Surfeit locus gene homologs are widely distributed in invertebrate genomes.

The mouse Surfeit locus contains six sequence-unrelated genes (Surf-1 to -6) arranged in the tightest gene cluster so far described for mammals. The organization and juxtaposition of five of the Surfeit genes (Surf-1 to -5) are conserved between mammals and birds, and this may reflect a functional or regulatory requirement for the gene clustering. We have undertaken an evolutionary study to determine whether the Surfeit genes are conserved and clustered in invertebrate genomes. Drosophila melanogaster and Caenorhabditis elegans homologs of the mouse Surf-4 gene, which encodes an integral membrane protein associated with the endoplasmic reticulum, have been isolated. The amino acid sequences of the Drosophila and C. elegans homologs are highly conserved in comparison with the mouse Surf-4 protein. In particular, a dilysine motif implicated in endoplasmic reticulum localization of the mouse protein is conserved in the invertebrate homologs. We show that the Drosophila Surf-4 gene, which is transcribed from a TATA-less promoter, is not closely associated with other Drosophila Surfeit gene homologs but rather is located upstream from sequences encoding a homolog of a yeast seryl-tRNA synthetase protein. There are at least two closely linked Surf-3/rpL7a genes or highly polymorphic alleles of a single Surf-3/rpL7a gene in the C. elegans genome. The chromosomal locations of the C. elegans Surf-1, Surf-3/rpL7a, and Surf-4 genes have been determined. In D. melanogaster the Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs and in C. elegans the Surf-1, Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs are located on completely different chromosomes, suggesting that any requirement for the tight clustering of the genes in the Surfeit locus is restricted to vertebrate lineages.     

Isolation of a mouse heat-shock gene (hsp68) by recombinational screening

We have used cloned fragments from a Drosophila melanogaster hsp70 gene and a mouse hsp68 cDNA in recombinational screens of mouse genomic libraries. Using the mouse probe we have isolated two overlapping recombinant lambda phages comprising 22 kb of cloned DNA. Southern analysis has localized the homology with the Drosophila hsp70 coding region to a 2.2-kb fragment containing the mouse heat-shock gene. Insertion accompanying recombinational screening can disrupt interesting sequences; we have overcome this inconvenience by developing a simple one-step genetic selection for phage which have precisely excised the microplasmid probe.     

High-frequency P element loss in Drosophila is homolog dependent

P transposable elements in Drosophila melanogaster can undergo precise loss at a rate exceeding 13% per generation. The process is similar to gene conversion in its requirement for a homolog that is wild type at the insertion site and in its reduced frequency when pairing between the homologs is inhibited. However, it differs from classical gene conversion by its high frequency, its requirement for P transposase, its unidirectionality, and its occurrence in somatic and premeiotic cells. Our results suggest a model of P element transposition in which jumps occur by a "cut-and-paste" mechanism but are followed by double-strand gap repair to restore the P element at the donor site. The results also suggest a technique for site-directed mutagenesis in Drosophila.     

Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionarily distant rodent genomes.

Sequences related to mouse intracisternal A-particle (IAP) genes have been isolated from rat and Syrian hamster gene libraries as recombinants in lambda phage. The sequences are moderately reiterated in both these genomes but their sequence organization in the hamster genome is different from that in the rat genome. Restriction analysis and electron microscopy indicate that the Syrian hamster IAP sequences represent a family of relatively homogeneous well-conserved units; in this, they resemble the mouse IAP genes. The rat sequences, in contrast, are heterogeneous. Both the hamster and rat IAP sequences contain regions homologous to mouse IAP genes interspersed with regions of apparent non-homology. The interspersed regions range in size from 0.5-1.0 kilobases (Kb). The regions of homology among the mouse, rat and Syrian hamster IAP sequences have been mapped to a 5-6 Kb internal region on the mouse IAP genes. Mouse IAP long terminal repeat (LTR) sequences were not detected in the rat and Syrian hamster genomes. We used the thermal stability of hybrids between cloned and genomic IAP sequences to measure family homogeneity. Mouse and Syrian hamster IAP sequences are homogeneous by this criterion, but the rat IAP sequences are heterogeneous with a Tm 6 degrees C below the self-hybrid. The contrasting organization of IAP-related elements in the genomes of these rodents indicates that amplification or homogenization of this sequence family has occurred independently and at different periods of time during their evolution.     

Zinc finger protein gene complexes on mouse chromosomes 8 and 11

Two murine homologs of the Drosophila Krüppel gene, a member of the gap class of developmental control genes that encode a protein with zinc fingers, were mapped to mouse chromosomes 8 and 11 by using somatic cell hybrids and an interspecific backcross. Surprisingly, both genes were closely linked to two previously mapped, Krüppel-related zinc finger protein genes, suggesting that they are part of gene complexes.     

