The HOX-5 and surfeit gene clusters are linked in the proximal portion of mouse chromosome 2

Using an interspecies backcross, we have mapped the HOX-5 and surfeit (surf) gene clusters within the proximal portion of mouse chromosome 2. While the HOX-5 cluster of homeobox-containing genes has been localized to chromosome 2, bands C3-E1, by in situ hybridization, its more precise position relative to the genes and cloned markers of chromosome 2 was not known. Surfeit, a tight cluster of at least six highly conserved “housekeeping” genes, has not been previously mapped in mouse, but has been localized to human chromosome 9q, a region of the human genome with strong homology to proximal mouse chromosome 2. The data presented here place HOX-5 in the vicinity of the closely linked set of developmental mutations rachiterata, lethargic, and fidget and place surf close to the proto-oncogene Abl, near the centromere of chromosome 2.     

Cloning and Gene Mapping of the Mouse Homologue of theCBFA2T1Gene Associated with Human Acute Myeloid Leukemia

The humanCBFA2T1(also known asMTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of theCBFA2T1gene,Cbfa2t1h.Nucleotide sequence analysis ofCbfa2t1hcDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5′- and 3′-untranslated regions (87.0, 92.0, and 93.7% identities for 5′-untranslated, coding, 3′-untranslated regions, respectively). The 3′-untranslated region ofCbfa2t1hcontains a (CA)_ndinucleotide repeat, and the polymerase chain reaction amplification of the (CA)_nrepeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mappedCbfa2t1hto mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 × Mae) × Mae cross. The chromosomal location was also confirmed by fluorescencein situhybridization.     

Molecular Cloning, Expression Pattern, and Chromosomal Localization of Human CDKN2D/INK4d,an Inhibitor of Cyclin D-Dependent Kinases

Progression through the G1 phase of the cell cycle is dependent on the activity of holoenzymes formed between D-type cyclins and their catalytic partners, the cyclin-dependent kinases cdk4 and cdk6. p16^INK4a,p15^INK4b, and p18^INK4c, a group of structurally related proteins, function as specific inhibitors of the cyclin D-dependent kinases and are likely to play physiologic roles as specific regulators of these kinasesin vivo.A new member of the INK4 gene family, murineINK4d,has recently been identified. Here we report the isolation of humanINK4d(gene symbolCDKN2D), which is 86% identical at the amino acid level to the murine clone and 44% identical to each of the other human INK4 family members. TheINK4dgene is ubiquitously expressed as a single 1.4-kb mRNA with the highest levels detected in thymus, spleen, peripheral blood leukocytes, fetal liver, brain, and testes. The abundance ofINK4dmRNA oscillates in a cell-cycle-dependent manner with expression lowest at mid G1 and maximal during S phase. Using a P1-phage genomic clone ofINK4dfor fluorescencein situhybridization analysis, the location of this gene was mapped to chromosome 19p13. No rearrangements or deletions of theINK4dgene were observed in Southern blot analysis of selected cases of pediatric acute lymphoblastic leukemia (ALL) containing a variant (1;19)(q23′3) translocation that lacks rearrangement of eitherE2AorPBX1, or in ALL cases containing homozygous or hemizygous deletions of the related genes,INK4aandINK4b.     

Localization of the genes for the 100-kDa complement-activating components of Ra-reactive factor (CRARF and Crarf) to human 3q27–q28 and mouse 16B2–B3

Human and mouse genes for the complement-activating component (P100) of Ra-reactive factor, a novel bactericidal factor (CRARF and Crarf), were mapped to R-banded metaphase chromosomes by fluorescence in situ hybridization with human and mouse P100 cDNA 2.7 and 2.0 kb long, respectively. The localization of fluorescent signals showed that CRARF and Crarf mapped to human 3q27–q28 and mouse 16B2–B3, respectively. This evidence is consistent with the previous assumption that the distal portion of the long arm of human chromosome 3 is homologous to the proximal portion of mouse chromosome 16.     

