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Several Galactosyltransferase Activities Are Associated with Mouse Chromosome 17

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Several Galactosyltransferase Activities Are Associated with Mouse Chromosome 17 Copyright 0 1986 by the Genetics Society of America SEVERAL GALACTOSYLTRANSFERASE ACTIVITIES ARE ASSOCIATED WITH MOUSE CHROMOSOME 17 KIYOSHI FURUKAWA,**' STEPHEN ROTH* AND JANET SAWICKI? *Department of Biology, University of Pennsylvania, and +Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 Manuscript received May 20, 1986 Accepted July 24, 1986 ABSTRACT Indirect evidence suggests that some major histocompatibility complex (MHC) proteins are glycosyltransferases. No sequence or mapping information is avail- able for transferases, although ganglioside variations in mice are linked to the H-2 complex on chromosome 17, and one galactosyltransferase activity on mouse sperm varies with T/t complex genotypes, also on chromosome 17. In the present experiments, diploid and trisomy 17 mouse embryos were assayed for four dif- ferent galactosyltransferase activities. The same preparations were assayed for isocitrate dehydrogenase (ld-1, chromosome I) and glyoxalase-1 (Glo-1, chro- mosome 17). Galactosyltransferase specific activities in trisomy 17 embryos are almost 1.5 times higher than in diploid embryos. The correlation between gal- actosyltransferase activities and chromosome 17 dosage indicates that the struc- tural or regulatory gene for these enzymes are located on chromosome 17. LYCOSYLTRANSFERASES transfer sugars from activated donors to an G exceedingly wide variety of acceptors. Several lines of evidence suggest a relationship between glycosyltransferases and the products of the major his- tocompatibility complex (MHC) family of genes (ROTH 1985). First, like MHC products, some transferases have intercellular recognition functions (ROTH, MCGUIREand ROSEMAN197 1; SHUR 1982). Second, when carefully purified, glycosyltransferases appear to exist in structurally related groups (NAGAI, MUENSCHand YOSHIDA1976; NACAI et al. 1978a, b; FURUKAWAand ROTH 1985). Third, because each different glycosidic linkage is thought to be cata- lyzed by a different transferase (BEYERet al. 1981), the number of different transferases must be very large. Fourth, two murine transferase activities vary according to changes in the T/t and H-2 regions (SHUR and BENNETT 1979; HASHIMOTOet al. 1983), both of which are located on chromosome 17. Fifth, polyclonal and monoclonal antibodies raised against purified galactosyltransfer- ases cross-react with immunoglobulins (WILSON et al. 1982; PODOLSKYand ISSELBACHER1984) that share sequence homology with MHC proteins (HONJO 1983). Gene dosage effects on enzyme specific activities have been demonstrated Current address: The Institute of Medical Science, The University of Tokyo, Tokyo 108, Japan. Genetics 114 983-991 November, 1986. 984 K. FURUKAWA, S. ROTH AND J. SAWICKI for a number of mouse aneuploids (EPSTEIN et al. 1977; COX, TUCKERand EPSTEIN1980). Because no transferase has yet been mapped, a study of gal- actosyltransferase specific activities in diploid and trisomy 17 mice was initiated. Trisomy 17 embryos can be produced, although they die at about 10-12 days of gestation (GROPP 1978; EPSTEINet aE. 1982). TO test the relationship be- tween chromosome 17 and glycosyltransferase specific activities, l l-day em- bryos were karyotyped and assayed for galactosyltransferases, as well as isocit- rate dehydrogenase (chromosome I) (HENDERSON1965; HUTTONand RODER- ICK 1970) and glyoxalase-1 (chromosome 17) (MEo, DOUGLASand RUNBEEK 1977). The data show that four galactosyltransferase specific activities vary in proportion to chromosome I7 dosage. MATERIALS AND METHODS Materials: UDP-[6-'H]-GaI (16.3 Ci/mmol) was purchased from Amersham (Arling- ton Heights, Illinois), and its purity was monitored periodically by high-voltage paper electrophoresis in 1% sodium tetraborate. Human a1 -acid glycoprotein was generously donated by M. WICKERHAUSER(American Red Cross Blood Services Laboratories, Be- thesda, Maryland). All other reagents were purchased from Sigma Chemical Company (St. Louis, Missouri). Mice: Litters containing diploid and trisomy 17 mouse embryos were generated by mating mice with the normal complement of 40 acrocentric chromosomes to mice carrying two different Robertsonian fusion metacentric chromosomes; the two fusion chromosomes carry chromosome 17 as a common arm (WHITE et al. 1974; GROPP, KOLBUSand GIERS 1975). Thus, when B6D2F1 mice are crossed to mice doubly het- erozygous for two Robertsonian translocation chromosomes-Rb(2.17)11 Rma and Rb(8.17)l Iem-the two metacentric chromosomes often segregate together during meiosis. The result is a trisomy 17 embryo with cells that carry 39 chromosomes, including the two metacentric chromosomes, and a total of 41 chromosome arms. Although Rb(2.17) 11 Rma/Rb(8.17)1 Iem females and males are fertile, and can be mated to B6D2F1 mice of the appropriate gender to produce embryos, males doubly heterozygous for a different pair of Robertsonian translocation chromosomes- Rb( 16.17) 7Bnr/Rh(8.17)1 Iem-are sterile. Some of the trisomy 17 embryos