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Highly efficient phage-based Escherichia coli homologous recombination systems have recently been developed that enable genomic DNA in bacterial artificial chromosomes to be modified and subcloned, without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombinogenic engineering or recombineering, is efficient and greatly decreases the time it takes to create transgenic mouse models by traditional means. Recombineering also facilitates many kinds of genomic experiment that have otherwise been difficult to carry out, and should enhance functional genomic studies by providing better mouse models and a more refined genetic analysis of the mouse genome.

The introduction of two transgenes into one animal is increasingly common as transgenic experiments become more sophisticated. In this study we examine two strategies for creating double transgenic founders from a single microinjection. In the first approach, two constructs, each with its own promoter element, were coinjected into the pronucleus. In the second approach, both transgenes were cloned into one vector, separated by an internal ribosomal entry site (IRES), and placed under control of a single promoter. Both strategies save time and increase the percentage of double transgenic offspring over the standard method of mating single transgenic lines. However, despite high transgene copy numbers, the bicistronic lines did not show robust expression of either protein. Copy number and protein expression correlated much better in the coinjected lines, with expression levels in one line approaching that observed in some of our best single transgenic controls. Thus we recommend coinjection of individual plasmids for the generation of multiply transgenic founders.

Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25., (Copyright 1999 Academic Press.)

Paralemmin is a newly identified protein that is associated with the plasma membrane and with intracellular membranes through a lipid anchor. It is abundant in brain, is expressed at intermediate levels in the kidney and in endocrine cells, and occurs at low levels in many other tissues. As it is a candidate for genetic disorders that affect membrane functions, we have determined the structure of the human paralemmin gene, PALM, showing that it is organized into nine exons. Moreover, we have performed chromosomal assignments of the human and mouse paralemmin genes, localizing them to regions of homology at human 19p13.3 and the central mouse chromosome 10. Finally, mutation analysis using RNA from mice homozygous for the mutant genes grizzled (gr), mocha (mh), mocha 2J (mh2J), jittery (ji) and hesitant (ji(hes)), which map to this area, excluded mutations in their Palm coding sequences.

The evolutionarily conserved polycomb and trithorax-group genes are required to maintain stable expression patterns of homeotic genes and other target genes throughout development. Here, we report the cloning and characterization of a novel mouse polycomb homolog, MPc2, in addition to the previously described M33 polycomb gene. Co-immunoprecipitations and subnuclear co-localization studies show that MPc2 interacts with the mouse polycomb-group oncoprotein Bmi1 and is a new member of the mouse polycomb multiprotein complex. Gal4DB-MPc2 or -M33 fusion proteins mediate a five- to tenfold repression of stably integrated reporter constructs carrying GAL4 binding sites, demonstrating that these proteins are transcriptional repressors. The MPc2 gene is localized on chromosome 11, in close proximity to the classical mouse mutations tail short (Ts) and rabo torcido (Rbt). Ts and Rbt hemizygous mice display anemia and transformations of the axial skeleton reminiscent of phenotypes observed in mice with mutated polycomb or trithorax-group genes, suggesting that MPc2 is a candidate gene for Ts and Rbt., (Copyright 1997 Academic Press Limited.)

We have determined the sequence and structure of the mouse Müllerian-inhibiting substance (MIS) type II receptor gene. Sequence comparisons demonstrate that the mouse, rat, rabbit, and human MIS type II receptors are highly conserved. The mouse MIS type II receptor gene is encoded by 11 exons and spans approximately 9-kb. Only half of the intron/exon boundaries of its kinase domain are conserved in comparison to the kinase domain of the related activin type II receptor. Whereas the activin type II receptor gene contains large introns (> 40-kb), the largest intron of the MIS type II receptor gene is only 4.3-kb. The MIS type II receptor gene (Amhr) is closely linked to Hoxc on mouse chromosome 15. Knowledge of the sequence and genomic structure of Amhr provides important information for the genetic manipulation of the Amhr locus.

