Biomedical subjects

E M Rubin

Publications and source records attributed to E M Rubin.

At least 19 recordsLinked to original sources

PubMed2001

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.

ATP Binding Cassette Transporter 1↗

PubMed2001

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

ATP Binding Cassette Transporter 1↗

PubMed2000

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.

Angiotensin II↗

PubMed2000

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.

Animals↗

PubMed2000

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons.

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

Monocyte chemoattractant protein-1 but not tumor necrosis factor-alpha is correlated with monocyte infiltration in mouse lipid lesions.

BACKGROUND: Apolipoprotein (apo)(a) transgenic mice and C57BL/6 mice fed a high fat diet develop similar-sized lipid lesions, but lesions in apo(a) mice are devoid of macrophages. We used this observation to identify which proinflammatory proteins might be involved in mediating monocyte recruitment during atherogenesis. METHODS AND RESULTS: Macrophage-deficient apo(a) transgenic mouse lesions contained similar levels of several different proinflammatory proteins, both adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and cytokines (tumor necrosis factor-alpha [TNF-alpha] and macrophage inflammatory protein-1alpha [MIP-1alpha]), similar to the macrophage-rich lesions of C57BL/6 mice. CONCLUSIONS: From this we conclude that ICAM-1, VCAM-1, TNF-alpha, and MIP-1alpha may all be necessary for vascular monocyte recruitment in vivo, but they cannot be sufficient. Monocyte chemoattractant protein-1 (MCP-1) protein was undetectable in the vessel wall taken from apo(a) transgenic mice fed a high fat diet compared with high expression in mice with lipid lesions (C57BL/6 and apoE knockout mice). Therefore elevated expression of MCP-1 but not TNF-alpha, MIP-1alpha, ICAM-1, or VCAM-1 is correlated with vascular macrophage accumulation. To test the hypothesis that monocyte infiltration during atherogenesis is MCP-1 dependent, it will be necessary to develop specific pharmacological inhibitors of MCP-1 activity.

Animals↗

PubMed1999

Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice.

Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins.

Animals↗

PubMed1999

A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.

We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD). YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration. By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus. Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death. These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration.

Adaptation, Physiological↗

PubMed1999

Functional screening of an asthma QTL in YAC transgenic mice.

Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.

Animals↗