and their diploid littermates were derived, therefore, from mating Rb( 16.17)7 Bnr/Rb(8.17)1 Iem females to B6D2F1 males. Females bearing trisomy 17 and diploid embryos were dis- sected at 11 days of gestation, using plug day as day 0. Litters containing diploid and trisomy 8 embryos were generated in a similar manner. Rb(8.12)22 Lub/Rb(8.17)1 Iem females were mated to B6D2F1 males; pregnant females were dissected at 9 days of gestation. B6D2F 1 mice and homozygous translocation stocks were obtained from Jack- son Laboratories, Bar Harbor, Maine. Chromosome analysis: To determine the karyotype of each embryo, amnionic mem- brane cells were arrested in metaphase, and their chromosomes were subsequently spread (EVANS,BURTENSHAW and FORD 1972). Each membrane was incubated in Dul- hecco's Modified Eagle's Medium (I g of dextrose/liter) containing 5% fetal calf serum and 0.0 15 pg vinblastin (Velban, Grand Island Biological Company, Grand Island, New York) per milliliter for 6 hr at 37" in humidified air containing 5% CO2. Membranes were then incubated in 0.56% KCI for 8.5 min, ethanol/acetic acid (3:l) for 30 min and 60% acetic acid for 5 min. Chromosomes were then spread on glass slides pre- warmed to 50-70". Sonication and homogenization: Dissected embryos were suspended in 300 pl phos- phate-buffered saline containing 0.1% Triton X-1 00. Embryos were either homogenized with a glass pestle fitted to a plastic centrifuge tube or sonicated 3 X 90 sec at 0" with a Fischer Sonic Dismembrator at one-third energy level. The method of embryo dis- ruption had no effect on any of the enzyme assays. MOUSE CHROMOSOME I7 TRANSFERASES 985 Galactosyltransferase assays: Galactosyltransferase assays were conducted as de- scribed in detail (FURUKAWAand ROTH1985) in a final volume of 50 p1 containing the following reagents: 23 PM UDP-['HI-Gal (390 mCi/mmol), 3 mM 5'-AMP, 0.1% Triton X-100, 15 mM MnC12, 20 mM 2[N-morpholino]ethane sulfonic acid, pH 6.5, 10 pl of embryo homogenate, and 250 pg asialo-, agalacto-al-acid glycoprotein (AsAgAGP), which was prepared as described (FURUKAWAand ROTH 1985). All galactosyltransferase assays on trisomy 17 embryos, and their littermates were incubated for 3 hr. All trans- ferase assays on trisomy 8 embryos and littermates were incubated for 6 hr. After terminating the reactions, the incubation mixtures were subjected to high voltage elec- trophoresis on borate-impregnated paper, and the galactosylated product was deter- mined by excising the dried origins and counting them in a liquid scintillation counter. To assay Gal transfer to other acceptors, these were added in place of AsAgAGP, and at the following concentrations: asialo-ovine submaxillary mucin (AsOSM), 250 fig; GlcNAc, 10 mM; GalNAc, 10 mM; ganglioside GM2, 8 mM. For transferase assays using AsOSM, which was prepared as described (FURUKAWAand ROTH 1985), GalNAc, and GM2, manganese concentrations were 10 mM. Transfer to endogenous Gal acceptors was determined by omitting added acceptor. Isocitrate dehydrogenase assay: Isocitrate dehydrogenase activity was determined colorimetrically by incubating 10 PI of embryo homogenate in a total volume of I ml containing 1 mM isocitrate, 5 mM MnCI2, 0.5 mM NADP, and 0.02% bovine serum albumin in 50 mM sodium cacodylate, pH 7.5, at 37" for 5 min. After termination with EDTA, the a-ketoglutarate produced was reacted with 2,4-dinitrophenylhydrazine and converted to the chromogen phenylhydrazone in an alkaline solution (AMADORand WACKER1965). Glyoxylase-1 assay: Glyoxalase-1 activity was determined spectrophotometrically by measuring the increase in A240 nm in 1.5 ml of a solution containing 10 ~1 of embryo homogenate, 0.03% reduced glutathione and 0.04% methylglyoxal, based on the method described by RACKER(1 95 1). UDP-Gal hydrolysis: To determine the extent of hydrolysis of the sugar donor, UDP-Gal, pyrophosphorylase activity was determined by the method of SPIK,SIX and MONTREUIL(1 979). After incubation for 3 hr, galactosyltransferase assay mixtures were subjected to paper chromatography in pyridine:ethylacetate:water:acetic acid (5:5:3:1) for 24 hr. For standards, authentic UDP-Gal, Gal-1-phosphate and Gal were included. The areas to which Gal-1-P and Gal migrated were cut from the paper and counted in a liquid scintillation counter. Protein was determined by the method of LOWRYet al. (1951) using bovine serum albumin as a standard. RESULTS Characteristic metaphase chromosome spreads from diploid and trisomy 17 amnionic membranes are shown in Figure la and b. The presence of two metacentric chromosomes is indicative of trisomy. The karyotype of each em- bryo from five litters was determined. At least five unambiguous spreads were examined for each embryo. Figure 2 shows the incorporation of Gal to AsAgAGP and AsOSM by tri- somy 17 and diploid embryo homogenates as a function of time. Although transfer to AsAgAGP is not linear after 1 hr, trisomy 17 specific activities are consistently higher than diploid activities. Gal transfer to AsOSM is linear with time for up to 3 hr, and is also higher in trisomy 17 embryos, although the transfer rates by both embryo types are much reduced compared to those for AsAgAGP.
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