The Belt mutation in pigs is an allele at the Dominant white (I/KIT) locus.

A white belt is a common coat color phenotype in pigs and is determined by a dominant allele (Be). Here we present the result of a genome scan performed using a Hampshire (Belt)/Pietrain (non-Belt) backcross segregating for the white belt trait. We demonstrate that Belt maps to the centromeric region of pig Chromosome (Chr) 8 harboring the Dominant white (I/KIT) locus. Complete cosegregation between Belt and a single nucleotide polymorphism in the KIT gene was observed. Another potential candidate gene, the endothelin receptor type A gene (EDNRA), was excluded as it was assigned to a different region (SSC8q21) by FISH analysis. We argue that Belt is a regulatory KIT mutation on the basis of comparative data on mouse KIT mutants and our previous sequence analysis of the KIT coding sequence from a Hampshire pig. Quantitative PCR analysis revealed that Belt is not associated with a KIT duplication, as is the case for the Patch and Dominant white alleles. Thus, Belt is a fourth allele at the Dominant white locus, and we suggest that it is denoted I(Be).

Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin.

Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

Ricin B chain fragments expressed in Escherichia coli are able to bind free galactose in contrast to the full length polypeptide.

Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced in E. coli and targeted to the periplasm by fusion to the ompA or ompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced in Xenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced in E. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced in Xenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced in E. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility to E. coli proteases.

The metabolism of glutamine by the preimplantation sheep conceptus and its interaction with glucose.

The metabolism of glutamine and glucose, separately and in combination, by the sheep conceptus recovered on Days 2, 6, 13, 15, 17, and 19 of pregnancy was assessed over 2.5 h. At Day 2, the production of CO2 from glutamine was similar to that from glucose, with additive effects seen when both substrates were present. Between Day 2 and Day 6, there was a three-fold increase in glucose oxidation but no change in the oxidation of glutamine. From Day 13 to Day 19, the oxidation of glutamine was relatively high in embryonic tissue, low in trophoblastic tissue and intermediate in the yolk sac but in all tissues decreased as development progressed. Over this latter period the oxidation of glutamine was reduced to approximately 50% by the addition of glucose to the medium but glucose oxidation was unaffected by the addition of glutamine. At the early stages of development, the incorporation of substrate carbon from glutamine was less than that from glucose but in each case, incorporation into the acid-insoluble macromolecular fraction increased 2-3 times between Day 2 and Day 6. Incorporation of glutamine into the Day-17 and Day-19 conceptus was also measured; embryonic tissue exhibited the highest rate of incorporation and trophoblastic tissue the lowest. Incorporation was lower on Day 19 than on Day 17 and the proportion of carbon isolated in the acid-insoluble fraction represented 20% of the total incorporated. At no time did the addition of glucose to the medium alter incorporation of glutamine into either embryonic tissue or extraembryonic membranes.

Oxidation of [U-14C]acetate by the sheep conceptus between days 13 and 19 of pregnancy.

Acetate metabolism by the sheep conceptus was assessed by measuring CO2 production during a 2.5-h incubation of embryos and samples of the extraembryonic membranes in HEPES-buffered media containing 1.12 mM [U-14C]acetate. The rate of oxidation of acetate by embryonic tissue showed little change between Days 13 and 15 of pregnancy but greatly decreased by Days 17 and 19. By contrast, oxidation of the substrate by the trophoblast increased substantially with development and was five times the early rate by Day 19. Oxidation of acetate by the yolk sac also increased 4-fold between Days 17 and 19. The addition of glucose to incubations of extraembryonic membranes resulted in some reduction in the oxidation of acetate by the yolk sac and allantois but had little effect on the trophoblast. At Days 13 and 15, the rate of oxidation of acetate by the embryonic disc was 6-7 times that by the trophoblast. As development progressed, this situation was reversed and by Day 19 the trophoblast metabolized more than five times the amount of acetate per microgram than did the Day-19 embryo. Although acetate metabolism by yolk sac and allantois on Day 17 was low, its metabolism by the yolk sac increased to values similar to those for the trophoblast at Day 19 but its utilization by the allantoic membrane remained low. Comparison of the estimates of ATP generated from acetate by these tissue with those published for glucose demonstrates that acetate is much less effective than glucose for the provision of metabolic energy.

Catabolic utilization of glucose by the sheep conceptus between days 13 and 19 of pregnancy.

