Mouse Genomic Fragment Research Articles

Human homolog of a mouse sequence from the dystonia musculorum locus is on Chromosome 6p12

Dystonia musculorum is a hereditary neurodegenerative disease in mice that affects sensory neurons. In an effort to clone the gene responsible for this disorder, we have assembled a genomic contig spanning 75 kb of the dystonia musculorum (dt) locus. Within this genomic contig, we have identified a small restriction fragment that shows evolutionary conservation to rat, hamster, rabbit, and human genomic DNA. Using this mouse sequence, we have cloned the conserved human genomic fragment. Sequence analysis of the mouse and human genomic fragments revealed that they share a sequence similarity of 82% over 175 bp. A panel of human/rodent somatic cell hybrids was used to map the human genomic sequence to Chromosome (Chr) 6, and high-resolution in situ hybridization (FISH) allowed it to be sublocalized to 6p12. The human homolog of the mouse Bpag1 gene, a gene tightly linked to the mouse dt gene, also maps to Chr 6. Thus, this comparative mapping reveals a new region of conserved synteny between the chromosomes of mouse and human. Mapping the human homolog of the mouse dt gene enables us to initiate linkage studies to identify neurodegenerative disorders that may be caused by mutations in this gene.

Structures of Replacement Vectors for Efficient Gene Targeting1

The relationship between DNA structure of replacement vectors and gene targeting efficiency was studied using positive-negative selection. The vectors contained pBR322 DNA, a bacterial neomycin-resistance gene (neo) for positive selection, a herpes simplex virus (HSV) thymidine kinase gene (tk) for negative selection, and a mouse genomic fragment, including exons 1 to 3 of the transthyretin (ttr) gene. The neo gene that confers G418 resistance was inserted into the second ttr exon, and the HSV-tk gene that confers gancyclovir (GANC) sensitivity was added to the 3' end of the ttr fragment. The vectors were linearized by digesting with restriction enzyme(s) and transfected into mouse embryonal carcinoma F9 cells. In this system, the enrichment by GANC selection as well as the frequency of gene targeting was increased by placing the pBR322 DNA at the 3' end of the HSV-tk gene. Adding one more HSV-tk gene at the 5' end of the ttr fragment did not increase the enrichment by GANC selection. This enrichment factor was also increased by reducing the size of the ttr fragment present between the two selection markers. However, it decreased the frequency of gene targeting and, overall, it did not increase the efficiency of isolating targeted clones. When structures of the vector DNA fragments present in 20 G418-resistant and GANC-resistant non-targeted clones were examined by Southern blot analysis, the inefficiency of GANC selection proved to be mostly caused by exonucleolytic degradation of HSV-tk genes progressing from ends of the vectors.

Isolation and characterization of the mouse heme oxygenase-1 gene. Distal 5' sequences are required for induction by heme or heavy metals.

Mouse genomic fragments encoding heme oxygenase-1 (HO-1) were isolated from a recombinant lambda library by in situ plaque hybridization. The mouse HO-1 gene, approximately 7 kilobase pairs (kbp) in length, is organized into 5 exons and 4 introns. The primary structure of the exons and 1287 base pairs (bp) of the 5'-flanking region was determined. The deduced amino acid sequence of the mouse HO-1 gene is identical to that of p32, initially identified as a stress-induced protein in mouse BALBc/3T3 cells. A single, major transcription initiation site is utilized for constitutive and heme- or metal-induced expression of the HO-1 gene in mouse hepatoma (Hepa) cells. The transcriptional activity of the 5'-flanking region was examined by transient expression assays using the chloramphenicol acetyltransferase gene as the reporter gene. Basal promoter activity in several cell lines was localized to within 149 bp of the upstream sequence by deletion analysis. This proximal promoter region of the mouse HO-1 gene contains several sequence elements that are not only conserved in both the rat and human HO-1 genes but also resemble consensus binding sites of various transcription factors including AP-1, AP-4, C/EBP and c-Myc:Max/USF. Heavy metals activate HO-1 gene transcription and the rat gene contains a putative metal regulatory element (Müller, R. M., Taguchi, H., and Shibahara, S. (1987) J. Biol. Chem. 262, 6795-6802) that is completely conserved in the mouse gene. Transient expression analyses, however, indicate that this sequence, which contains a core heptanucleotide, TGCACTC, identical to that of the strongest metal regulatory element of the mouse metallothionein-1 gene, is not responsive to Cd2+ or Zn2+. Stable transfection of constructs containing the entire mouse HO-1 gene and various portions of the 5'-flanking region into rat C6 glioma cells and simultaneous, quantitative analysis of the mouse and rat HO-1 mRNAs indicate that distal 5' sequences, between positions -3.5 and -12.5 kbp, are required for induction of mouse HO-1 gene transcription by both heme and heavy metals. A 5-7-fold difference in the levels of induction between stably integrated and transiently expressed mouse HO-1 gene constructs is observed in this cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat and mouse proopiomelanocortin gene sequences target tissue-specific expression to the pituitary gland but not to the hypothalamus of transgenic mice.