Human and rat chromosomal localization of two genes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization

Two genes encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were localized in human and rat chromosomes. PFKFB1 (previously PFRX), which encodes the liver and muscle isozymes, was assigned to Xq22-q31 in the rat and to Xq27–q28 in the human by in situ hybridization using probes generated by the polymerase chain reaction. PFKFB2, which encodes the heart isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was assigned to chromosome 13 in the rat and to chromosome 1 in the human by hybridization of DNA from somatic cell hybrids. By in situ hybridization, this gene was localized to the regions 13q24–25 in the rat and 1q31 in the human.     

Localization ofHuC(ELAVL3) to Chromosome 19p13.2 by Fluorescencein SituHybridization Utilizing a Novel Tyramide Labeling Technique

HuC is a neural-specific member of the Elav family of RNA-binding proteins. This highly conserved gene family plays a crucial role in neurogenesis, andHuC(HGMW-approved symbol ELAVL3) is expressed at an early stage of neural development. Using a novel tyramide fluorescencein situhybridization (T-FISH) technique, we localizedHuCto chromosome 19p13.2. This localization was confirmed by radiation hybrid mapping and coincides with that ofHuR(HGMW-approved symbol ELAVL1), anotherelavfamily member. Dual T-FISH analysis withHuCandHuRprobes, however, indicated distinct loci, withHuCbeing centromeric to HuR. This study demonstrates the utility of T-FISH in colocalizing two genes on the same chromosomal preparation using only biotinylated probes.     

Comparative mapping of mouse and rat chromosomes by fluorescence in situ hybridization

Comparative fluorescence in situ hybridization mapping using DNA libraries from flow-sorted mouse chromosomes and region-specific mouse BAC clones on rat chromosomes reveals chromosomal homologies between mouse (Mus musculus, MMU) and rat (Rattus norvegicus, RNO). Each of the MMU 2, 3, 4, 6, 7, 9, 12, 14, 15, 16, 18, 19, and X chromosomes paints only a single rat chromosome or chromosome segment and, thus, the chromosomes are largely conserved between the two species. In contrast, the painting probes for MMU chromosomes 1, 5, 8, 10, 11, 13, and 17 produce split hybridization signals in the rat, disclosing evolutionary chromosome rearrangements. Comparative mapping data delineate several large linkage groups on RNO 1, 2, 4, 7, and 14 that are conserved in human but diverged in the mouse. On the other hand, there are linkage groups in the mouse, i.e., on MMU 1, 8, 10, and 11, that are disrupted in both rat and human. In addition, we have hybridized probes for Nap2, p57, Igf2, H19, and Sh3d2c from MMU 7 to RNO 1q and found the orientation of the imprinting gene cluster and Sh3d2c to be the same in mouse and rat. Hybridization of rat genomic DNA shows blocks of (rat-specific) repetitive sequences in the pericentromeric region of RNO chromosomes 3-5, 7-13, and 20; on the short arms of RNO chromosomes 3, 12, and 13; and on the entire Y chromosome.     

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P₁, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.     

Assignment of the CD45-AP Gene to the Centromeric End of Mouse Chromosome 19 and Human Chromosome 11q13.1–q13.3

CD45-AP is a recently identified phosphorylated protein that specifically associates with the leukocyte-specific transmembrane glycoprotein CD45. The gene for CD45-AP,Ptprcap(protein tyrosine phosphatase, receptor type c polypeptide associated protein), was mapped in mouse by typing the progeny of two multilocus crosses using the mouse CD45-AP cDNA as a Southern hybridization probe. The CD45-AP gene mapped to the centromeric region of Chr 19 proximal to the genesFth, Cd5,andPcna-rs.The gene for the human CD45-AP homologue,PTPRCAP,was localized to chromosome band 11q13.1–q13.3 by fluorescencein situhybridization using human genomic CD45-AP DNA as a hybridization probe. The genetic mapping of thePtprcap/PTPRCAPgenes extends the previously defined synteny conservation of various genes that have been assigned to these regions of the mouse and the human chromosomes.     