RP105 is a mouse B cell surface molecule that transmits a growth-promoting signal and is implicated in the life/death decision of B cells. RP105 has tandem repeats of a leucine-rich motif in the extracellular domain that is expected to be involved in protein-protein interactions. In the present study, a cDNA clone encoding the human homologue of RP105 was isolated. The amino acid sequence of human RP105 is highly homologous to that of mouse RP105 with 74% identity, and the leucine-rich repeats are well conserved. The expression of the human RP105 transcript was detected in some B cell lines, a histiocytic leukemia cell line, and peripheral blood leukocytes. We also determined the chromosomal locations of the mouse RP105 gene (Ly64 locus) and the human RP105 gene (LY64 locus). Interspecific mouse backcross analysis was used to map the Ly64 locus at the distal region of chromosome 13. The human LY64 locus was localized to 5q12 by fluorescence in situ hybridization, confirming the syntenic relationship between these regions of the mouse and human chromosomes.

Mouse annexin XI (anx11)2 was cloned from a macrophage cDNA library and characterized by genetic linkage mapping, DNA sequencing, and structural comparison with other annexins. The Anx11 gene localized to mouse chromosome 14 in close linkage with the Rarb, Plau, and Wnt5a genes near the centromere and 1.8 cM distal from the Anx7 gene. The open reading frame was flanked by long, untranslated regions and encoded a 503-amino-acid protein with 93.1% identity to its human orthologue. Its 189-aa amino terminus corresponded to the widely expressed variant 1 of two possible, alternatively spliced forms. A previously described peptide from Aplysia brasiliana was identified as a closely related invertebrate homologue. Since annexin XI is known to be localized in the nucleus at certain stages of development, the identification of a region in tetrad repeats 3 and 4 resembling the "chromo box" domain may be relevant to a nuclear regulatory function of annexin XI. Knowledge of the mouse cDNA sequence and genetic map location will assist in the analysis of genomic organization and expression and provide a useful animal model to investigate gene function and hereditary phenotype for annexin XI.

A mouse histamine H2 receptor (H2R) gene was isolated from a mouse ES cell genomic DNA library. The gene encoded a 358-amino-acid protein and displayed a 95% homology with the rat histamine H2 receptor at the amino acid level. It had features characteristic of other G-protein-coupled receptors. Reverse transcriptase-polymerase chain reaction analysis of total RNA prepared from mouse tissues showed that the gene was highly expressed in stomach and moderately in brain and heart. A weak expression was also detected in liver. An interspecific backcross analysis revealed that the mouse H2R gene, designated Hrh2, was located in the central region of chromosome 13.

The eph-related receptors are the largest subfamily of receptor tyrosine kinases. Recently, we and others have identified seven different, but related, cDNAs encoding membrane-bound ligands for this family of receptors. One member, LERK-7, is attached to the cell membrane via glycosyl-phosphatidylinositol linkage and has been found to be a ligand for the eph-family receptors hek, elk, eck, and rek. Using PCR-based screening of human x rodent somatic cell hybrid DNAs, we have assigned the gene that encodes LERK-7 (EPLG7) to human chromosome 5. Fluorescence in situ hybridization to metaphase chromosome preparations using a genomic clone from the locus refined this localization to chromosome 5, band q21. In addition, Southern blot analysis of DNAs from interspecific backcross mice indicated that the mouse homologue Epl7 maps to a homologous region on chromosome 17.

Atm, the mouse homolog of the human ATM gene defective in ataxia-telangiectasia (A-T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescence in situ hybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22-q23.

The Ikaros gene is an essential regulator in the development and homeostasis of the mouse lymphopoietic system. To study the role of the Ikaros gene in the human lymphopoietic system, we cloned and characterized human Ikaros cDNAs. In the human, as in the mouse, differential splicing of Ikaros primary transcripts generates a family of lymphoid-restricted zinc finger DNA binding proteins, highly conserved in sequence composition and relative expression to the mouse homologues. Expression of Ikaros isoforms is highly restricted to the lymphopoietic system and is particularly enriched in maturing thymocytes. The Ikaros gene maps at a syntenic locus located on the short arm of human chromosome 7 and on mouse chromosome 11 next to the epidermal growth factor receptor (Egfr). The high degree of conservation of the Ikaros gene at the genetic and expression levels strongly suggests that it plays a fundamental role in the ontogeny of the lymphopoietic system across species.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

By using primers complementary to the rat beta B1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences. CRYBB1 was assigned to the group 5 region in 22q11.2-q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products of CRYBB1 were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other beta crystallin genes (beta B2(-1), beta B3, and beta A4) have previously been mapped to the same regions in human and mouse. We demonstrate that the beta B1 and beta A4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major beta crystallins genes that are expressed in the bovine lens.

