase in error rates.^ For all sample ascertainment costs, for recent mutations ($<$100 generations) linkage disequilibrium tests were less expensive than the variance component test to carry out. Candidate gene scans saved even more money. The use of recently admixed populations also decreased the cost of performing a linkage disequilibrium test. ^

Familial aggregation, candidate genes and genome scans: Analyzing the role of genetics in stroke

The genetic etiology of stroke likely reflects the influence of multiple loci with small effects, each modulating different pathophysiological processes. This research project utilized three analytical strategies to address the paucity of information related to the identification and characterization of genetic variation associated with stroke in the general population. ^ First, the general contribution of familial factors to stroke susceptibility was evaluated in a population-based sample of unrelated individuals. Increased risk of subclinical cerebral infarction was observed among individuals with a positive parental history of stroke. This association did not appear to be mediated by established stroke risk factors, specifically blood pressure levels or hypertension status. ^ The need to identify specific gene variation associated with stroke in the general population was addressed by evaluating seven candidate gene polymorphisms in a population-based sample of unrelated individuals. Three polymorphisms were significantly associated with increased subclinical cerebral infarction or incident clinical ischemic stroke risk. These relationships include the G-protein β3 subunit 825C/T polymorphism and clinical stroke in Whites, the lipoprotein lipase S/X447 polymorphism and subclinical and clinical stroke in men, and the angiotensin I-converting enzyme Ins/Del polymorphism and subclinical stroke in White men. These associations did not appear to be obfuscated by the stroke risk factors adjusted for in the analysis models specifically blood pressure levels or anti-hypertensive medication use. ^ The final research strategy considered, on a genome-wide scale, the idea that genetic variation may contribute to the occurrence of hypertension or stroke through a common etiologic pathway. Genomic regions were identified for which significant evidence of heterogeneity was observed among hypertensive sibpairs stratified by family history of stroke information. Regions identified on chromosome 15 in African Americans, and chromosome 13 in Whites and African Americans, suggest the presence of genes influencing hypertension and stroke susceptibility. ^ Insight into the role of genetics in stroke is useful for the potential early identification of individuals at increased risk for stroke and improved understanding of the etiology of the disease. The ultimate goal of these endeavors is to guide the development of therapeutic intervention and informed prevention to provide a lasting and positive impact on public health. ^

GCN5 maintains genome stability during development

The histone acetyltransferase, GCN5, is essential for survival of mice during embryogenesis. GCN5 null embryos die early during development due to increased apoptosis. We have demonstrated that the increased apoptosis in associated with increased p53 protein levels. Loss of p53 rescues the embryonic apoptosis in the GCN5 null embryos. These results raised the question of what molecular trigger leads to p53 stabilization and cell death in the absence of GCN5. p53 is generally referred to as the gatekeeper of the cell, monitoring cellular responses to DNA damage, genotoxic stress, and other unfavorable conditions in the cell. Therefore, we examined individual cells in wild type and mutant embryos for gross chromosomal aberrations that might trigger a genome integrity checkpoint. Karyotype analysis indicates that approximately 30% of the cells in an E8.5 GCN5 null embryo display chromosomal aberrations, predominantly chromosomal end adhesions and associations. In wild type E8.5 embryos, only 6% of the cells have chromosomal aberrations. Recent data using telomeric FISH demonstrates that cells from GCN5 null embryos have a decreased telomeric signal. Telomere maintenance is essential for maintaining genome integrity. Telomeric defects are associated with loss of chromosomes and chromosomal rearrangements that can lead to detrimental gene fusions involved in many types of cancers. Little is known about the chromatin structures present near the telomeric ends, or whether any of the telomere-associated proteins are subject to post-translational modification such as acetylation. Our results are the first data to demonstrate the involvement of a histone acetyltransferase, GCN5, in maintaining genome integrity through telomere maintenance and/or capping. ^