Analysis of adenovirus DNA detected in rodent species from the Democratic Republic of the Congo indicates potentially novel adenovirus types.

Different species of adenoviruses (AdVs) infect humans and animals and are known for their role as pathogens, especially in humans, with animals, primarily rodents, often serving as model systems. However, although we know over 100 types of human AdVs, we know comparatively little about the diversity of animal AdVs. Due to the fact that rodents are the most diverse family of mammals and a standard model system for human disease, we set out to sample African rodents native to the Democratic Republic of the Congo and test them for AdV DNA using a semi-nested consensus PCR. A total of 775 animals were tested, and viral DNA was detected in four of them. The AdV DNA found belongs to three different AdVs, all being closely related to murine adenovirus 2 (MAdV-2). Considering the genetic differences of the amplicon were 9%, 11% and 19% from MAdV-2 and at least 10% from each other, they seem to belong to up to three different novel types within the Murine mastadenovirus B species. This evidence of genetic diversity highlights the opportunities to isolate and study additional AdVs that infect rodents as models for AdV biology and pathology.

Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing.

Determining how an organism responds to its environment by altering gene expression is key to understanding its ecology. Here, we used RNA-seq to comprehensively and quantitatively assess the transcriptional response of the bacterial opportunistic cystic fibrosis (CF) pathogen and endemic soil dweller, Burkholderia cenocepacia, in conditions mimicking these 2 environments. By sequencing 762 million bases of cDNA from 2 closely related B. cenocepacia strains (one isolated from a CF patient and one from soil), we identified a number of potential virulence factors expressed under CF-like conditions, whereas genes whose protein products are involved in nitrogen scavenging and 2-component sensing were among those induced under soil-like conditions. Interestingly, 13 new putative noncoding RNAs were discovered using this technique, 12 of which are preferentially induced in the soil environment, suggesting that ncRNAs play an important role in survival in the soil. In addition, we detected a surprisingly large number of regulatory differences between the 2 strains, which may represent specific adaptations to the niches from which each strain was isolated, despite their high degree of DNA sequence similarity. Compared with the CF strain, the soil strain shows a stronger global gene expression response to its environment, which is consistent with the need for a more dynamic reaction to the heterogeneous conditions of soil.

An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.

Comparison of genomic DNA sequences from human and mouse revealed a new apolipoprotein (APO) gene (APOAV) located proximal to the well-characterized APOAI/CIII/AIV gene cluster on human 11q23. Mice expressing a human APOAV transgene showed a decrease in plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking Apoav had four times as much plasma triglycerides as controls. In humans, single nucleotide polymorphisms (SNPs) across the APOAV locus were found to be significantly associated with plasma triglyceride levels in two independent studies. These findings indicate that APOAV is an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease.

Deletion of a coordinate regulator of type 2 cytokine expression in mice.

Mechanisms that underlie the patterning of cytokine expression in T helper (T(H)) cell subsets remain incompletely defined. An evolutionarily conserved approximately 400-bp noncoding sequence in the intergenic region between the genes Il4 and Il13, designated conserved noncoding sequence 1 (CNS-1), was deleted in mice. The capacity to develop T(H)2 cells was compromised in vitro and in vivo in the absence of CNS-1. Despite the profound effect in T cells, mast cells from CNS-1(-/-) mice maintained their capacity to produce interleukin 4. A T cell-specific element critical for the optimal expression of type 2 cytokines may represent the evolution of a regulatory sequence exploited by adaptive immunity.

A common phenotype associated with atherogenesis in diverse mouse models of vascular lipid lesions.

The introduction of a range of different genetic modifications in mice results in altered lipoprotein metabolism and the development of vascular lipid lesions. At present, however, it is unclear to what extent the molecular events underlying lipid lesion formation are similar in these different mouse models of atherosclerosis. The aim of this study was to compare the protein expression pattern of lipid lesions from seven different mouse lines with varying susceptibility to vascular lipid lesion development, to determine to what extent lesions induced by different genetic interventions have a similar composition. The proteins we have measured, using quantitative immunofluorescence, are proteins whose expression is known to be modulated during atherogenesis in humans, including plasminogen activator inhibitor (PAI)-1, transforming growth factor (TGF)-beta 1, osteopontin and the macrophage marker CD11b. In all the mice lines we have investigated, PAI-1 was elevated wherever lesions developed. Active TGF-beta was depressed in the vessel wall of mice which developed lipid lesions, particularly in the intima. In contrast, TGF-beta 1 antigen (active plus latent TGF-beta 1) was increased at lesion sites. Accumulation of osteopontin and, with the marked exception of apolipoprotein(a) transgenic mice, tissue macrophages occurred at sites of lipid deposition in the vessel wall. Each lesion, irrespective of its size and the mouse strain in which it developed, had similar amounts of PAI-1, active TGF-beta and osteopontin per unit area of lesion. These data are consistent with a common phenotype accompanying atherogenesis, irrespective of the genetic basis of susceptibility.