Elevated galactose levels can be caused by several enzyme defects, one of which is galactokinase. Galactokinase deficiency cause congenital cataracts during infancy and presenile cataracts in the adult population. We have isolated the mouse cDNA for galactokinase, which shares extensive amino acid sequence homology, 88% identity, with a recently cloned human galactokinase. It is expressed in all tissues examined. In an interspecific backcross analysis galactokinase maps to the distal region of mouse chromosome 11, a region that is homologous to human chromosome 17q22-25. The availability of the mouse gene provides an opportunity to make a knockout model for galactokinase deficiency.

There are four known members of the C/EBP family of basic region-leucine zipper transcription factors: Cebpa, Cebpb, Cebpd, and Cebpe. Cebpa has previously been mapped to mouse chromosome 7. Here, we show that Cebpb maps to mouse chromosome 2, Cebpd to chromosome 16, and Cebpe to chromosome 14. The assignment of Cebpd to chromosome 16 identifies a new region of homology between mouse chromosome 16 and human chromosome 8.

The genes for the bHLH-Zip transcription factors Tfap4, Mxi1, Tcfeb, Usf1, and Usf2 have been mapped in mouse by interspecific backcross analysis. Mxi1, Usf1, and Usf2 have been mapped previously by in situ hybridization, but their positions on the meiotic linkage map had not been determined. The other two genes have not previously been mapped in mouse. These transcription factors belong to a growing family of transcriptional regulators, some of which are known to form a complex network of interacting proteins that control cell proliferation and apoptosis. As expected, based on mapping studies of other bHLH-Zip genes, these loci were well distributed among mouse chromosomes. In addition, some of the probes used in this study detected multiple, independently segregating loci, suggesting the possible existence of additional family members or species-specific pseudogenes.

We have previously cloned the murine Lhx3 cDNA that encodes a predicted protein containing two tandemly repeated LIM domains and a homeodomain. Early expression of Lhx3 in oral ectoderm that is committed to contribute to the anterior and intermediate lobes of the pituitary and its perseverance in the adult gland strongly suggest an involvement of the gene in mediating and maintaining the differentiation program of this important endocrine system. Additional functions are suggested by the fact that Lhx3 is also expressed bilaterally along the spinal cord and the hindbrain at early stages of mouse development. Here we report the structural organization and chromosomal localization of the Lhx3 gene. The gene is composed of six exons and five introns. Two different exons, Ia and Ib, appear to be alternatively spliced to exon II. The first LIM domain is encoded by exon II and the second by exon III. The homeobox is shared by exons IV and V. We have mapped Lhx3 to the proximal region of mouse chromosome 2 in a region that shares homology with human chromosomes 9q and 10p.

The interleukin-3 receptor (IL-3R) is composed of alpha and beta subunits, members of the class I cytokine receptor family. Here we describe isolation and characterization of the chromosomal gene for the mouse IL-3R alpha subunit (mIL-3R alpha). Whereas the human IL-3R alpha gene is tightly linked with the granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-CSFR alpha) gene in the pseudoautosomal region of the X and Y chromosomes, the mIL-3R alpha gene (II3ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFR alpha gene, which is on chromosome 19. The mIL-3R alpha gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (5'GT-AG3'), and the whole genomic structure is similar to those of the previously reported class I cytokine receptor genes. There are two major transcription initiation sites that are located at 215 and 188 nucleotides upstream of the initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c-myb,, Sp1, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 5' noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoter functions in a cell type-specific manner. Further sequential deletion of the 5' region suggests two potential regulatory regions for transcription of the mIL-3R alpha gene.

