Genome-tagged mice (GTM): two sets of genome-wide congenic strains.

An important approach for understanding complex disease risk using the mouse is to map and ultimately identify the genes conferring risk. Genes contributing to complex traits can be mapped to chromosomal regions using genome scans of large mouse crosses. Congenic strains can then be developed to fine-map a trait and to ascertain the magnitude of the genotype effect in a chromosomal region. Congenic strains are constructed by repeated backcrossing to the background strain with selection at each generation for the presence of a donor chromosomal region, a time-consuming process. One approach to accelerate this process is to construct a library of congenic strains encompassing the entire genome of one strain on the background of the other. We have employed marker-assisted breeding to construct two sets of overlapping congenic strains, called genome-tagged mice (GTMs), that span the entire mouse genome. Both congenic GTM sets contain more than 60 mouse strains, each with on average a 23-cM introgressed segment (range 8 to 58 cM). C57BL/6J was utilized as a background strain for both GTM sets with either DBA/2J or CAST/Ei as the donor strain. The background and donor strains are genetically and phenotypically divergent. The genetic basis for the phenotypic strain differences can be rapidly mapped by simply screening the GTM strains. Furthermore, the phenotype differences can be fine-mapped by crossing appropriate congenic mice to the background strain, and complex gene interactions can be investigated using combinations of these congenics.

Molecular phenotype of the human oocyte by PCR-SAGE.

Consecutive application of PCR and serial analysis of gene expression (SAGE) was used to generate a catalog of approximately 50, 000 SAGEtags from nine human oocytes. Matches for known genes were identified using the National Institutes of Health SAGEtag database. This database links directly to the UniGene database, providing rapid discrimination between SAGEtags that match known genes and expressed sequence tags and those that currently have no match. Matches in the oocyte SAGE catalog were found for surface receptors, second-messenger systems, and cytoskeletal, apoptotic, and secreted proteins. Many of these proteins were not previously known to be expressed in mammalian oocytes. The relative abundances of transcripts for cytoskeletal proteins and proteins known to be in oocytes are consistent with their documented expression, suggesting an absence of representational distortion by the PCR step. The expression profile of the human oocyte may help identify factors that reprogram somatic cell nuclei to totipotency.

Cell biology of tubotympanum in relation to pathogenesis of otitis media - a review.

The sterility of the eustachian tube and tympanic cavity of normal individuals is maintained not only by the adaptive immune system, but also by the mucociliary system and the antimicrobial molecules of innate immunity. Mucin production and periciliary fluid homeostasis are essential for normal mucociliary function and dysfunction of this system is an important risk factor for otitis media. The secreted antimicrobial molecules of the tubotympanum include lysozyme, lactoferrin, beta defensins, and the surfactant proteins A and D (SP-A, SP-D). Defects in the expression or regulation of these molecules may also be the major risk factor for otitis media.

Genetic control of the inflammatory response induced by oxidized lipids.

The fatty streak begins with entrapment of apolipoprotein B (apoB)-containing lipoproteins in the subendothelial space at susceptible sites in the arterial wall. Minimally oxidized low density lipoprotein (MM-LDL) induces endothelial cells to bind monocytes and produce message and protein for monocyte chemotactic protein-1 and macrophage colony-stimulating factor. In culture, human endothelial and smooth muscle cells in arterial wall configuration sequester LDL, protecting it from antioxidants and giving rise to MM-LDL-like species. In mice, MM-LDL induces monocyte binding at susceptible aortic sites; the monocytes may then differentiate into macrophages that release reactive oxygen and active aldehydes, resulting in highly oxidized LDL leading to foam cell formation. Feeding mice an atherogenic diet induces expression of several inflammatory and oxidative stress genes, including serum amyloid A, which binds exclusively to HDL. This may contribute to a decrease in protective HDL levels seen in mice susceptible to fatty streak formation.

Role of the GRO family of chemokines in monocyte adhesion to MM-LDL-stimulated endothelium.

