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Oligosyndactylism (Os) is a radiation-induced mouse mutation associated with recessive lethality and a dominant effect on limb and kidney development. The lethal effect of the mutation is due to a cell-autonomous block in the transition from metaphase to anaphase. We have previously characterized two transgene-induced mutations, 94-A and 94-K, which are allelic with Os. These mutations facilitated the identification of genomic segments and transcribed sequences in the affected region. One of the transcripts in this region corresponds to the mouse homolog of the anaphase-promoting complex component APC10/DOC1. The disruption of this gene can explain the mitotic arrest phenotype of all three alleles of Os., (Copyright 2001 Academic Press.)

CpG islands are GC-rich regions located in the promoter regions of housekeeping genes and many tissue-specific genes. While most CpG islands are normally unmethylated, island methylation can occur and is associated with silencing of the corresponding gene. Experiments with transgenic mice and DNA transfection in pluripotential embryonic cells have led to the conclusion that the information required for protecting the islands from methylation is contained within the CpG islands themselves and have identified Sp1 binding sites as an important element in establishing and/or maintaining the methylation-free state of CpG islands. To examine the generality of these observations, we analyzed the methylation of one of the mouse Igf2 CpG islands and its flanks in transgenic mice. We observed that the undermethylated state of this region is dependent on the presence of a separate cis-regulatory element, the H19 enhancers. These tissue-specific enhancers had a ubiquitous, non-tissue-specific effect on island region methylation. Structural alterations outside of the island and these enhancers also affected this region's methylation. These findings indicate that the methylation of some CpG island-containing regions is more sensitive than previously believed to the activity of distant cis-regulatory elements and to structural alterations in nonisland sequences in cis., (Copyright 1999 Academic Press.)

The insulin-like growth factor II is mitogenic for a number of cell types and can inhibit apoptosis. The frequent expression of this gene in human and experimental animal tumors indicates that insulin-like growth factor-2 may play an important role in tumor development. It has also been hypothesized that overexpression of this growth factor may be responsible for the increased incidence of childhood tumors in patients with Beckwith-Wiedemann syndrome. To assess the effects of Igf2 on tumor development we produced six transgenic mouse lines that express the gene under the control of the H19 enhancers. Transgenic expression was initiated in the embryonic period but remained high in several adult tissues, including the mammary gland, lung, and liver. Adult transgenic females from five of the transgenic lines developed often multiple mammary tumors that had the ability to metastasize. Increased incidence of other solid tumors was also noted in older mice. These findings indicate that Igf2 expression increases the probability of malignant transformation and that the mammary gland is at a particularly high risk of tumor development in response to chronic increase in Igf2 gene dosage.

Oligosyndactylism (Os) is a radiation-induced mutation on mouse chromosome 8 associated with early postimplantation lethality in homozygotes and abnormal development of the limbs and kidneys in heterozygotes. The recessive lethal effect of Os is due to a mitotic block of the embryonic cells that becomes apparent at the blastocyst stage, but it is not known if the heterozygous effect of Os is due to haploinsufficiency of the gene responsible for the mitotic arrest, or is due to mutation(s) of other gene(s). We have recently described a transgene-induced recessive mutation, 94-A/K, that results in early postimplantation death of the embryos, and we have mapped this mutation to the same region of chromosome 8 where Os has been assigned. On the basis of complementation tests between transgenic and Os/+ mice, in vitro growth characteristics and increased mitotic index of 94-A/K embryos, and molecular structural analysis of 94-A and 94-K transgenic and Os/+ mice, we conclude that the 94-A/K mutation represents a new allele of Os. This insertional mutation should facilitate the isolation of a mammalian gene essential for normal progression of the cell cycle beyond metaphase.