The human beta crystallin genes CRYBB2, CRYBB2P1, CRYBB3, and CRYBA4 are located in 22q11.2. Using interspecific backcross analysis, we mapped the mouse homologues of CRYBB2, CRYBB3, and CRYBA4 (i.e., Crybb2, Crybb3, and Cryba4) to the central region of mouse chromosome 5. The homologue of human CRYBB2P1 is absent in mouse. These assignments define a new region of homology in human and mouse.

A 100-amino-acid DNA-binding motif, known as the winged helix, was first identified in the mammalian hepatocyte nuclear factor-3 (HNF-3) and Drosophila fork head family of transcription factors. Subsequently, more than 40 different genes that contain the winged helix motif have been identified. In the studies described here, we have determined the murine chromosomal location of eight members of this gene family, HFH-1, HFH-3, HFH-4, HFH-5, HFH-6, HFH-8, BF-1, and BF-2, by interspecific backcross analysis. These genes, designated HNF-3 fork head homolog 1 (Hfh1), Hfh3, Hfh4, Hfh5, Hfh6, Hfh8, Hfh9, and Hfh10, respectively, mapped to 6 different mouse autosomes and are thus well dispersed throughout the mouse genome. Based on this mapping information, we predict the chromosomal location of these genes in humans and discuss the potential of these genes as candidates for uncloned mouse mutations.

Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.

MDK and PTN are two members of a family of heparin-binding cytokines thought to be involved in a number of developmental processes. The locations for these genes were determined in man and mouse using somatic cell hybrid analysis and interspecific backcross analysis. Human MDK was mapped to 11p13-->p11. MDK in the mouse (Mdk) was mapped to a syntenic region of mouse Chromosome 2. A pseudogene of Mdk was mapped to mouse Chromosome 11. The closely related human gene PTN was mapped to a separate location on human chromosome region 7q22-->qter.

The Gfi1 gene encodes a zinc finger protein which binds DNA and is involved in transcriptional regulation. Gfi1 was assigned to the central portion of mouse Chr 5 by interspecific backcross mapping and to human chromosome band 1p22 and rat chromosome band 14p22 by fluorescence in situ hybridization (FISH). Comparative mapping data presented here describes a new syntenic region between man and rodents.

The murine homolog of the human anaplastic lymphoma kinase gene, which encodes a membrane-spanning receptor tyrosine kinase in the insulin receptor kinase subfamily, was assigned to mouse Chromosome 17 by interspecific backcross analysis. This assignment further confirms the homology between a portion of the distal Chromosome 17 and the short arm of human chromosome 2 and extends this region in the mouse by an additional 3 cM.

By subtraction cloning we previously identified a set of mouse genes (named Nedd1 through Nedd10) with developmentally down-regulated expression in brain. We now show that one such gene, Nedd2, encodes a protein similar to the mammalian interleukin-1 beta-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3 (CED-3). Both ICE and CED-3 are known to encode putative cysteine proteases and induce apoptosis when overexpressed in cultured cells. Overexpression of Nedd2 in cultured fibroblast and neuroblastoma cells also resulted in cell death by apoptosis, which was suppressed by the expression of the human bcl-2 gene, indicating that Nedd2 is functionally similar to the ced-3 gene in C. elegans. We also show that during embryonic development, Nedd2 is highly expressed in several types of mouse tissue undergoing high rates of programmed cell death such as central nervous system and kidney. Our data suggest that Nedd2 is an important component of the mammalian programmed cell death machinery.

The genes for insulin-like growth factor binding proteins (IGFBPs) encode secreted proteins that bind insulin-like growth factors I and II with high affinity and modulate their biological activities. In this report we have used interspecific backcross mapping and gene cloning to define the chromosomal locations of 4 mouse Igfbp genes. Igfbp1 and 3 are found in the proximal part of chromosome 1. In the human genome these two loci map within 20 kb of one another on chromosome 7p14-p12, and the genes are organized in a tail-to-tail configuration. Mouse Igfbp2 and 5 colocalize to a proximal region of chromosome 1 that is syntenic with human chromosome 2q33-q36, and the two genes are 5 kb apart in a tail-to-tail orientation. These results suggest an evolutionary scheme in which a primordial IGFBP gene duplicated to form a cluster that was later replicated to create second linkage group.

