The pattern of gene expression in mouse Gr-1(+) myeloid progenitor cells

To understand the pattern of gene expression in mouse myeloid progenitor cells, we carried out a genome-wide analysis of gene expression in mouse bone marrow Gr-1(+) cells using SAGE and GLGI techniques. We identified 22,033 unique SAGE tags with quantitative information from 73,869 collected SAGE tags. Among these unique tags, 64% match known sequences, including many genes important for myeloid differentiation, and 36% have no matches to known sequences and are likely to represent novel genes. We compared the expression of mouse Gr-1(+) and human CD15(+) myeloid progenitor cells and showed that the pattern of gene expression of these two cell populations had some similarities. We also compared the expression of mouse Gr-1(+) myeloid progenitor cells with that of mouse brain tissue and found a highly tissue-specific manner of gene expression in these two samples. Our data provide a basis for studying altered gene expression in myeloid disorders using mouse models.     

Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver

After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.     

Apoptosis-associated changes in the glycerophospholipid composition of hematopoietic progenitor cells monitored by 31P NMR spectroscopy and MALDI-TOF mass spectrometry

Apoptosis, or programmed cell death, plays an important role in development and in tissue homeostasis and is assumed to be accompanied by changes in the composition of cellular glycerophospholipids (GPL). We have applied a combination of ³¹P nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to the analysis of organic extracts of hematopoietic progenitor cells undergoing the physiologically relevant process of apoptosis following growth factor withdrawal. The combined application of these methods enables the quantitative analysis of all glycerophospholipid classes and reveals changes in the acyl chain compositions from crude cell extracts. Using these techniques, an increase in the ratio of ether-linked glycerophospholipids to diacyl-glycerophospholipids during apoptosis was observed. The relative decrease in the membrane diacyl-phosphatidylcholine (PC) levels was found to correlate with increased concentrations of the corresponding lysophosphatidylcholine (LPC) present in the medium.     

Genomic changes following host restriction in bacteria

Many genomic sequences have been recently published for bacteria that can replicate only within eukaryotic hosts. Comparisons of genomic features with those of closely related bacteria retaining free-living stages indicate that rapid evolutionary change often occurs immediately after host restriction. Typical changes include a large increase in the frequency of mobile elements in the genome, chromosomal rearrangements mediated by recombination among these elements, pseudogene formation, and deletions of varying size. In anciently host-restricted lineages, the frequency of insertion sequence elements decreases as genomes become extremely small and strictly clonal. These changes represent a general syndrome of genome evolution, which is observed repeatedly in host-restricted lineages from numerous phylogenetic groups. Considerable variation also exists, however, in part reflecting unstudied aspects of the population structure and ecology of host-restricted bacterial lineages.     

Circling,deafness, and yellow coat displayed by yellow submarine (ysb) and light coat and circling (lcc) mice with mutations on chromosome 3

We describe here two mouse mutants, yellow submarine (Ysb) and light coat and circling (Lcc). Ysb arose as the result of insertions of a transgene, pAA2, into the genome. Lcc is an independent, radiation-induced mutation. Both mutants are characterized by recessive circling behavior and deafness, associated with a non-segregating, semi-dominant yellow coat color. Complementation tests showed that Ysb and Lcc are allelic. We attribute the yellow coat in Ysb and Lcc mice to the absence of black awl overhairs, increased agouti zigzag underhairs, and the presence of agouti awls with long subapical yellow pigment. Chromosomal mapping and genomic characterization showed the Ysb and Lcc mutations involve complex chromosomal rearrangements in overlapping regions of mouse chromosome 3, A2/A3-B/C and B-E1, respectively. Ysb and Lcc show for the first time, to our knowledge, the presence of genes in the B-C region of chromosome 3 important for balance and hearing and the pigmentation and specification of coat hair.     

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20 days on 3 μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β₂-integrins, but not β1- or β₃-integrins. Migration from the subendothelial compartment was supported by β1- and β₂-integrins for all cultures, but blockade of β₃-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β₃-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.     

Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: implication in multinucleation and chemosensitization

The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC−/−p53−/− mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.     

Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells

Apoptotic cell death eventually results in secondary necrotic cell death, whereas caspase-independent primary necrotic cell death has been reported and its mechanism involving RIP1 and RIP3 has been recently elucidated. Dual staining with fluorescent Annexin V and propidium iodide (PI) has been used to discriminate apoptotic and necrotic cell death, in which Annexin V-positive/PI-negative staining is regarded as apoptosis and PI-positive staining as necrosis. Here we demonstrate that primary necrotic cells unexpectedly show Annexin V-positive/PI-negative staining before they become PI-positive, and that primary necrotic and apoptotic Annexin V-positive/PI-negative cells can be discriminated by necrostatin-1, an inhibitor of primary necrosis by inhibition of RIP1.     

Nucleotide Sequence, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR3A and NR3B

The N-methyl-D-aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13-34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.     

Physical mapping of the mouse tilted locus identifies an association between human deafness loci DFNA6/14 and vestibular system development

The tilted (tlt) mouse carries a recessive mutation causing vestibular dysfunction. The defect in tlt homozygous mice is limited to the utricle and saccule of the inner ear, which completely lack otoconia. Genetic mapping of tlt placed it in a region orthologous with human 4p16.3-p15 that contains two loci, DFNA6 and DFNA14, responsible for autosomal dominant, nonsyndromic hereditary hearing impairment. To identify a possible relationship between tlt in mice and DFNA6 and DFNA14 in humans, we have refined the mouse genetic map, assembled a BAC contig spanning the tlt locus, and developed a comprehensive comparative map between mouse and human. We have determined the position of tlt relative to 17 mouse chromosome 5 genes with orthologous loci in the human 4p16.3-p15 region. This analysis identified an inversion between the mouse and human genomes that places tlt and DFNA6/14 in close proximity.     

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA.     

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.     

Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

RGSZ1 and Ret RGS: Two of Several Splice Variants from the Gene RGS20

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.     

Cloning, Pharmacology, and Tissue Distribution of G-Protein-Coupled Receptor GPR105 (KIAA0001) Rodent Orthologs

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.     

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.