Biomedical subjects
Publications and source records attributed to R D Wesley.
Commercially-processed broilers were held at 4 C in carbon dioxide(CO2)-flushed bags or in natural atmosphere for nine days prior to determining the most probable number of Campylobacter jejuni on individual carcasses. Fifty broilers were evaluated. The CO2-enriched atmosphere packaging of broilers had no detectable effect on the C. jejuni populations.
The preparation of wild-type African swine fever (ASF) virus DNA from small amounts of viremic blood from acutely febrile pigs is outlined. The extracted DNA is viral and not host-cell DNA, because of specific homology with cell culture grown and purified ASF virus and because no DNA bands are obtained with an equal amount of nonviremic pig blood. Thus, in the absence of suitable serologic methods for strain identification, it is now possible to catalogue wild-type isolates by characteristic DNA restriction patterns. The wild-type virus genome contains terminal single-stranded DNA cross-links and has the largest genome size (180 kilobase pairs) reported for the ASF virus. Experimental passage of the virus in contact-infected pigs and buffy coat cultures appears to confirm the stable nature of the ASF genome in the field.
A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)
DNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 x 10(6) to 100 x 10(6). Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.
The low-complexity "gene-sized" linear DNA duplexes of the Oxytricha macronucleus sport short inverted terminal repeats; thus, each single strand is capable of forming a circle held together by a duplex "neck" [Wesley, R. D. (1975) Porc. Natl. Acad. Sci. USA 72, 678--682]. We have isolated necks from total, circularized, single-stranded macronuclear DNA by treatment with nuclease S1. Necks represent at least 2.2% of the total DNA, are homogeneous in size (23 base pairs), melt at 55 degrees in 0.18 M Na+, and reassociate extremely rapidly at 22 degrees (Cot1/2 = 1.1 X 10(-5) mol-liter-1.sec) to form hybrid necks of the same thermal stability. From these and other results, we conclude that all necks on all the many thousands of different single-stranded circles are the same. The neck sequence is therefore highly repetitious--found in multiple copies (as inverted terminal repeats at flush duplex ends and probably also internally) on each natural "gene-sized" macronuclear DNA molecule--implying the possible participation of this sequence both in the general vegetative metabolism of macronuclear DNA and in the pre-vegetative process whereby macronuclear DNA is excised from the total Oxytricha genome.
The low-molecular-weight macronuclear DNA of the hypotrichous ciliates Oxytricha, Euplotes, and Paraurostyla contains inverted repetitious sequences. Up to 89% of the denatured macronuclear DNA molecules form single-stranded circles due to intramolecular renaturation of complementary sequences at or near the ends of the same polynucleotide chain. Other ciliated protozoans, such as Tetrahymena, with high-molecular-weight macronuclear DNA and an alternative mode of macronuclear development, appear to lack these selfcomplementary sequences. The denatured macronuclear molecules of hypotrichs are held in the circular conformation by a hydrogen-bonded duplex region, which is probably less than 50 base pairs in length, since the duplex regions are not visible by electron microscopy and since the circles in 0.12 M phosphate buffer are not retained during hydroxylapatite chromatography at 60 degrees. The existence of extremely small circles, with contour lengths shorter than the smallest pieces of native DNA, suggests that inverted repetitions containing nicks (broken phosphodiester bonds in duplex DNA) or gaps (interruptions with missing nucleotides) are present at internal positions as well as at the ends of native molecules. Estimates from length measurements of native and denatured Oxytricha macronuclear DNA indicate an average of 1.7 nicks per duplex molecule. Thus, in order to account for the high frequency of circle formation, a restriction-type enzyme(s) must exist which inserts single-strand, site-specific nicks or gaps at internal positions in the macronuclear DNA of Oxytricha.
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