Mouse cDNA clones coding for a desmocollin and a desmoglein, desmosomal cadherins that are putative adhesion molecules of the desmosome type of cell-cell junction characteristically found in epithelial tissues, have been isolated and sequenced. From sequence comparisons with the known human and bovine desmosomal cadherins, these clones represent a mouse Dsc3 and Dsg1. By interspecific backcross analysis, these genes were found to be closely linked in the proximal region of mouse chromosome 18, a region having conserved synteny with human chromosome 18. From these results, and recently reported linkage of DSG1 and DSG2 on human chromosome 18 at 18q12.1 in a deletion panel of somatic cell hybrids, all the desmosomal cadherins genes so far examined are clustered on chromosome 18 in human and mouse, which may have implications for gene expression. We further show that the human DSC3 gene, previously reported to be located on chromosome 9, also maps to human chromosome 18.

A murine transcription factor, PEBP2, is composed of two subunits, alpha and beta. There are two genes in the mouse genome, PEBP2 alpha A and PEBP2 alpha B, which encode the alpha subunit. Two types of the alpha B cDNA clones, alpha B1 and alpha B2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8- and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described alpha B protein referred to as alpha B1, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed alpha B2, which is identical to alpha B1 except that it has an internal deletion of 64 amino acid residues. Both alpha B1 and alpha B2 associate with PEBP2 beta and form a heterodimer. The alpha B2/beta complex binds to the PEBP2 binding site two- to threefold more strongly than the alpha B1/beta complex does. alpha B1 stimulates transcription through the PEBP2 site about 40-fold, while alpha B2 is only about 25 to 45% as active as alpha B1. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that alpha A, alpha B, and beta genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AML1 on chromosome 21q22 and PEBP2 beta/CBF beta on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that previously described chimeric gene products, AML1/MTG8(ETO) and AML1-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AML1.

The extent of the semidominant coloboma (Cm) mutation on mouse Chromosome 2 was determined by deletion mapping using interspecific hybrid mice. The Cm deletion mutation results in ophthalmic dysmorphology and behavioral deficits, including profound hyperactivity, and has been shown to encompass the gene Snap. In addition to Snap, the gene encoding phospholipase C beta-1 (Plcb-1), which maps 0.60 +/- 0.60 cM proximal to Snap, and simple sequence repeat (SSR) loci D2Mit19, D2Mit46, D2Mit28, and D2Mit136 were shown to be deleted at the Cm locus. In contrast, analysis of other closely linked SSRs and genes either proximal (Bmp-2a) or distal (Nec-1) to Snap, as well as a complementation test with the closely linked mutation lethal milk (lm), indicates that these gene sequences are unaffected by the Cm mutation. These data demonstrate that the Cm deletion represents a contiguous gene defect encompassing 1.1 to 2.2 cM that may be probed for genes, both in the mouse and in the syntenic region of human Chr 20, that independently affect elements of neurological behavior and eye development.

The Grg gene encodes a 197-amino-acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. We describe here the genomic organization of the mouse Grg gene. It spans approximately 7 kb on chromosome 10 and consists of seven exons. The 3' region of the Grg gene contains two functional polyadenylation sites that give rise to two transcripts that are differentially expressed among adult mouse tissues. The promoter region is very GC rich and lacks TATA box and "initiator" sequences. Primer extension analysis and ribonuclease protection assays show that Grg has a major transcription start site situated down-stream of putative binding motifs for the transcription factors Sp1, E2A, and PuF.