Genomic strategies to identify mammalian regulatory sequences.

With the continuing accomplishments of the human genome project, high-throughput strategies to identify DNA sequences that are important in mammalian gene regulation are becoming increasingly feasible. In contrast to the historic, labour-intensive, wet-laboratory methods for identifying regulatory sequences, many modern approaches are heavily focused on the computational analysis of large genomic data sets. Data from inter-species genomic sequence comparisons and genome-wide expression profiling, integrated with various computational tools, are poised to contribute to the decoding of genomic sequence and to the identification of those sequences that orchestrate gene regulation. In this review, we highlight several genomic approaches that are being used to identify regulatory sequences in mammalian genomes.

Regulation and activity of the human ABCA1 gene in transgenic mice.

The ABCA1 transporter is one of the limiting steps in cellular cholesterol efflux. To study the expression and activity of the human ABCA1 gene in vivo we have examined mice containing two human BAC transgenes with different 5' ends. Mice containing a 255-kilobase (kb) BAC transgene, including 70 kb upstream of the previously defined exon 1, demonstrated a pattern of tissue-specific expression mimicking that of the endogenous mouse gene. Compared with macrophages from control mice, macrophages from these transgenics had increases in apoA-I cholesterol efflux heightened in response to increases in cell cholesterol content. The observed increase in macrophage apoA-I-mediated cholesterol efflux was not accompanied by alterations in plasma high density lipoprotein in the transgenics. Although mice containing a smaller 171-kb human BAC transgene, lacking the previously described exon 1 and ABCA1 promoter, did not express human ABCA1 in macrophages, they did express the human transgene in liver at levels comparable with those of the orthologous mouse gene. Analysis by 5' rapid amplification of cDNA ends of liver mRNA from these animals revealed a new ABCA1 exon 1 (exon 1A) and a previously unrecognized promoter. Analysis of human tissue revealed that exon 1A containing transcripts accounted for a high proportion of the ABCA1 mRNAs present in human liver. This analysis of ABCA1 transgenics showed that the expression of human ABCA1 transgenes can result in increased cholesterol efflux from macrophages, unaccompanied by changes in plasma high density lipoprotein, and identified a new ABCA1 promoter in humans.

Microarray expression profiling identifies genes with altered expression in HDL-deficient mice.

Based on the assumption that severe alterations in the expression of genes known to be involved in high-density lipoprotein (HDL) metabolism may affect the expression of other genes, we screened an array of >5000 mouse expressed sequence tags for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apoAI)-knockout mice, scavenger receptor BI (SR-BI) transgenic mice, and control mice were cohybridized to microarrays. Two-sample t statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared with control mice. In the SR-BI group we found nine array elements representing at least five genes that were significantly altered on the basis of an adjusted P value < 0.05. In the apoAI-knockout group, eight array elements representing four genes were altered compared with the control group (adjusted P < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments.

Genome scan identifies a locus affecting gamma-globin level in human beta-cluster YAC transgenic mice.

Genetic factors affecting postnatal gamma-globin expression--a major modifier of the severity of both beta-thalassemia and sickle cell anemia--have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human gamma-globin. To model the human beta-cluster in mice, with the goal of screening for loci affecting human gamma-globin expression in vivo, we introduced a human beta-globin cluster YAC transgene into the genome of FVB/N mice. The beta-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) gamma allele, resulting in postnatal expression of human gamma-globin in transgenic mice. The level of human gamma-globin for various F1 hybrids derived from crosses between the FVB/N transgenics and other inbred mouse strains was assessed. The gamma-globin level of the (C3HeB/FeJ x FVB/N)F1 transgenic mice was noted to be significantly elevated. To map genes affecting postnatal y-globin expression, we performed a 20-centiMorgan (cM) genome scan of a (C3HeB/FeJ x FVB/N)F1 transgenics x FVB/N backcross, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within an 18-cM interval of mouse Chromosome (Chr) 1 (LOD = 4.3) that contributes 10.9% of variation in gamma-globin level. Combining transgenic modeling of the human beta-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human gamma-globin level in vivo.

Genetic dissection of region associated with behavioral abnormalities in mouse models for Down syndrome.

