Browsing by Author "Locke, John"
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ItemGenomic organization of the rat alpha(2u)-globulin gene cluster(1999) McFadyen, David A.; Addison, William; Locke, JohnThe α2u-globulins are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many α2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 α2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the α2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the α2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing α2u-globulin genes indicated that the α2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the α2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes. ItemHigh-resolution FISH mapping of the rat alpha(2u)-globulin multigene family(2000) McFadyen, David A.; Locke, JohnThe rat α2u-globulins are a group of similar proteins that are encoded by a family of approximately 20 genes located a single locus of ≤880 kbp on Chromosome (Chr) 5q. Individual members of this gene family demonstrate complex tissue, hormonal, and developmental expression patterns despite a high degree of sequence similarity among the members and consequently provide an interesting system for studying the evolution of differential gene expression. Hybridization analysis indicated that gene classes, similar to those identified at the homologous MUP locus in the mouse, do not exist within the rat α2u-globulin locus. Furthermore, cross-hybridization analysis revealed the presence of conserved sequences in the 5′ and 3′ regions flanking the α2u-globulin genes, some of which were present in an inverted orientation. We have used high-resolution fiber FISH to examine the structural organization of the α2u-globulin locus, and found the genes to be arranged as an array of both direct and inverted repeats. The organization of the rat α2u-globulin genes differs from the MUP genes and suggests different evolutionary events have reorganized these homologous sets of genes. ItemThree subsets of genes whose tissue specific expression is sex and age-dependent can be identified within the rat alpha(2u)-globulin family(1997) Wang, Kathy S.; McFadyen, David A.; Locke, John; Hodgetts, Ross B.The rat α2u‐globulins are encoded by a multigene family whose 20–25 members are subjected to multihormonal regulation that is dependent upon the sex of the animal, the developmental stage and the tissue being examined. Using RT‐PCR and diagnostic restriction analysis of the products, we have examined the specificity of the expression of different members of the gene family. All family members can be classified into three subsets, depending on how the amplified cDNA responds to digestion with ApaLI, SstI and VspI. Subset A contains the restriction sites for both ApaLI and SstI but not VspI and typifies the genes expressed in the salivary glands of both mature and juvenile animals of both sexes, where it is the only subset expressed. This subset of genes also accounts for all the transcripts observed in the kidneys and mammary glands of juvenile males. Although subset A was represented in the transcript populations of all the other tissues examined, its proportion relative to the total varied greatly. The two other subsets were subset V, which contains only the restriction site for VspI, and subset N, which lacks all three restriction sites. In all the other tissues examined, two or all three of the subsets were expressed, usually in a manner that was unique to the sex and age of the tissue in question. The proportion of each of the three α2u‐globulin subsets in the α2u‐globulin gene family was determined by quantitation of the restriction products of amplified genomic DNA. Interestingly, the most prevalent subset in the genome (N) has the most limited tissue expression pattern, but is found in liver and preputial glands, the tissues expressing the most substantial quantities of α2u‐globulin. These results indicate the complexity of the regulation of the α2u‐globulins and point to the necessity for gene specific analyses if the expression of the family is to be understood in molecular terms.