Insulin-like growth factor binding proteins (IGFBPs) constitute a family of at least six secreted proteins that bind insulin-like growth factors I and II (IGF-I and -II) and are capable of modifying IGF actions on target cells. We previously have purified an approximately 29-kDa IGFBP that is secreted by myoblasts during their terminal differentiation, have identified the protein as mouse IGFBP-5, and have cloned its cDNA (James et al., 1993). In this study, we have characterized the mouse IGFBP-5 gene, established its pattern of expression in the adult mouse, and defined its chromosomal location. The 17-kb gene was isolated on overlapping cosmid and lambda clones, and the four exons encoding the 5914-bp mRNA were sequenced. The 5' end of the gene was mapped by solution-hybridization ribonuclease protection assay to two discrete sites in exon 1 that were separated by 21 bp. The relative use of each transcription start site was found to vary among different mouse tissues. By interspecies backcross mapping using progeny derived from matings of [(C57BL/6J X Mus spretus)F1 x C57BL/6J] mice, the IGFBP-5 gene was localized to the proximal region of chromosome 1 in tight linkage with fibronectin 1 (0 recombinants in 168 mice analyzed). Since this part of chromosome 1 shares homology with human chromosome 2q, and since fibronectin has been mapped to 2q34-q36, it is likely that human IGFBP-5 will reside on 2q as well. Characterization of the mouse IGFBP-5 gene now provides a starting point for studying the roles and regulation of this protein in development.

The human and murine chromosomal localization for the gene for the retinal pigment epithelium-specific protein RPE65 was determined. Using interspecific backcross analysis, we mapped Rpe65 to the distal end of mouse chromosome 3. In the human, using a human-hamster hybrid panel, RPE65 was mapped to chromosome 1. By the use of fluorescence in situ hybridization, this localization was refined to 1p31. The mouse and human loci for this potential candidate gene for hereditary retinal disease do not match those of any known disease in mouse or man.

The activation of cyclic AMP-dependent protein kinase is controlled by the regulatory (R) subunits of the holoenzyme. Here we present a characterization of the mouse RI beta subunit gene, which in contrast to other subunit genes of cyclic AMP-dependent protein kinase is expressed almost exclusively in neurons. It was determined that RI beta is relatively large with 11 exons spanning a minimum 75 kb. The mouse chromosomal locus (designated Prkar1b) was determined by interspecific backcross mapping and found to reside on the distal arm of chromosome 5. Previously, it was shown that 3.5 kb of DNA encompassing the RI beta promoter could direct neural-specific gene expression in transgenic mice. Analysis of this DNA suggests the presence of an unusually large number of binding sites for transcription factors ranging from tissue-specific regulators, immediate-early genes, and mediators of hormone action. In addition to 18 putative SP1 sites, we identified 27 consensus sequences for basic Helix-Loop-Helix, POU, and Pax family members, 5 AP1 sites, and over 40 half-sites for the superfamily of steroid hormone receptor. Gel mobility-shift assays employing brain nuclear extract and pure transcription factor protein established that many of these DNA sequences are functional in binding protein. The abundance and configuration of transcription factor binding sites within the promoter region of RI beta suggests that this gene is subject to complex modes of regulation in neurons.

A gene has been characterized that is required for postimplantation mouse development. The gene, designated fug1, was disrupted in embryonic stem cells by the U3Neo gene trap retrovirus, and the disrupted allele was introduced into the germ line. Homozygous mutant embryos arrest at the egg cylinder stage at about embryonic day 6 and are mostly resorbed by day 8.5. The appearance of the proamniotic cavity is delayed, and epiblast cells that surround the cavity are disorganized. fug1 transcripts are undetectable at E6 but are induced throughout the embryo after E6.5. The gene is expressed at low levels in all adult tissues examined, maps to chromosome 15, and is conserved among mammals. The cDNA sequence encodes a protein of 589 amino acids, the first 400 of which are 38% identical to the Saccaromyces cerevisiae RNA1 gene. Regions of greatest similarity include a long acidic domain and 11 leucine-rich motifs, thought to mediate high affinity protein-protein interactions. These similarities suggest that Fug1 may be required for developmental changes in RNA processing or chromatin structure prior to gastrulation.