We have previously shown that treatment of endothelial cells with minimally modified LDL (MM-LDL) induces the binding of monocytes to unknown endothelial receptor molecules. We now report that a member of the GRO family of chemokines plays a role in MM-LDL-induced monocyte binding. A cDNA library made from rabbit aortic endothelial cells (RAEC) treated with MM-LDL was expression screened for molecules inducing binding of a human monocyte cell line (THP-1). A cDNA was isolated with 75% homology to GRO. GRO mRNA levels were significantly elevated after exposure of RAEC or human aortic endothelial cells (HAEC) to MM-LDL. HAEC treated with MM-LDL displayed an increase in a surface-associated protein that bound to antibody against GRO despite low levels of GRO in the medium. Antibody to GRO significantly inhibited the binding of monocytes to MM-LDL-treated RAEC and HAEC. The increase in GRO expression and monocyte binding were reduced by incubating MM-LDL-treated endothelial cells with heparin (in a method that releases heparan sulfate bound molecules from the cell surface). These results suggest that GRO related chemokines are bound to the surface of MM-LDL-treated endothelial cells and may contribute to the monocyte adhesion induced by MM-LDL.

Genetic evidence for a common pathway mediating oxidative stress, inflammatory gene induction, and aortic fatty streak formation in mice.

In a previous survey of inbred mouse strains on an atherogenic diet, we observed that the susceptibility to aortic atherosclerotic lesion formation was associated with the accumulation of lipid peroxidation products, induction of inflammatory genes, and the activation of NF-kB-like transcription factors (Liao, F., A. Andalibi, F. C. deBeer, A. M. Fogelman, and A.J. Lusis. 1993. J. Clin. Invest. 91:2572-2579). We hypothesized that the inflammation-related processes were stimulated by oxidized lipids, since injection of minimally oxidized LDL (MM-LDL) activated the same set of genes. We now report that the induction of inflammatory genes and activation of NF-kB-like transcription factors cosegregate with aortic atherosclerotic lesion formation in BXH recombinant inbred strains derived from parental C57BL/6J (susceptible) and C3H/HeJ (resistant) mice. In addition, the accumulation of hepatic conjugated dienes exhibited a significant correlation with inflammatory gene activation. These results provide strong evidence for the role of inflammatory mediators inducible by oxidative stress in atherogenesis. They also suggest that a major gene contributing to aortic lesion development in this mouse model, designated Ath-1, may control either the accumulation of lipid peroxides in tissues or the cellular responses to such lipid peroxides.

Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate.

We have previously shown that minimally oxidized LDL (MM-LDL) activated endothelial cells to increase their interaction with monocytes but not neutrophils, inducing monocyte but not neutrophil binding and synthesis of monocyte chemotactic protein-1 and monocyte colony-stimulating factor (M-CSF). In the present studies we have examined the signaling pathways by which this monocyte-specific response is induced. Both induction of monocyte binding and mRNA levels for M-CSF by MM-LDL were not inhibited in protein kinase C-depleted endothelial cells. A number of our studies indicate that cAMP is the second messenger for the effects of MM-LDL cited above. Incubation of endothelial cells with MM-LDL caused a 173% increase in intracellular cAMP levels. Agents which increased cAMP levels, including cholera toxin, pertussis toxin, dibutyryl cAMP, and isoproterenol mimicked the actions of MM-LDL. Agents which elevated cAMP were also shown to activate NF kappa B, suggesting a role for this transcription factor in activation of monocyte-endothelial interactions. Although endothelial leukocyte adhesion molecule (ELAM) mRNA synthesis can be regulated by NF kappa B, ELAM was not expressed and ELAM mRNA was only slightly elevated in response to MM-LDL. We present evidence that induction of neutrophil binding by LPS is actually suppressed by agents that elevated cAMP levels.

Genetic control of inflammatory gene induction and NF-kappa B-like transcription factor activation in response to an atherogenic diet in mice.

A high fat, high cholesterol "atherogenic" diet induced considerably greater hepatic levels of conjugated dienes and expression of several inflammatory and oxidative stress responsive genes (JE, the mouse homologue of monocyte chemotactic protein-1, colony-stimulating factors, heme oxygenase, and members of the serum amyloid A family) in fatty streak susceptible C57BL/6 mice compared to fatty streak resistant C3H/HeJ mice. Since serum amyloid A proteins bind exclusively to HDL and influence the properties of HDL, serum amyloid A expression may contribute to the decrease in HDL levels seen in the susceptible strains. Induction of a similar set of genes was observed upon injection of minimally oxidized low density lipoprotein. The transcription factor NF-kappa B is known to be activated by oxidative stress and is involved in the transcriptional regulation of several of these genes. On the atherogenic diet the susceptible C57BL/6 mice exhibited significant NF-kappa B-like activation whereas the resistant C3H/HeJ mice exhibited little or no activation. These results are consistent with the hypothesis that the atherogenic diet resulted in the accumulation of oxidized lipids in certain tissues (e.g., liver and arteries) and the resulting inflammatory response to this oxidative stress was genetically determined.