We have produced three lines of transgenic mice that contain additional copies of the mouse phosphoglycerate kinase 1 (Pgk1) gene. Two of these lines, 94-A and 94-K, which are descendants of a common founder, did not produce liveborn progeny carrying two copies of these transgenes (i.e., A/A, K/K, or A/K). Genotyping of midgestation embryos showed that A/K embryos are dead by Embryonic Day 10. Comparison of the level of transgene expression in the three transgenic lines ruled out PGK1 toxicity as the cause of death of A/A, A/K, and K/K embryos. The death of A/A, K/K, and A/K transgenic mice was therefore attributed to an insertional mutation disrupting a gene or genes essential for normal embryogenesis. Analysis of the structure of the 94-A and 94-K transgenes indicated that they differ in the number of tandem repeats and in the positions of the transgene-cellular DNA junctions. To determine if the two transgenes represent a single integration followed by a rearrangement or two independent integration events, we cloned the endogenous sequences surrounding the 94-A and 94-K transgene insertion sites. Restriction analysis of the isolated genomic clones indicated that the endogenous sequences abutting the 3' ends of the 94-A and 94-K transgenes are separated by less than 20 kb, providing strong support for the single integration model. Further analysis indicated that the 94-A transgene is associated with a deletion of at least 18 kb and is located in the vicinity of a widely transcribed endogenous gene. Chromosomal mapping of the endogenous sequences flanking the 94-A and 94-K transgene insertions using mouse-hamster somatic cell hybrids and a (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei backcross panel allowed us to assign the 94-A(K) transgene insertion to the subcentral region of mouse chromosome 8.

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to be encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.

The sensitivity of small transgenes to position effects on their expression suggests that they could serve as indicators of the chromatin properties at their integration site. In particular, they might be expected to provide information on the functional properties of mammalian heterochromatin. We have produced a transgenic line that carries a mouse Hprt minigene on the Y chromosome. In situ hybridization localized the transgene to the heterochromatic portion of the Y. Analysis of transgene expression by isoelectric focusing indicated that the transgene is expressed in a mosaic pattern, and expressing cells have different levels of transgene activity. These findings can be explained as a position effect variegation induced by Y heterochromatin. However, two other transgenes, located at autosomal sites, also showed mosaic activity. If the mosaic transgene expression is attributed to the influence of the chromatin at the insertion site, the Y heterochromatin would appear less potent than some autosomal regions at inducing variegation. An alternative explanation consistent with our results is that the mosaic expression is a semi-autonomous characteristic of these transgene loci. Transgene-expressing and non-expressing cells differed in their ability to grow and be cloned in vitro, indicating that cellular differentiation affected the chromatin structure of the transgene locus on the Y. Karyotype analysis of male mice with the Y-linked transgene and from control male mice carrying the human HPRT transgene, or the mouse Pgk-1 gene at autosomal sites, indicated that the transgene-carrying Y is prone to non-disjunction, generating cells with two (or more) or no Y chromosomes in equal proportion. Further studies will determine if the propensity of this Y chromosome to mitotic errors is also observed in vivo.

The murine glial fibrillary acid protein (GFAP) gene is located on chromosome 11 in close proximity to the genes encoding transforming protein p53 (Trp53) and myeloperoxidase (Mpo). Both Trp53 and Mpo have been mapped to human chromosome 17, but the chromosomal assignment of human GFAP has not been previously determined. In this report, we have amplified a cDNA fragment encoding a portion of GFAP from human brain and have used this probe to screen a mouse x human somatic cell hybrid panel. The results show that a human-specific GFAP species of approx 3.7 kb maps to one of these lines, TMS5, which contains chromosome 17 as its only human chromosome. On the basis of these data we speculate that there may be evolutionary relatedness between GFAP and other genes that map to both murine chromosome 11 and human chromosome 17.

