Characterization and expression of the human leukocyte-common antigen (CD45) gene contained in yeast artificial chromosomes.

The leukocyte-common antigen (CD45) is a transmembrane protein tyrosine phosphatase expressed uniquely by cells of hematopoietic origin. There are multiple isoforms of CD45 that are generated by the variable use of three exons (exons 4-6). The use of the variable exons results in changes near the amino-terminus of the mature glycoprotein. The gene is located on chromosome 1 for both human and mouse in a region that is homologous between these two species. This conserved linkage group contains a number of genes of immunological interest, such as the genes for complement regulatory proteins and the FCG2 receptor. Yeast artificial chromosomes provide a vector system in which large fragments of foreign DNA can be isolated and are suited to long-range physical mapping. To this end, three yeast artificial chromosomes containing the human CD45 gene have been isolated and characterized. They overlap to span 475 kb, establishing the largest physical map for DNA within the conserved linkage group. The CD45 gene is entirely encoded within one yeast artificial chromosome clone as determined by mapping with cDNA probes. A mouse B cell line transfected with this YAC clone expressed the low-molecular-weight isoform of the protein into the cell surface. The size of the human CD45 gene was determined to be approximately 120 +/- 10 kb.     

Murine equivalent of the human histo-blood group ABO gene is a cis-AB gene and encodes a glycosyltransferase with both A and B transferase activity

We have cloned murine genomic and complementary DNA that is equivalent to the human ABO gene. The murine gene consists of at least six coding exons and spans at least 11 kilobase pairs. Exon-intron boundaries are similar to those of the human gene. Unlike human A and B genes that encode two distinct glycosyltransferases with different donor nucleotide-sugar specificities, the murine gene is a cis-AB gene that encodes an enzyme with both A and B transferase activities, and this cis-AB gene prevails in the mouse population. Cloning of the murine AB gene may be helpful in establishing a mouse model system to assess the functionality of the ABO genes in the future.     

Genomic structure, alternative splicing, and physical mapping of the killer cell lectin-like receptor G1 gene (KLRG1), the mouse homologue of MAFA

The mouse killer cell lectin-like receptor G1 (KLRG1), the mouse homologue of the mast cell function-associated antigen (MAFA), is an inhibitory C-type lectin expressed on natural killer (NK) cells and activated CD8 T cells. Here we report the complete nucleotide sequence, alternatively spliced variants, and the physical mapping of the KLRG1 gene in the mouse. The gene spans about 13 kb and consists of five exons. Short interspersed repeats of the B1 and B2 family, a LINE-1-like element, and a (CTT)170 triplet repeat were found in intron sequences. In contrast to human KLRG1 and to the murine KLR family members, mouse KLRG1 locates outside the NK complex on Chromosome 6 between the genes encoding CD9 and CD4.     

Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13 [corrected]

Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.     

The gene encoding disabled-1 (DAB1), the intracellular adaptor of the Reelin pathway,reveals unusual complexity in human and mouse

The Disabled-1 (Dab1) gene encodes a key regulator of Reelin signaling. Reelin is a large glycoprotein secreted by neurons of the developing brain, particularly Cajal-Retzius cells. The DAB1 protein docks to the intracellular part of the Reelin very low density lipoprotein receptor and apoE receptor type 2 and becomes tyrosine-phosphorylated following binding of Reelin to cortical neurons. In mice, mutations of Dab1 and Reelin generate identical phenotypes. In humans, Reelin mutations are associated with brain malformations and mental retardation; mutations in DAB1 have not been identified. Here, we define the organization of Dab1, which is similar in human and mouse. The Dab1 gene spreads over 1100 kb of genomic DNA and is composed of 14 exons encoding the major protein form, some alternative internal exons, and multiple 5'-exons. Alternative polyadenylation and splicing events generate DAB1 isoforms. Several 5'-untranslated regions (UTRs) correspond to different promoters. Two 5'-UTRs (1A and 1B) are predominantly used in the developing brain. 5'-UTR 1B is composed of 10 small exons spread over 800 kb. With a genomic length of 1.1 Mbp for a coding region of 5.5 kb, Dab1 provides a rare example of genomic complexity, which will impede the identification of human mutations.     

Genomic organization and chromosomal localization of the murine epididymal retinoic acid-binding protein (mE-RABP) gene

The murine epididymal retinoic acid-binding protein (mE-RABP) is specifically synthesized in the mouse mid/distal caput epididymidis and secreted in the lumen. In this report, we have demonstrated by Southern blot analysis of genomic DNA that mE-RABP is encoded by a single-copy gene. A mouse 129/SvJ genomic bacterial artificial chromosome (BAC) library was screened using a cDNA encoding the minor form of mE-RABP. One positive BAC clone was characterized and sequenced to determine the nucleotide sequence of the entire mE-RABP gene. The molecular cloning of the mE-RABP gene completes the characterization of the 20.5-kDa–predicted preprotein leading to the minor and major forms of mE-RABP. Comparison of the DNA sequence of the promoter and coding regions with that of the rat epididymal secretory protein I (ESP I) gene showed that the mE-RABP gene is the orthologue of the ESP I gene that encodes a rat epididymal retinoic acid-binding protein. Several regulatory elements, including a putative androgen receptor binding site, “CACCC-boxes,” NF-1, Oct-1, and SP-1 recognition sites, are conserved in the proximal promoter. Analysis of the nucleotide sequence of the mE-RABP gene revealed the presence of seven exons and showed that the genomic organization is highly related to other genes encoding lipocalins. The mE-RABP gene was mapped by fluorescent in situ hybridization to the [A3-B] region of the murine chromosome 2. Our data, combined with that of others, suggest that the proximal segment of the mouse chromosome 2 may be a rich region for genes encoding lipocalins with a genomic organization highly related to the mE-RABP gene. Mol. Reprod. Dev. 50:387–395, 1998. © 1998 Wiley-Liss, Inc.     

Genomic structure of murine mitochondrial DNA polymerase-gamma

We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron-exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron-exon borders are in almost identical locations for the 22 introns. The 5' upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 5' flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.