The proopiomelanocortin (POMC) gene is expressed predominantly in corticotrophs of the pituitary anterior lobe, melanotrophs of the intermediate lobe and neurons of the arcuate nucleus of the hypothalamus. The different ontogeny of POMC mRNA as well as the complicated hormonal regulation of POMC gene expression in the three different cell types suggests a concerted interaction between several cis-acting elements in the POMC gene and transcription factors located in each of the three cell types. To investigate cell-specific elements in the POMC gene we tested two different constructs in transgenic mice. The construct -4000rPOMCLacZ, carrying 4 kb of the rat POMC promoter fused to the Escherichia coli beta-galactosidase gene, showed appropriate expression in melanotrophs in 50% of the mice analyzed. beta-Galactosidase activity was less evident in corticotrophs under basal environmental conditions. In brain, 7 out of 15 independently derived transgenic founders had ectopic expression of the transgene in different areas; however, none of the animals analyzed expressed beta-galactosidase in neurons of the arcuate nucleus. The construct HAL*, a 'tagged' 10.2-kb mouse genomic fragment, was more efficiently targeted to the pituitary. Using in situ hybridization, we detected uniform expression of HAL* in melanotrophs in 100% of the 6 pedigrees analyzed and transgenic mRNA levels paralleled those of the endogenous POMC mRNA. In corticotrophs, basal expression was low but after adrenalectomy HAL* mRNA levels were comparable to those of POMC. None of the 6 pedigrees had appropriate expression of HAL* in the brain; however, 2 lines had ectopic expression in the dentate gyrus of the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of dystrophin brain promoter: A deletion of this region is compatible with normal intellect

Using a mouse genomic fragment containing the brain-specific promoter region of the dystrophin gene, we have located the brain promoter 75–300 kb proximal of the muscle promoter. Within our DMD-families we detected a patient who lacks both the brain-specific and muscle-specific promoter sequences. The normal intellectual capabilities of the patient argue against an indispensable role of the brain-specific first exon in mental functioning. The possibility exists that a NH 2-terminally truncated dystrophin has taken over the function of the normal dystrophins in brain and/or muscle.

Preimplantation mouse embryos and liver express the same type I keratin gene product.

The cytoskeletal B protein isolated from extraembryonic endodermal cells (Endo B) is a 50-kDa subunit of intermediate filaments that is expressed in trophoblast and extraembryonic endoderm of early mouse embryos. Endo B was compared to cytokeratin D of adult mouse liver by immunoprecipitation, two-dimensional gel electrophoresis, and peptide mapping. The two proteins were indistinguishable. A cDNA probe for Endo B mRNA identified mRNA species of similar size in liver and endoderm, and primer extension analysis indicates that the Endo B mRNAs from the two cell types have similar 5' ends. An internal fragment of the Endo B cDNA was found to cross-hybridize with a conservative domain of a human type I keratin cDNA under low stringency conditions, demonstrating that Endo B is related to type I keratins. However, under stringent conditions necessary for genomic Southern analysis, mouse and human genomic fragments homologous to the Endo B cDNA were distinct from those defined by hybridization with the type I keratin cDNA. These results indicate that Endo B is related to the type I keratin family and expands the number of type I keratin genes identified in both the mouse and human genomes. It is likely that extraembryonic endoderm, one of the first differentiated cell types of the mammalian embryo, and adult liver express the same Endo B gene.

Identification of a Balb/c mouse pro alpha 1(I) procollagen gene: evidence for insertions or deletions in gene coding sequences.

We report the first isolation and identification of a mouse genomic fragment encoding amino acid sequences for the pro alpha 1(I) chain of type I procollagen. The DNA sequence of eight coding sequences is presented; five of these are 54 bp and three are 108 bp in length. Together these specify 198 amino acids which are 94% homologous to the corresponding bovine pro alpha 1(I) chain protein sequences. Each of the eight coding sequences is flanked by appropriate splice-junction sequences that exhibit considerable sequence complementarity to the rat small nuclear U1a RNA. In the 198 codons examined in this mouse genomic clone, the preferred codons for glycine and alanine are GGU (46/67) and GCU (23/30), respectively. This is in contrast to the codon usage reported for the chicken pro alpha 1(I) cDNA clone (Fuller and Boedtker, 1981). The examined coding sequences exhibit considerable nucleotide homology in both end-to-end and in staggered alignments. Based on an analysis of this homology data, a model is presented for the generation of 108-bp coding sequences from 54-bp units by two successive homologous recombinational events within coding sequences. Alternatively, the 108-bp units may have arisen by precise deletions of an intervening sequence between 54-bp coding sequences. Evidence supporting this is provided by a comparison of pro alpha 1(I) and pro alpha 2(I) genes. In the mouse pro alpha 1(I) gene amino acids 856-891 are encoded in a 108-bp unit; the chicken pro alpha 2(I) gene these residues are encoded in two 54-bp coding sequences. In addition, the coding sequences for nearly 50% of the alpha domain are condensed in the pro alpha 1(I) gene into a region approximately one half the size occupied by the comparable sequences in the pro alpha 2(I) gene.