In mouse development, the paternal allele of the X-linked gene Pgk-1 initiates expression on day 6, two days later than the maternal allele, which is activated on day 4. The different timing of expression of the maternal and paternal alleles may be determined by (i) imprinting of the chromosome region in which the gene resides, but not aimed specifically at the Pgk-1 gene; (ii) gene specific imprinting, acting on Pgk-1 irrespective of the chromosomal localization of the gene; (iii) an interplay between embryo cell differentiation, timing of X-inactivation and Pgk-1 expression, without the involvement of imprinting at the Pgk-1 locus itself (Fundele R., Illmensee, K., Jagerbauer, E. M., Fehlau, M. and Krietsch, W. K. (1987) Differentiation 35, 31-36). Our findings in transgenic mouse lines, carrying Pgk-1 on autosomes, indicate the importance of the X chromosomal location for the delayed expression of the paternal Pgk-1 allele, and are in agreement with the first of the explanations listed above. We propose that the late activation of the paternal Pgk-1 locus is a consequence of imprinting targeted at, and centered around, the X chromosome controlling element.

The effect of a normal mouse X chromosome on the chromosome segregation of mouse-Chinese hamster somatic cell hybrids was determined by (i) producing hybrids between the mouse sarcoma line CMS4 and a microcell hybrid (mfe4) of the hamster line E36, containing a mouse X chromosome from a normal cell; (ii) isolating hybrids between CMS4 and a 6-thioguanine selected (X minus) mfe4 subpopulation; (iii) comparing the direction of segregation in the two sets of hybrids. It was found that the normal X chromosome, like the X chromosomes from two MCA-transformed sarcoma lines reported previously [9], has the ability to switch the chromosome segregation of mouse-Chinese hamster somatic cell hybrids. We conclude that the reversal in chromosome segregation is mediated by factors located on the X chromosome. We designate these genetic elements as segregation reversal genes or sr genes.

The chromosomal location of the murine lambda 5 gene was analyzed by Southern hybridization using restriction enzyme-digested DNA from a panel of 15 mouse X hamster somatic cell hybrids. Sequences homologous with those of lambda 5 DNA were detected in DNA of 5 hybrids. In all 5 hybrids lambda 5 was contained in restriction fragments of equal sizes, the lengths of which indicated that the germline configuration of lambda 5 with three exons and the restriction sites expected from its genomic structure were present. Southern hybridization with the murine lambda 1 gene as a probe detected the same 5 hybrids as positive. The only mouse chromosome present on all of the positive hybrids, and absent from negative ones, was number 16. We conclude that lambda 5 is situated on the same chromosome as lambda 1, i.e., on the murine chromosome 16.

The murine homebox gene 4.1 was assigned to chromosome 2 by Southern analysis of somatic cell hybrids and by in situ hybridization. This assignment and the report of Featherstone et al. (M. S. Featherstone, A. Baron, S. J. Gaunt, M. G. Mattei, and D. Duboule, 1988, Proc. Natl. Acad. Sci. USA, 85, 4760-4764) indicate that a fourth group of homeobox genes is located on chromosome 2 in the mouse (in addition to the homeobox gene clusters on chromosomes 6, 11, and 15).

The 202 gene which specifies a 56.5 kD protein can be induced in Ehrlich ascites tumor cells by treatment with mouse beta interferon. This treatment increases the level of the gene 202-specific mRNA at least 12-fold. For determining the chromosomal location of this gene a 1.5 kb fragment of the gene was hybridized to EcoR1 digested DNA samples from a set of mouse-hamster somatic cell hybrids. Each of the cell hybrids used contained a complete array of hamster chromosomes and one or more mouse chromosomes. The 202 gene fragment hybridized to every DNA sample from cell hybrids containing mouse chromosome 1 (8 hybrids in total) and to none of the DNA samples from hybrids lacking this chromosome (7 hybrids in total). These and other data indicate that the 202 gene is located on mouse chromosome 1.

The actins are a group of highly conserved proteins encoded by a multigene family. We have previously reported that the skeletal muscle actin gene is located on mouse chromosome 3, together with several other unidentified actin DNA sequences. We show here that the gene coding for the cardiac muscle actin, which is closely related to the skeletal muscle actin (1.1% amino acid replacements), is located on mouse chromosome 17. The gene coding for the cytoplasmic beta-actin is located on mouse chromosome 5. Thus, these three actin genes are located on three different chromosomes.