The production of carbon dioxide and lactate from glucose by sheep embryos and samples of extraembryonic membranes was measured during a 2.5 h incubation period. Both embryos and their membranes were active in the glycolytic and oxidative utilization of glucose and, in general, the utilization of glucose per unit weight fell as development progressed from Day 13 to Day 19 of pregnancy. Both oxidation of glucose and glycolysis by the extraembryonic tissues, expressed as activity per microgram dried tissue, fell progressively with development. The rate of decline in CO2 production was greater than the rate for glycolysis and, as a consequence, the contribution of glycolysis to the estimated energy yield from the catabolism of glucose rose with time. In the embryo, both glucose oxidation and glycolysis peaked on Day 15 with estimates of adenosine triphosphate (ATP) production from glucose per microgram dried tissue on this day being 50% above those on Day 13 and 100% above those on Day 17. In general, the estimated yields of ATP from glucose were similar for structures of the same developmental age except that, at Day 19, it was calculated that the rate of ATP production by embryos was double that by the extraembryonic membranes. In incubations using 5.56 mM glucose as sole exogenous energy source, glucose turnover by embryos and embryonic membranes tended to be higher in a bicarbonate-buffered medium than in HEPES (4-(2-hydroxyethyl)-1-piperazincethane sulfonic acid) and phosphate-buffered media. As a result, the estimate of ATP yield plus the contribution of oxidative pathways to this yield were significantly higher in this medium than in the others. Glucose turnover by the embryo and its membranes in bicarbonate-buffered medium containing 0.56 mM glucose plus the alternate substrates, lactate and pyruvate, was severely depressed. Further experiments using samples of trophoblast and yolk sac indicated that both reduction in glucose concentration and the presence of the other substrates contributed to this suppression. Furthermore, an interaction between these factors was evident with the effects of alternative substrates being exaggerated when glucose concentration was low.

Glycolysis and glucose oxidation by the sheep conceptus at different oxygen concentrations.

The effect of changes in oxygen concentration on the catabolic utilization of glucose by the sheep conceptus at selected periods between Days 3 and 19 of preimplantation development was examined by estimating the production of CO2 and lactate from [U-14C]glucose during a 2.5-h culture in vitro in the presence of 20%, 5% and 1% O2. In general, lowering O2 significantly altered the catabolism of glucose with a changing pattern of response depending on the stage at which the conceptus was explanted. For embryos at Days 3 and 6 post insemination, reduced O2 caused no significant change in oxidative utilization of glucose and a small decrease in conversion of the substrate to lactate. By contrast, lowering O2 concentration during incubation of the structures of the advanced conceptus from Day 13 through to Day 19 of pregnancy significantly restrained oxidative utilization of glucose but stimulated its conversion to lactate. The effects of these changing levels of O2 on the generation of energy from glucose in the form of ATP was estimated. Except for the Day-13 conceptus, reduction in O2 concentration had little influence on the calculated amount of ATP produced from glucose, with glycolysis making up the deficit in energy production when reduced O2 inhibits oxidation of the substrate at the later stages of development. At Day 13, the switch in the metabolism of glucose to glycolysis is not fully effective and energy production fell as O2 concentration was reduced. The results indicate a major shift towards dependence by the preimplantation sheep conceptus on the glycolytic pathway for energy generation from glucose as development progresses. This move to glycolysis is increased by low O2 concentration. As a low concentration of O2 most probably exists in the lumen of the sheep uterus, the results indicate that, in utero, the energy required for the rapid growth of the conceptus depends progressively more on glycolysis than oxidative metabolism of glucose. The finding that the Day-13 conceptus has not fully adapted to this method of ATP generation at low O2 concentrations may make it especially vulnerable during development in utero.

Metabolism of glucose by vesicles derived from sheep trophoblast and embryonic discs.

