Michael G. Raducha

ANCHORING OF CANINE LINKAGE GROUPS WITH CHROMOSOME-SPECIFIC MARKERS

Abstract. A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest.

Tissue distribution of mammalian aldose reductase and related enzymes

Activities of aldose reductase (AR) and related NADPH-dependent enzymes were examined in extracts of human, cat, dog, guinea pig, mouse, monkey, pig, rabbit, rat and sheep lenses and a variety of other tissues. The activity of the tissues against DL-glyceraldehyde, D-glucuronic acid, and 3-pyridinecarboxaldehyde (PCA) was determined. High glyceraldehyde:glucuronic acid activity ratios, a characteristic of aldose reductase, were found in all lenses, except from mouse. An analytical thin-layer isoelectric focusing system which separates the mammalian NADPH-dependent enzymes was developed. AR appears to be present as two or more isozymes in all mammalian lenses studied with the exception of mouse. Other tissues contain one or more isozymes which have the same isoelectric point and substrate specificity as the AR present in the lens of that species. This AR activity, however, may represent only a small proportion of the total NADPH reducing activity present. AR and HDH isozymes reduce the aromatic substrate, PCA, and thus have the general characteristics of an aldehyde reductase.

Production of a monoclonal antibody to human liver alkaline phosphatase

A monoclonal antibody to human liver alkaline phosphatase (ALP) has been produced by the mouse-hybridoma method using a partially purified enzyme preparation as antigen. The particular hybridoma secreting the antibody was detected by a screening procedure based on the retention of enzyme activity by the enzyme/antibody complex. The antibody cross-reacts strongly with human kidney and bone ALPs but not with human placental or intestinal ALPs. It also cross-reacts with liver and kidney ALPs from gorilla, chimpanzee and orangutan. It shows no significant reaction, under the conditions used, with liver or kidney ALPs from several lower primates. An antibody affinity column was prepared and shown to be effective for the final stages of liver ALP purification.

A novel locus for dilated cardiomyopathy maps to canine chromosome 8.

Dilated cardiomyopathy (DCM), the most common form of cardiomyopathy, often leads to heart failure and sudden death. While a substantial proportion of DCMs are inherited, mutations responsible for the majority of DCMs remain unidentified. A genome-wide linkage study was performed to identify the locus responsible for an autosomal recessive inherited form of juvenile DCM (JDCM) in Portuguese water dogs using 16 families segregating the disease. Results link the JDCM locus to canine chromosome 8 with two-point and multipoint lod scores of 10.8 and 14, respectively. The locus maps to a 3.9-Mb region, with complete syntenic homology to human chromosome 14, that contains no genes or loci known to be involved in the development of any type of cardiomyopathy. This discovery of a DCM locus with a previously unknown etiology will provide a new gene to examine in human DCM patients and a model for testing therapeutic approaches for heart failure.

Restriction fragment length polymorphism of the T-cell receptor beta-chain gene in dogs

Since T-cells and the T-cell receptor (TCR) play a pivotal role in the response of the immune system, they are a target for pathogenesis studies in immune mediated diseases and have been used to generate markers for T-cell dependent diseases in humans and dogs. TCR rearrangement is generated at the genomic DNA level and can be analyzed by Southern blotting techniques. In the present study this method to detect rearrangement of the TCR beta chain in the dog was critically examined. To search for restriction fragment length differences due to either inherited polymorphism or in diseases with suspected superantigen influence (X-linked severe combined immune deficiency and canine juvenile polyarteritis syndrome) 13 dog families of three different breeds were examined. In addition primary spleen cell cultures, stimulated with either phytohemagglutinin A (PHA) or staphylococcus enterotoxin A (SEA) and B (SEB) were studied. The germline digest pattern of the enzymes Pst I, Sst I, Bgl II, Eco RI and Eco RV were identical in all dogs examined with the exception of one dog with canine juvenile polyarteritis syndrome. In this dog an additional band was found in the Bgl II and Eco RV digestion suggestive of specific TCR rearrangement. Bam HI digestion revealed restriction fragment length polymorphisms (RFLPs) showing Mendelian inheritance. After digestion of the genomic DNA extracted from PHA, SEA or SEB stimulated spleen cells and Southern blot analysis, no differences in fragment patterns between the unstimulated cells and the stimulated cells could be detected. An important point to consider before a specific pattern variation between dogs is classified to be a marker for a specific disease or is used in pathogenesis studies, is the possibility of an inherited RFLP, especially after Bam HI digestion. In such studies the combined examination of the parents and the offspring must be recommended.