Centromere mapping of mouse chromosomes has been problematic due to a paucity of appropriate markers. As a result, the mapping of centromeres has most often relied on the use of Robertsonian chromosomes to mark chromosome ends. Many Robertsonian translocations have been shown to suppress recombination in pericentric regions; therefore, centromere mapping data generated by using Robertsonian chromosomes must be interpreted with caution. We have utilized a new tool for centromere mapping that is applicable to all mouse chromosomes (except the Y chromosome) and that potentially overcomes the inherent limitations of using Robertsonian translocations. Briefly, an interspecific backcross mapping panel was constructed from crosses of C57BL/6Ros and Mus spretus mice. The centromere of each chromosome was subsequently typed by in situ hybridization, using a major satellite probe that uniformly labels C57BL/6Ros centromeres but hybridizes only weakly to M. spretus centromeres. Genetic markers that were already known to map in the proximal region of each of the mouse chromosomes were then typed by segregation analyses of restriction fragment length polymorphisms. These studies have made it possible to align the interspecific genetic map of each of the mouse autosomes and the X chromosome with respect to the centromere. They also provide a basis for comparison with centromere mapping data generated previously by other means.

In many mammalian species antibodies transmitted from the mother provide humoral immunity to the young. Maternal IgG from milk is transported across the intestinal epithelium of neonatal rats by an Fc receptor (FcRn) that comprises an alpha-chain similar to the class I Ag of the MHC and beta 2-microglobulin. Suckling mice also acquire antibodies by uptake from the gut. We made a neonatal mouse intestinal cDNA library and screened it with a probe encoding rat FcRn alpha-chain. The nucleotide and predicted amino acid sequences of the two positive clones were very similar to those of rat FcRn. Comparison of the FcRn domains to various MHC class I and CD1 molecules suggests a divergence of FcRn from MHC early in the mammalian lineage. We expressed one of these cDNA in mouse 3T3 fibroblasts. Cells that expressed the cDNA product bound the Fc fragment of IgG with the same pH dependence as neonatal rat intestinal epithelium. We detected RNA that hybridize with the mouse cDNA only in neonatal small intestine and fetal yolk sac, two tissues involved in IgG transport. These data show that the mouse cDNA code for FcRn alpha-chain. The mouse FcRn alpha-chain is similar in sequence to the class I MHC Ag, encoded on chromosome 17 in the mouse. However, we find that the mouse FcRn gene lies outside the MHC, on chromosome 7.

Technological advances have made possible the development of high-resolution genetic linkage maps for the mouse. These maps in turn offer exciting prospects for understanding mammalian genome evolution through comparative mapping, for developing mouse models of human disease, and for identifying the function of all genes in the organism.

R-cadherin was originally identified as a chicken cadherin expressed by the retina. Here, we describe the identification of a mouse homologue of R-cadherin. We isolated mouse cDNAs encoding a cadherin with 94% identity in amino acid sequence to the chicken R-cadherin, and defined this molecule as mouse R-cadherin. L cells transfected with the mouse R-cadherin cDNA acquired a cadherin-mediated cell-cell adhesiveness as found for other cadherins. To examine the binding specificity of mouse R-cadherin, L cells expressing this cadherin (mRL) were mixed with L cells expressing chicken R-cadherin (cRL), mouse N-cadherin (mNL), mouse E-cadherin (mEL) and mouse P-cadherin (mPL). While mRL cells randomly intermixed with cRL cells, those cells aggregated separately from mEL or mPL cells. Mixing of mRL with mNL cells gave an intermediate result; that is, they formed both separate and chimeric aggregates, suggesting that R- and N-cadherin can interact with each other although each has a preference to bind to its own type. Similar properties were previously found for chicken R-cadherin. Thus, the cell binding specificity of R-cadherin is entirely conserved between the two species, suggesting a conserved role for this protein in morphogenesis. We also located the mouse R-cadherin gene to chromosome 2.

We have cloned cDNAs from a novel gene designated Unp. Unp cDNAs contain a large open reading frame that would encode a protein of 89 kDa. The predicted protein contains a putative nuclear localization signal, as well as consensus sequences for binding to the retinoblastoma gene product. The latter elements are contained within a region having strong similarity to the human tre oncogene. We have localized the Unp gene to mouse chromosome 9 in a region of homology with human chromosome 3p. This region has been implicated in a number of human malignancies.