Christine A. Kozak

Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family

A cDNA was isolated from rat brain by low stringency hybridization with the rat μ opioid receptor cDNA. Sequence analysis of this clone indicated that it contains an open reading frame capable of encoding a 367 amino acid protein. The deduced amino acid sequence of this protein shows high degrees of homology to all three opioid receptors, μ, κ, and δ, suggesting that it is a member of the opioid receptor gene family. RNA blot analysis detected high level expression of the receptor mRNA in the brain. Southern blot analysis suggests that it is a single‐copy gene, and mapping studies localized the gene on mouse chromosome 2. Despite the high sequence homologies between this protein and the other opioid receptors, expression studies of this clone in COS‐7 cells did not show binding to [3H]diprenorphine, a ligand that binds to the other three opioid receptors. Furthermore, co‐expression of this receptor with a G protein‐activated potassium channel in Xenopus oocytes did not show functional coupling upon stimulation with μ, κ and δ agonists. Given the similar degrees of high homology to the μ, κ and δ opioid receptors and the lack of apparent affinity for their ligands, this receptor does not appear to belong to any of the three known classes of opioid receptors. Rather, it represents a novel member of the opioid receptor gene family, not identified from previous pharmacological studies.

Identification of CRAMP, a Cathelin-related Antimicrobial Peptide Expressed in the Embryonic and Adult Mouse

Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, namedCramp for cathelin-relatedantimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic α-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5–8.0 μm) and by permeabilizing the inner membrane of Escherichia colidirectly at 1 μm. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair.

Targeted Deletion of the Vgf Gene Indicates that the Encoded Secretory Peptide Precursor Plays a Novel Role in the Regulation of Energy Balance

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map.

The mechanisms that establish the dorsal-ventral (D-V) axis of the eye are poorly understood. We isolated two homeobox genes from mouse and chicken, mVax2 and cVax, whose expression during early eye development is restricted to the ventral retina. In chick, ectopic expression of either Vax leads to ventralization of the early retina, as assayed by expression of the transcription factors Pax2 and Tbx5, and the Eph family members EphB2, EphB3, ephrinB1, and ephrinB2, all of which are normally dorsally or ventrally restricted. Moreover, the projections of dorsal but not ventral ganglion cell axons onto the optic tectum showed profound targeting errors following cVax misexpression. mVax2/cVax thus specify positional identity along the D-V axis of the retina and influence retinotectal mapping.

Molecular Cloning of Microtubule-Associated Protein 1 (MAP1A) and Microtubule-Associated Protein 5 (MAP1B): Identification of Distinct Genes and Their Differential Expression in Developing Brain

Abstract: cDNA clones encoding microtubule‐associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10‐kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11‐kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse‐hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap‐1, and the MAP5 gene to chromosome 13, designated Mtap‐5 MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.

The mouse homolog of the Gibbon ape leukemia virus receptor: Genetic mapping and a possible receptor function in rodents

The mouse homolog of the Gibbon ape leukemia virus (GALV) receptor (Glvr-1) was mapped to mouse Chromosome 2 (Chr 2) by Southern blot analysis of somatic cell hybrids and positioned on this chromosome using an interspecies genetic cross. Mouse Chr 2 also encodes a receptor (Rec-2) for the wild mouse virus M813. To investigate whether Glvr-1 and Rec-2 could be the same gene, we sought evidence for sequence homology between the env- genes of their respective viruses. Southern blot hybridization with GALV-derived env and pol-env probes failed to detect any homology between GALV and M813, but did show that all mouse species tested carry numerous copies of GALV-related sequences. We speculate that a functional receptor for GALV-related viruses was expressed during Mus evolution.

Molecular genetic markers spanning mouse chromosome 10

Somatic cell hybrids, recombinant inbred (RI) strains, and progeny of an intersubspecific backcross were typed by Southern blot analysis to prepare a linkage map of mouse chromosome 10. The seven genetic markers in this map, four of which had not previously been positioned, include genes involved in oncogenesis (Gli, Myb, Tra-1), proviral integration (Emv-25), and immune responses (Ifg, Ifgr, Pfp). The linkage map spans much of the chromosome and covers a region of the mouse genome with few molecular markers. The gene order established here demonstrates that the genes for murine interferon-gamma (Ifg) and its receptor (Ifgr) are at opposite ends of the chromosome and that Ifgr and the Myb oncogene are closely linked, a factor that may be related to their joint transcriptional enhancement in some plasmacytoid lymphosarcomas.

Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping.

Bone sialoprotein (BSP) is a small ( -70 ,000 Mr), highly post-translationally modified protein that is an abundant noncollagenous component of the bone matrix (Fisher et al. 1983). With a combination of immunohistochemistry and in situ hybridization, it has been shown that in the human and rat, BSP is produced by bone-forming cells and mature osteoblasts, recently entombed osteocytes, osteoclasts, and hypertrophic chondrocytes within the growth plate (Bianco et al. 1991; Chen et al. 1991). The only nonskeletal source that shows expression of BSP is the mononucleated trophoblast cells and their multinucleated syncytia in the developing placenta. Studies in the rat also showed that BSP is expressed in the odontoblasts of the developing mandibular incisor (Chen et al. 1992b). In an in vitro model of differentiating fetal bovine osteoblasts, BSP mRNA was increased 120-fold at a time point that corresponded to a dramatic formation of mineralizing nodules and trabecular-like structures (Ibaraki et al. 1992). This localization of BSP to sites of new mineral formation implicates BSP in this process. To determine the structure and describe the expression of BSP in the mouse, we have cloned and sequenced mouse BSP cDNA. In this study we report the nucleotide sequence of the murine cDNA, its deduced amino acid sequence, and we report the mapping of the mouse BSP gene (Ibsp) to Chromosome (Chr) 5. Mouse BSP cDNA was cloned from a 16-day mouse embryo cDNA library in a lambda SHlox vector (Novogen Cat #69641-1) with a 1.8-kb insert of rat cDNA (Oldberg et al. 1988a) as a probe under moderate hybridization conditions (Sambrook et al. 1989). One clone contained an insert size of 1.3 kb and was sequenced completely through-

The mouse genome encodes a single homolog of the antimicrobial peptide human β-defensin 1

The cysteine‐rich β‐defensin peptides are broad‐spectrum bactericidal agents expressed in epithelial and myeloid tissues. The human β‐defensin‐1 (hBD‐1) gene maps adjacent to the human α‐defensin cluster and is expressed in the respiratory, gastrointestinal and genitourinary tracts. Here, we characterize a mouse β‐defensin gene (mBD‐1) which is: (1) closely related to hBD‐1 both in sequence and gene organization; (2) expressed at high levels in the mouse kidney and at lower levels in brain, heart, lung, uterus, spleen, skeletal muscle, stomach, and small intestine; and (3) maps to mouse chromosome 8 at or near the location of the mouse α‐defensin genes. These data indicate that mBD‐1 is a close homolog of hBD‐1, and suggest that analysis of its role in mouse host defense may provide significant insights into human epithelial innate immunity.

The Lake Casitas wild mouse:: Evolving genetic resistance to retroviral disease

A small colony of feral mice from California continues to flourish in spite of a virulent epizootic of pathological retrovirus. Epidemiological and genetic studies revealed that the viral infection is strongly balanced by the polymorphic resistance locus, Fv-4, a transcriptionally active but truncated provirus that originated in the East Asian ancestors of the Californian mice. The natural history of these populations represents a graphic example of genomic adaptation in free-ranging populations to regulate and delimit infectious disease.