Structure and chromosomal localization of the murine gene encoding GLYCAM 1. A mucin-like endothelial ligand for L selectin.

We recently described the molecular cloning of a murine cDNA encoding an endothelial cell surface ligand for the leukocyte adhesion molecule, L Selectin (Lasky, L. A., Singer, M., Dowbenko, D., Ima, Y., Henzel, W., Grimley, C., Gennie, C., Gillett, N., Watson, S., and Rosen, S. D (1992) Cell 69, 927-938). This glycoprotein ligand was found to resemble mucins in that it contained a large percentage of serine and threonine residues that were apparently O-glycosylated. At least one of the O-linked carbohydrates found on this endothelial ligand interacts with the lectin domain of L Selectin. These data suggest that this endothelial ligand is an adhesion molecule that accomplishes cell binding by presenting carbohydrate(s) to the lectin domain of L Selectin, and the name GLYCAM 1 (GLY-cosylation-dependent Cell Adhesion Molecule 1) has been proposed. In this paper we describe the genomic structure and chromosomal localization of this unique Selectin ligand. The gene has been found to be encoded on four separate exons, and it thus differs from the cell surface mucin leukosialin, whose coding region is contained on one exon, but is similar to glycophorin and CD34, other cell surface mucins whose genes are divided into multiple coding exons. While there is some correlation between exon division and protein domain structure, these relationships are not as clear as they are in other genes. The gene encoding GLYCAM 1 was found to map to murine chromosome 15.

Mapping of multiple mouse loci related to the farnesyl pyrophosphate synthetase gene.

The prenyltransferases are a class of enzymes involved in the synthesis of sterol and nonsterol isoprene compounds. We report here the chromosomal mapping of nine loci in the mouse that hybridize to the cDNA for the enzyme farnesyl pyrophosphate synthetase (FPS), a prenyltransferase that catalyzes the synthesis of an intermediate common to both the sterol and nonsterol branches of the isoprene biosynthetic pathway. Mapping was performed with genomic DNA from a mouse-hamster somatic cell hybrid panel, and by linkage analysis with recombinant inbred strains and the progeny of an interspecific backcross. The mapped loci have been designated farnesyl pyrophosphate synthetase-like-1 (Fpsl-1) on mouse Chromosome (Chr) 3; Fpsl-2 on Chr 4; Fpsl-3, Fpsl-4, and Fpsl-5, dispersed on Chr 10; Fpsl-6 on Chr 12; Fpsl-7 on Chr 13; Fpsl-8 on Chr 17; and Fpsl-9 on Chr X. It is presently unclear which of these loci encode active prenyltransferases and which may correspond to pseudogenes. The strongly hybridizing loci provide convenient genetic markers for seven mouse chromosomes.

Minimally modified lipoproteins in diabetes.

Studies from several laboratories suggest that oxidized LDL may play an important role in atherogenesis. Our group previously showed that treatment of aortic endothelial cells with low levels of MM-LDL caused increased expression of MCP-1, M-CSF, tissue factor, and a monocyte-binding protein. In these studies MM-LDL was produced by storage of native LDL. We now show that cocultures of endothelial and smooth muscle cells can also produce MM-LDL from native LDL. This production of MM-LDL by cells is prevented by preincubating the LDL with probucol or vitamin E. However, addition of antioxidants to MM-LDL did not block its action. In past studies we also showed that endothelial cells exhibit differential sensitivity to the effects of MM-LDL. We report herein that in resistant cells there is no elevation of catalase, glutathione peroxidase, or copper-zinc-dependent SOD. However, manganese-dependent SOD is elevated in resistant cells. Ways in which MM-LDL production may be elevated in poorly controlled diabetics subjects are discussed.

Defective elongation of fatty acids in a recessive 25-hydroxycholesterol-resistant mutant cell line.

