Restriction fragment analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure of the migratory grasshopper, Melanoplus sanguinipes. The total DNA of 89 individuals was digested with 10 restriction enzymes and probed with three cloned EcoRT fragments representing the entire mitochondrial genome (total length 16.1 kb). Five endonucleases revealed polymorphism, yielding 12 haplotypes in combination. Nucleotide diversity indices (6) between haplotypes ranged from 0.18 to 0.92 per cent with an average value (pr) of 0.27 per cent, a comparatively low figure that possibly reflects a history of chronically small population numbers prior to the species' colonization of a suitable habitat engendered by agricultural settlement. Little, if any, differentiation was evident among locations or between prairie and parkland regions, areas spanned by a colour trait dine. Selective forces with respect to the latter must, therefore, be sufficiently powerful to withstand the attenuating effects of widespread gene flow, implied by the mtDNA data.
The alpha 2u-globulin 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 alpha 2u-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 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-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 alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-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.
The rat alpha 2u-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 alpha 2u-globulin subsets in the alpha 2u-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 alpha 2u-globulin. These results indicate the complexity of the regulation of the alpha 2u-globulins and point to the necessity for gene specific analyses if the expression of the family is to be understood in molecular terms.
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