Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.     

A digitonin-based procedure for the isolation of mitochondrial DNA from mammalian cells

A procedure for the isolation of closed circular mitochondrial DNA (mtDNA) from cells permeabilized with digitonin is described. Compared to the standard procedure in which cells are broken after osmotic swelling, the digitonin-based procedure is more consistent and results in higher mtDNA yields.     

Rapid identification of hybridization probes for chromosomal walking

The presence of repeated elements in restriction fragments used as hybridization probes for chromosomal walking poses a major obstacle to the success of this gene-cloning strategy. This report describes a simple and rapid means of identifying restriction fragments devoid of repeated sequences and therefore useful as hybridization probes for chromosomal walking. Restriction fragments derived from a genomic DNA clone are Southern blotted and hybridized to nick-translated total genomic [32P]DNA. Fragments of the genomic clone that contain high abundance sequences (i.e., repeated elements) hybridize strongly to their nick-translated counterparts, which, due to their high copy number, comprise a significant proportion of the total genomic DNA probe. Conversely, fragments containing single-copy or low-abundance sequences do not hybridize, as their nick-translated counterparts are poorly represented in the total genomic DNA probe. These latter fragments, by virtue of their low-abundance sequences, are well suited as probes for chromosomal walking. Ensuring the absence of repeated elements in restriction fragments prior to their purification and utilization as chromosomal walking probes results in marked savings of time, effort and materials.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

American Black Duck Nesting Ecology in North Carolina

North Carolina, USA, is the southernmost extent of the American black duck's (Anas rubripes) breeding range; however, little is known about their nesting ecology in this region. We located and monitored 140 nesting black ducks over 2 years (2017–2018) to quantify preferred nesting habitat and assess nesting productivity within coastal North Carolina. We located nests in brackish marshes (75%) and man-made dredge spoil islands (25%) at a density of 1 nest/22 ha. Black ducks selected high marsh and nested an average of 21.81 m from open water at a mean elevation of 1.36 m. In preferred nesting habitat, visual obstruction readings were 0.50 m with a maximum mean vegetation height of 0.81 m and land cover consisted largely of grasses (84.6%). Apparent nest success rates varied from 31% (2017) to 63% (2018) across years. The majority (72.2%) of variability in nest success was best predicted by nest location (mainland marsh, natural island, or spoil island), vegetation density, maximum vegetation height, and year. Management for breeding black ducks in coastal North Carolina should focus on promoting selected nesting habitat and reducing nest predators. Prescribed burns, used to set back succession on spoil islands and in brackish marshes should be conducted in the winter or in the early growing season not to exceed the twenty-fifth quantile date of black duck nest initiation (2 Apr). © 2021 The Wildlife Society.