The gene encoding the mouse placental alkaline phosphatase (ALP; orthophosphoric-monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1) is mapped to chromosome 4, based on Southern blot hybridization of the mouse cDNA with DNAs from mouse-Chinese hamster somatic cell hybrids. This assignment is consistent with the genetic analysis of the Akp-2 locus, which is responsible for the genetic variation of alkaline phosphatase enzyme in placenta as well as in liver, kidney, and bone.

Mouse-Chinese hamster somatic cell hybrids containing various combinations of mouse chromosomes were analyzed for the presence of the mouse c-Ha-ras (1) sequences after restriction endonuclease digestion and hybridization with a 32P-labeled Ha-ras specific probe according to the procedure of Southern (2). The presence of the mouse c-Ha-ras containing fragment was correlated with the presence of mouse chromosome 7 in the hybrids.

The locus recognized by the probe OS-3 is assigned to chromosome 10 both by Southern blot analysis of a panel of somatic cell hybrid DNAs and by genetic linkage to markers already assigned to chromosome 10. In Caucasians this probe recognizes a three-allele TaqI RFLP as well as two-allele BanII and RsaI RFLPs which are both in strong linkage disequilibrium with each other and with the TaqI RFLP. The D10S20 locus defined by this probe maps 5.5 cM distal to D10S4 on the long arm of chromosome 10. Because this human clone hybridizes with mouse genomic DNA, it will be useful in comparative mapping studies.

Two independent methods were used to identify the mouse chromosomes on which are located two families of immunoglobulin (Ig)-like genes that are rearranged and expressed in T lymphocytes. The genes coding for the alpha subunit of T-cell receptors are on chromosome 14 and the gamma genes, whose function is yet to be determined, are on chromosome 13. Since genes for the T-cell receptor beta chain were previously shown to be on mouse chromosome 6, all three of the Ig-like multigene families expressed and rearranged in T cells are located on different chromosomes, just as are the B-cell multigene families for the Ig heavy chain, and the Ig kappa and lambda light chains. The findings do not support earlier contentions that genes for T-cell receptors are linked to the Ig heavy chain locus (mouse chromosome 12) or to the major histocompatibility complex (mouse chromosome 17).

The direction of chromosome loss in two sets of mouse-Chinese hamster hybrids was compared with the direction of segregation of the same hybrids, to which an additional X chromosome derived from either of the mouse sarcoma lines MethAa, MethAs, or CMS4, was introduced at the time of the fusion. The addition of the X chromosome was carried out by substituting in place of the Chinese hamster parent a mouse X containing microcell hybrid of the latter. It was found that the addition of an X chromosome reverses the direction of chromosome segregation, but it can do so only if the mouse parent in the hybridization is different from the line from which the X originated. The possible reasons for recognition by the cells of a native and a foreign X are discussed. The existence of a multigene family on the X chromosome, involved in this recognition, is proposed.

Mouse spermatozoa contain two protamines with different amino acid sequences. By hybridizing Southern blots of a series of mouse-hamster somatic cell hybrids containing subsets of mouse chromosomes and a complete set of hamster chromosomes with 32P-labeled cDNAs for each mouse protamine, we assign the two mouse protamine genes to chromosome 16. This report presents the first evidence for chromosomal linkage of two sperm-specific, haploid regulated gene products.