Two animal models of Down syndrome (human trisomy 21) with segmental trisomy for all (Ts65Dn) or part (Ts1Cje) of human chromosome 21-homologous region of mouse chromosome 16 have cognitive and behavioral abnormalities. To compare these trisomies directly and to assess the phenotypic contribution of the region of difference between them, Ts65Dn, Ts1Cje, and a new segmental trisomic (Ms1Ts65) for the region of difference (APP: to Sod1) have been generated as littermates and tested in parallel. Although the performance of Ts1Cje mice in the Morris water maze is similar to that of Ts65Dn mice, the reverse probe tests indicate that Ts65Dn is more severely affected. By contrast, the deficits of Ms1Ts65 mice are significantly less severe than those of Ts65Dn. Therefore, whereas triplication of Sod1 to Mx1 plays the major role in causing the abnormalities of Ts65Dn in the Morris water maze, imbalance of APP: to Sod1 also contributes to the poor performance. Ts65Dn mice are hyperactive and Ts1Cje mice are hypoactive; the activity of Ms1Ts65 mice is not significantly above normal. These findings indicate that genes in the Ms1Ts65 trisomic region must interact with others in the Ts1Cje region to produce hyperactivity in Ts65Dn mice.

Perspectives for vascular genomics.

Diseases of the vascular system result from a complex mixture of genetic and environmental factors. Data sets, technologies and strategies emanating from the human genome programme have been applied to the analysis of both rare single-gene and common multigenic vascular disorders. Genomic approaches including inter- and intraspecies sequence comparisons, genotyping with dense marker sets spanning the genome, large-scale mutagenesis screens of model organisms, and genome-wide expression profiling have all begun to contribute to the identification of new genes and mechanisms that are central to cardiovascular disease processes.

Active conservation of noncoding sequences revealed by three-way species comparisons.

Human and mouse genomic sequence comparisons are being increasingly used to search for evolutionarily conserved gene regulatory elements. Large-scale human-mouse DNA comparison studies have discovered numerous conserved noncoding sequences of which only a fraction has been functionally investigated A question therefore remains as to whether most of these noncoding sequences are conserved because of functional constraints or are the result of a lack of divergence time.

Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.

The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.

Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy.

Although cardiac hypertrophy has been the subject of intensive investigation, regression of hypertrophy has been significantly less studied, precluding large-scale analysis of the relationship between these processes. In the present study, using pharmacological models of cardiac hypertrophy in mice, expression profiling was performed with fragments of more than 4,000 genes to characterize and contrast expression changes during induction and regression of hypertrophy. Administration of angiotensin II and isoproterenol by osmotic minipump produced increases in heart weight (15 and 45%, respectively) that returned to preinduction size after drug withdrawal. From multiple expression analyses of left ventricular RNA isolated at daily time-points during cardiac hypertrophy and regression, we identified sets of genes whose expression was altered at specific stages of this process. While confirming the participation of 25 genes or pathways previously shown to be altered by hypertrophy, a larger set of 30 genes was identified whose expression had not previously been associated with cardiac hypertrophy or regression. Of the 55 genes that showed reproducible changes during the time course of induction and regression, 32 genes were altered only during induction, and 8 were altered only during regression. This study identified both known and novel genes whose expression is affected at different stages of cardiac hypertrophy and regression and demonstrates that cardiac remodeling during regression utilizes a set of genes that are distinct from those used during induction of hypertrophy.

Faithful expression of the human 5q31 cytokine cluster in transgenic mice.

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.

Relationship between expression levels and atherogenesis in scavenger receptor class B, type I transgenics.

Both in vitro and in vivo studies of scavenger receptor class B type I (SR-BI) have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate the effect of SR-BI on atherogenesis, we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) SR-BI expression in an inbred mouse background hemizygous for a human apolipoprotein (apo) B transgene. Unlike non-HDL cholesterol levels that minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol, whereas the high expression SR-BI transgene was associated with 2-fold decreases in HDL cholesterol as well as dramatic alterations in HDL composition and size including the near absence of alpha-migrating particles as determined by two-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a 2-fold decrease in the development of diet-induced fatty streak lesions compared with the apo B transgenics (4448 +/- 1908 micrometer(2)/aorta to 10133 +/- 4035 micrometer (2)/aorta; p < 0.001), whereas the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692 +/- 7238 micrometer(2)/aorta) but 3-fold greater than the low SR-BI/apo B mice (p < 0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences, illustrating the complexity of the relationship between SR-BI and atherogenesis.

Genomic interval engineering of mice identifies a novel modulator of triglyceride production.

To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine, and hepatic triglyceride production is of particular importance because of the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.

VISTA : visualizing global DNA sequence alignments of arbitrary length.

SUMMARY: VISTA is a program for visualizing global DNA sequence alignments of arbitrary length. It has a clean output, allowing for easy identification of similarity, and is easily configurable, enabling the visualization of alignments of various lengths at different levels of resolution. It is currently available on the web, thus allowing for easy access by all researchers. AVAILABILITY: VISTA server is available on the web at http://www-gsd.lbl.gov/vista. The source code is available upon request. CONTACT: vista@lbl.gov