Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin.

Analyses of the peptidoglycan nucleotide precursor contents of enterococci and staphylococci treated with ramoplanin, tunicamycin, or vancomycin were carried out by high-pressure liquid chromatography coupled with mass spectrometry (MS). In all cases, a sharp increase in the UDP-N-actetylmuramoyl-pentapeptide or -pentadepsipeptide pool was observed. Concomitantly, new peptidoglycan nucleotide peptides of higher molecular masses with hexa- or heptapeptide moieties were identified: UDP-MurNAc-pentapeptide-Asp or pentadepsipeptide-Asp in enterococci and UDP-MurNAc-pentapeptide-Gly or -Ala and UDP-MurNAc-pentapeptide-Gly-Gly or -Ala-Gly in staphylococci. These new compounds are derivatives of normal UDP-MurNAc-pentapeptide or -pentadepsipeptide precursors with the extra amino acid(s) linked to the lysine epsilon-amino group as established by various analytical procedures (MS, MS-MS fragmentation, chemical analysis, and digestion with R39 D,D carboxypeptidase). Except for tunicamycin-treated cells, it was not possible to ascertain whether these unusual nucleotides were formed by direct addition of the amino acids to UDP-MurNAc-pentapeptide (or -pentadepsipeptide) or whether they arose by reverse reactions from lipid I intermediates to which the amino acids had been added.     

Investigation of the dynamics of the spread of human immunodeficiency virus to brain and other tissues by evolutionary analysis of sequences from the p17gag and env genes.

The time of spread of human immunodeficiency virus type 1 (HIV-1) from lymphoid to nonlymphoid tissues in the course of infection was investigated by sequence comparisons of variants infecting a range of lymphoid and nonlymphoid tissues from three individuals with AIDS in the pl7gag gene and regions flanking the V1/V2 hypervariable regions. Phylogenetic analysis in both regions revealed several lineages in each individual that contained sequences from both lymphoid and nonlymphoid tissues such as the brain. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. Although individual pairwise comparisons of relatively short sequences such as p17gag are subject to considerable stochastic error, we found that the diversity of gag sequences in variants from lymphoid tissue was consistently lower than that found among variants amplified from the brain. By estimating mean synonymous pairwise distances in the p17gag region, we were able to make an approximate calculation of the ages of populations in different tissues. Those from lymphoid tissue ranged from 2.65 to 5.6 years in the three study subjects, compared with 4.1 to 6.2 years for variants in the brain. Indeed, variants infecting the brain were no more closely related to each other than they were to variants infecting other tissues in the body. In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This study is the first in which it was possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into nonlymphoid tissues and its possible adaptation for replication in different cell types.     

Bacterial adaptation to low-nutrient conditions as studied with algal extracellular products

Kinetic analyses indicate that members of natural bacterial populations from 2 marine environments near Woods Hole, MA, possess enzyme-mediated transport systems which permit utilization of¹⁴C-labeled extracellular organic C (¹⁴C-EOC) prepared from the algae,Skeletonema costatum, Thalassiosira pseudonana, andDunaliella tertiolecta, and supplied over a concentration range of 15–150C·liter^–1. It is shown that previous exposure of the bacteria to the EOC from these algae cannot explain the linear kinetic patterns obtained. Therefore, the ability to utilize algal EOC at low concentrations is a general feature of metabolically active bacterial populations. Further, as the native bacteria do not restrict this ability to a specific EOC pool, the results are consistent with the hypothesis that bacteria adapted to low nutrient environments possess uptake systems of high substrate affinity and low substrate specificity. Elevation of substrate levels with as little as 10 mg·1^–1 peptone is shown to favor development of a bacterial population that lacks these adaptations. Standard enrichment techniques typically result in the isolation of bacteria that are poor models for evaluating the ecology of native microbiota.Contribution No. 1523 from the University of Maryland Center for Environmental and Estuarine Studies.     

The plasma protease inhibitor system (Pi) of Standardbred horses

The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles (PiF, G, I, L, N, S1, S2, U), another 10 alleles, designated PiH, J, K, O, P, Q, R, V, X, Z, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pi1, Pi2 classification of Juneja et al. (1979) were found. Limited family data demonstrated the genetic nature of the 'new' variants and confirmed the allelic inheritance of the 'new' Pi variants.     

Effect of dextran-charcoal treatment on the dissociation kinetics of glucocorticoid-receptor complexes: possible involvement of lipid

Dissociation kinetics were determined at 0 degrees C for molybdate-stabilized glucocorticoid-receptor complexes in rat thymus cytosol. Exposure of complexes to dextran-coated charcoal had no effect on their chromatographic properties or transformation status, but dissociation rates measured after charcoal treatment were significantly lower than those determined by displacement with excess competing steroid. The dissociation rate of the [2,4,6,7-3H]prednisolone-receptor complex was similarly modified by chromatography on Lipidex 1000, but not by chromatography on Sephadex G-25 or G-75. It is concluded that treatment of glucocorticoid-receptor complexes with dextran-charcoal or Lipidex 1000 brings about a change in dissociation rate as a consequence of the removal of a lipid component from the complex.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Nearest-neighbor and next-nearest-neighbor effects in the proton NMR spectra of the oligoribonucleotides ApGpX and CpApX

The variable-temperature proton nmr spectra of the oligoribonucleotides in the series CpApX and the series ApGpX, X = A, G, C, U, together with the parent dimers CpA and ApG have been measured. A complete analysis of all the nonexchangeable base proton resonances and ribose H-1′ proton resonances was made. The presence of trends in the shielding abilities of the various bases at both the nearest-neighbor and next-nearest-neighbor positions were identified. The observed shieldings could be used to predict the chemical shifts of protons in related systems. Based on the empirical results from ribodinucleoside monophosphates, the temperature-dependent behavior of the J_1′2′ coupling constants of the triribonucleotides suggested that the compounds in the CpApX series stacked from the 5′-end to the 3′-end, while those in the ApGpX series stacked from the 3′-end to the 5′-end.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.