Several interferon-activatable murine genes were mapped to murine chromosomes by hybridizing cDNA probes to Southern blots of genomic DNA samples from a panel of mouse-hamster somatic cell hybrid lines. The 12 gene is located on chromosome 12 and it specifies a 3.6-kb mRNA. The 204 gene (specifying a 2.5-kb mRNA), and three genes of the 203 gene family (hybridizing to five mRNAs of sizes between 2 and 4.5 kb), together with the 202 gene (specifying a 2-kb mRNA) are located on murine chromosome 1. By restriction fragment length polymorphism analysis of DNA samples prepared from a panel of recombinant inbred mouse lines (C57BL/6J D DBA/2J) and from 85 [C3H/HeJ-gld/gld x Mus spretus) F1 X C3H/HeJ-gld/gld] backcross mice we established a close linkage of the 202, 203, and 204 genes to the erythroid alpha-spectrin gene (Spna-1) on distal murine chromosome 1. Cosmids containing the 202, 203, and 204 genes were isolated from a library derived from AKR mouse DNA. Southern blot analysis of such cosmids revealed: (a) hybridization of a partial 203 cDNA to three genes of the 203 gene family; (b) cross-hybridization of the 202 and 204 genes with one another and with a third gene (designated as 201 gene), and (c) a close linkage of genes of the 203 family with the 201, 202, and 204 genes. These results indicate the existence of a cluster of at least six closely linked, interferon-activatable genes on distal murine chromosome 1 in the vicinity of the Spna-1 locus and also of the Minor lymphocyte stimulating locus (Mlsa).

The chromosomal assignments of genes in the PRL/GH family in the mouse have been determine in mouse-hamster hybrid cell lines. Mouse GH (mGH) appears to be encoded by a single copy gene located on chromosome 11 and is part of a highly conserved region between mouse chromosome 11 and human chromosome 17. All of the other genes in this hormone family, including those encoding mPRL, mouse placental lactogens I and II, and mouse proliferin and proliferin-related protein, map to chromosome 13.

The chromosomal distribution of murine genes expressed during differentiation of skeletal muscle cells was determined by Southern blot analysis of DNA from mouse-Chinese hamster hybrid cell lines containing incomplete subsets of mouse chromosomes. All detectable myosin heavy chain genes are located on chromosome 11. The gene for the myosin light chain 2 is located on chromosome 7. The skeletal muscle alpha-actin gene and several other actin genes, or pseudogenes, are located on chromosome 3. Additional actin DNA sequences are distributed on other mouse chromosomes.

Specific chromosomal abnormalities are regularly associated with many murine and human malignancies. In particular, the majority of murine plasmacytomas and human Burkitt's lymphomas contain a characteristic translocation which results in the juxtaposition of a cellular oncogene, c-myc, with the immunoglobulin heavy-chain gene locus, and this rearranged c-myc directs the synthesis of qualitatively and quantitatively abnormal transcripts which may have an aetiological role in the development of the transformed state in lymphoid malignancies. Similarly, rearrangement and abnormal expression of c-myb (ref. 10) and c-mos (ref. 11) has been reported in other murine lymphoid tumours. Here we describe a novel 6:10 chromosomal translocation in the murine plasmacytoma cell line NS-1 which juxtaposes the immunoglobulin Ck gene with a single-copy sequence of unknown function. The NS-1 plasmacytoma is a frequently used fusion partner in hybridoma production and is known to contain a rearranged c-myc gene and a genetic element which transforms normal mouse fibroblasts in DNA-mediated transfection assays. We conclude that individual B-cell tumours may contain multiple chromosomal translocations perhaps relevant to oncogenesis.

Cryptdin is a defensin-related peptide, and its mRNA accumulates to high abundance in epithelial cells of intestinal crypts beginning in the second week of postnatal development. The cryptdin (Defcr) locus was assigned to mouse chromosome 8 by Southern blotting of DNAs from mouse/hamster somatic hybrid cell lines. Analysis of somatic hybrid DNAs for mouse-specific restriction fragments showed zero discordance and perfect concordance with chromosome 8. The Defcr locus was localized on chromosome 8 by analysis of DNAs from recombinant inbred (RI) strains of mice after identification of three potential Defcr alleles based on restriction fragment length polymorphisms (RFLPs) in inbred strains. The strain distribution patterns of the Defcr locus were compared with those of chromosome 8 markers in five panels of RI strains. Analysis of cosegregation of Defcr with xenotropic proviral locus Xmv-26 and additional loci confirmed the chromosomal assignment and showed that Defcr is on proximal chromosome 8 within approximately 6 (1.3 to 21.3) cM of Xmv-26. The mouse Defcr locus and the human defensin gene(s) located on chromosome 8p23 appear to map to homologous regions.