Mapping of theARIXHomeodomain Gene to Mouse Chromosome 7 and Human Chromosome 11q13

The recently described homeodomain protein ARIX is expressed specifically in noradrenergic cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine β-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouseArixwas positioned approximately 50 cM distal to the centromere of chromosome 7, nearHbb.HumanARIXwas positioned through analysis of somatic cell hybrids and fluorescencein situhybridization of human metaphase chromosomes to chromosome 11q13.3–q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.     

Chromosomal Localization of the Human Urothelial “Tetraspan” Gene, UPK1B, to 3q13.3–q21 and Detection of aTaqI Polymorphism

The TI1/UPK1b gene codes for a protein of the “tetraspan” family and is expressed as a differentiation product of the mammalian urothelium. A partial genomic clone of the human homologue of the TI1/UPK1b gene was isolated and used as probe to localize the human gene to chromosome 3q13.3–q21 byin situhybridization. Using the same probe, aTaqI restriction fragment length polymorphism, with 29% heterozygosity, was identified by Southern analysis.     

A Novel Zinc Finger-Containing RNA-Binding Protein Conserved from Fruitflies to Humans

TheDrosophila larkgene encodes an essential RNA-binding protein of the RNA recognition motif (RRM) class that is required during embryonic development. Genetic analysis demonstrates that it also functions as a molecular element of a circadian clock output pathway, mediating the temporal regulation of adult emergence in the fruitfly. We now report the molecular characterization of a human gene with significant similarity tolark.Based on fluorescencein situhybridization and radiation hybrid mapping, the human gene has been localized to chromosome region 11q13; it is closely linked to several identified genes including the locus of Bardet–Biedl syndrome type 1. Thelark-homologous human gene expresses a single 1.8-kb size class of mRNA in most or all tissues including brain. Additional database searches have identified a mouse counterpart that is virtually identical to the human protein. Similar to lark protein, both mammalian proteins contain two copies of the RRM-type consensus RNA-binding motif. Unlike most RRM family members, however, theDrosophilaand mammalian proteins also contain a retroviral-type (RT) zinc finger that is situated 43 residues C-terminal to the second RRM element. Within a 184-residue segment spanning the RRM elements and the RT zinc finger, the human and mouse proteins are 61% similar to theDrosophilalark sequence. These common sequence features and comparisons among a large collection of RRM proteins suggest that the human and mouse proteins represent homologues ofDrosophilalark.     

Detection of nerve growth factor and its mRNA by separate and combined immunohistochemistry andin situ hybridization in mouse salivary glands

Summary Intense labelling of secretory cells in the male mouse submandibular gland was observed afterin situ hybridization using mouse nerve growth factor (NGF) cDNA probes. Under the same conditions, sparse less intensely labelled cells were also found in the sublingual gland. Hybridization to a chicken NGF cDNA probe gave weak labelling on the glands in accordance with a weak cross-hybridization between mouse NGF mRNA and chicken NGF cDNA probes, whereas no labelling was seen using pUC9 DNA as a hybridization probe. A combination ofin situ hybridization and immunohistochemistry was also carried out on the same sections of submandibular gland. A good correlation was seen between actively synthesizing and intensely immunoreactive cells in the gland. The technique described here allows the detection of individual cells synthesizing relatively low levels of NGF. The combination ofin situ hybridization and immunocytochemistry on the same section should be particularly useful in cases where NGF is transported away from its site of synthesis.     

Isolation and Characterization of a New Member of the HumanLy6Gene Family(LY6H)

TheLy6family of genes encodes glycosylphosphatidylinositol-anchored cell surface glycoproteins expressed on various types of cells. Intriguing patterns of expression ofLy6genes on specific subpopulations of lymphoid and myeloid cells suggest that Ly6 molecules may be involved in the development and homeostasis of hematopoietic cells. We have isolated a new member of the humanLy6gene family,LY6H,from a human fetal brain cDNA library. Fluorescencein situhybridization and radiation hybrid analyses assignedLY6Hto chromosome 8, where other members of theLy6gene family are also located. Northern analysis revealed thatLY6His highly expressed in particular subdivisions of human brain and also in MOLT-3 and -4 acute lymphoblastic leukemia cells. These data suggest that LY6H may play a role(s) in both the central nervous system and the immune system.