The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes

Drosophila glial cells missing (gcm) is a key gene that determines the fate of stem cells within the nervous system. Two mouse gcm homologs have been identified, but their function in the nervous system remains to be elucidated. To investigate their function, we constructed retroviral vectors harboring Drosophila gcm and two mouse Gcm genes. Expression of these genes appeared to influence fibroblast features. In particular, mouse Gcm1 induced the expression of astrocyte-specific Ca(2+)-binding protein, S100beta, in those cells. Introduction of the mouse Gcm1 gene in cultured cells from embryonic brains resulted in the induction of an astrocyte lineage. This effect was also observed by in utero injection of retrovirus harboring mouse Gcm1 into the embryonic brain. However, cultures from mouse Gcm1-deficient mouse brains did not exhibit significant reductions in the number of astrocytes. Furthermore, in situ hybridization analysis of mouse Gcm1 mRNA revealed distinct patterns of expression in comparison with other well-known glial markers. The mammalian homolog of Drosophila gcm, mouse Gcm1, exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells.     

Chromosomal localization of the human homeo box-containing genes, EN1 and EN2

The human homologs of the mouse homeo box-containing genes, En-1 and En-2, which show homology to the Drosophila engrailed gene, have been isolated. The human EN1 gene was mapped to chromosome 2 by analysis of mouse-human somatic cell hybrids. The human EN2 gene was localized to chromosome 7, 7q32-7qter, by analysis of rodent-human somatic cell hybrids and cell lines carrying portions of chromosome 7.     

Molecular cloning of retrovirus-like genes present in multiple copies in the Syrian hamster genome.

Endogenous retrovirus-like sequences homologous to intracisternal type-A particle (IAP) genes, which are present in the inbred mouse (Mus musculus) genome, were cloned from a Syrian hamster gene library. A typical hamster IAP gene was 7 kb long and segments homologous to long terminal repeat (IAP) sequences present in Mus musculus IAP genes were located at both ends of the gene. Contrary to the pattern found in the Mus musculus IAP genes, the organization of the cloned hamster IAP genes was not markedly polymorphic and deletion was not observed among these cloned genes. A sequence about 0.8 kb long and located close to the 3' end of the hamster IAP gene was well conserved in both IAP gene families, although they showed less overall homology with one another. The reiteration frequency of the hamster IAP genes was calculated to be 950 copies per haploid genome. Since such IAP genes with the above properties were not found in the genome of the Chinese hamster, whose progenitors diverged from those of the Syrian hamster about 7.5 Myr ago, the integration of a huge number of Syrian hamster IAP genes must have occurred subsequent to such divergence.     

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.     

Assignment of RAS proto-oncogenes in Chinese hamsters: implications for mammalian gene linkage conservation and neoplasia

HRAS and KRAS are the cellular homologs of the oncogenic transforming genes found in the Harvey strain of murine sarcoma virus and the Kirsten murine sarcoma virus, respectively. Phyla as diverse as insects, birds, and mammals possess distinct HRAS and KRAS sequences, suggesting that these genes are essential to metazoa. In this report, we used a clone panel of Chinese hamster X mouse C11D somatic cell hybrids segregating hamster chromosomes to map those genes. Southern filter hybridization analyses of the hybrids revealed that hamster HRAS and KRAS gene sequences are on chromosomes 3 and 8, respectively. These gene assignments are consistent with the conservation of autosomal gene linkage groups observed among hamsters, humans, and mice and may provide insight into specific chromosomal alterations that have been observed during the spontaneous neoplastic transformation of Chinese hamster fibroblasts in vitro.     

The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family.

The Drosophila segment polarity gene product Porcupine (Porc) was first identified as being necessary for processing Wingless (Wg), a Drosophila Wnt (Wnt) family member. Mouse and Xenopus homologs of porc (Mporc and Xporc) were identified and found to encode endoplasmic reticulum (ER) proteins with multiple transmembrane domains. In contrast with porc, four different types of Mporc and Xporc mRNA (A-D) are generated from a single gene by alternative splicing. Mporc mRNA is differentially expressed during embryogenesis and in various adult tissues, demonstrating that the alternative splicing is regulated to synthesize the specific types of Mporc. In transfected mammalian cells, all Mporc types affect the processing of mouse Wnt 1, 3A, 4, 6, and 7B but not 5A. Furthermore, all Mporc types are co-immunoprecipitated with various Wnt proteins. These results suggest that Mporc may function as a chaperone-like molecule for Wnt. Interestingly, all Mporc types can substitute for Porc, as they are able to rescue the phenotypes of Drosophila porc embryos. Consistent with this observation, Mporc, like Porc, modifies the processing of Wg expressed in mammalian cells. These results demonstrate that the porc gene family encodes the multitransmembrane ER proteins, which are evolutionarily well conserved and involved in processing the Wnt family.     

Functional and comparative genomic analysis of the piebald deletion region of mouse chromosome 14

Several developmentally important genomic regions map within the piebald deletion complex on distal mouse chromosome 14. We have combined computational gene prediction and comparative sequence analysis to characterize an approximately 4.3-Mb segment of the piebald region to identify candidate genes for the phenotypes presented by homozygous deletion mice. As a result we have ordered 13 deletion breakpoints, integrated the sequence with markers from a bacterial artificial chromosome (BAC) physical map, and identified 16 known or predicted genes and >1500 conserved sequence elements (CSEs) across the region. The candidate genes identified include Phr1 (formerly Pam) and Spry2, which are mouse homologs of genes required for development in Drosophila melanogaster. Gene content, order, and position are highly conserved between mouse chromosome 14 and the orthologous region of human chromosome 13. Our studies combining computational gene prediction with genetic and comparative genomic analyses provide insight regarding the functional composition and organization of this defined chromosomal region.