The Chinese hamster ovary recessive mutant, crB, has been selected for its resistance to the cytotoxic effects of 25-hydroxycholesterol in sterol-free media (Sinensky, M., Logel, J., and Torget, R. (1982) J. Cell. Physiol. 113, 314-319). Growth of crB in a chemically defined lipid-poor medium is very slow and is enhanced by a mixture of saturated and unsaturated fatty acids. Incorporation of [3H]acetate into total fatty acids is 4-fold lower in crB compared to that in parental Chinese hamster ovary K1 and in contrast to the wild-type cells, crB cells are unable to synthesize either stearate or oleate. In addition, crB cells can not elongate exogenous palmitate, while they are capable of desaturating exogenous stearate. The mutant cells are also pleiotropically defective in the regulation of mRNA levels for the enzymes of cholesterol biosynthesis. 25-Hydroxycholesterol is a poor regulator of the synthesis and degradation of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase in crB in comparison to the wild-type Chinese hamster ovary K1 cells. The defect in the elongation of fatty acids is reversed in revertants of crB selected for their ability to grow in lipid-poor medium. Such revertants exhibit normal regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by 25-hydroxycholesterol. Regulation of reductase activity in crB cells can also be restored by supplementing the culture medium with a mixture of fatty acids that restores normal growth rate. The defective regulation of reductase in crB does not appear to be due to nonspecific adverse effects of fatty acid starvation nor is it due to any gross change in the fatty acid composition of cellular phospholipids. These results strongly suggest a direct relationship between the fatty acid auxotrophy of crB and defective regulation of the enzymes of cholesterol biosynthesis.

Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins.

Oxidized lipoproteins have been identified in atherosclerotic plaques and in early lesions in humans as well as in animals. There is accumulating evidence that such oxidized lipoproteins have an important role in atherosclerosis. Treatment of endothelial cells with altered lipoproteins stimulates monocyte binding as well as the production of chemotactic factors for monocytes. Both these findings could be relevant to the accumulation of monocytes-macrophages in the arterial wall during the early stages of lesion development. We now report that treatment of endothelial cells (EC) with modified low-density lipoproteins obtained by mild iron oxidation or by prolonged storage, results in a rapid and large induction of the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (M-CSF) and granulocyte CSF (G-CSF). These growth factors affect the differentiation, survival, proliferation, migration and metabolism of macrophages/granulocytes, and G-CSF and GM-CSF also affect the migration and proliferation of EC. Because EC and macrophages are important in the development of atherosclerosis, the expression of the CSFs by these cells could contribute to the disease.

Organization of the human lipoprotein lipase gene and evolution of the lipase gene family.

The human lipoprotein lipase gene was cloned and characterized. It is composed of 10 exons spanning approximately equal to 30 kilobases. The first exon encodes the 5'-untranslated region, the signal peptide plus the first two amino acids of the mature protein. The next eight exons encode the remaining 446 amino acids, and the tenth exon encodes the long 3'-untranslated region of 1948 nucleotides. The lipoprotein lipase transcription start site and the sequence of the 5'-flanking region were also determined. We compared the organization of genes for lipoprotein lipase, hepatic lipase, pancreatic lipase, and Drosophila yolk protein 1, which are members of a family of related genes. A model for the evolution of the lipase gene family is presented that involves multiple rounds of gene duplication plus exon-shuffling and intron-loss events.

Identification of a zinc finger protein that binds to the sterol regulatory element.

Cholesterol balance in mammalian cells is maintained in part by sterol-mediated repression of gene transcription for the low density lipoprotein receptor and enzymes in the cholesterol biosynthetic pathway. A promoter sequence termed the sterol regulatory element (SRE) is essential for this repression. With the use of an oligonucleotide containing the SRE to screen a human hepatoma complementary DNA expression library, a clone for a DNA binding protein was isolated that binds to the conserved SRE octanucleotide in both a sequence-specific and a single-strand--specific manner. This protein contains seven highly conserved zinc finger repeats that exhibit striking sequence similarity to retroviral nucleic acid binding proteins (NBPs). We have designated the protein "cellular NBP" (CNBP). CNBP is expressed in a wide variety of tissues, is up regulated by sterols, and exhibits binding specificity that correlates with in vivo function. These properties are consistent with a role in sterol-mediated control of transcription.