Samples of trophoblast recovered from the sheep conceptus on Day 13 of pregnancy formed spherical vesicles during culture in medium 199. These continued to expand and increase in dry weight over the next 6 days in vitro. After 6 days' culture, the metabolism of glucose by these vesicles was compared with that of Day-13 and Day-19 fresh trophoblastic tissue. The production of CO2 and lactate by vesicles was similar, although not identical, to production by Day-13 fresh tissue and did not exhibit the marked decrease in glucose catabolism seen in Day-19 trophoblast. The tissue from vesicles reacted to reduction in oxygen tension in a manner similar to the reaction of fresh tissue, with decreased glucose oxidation and increased glycolysis. The activity of the pentose phosphate pathway in vesicles was higher, and the activity in Day-19 fresh tissue was much lower, than that in Day-13 fresh trophoblast. Incorporation of glucose into the intracellular biochemical pools by vesicles was similar to incorporation into Day-13 fresh tissue. Limited observations were also made with vesicles derived from embryonic disc. Production of CO2 by these vesicles was intermediate between that detected in fresh Day-13 and Day-19 embryonic tissue. There were not significant differences in lactate production between fresh and cultured samples of embryonic tissue. These results show that vesicles formed in vitro remain metabolically active but do not mimic the biochemical changes seen in the tissue during development in vivo.

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling.

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1 x 10(4) sperm/ml (P less than 0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zona-free oocytes had high rates of polyploidy (greater than or equal to 40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1 x 10(4)/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1 x 10(5) and 1 x 10(4) sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea.

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in lambda gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea.

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in λgt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca(2+)-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.

Effects of oestradiol and progesterone on the metabolism of [U-14C]glucose by mouse morulae and early blastocysts in vitro.

The addition of progesterone (10(-7) to 10(-5) M) and/or oestradiol (10(-10) M) during 24-h chase culture of pulse-labelled morulae-early blastocysts did not affect the degradation of radiolabelled glycogen or other biochemical fractions. The presence of a high concentration of progesterone (10(-5) M) during 5-h pulse culture significantly inhibited incorporation of substrate carbon from [U-14C]glucose into both the acid-soluble and acid-insoluble glycogen fractions, but had no effect on non-glycogen fractions. Catabolic utilization of glucose as estimated by the rate of carbon dioxide and lactate production was not affected by the presence of progesterone (10(-7) to 10(-5) M), oestradiol (10(-10) to 10(-8) M) or a combination of both. The results indicate that ovarian steroids at expected physiological concentrations do not directly influence embryonic energy metabolism.

Metabolism of glucose by preimplantation mouse embryos in the presence of glucagon, insulin, epinephrine, cAMP, theophylline and caffeine.

Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother. Insulin was found to stimulate incorporation of glucose into non-glycogen macromolecules during both pulse and chase culture. Thus, whilst an effect of insulin on glycogen metabolism was absent, the anabolic effects of this hormone appear to have been expressed in the embryo at this stage of development.

Effect of parenteral administration of oestrogen and progesterone on the glycogen metabolism of mouse morulae--early blastocysts in vivo.

Morulae--early blastocysts were pulse-labelled with radioactive glucose and subsequently transferred for 24 h to ovariectomized recipients maintained on different hormone regimens. Embryos transferred to recipients treated with progesterone utilized more glycogen than did those incubated in untreated controls. Treatment with oestrogen alone had no significant effect on glycogen turnover of transferred embryos and, when given in combination with progesterone, antagonized the effect of progesterone. Priming doses of oestrogen given greater than 4 days before progesterone had no significant effect on the response to progesterone. The results indicate that the high levels of progesterone circulating in the mother before implantation cause glycogen degradation in the embryo and account for the low glycogen content of uterine embryos compared to those cultured in vitro in the presence of glucose.

Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos.

Methods are described for preparing and analyzing single preimplantation mouse embryos for a variety of metabolites and cofactors (glucose-6-P, fructose-6-P, fructose-1,6-bisphosphate, ATP, AMP, Pi, citrate, isocitrate, alpha-ketoglutarate, and malate). Oil-well and enzymatic cycling techniques are combined to provide the sensitivity needed to measure the amounts present (10(-12) to 1o(-15) moles). After experimental treatment, embryos are collected on glass slides and freeze-dried. They can then be stored indefinitely under vacuum at -25 degrees C without deterioration. With these procedures, the embryos were collected at successive stages of development and subjected to starvation and refeeding with glucose, pyruvate or both. The results confirm the existence of a block at early stages at the P-fructokinase step. This may be due to inhibition by the very high citrate levels present. The data suggest that glycolysis is turned on late in preimplantation development by the rise in fructose-6-P, a deinhibitor of P-fructokinase. In the citrate cycle, no step between citrate and alpha-ketoglutarate is rate-limiting, but a step between alpha-ketaglutarate and malate appears to impede the flux at early embryonic stages.