Characterization of 24 porcine (dA-dC)n-(dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P<0.01%) in the Yorkshire breed than in the other three breeds. Linkage analyses with the CEPH linkage packet were performed in a backcross family comprising 45 animals, of which 43 had informative meioses. Ten of the microsatellites could be assigned to six different linkage groups, demonstrating that linkage mapping with microsatellites can be carried out with great efficiency in a relatively small number of animals. Four of the linkage groups represent Chromosomes (Chrs) 4, 6, 7, and 8 respectively, while two linkage groups are unassigned.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers listed in Table 1.     

Batch and continuous production of lactic acid from salt whey using free and immobilized cultures of lactobacilli

Salt whey permeate was used as a substrate for lactic acid production by different strains of homofermentative lactobacilli. An isolate from Egyptian Cheddar cheese proved to be the most effective lactic acid producer. The salt whey permeate was optimized by addition of yeast extract and minerals to enable exponential growth of organisms. The lactic acid productivity of free and immobilized cells was compared and fermentation conditions were improved. Continuous lactic acid fermentation from salt whey permeate with cells immobilized in agarose beads was successfully carried out in a chemostat with a steady lactic acid concentration of 33.4 mg/ml.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.     

DNA binding properties of the Saccharomyces cerevisiae DAT1 gene product.

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein (Dat1p) that specifically recognizes the minor groove of non-alternating oligo(A).oligo(T) tracts. Sequence-specific recognition requires arginine residues found within three perfectly repeated pentads (G-R-K-P-G) of the Dat1p DNA binding domain [Reardon, B. J., Winters, R. S., Gordon, D., and Winter, E. (1993) Proc. Natl. Acad. Sci. USA 90, 11327-1131]. This report describes a rapid and simple method for purifying the Dat1p DNA binding domain and the biochemical characterization of its interaction with oligo(A).oligo(T) tracts. Oligonucleotide binding experiments and the characterization of yeast genomic Dat1p binding sites show that Dat1p specifically binds to any 11 base sequence in which 10 bases conform to an oligo(A).oligo(T) tract. Binding studies of different sized Dat1p derivatives show that the Dat1p DNA binding domain can function as a monomer. Competition DNA binding assays using poly(I).poly(C), demonstrate that the minor groove oligo(A).oligo(T) constituents are not sufficient for high specificity DNA binding. These data constrain the possible models for Dat1p/oligo(A).oligo(T) complexes, suggest that the DNA binding domain is in an extended structure when complexed to its cognate DNA, and show that Dat1p binding sites are more prevalent than previously thought.     

The PiGMaP consortium linkage map of the pig (Sus scrofa)

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (16.5 Morgans) than the genetic map of the homogametic sex (female) (21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.     

Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.