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Biomedical subjects

F E Young

Publications and source records attributed to F E Young.

At least 55 records · Page 3Linked to original sources
PubMed1984

Presence of two unique genes encoding macrolide-lincosamide-streptogramin resistance in members of the Bacteroides fragilis group as determined by DNA-DNA homology.

The relationship of a previously described, plasmid-encoded macrolide-lincosamide-streptogramin (MLS) resistance determinant to Bacteroides fragilis group clinical isolates from 11 different patients with bacteroides infections was assessed using in situ hybridization. Most of the strains were found to have DNA sequences homologous with the bacteroides MLS plasmid, pBF4, but this homology was exclusively to chromosomal DNA. Two organisms isolated from different sites in a single patient did not share homology with the plasmid, and probably represent a second type of MLS gene. The MLS resistance plasmids, pBF4 (from Bact. fragilis) and pE194 (from Staphylococcus aureus), did not share homologous sequences; therefore at least one of the bacteroids MLS genes is different from that found in Gram-positive organisms.

Bacteroides fragilis↗
PubMed1984

Characterization of Bacillus subtilis DSM704 and its production of 1-deoxynojirimycin.

A Bacillus subtilis strain, DSM704, was characterized by genetic means, and its production of a human intestinal sucrase inhibitor, 1-deoxynojirimycin, was described. Synthesis of this compound is detected concomitant with the detection of heat-resistant spores. The amount of 1-deoxynojirimycin produced is highly dependent on the carbon source, with growth on substrates metabolized via glycolysis giving the greatest amount of production (up to 1 mg/ml). 1-Deoxynojirimycin appears to be nonmetabolizable by the producing strain in that it cannot serve as a sole carbon or nitrogen source.

1-Deoxynojirimycin↗
PubMed1984

Cloning genes for proline biosynthesis from Neisseria gonorrhoeae: identification by interspecific complementation of Escherichia coli mutants.

DNA from Neisseria gonorrhoeae KH45 was partially digested with Sau3A and inserted into the BamHI site of the cloning vector pLES2 . After introduction into Escherichia coli JM83 by transformation, two different size classes of plasmids were isolated that could complement the proAB deletion of JM83 . These plasmids ( pLES4 and pLES7 ) were characterized by restriction endonuclease digestion. Southern hybridization demonstrated that the inserts had sequence homology. Various deletions of these plasmids were constructed that had lost the ability to complement the proA lesion of chi 463, the proB lesion of chi 340, or both (plasmids pLES9 , pLES8 , and pLES10 , respectively). These deleted plasmids were introduced into a proline-requiring strain of N. gonorrhoeae, F62, with plasmids pLES4 , pLES7 , and pLES8 possessing the ability to correct the proline requirement of F62. Further analysis indicated that the hybrid plasmids were stably maintained as plasmids in N. gonorrhoeae.

Aldehyde Oxidoreductases↗
PubMed1984

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.

Bacillus↗
PubMed1983

Characterization of a chimeric beta-lactamase plasmid of Neisseria gonorrhoeae which can function in Escherichia coli.

A chimeric beta-lactamase encoding plasmid, containing the 4.4 Mdal beta-lactamase plasmid and the 2.6 Mdal cryptic plasmid of Neisseria gonorrhoeae has been characterized by physical and biological methods. Digestion with restriction enzymes indicates the presence of the following restriction sites: 1 site: AccI, AvaI, HgiAI, HincII, MstI, PstI, PvuII, XbaI and XorI; 2 sites: HindIII and BamHI; 3 sites: BclI, Sau96I and AvaII; 6 sites: HinfI; greater than 8 sites: AluI, BbvI, DdeI, HgaI, HhaI, HpaII, MspI, Sau3A, TacI and TaqI. No restriction sites were found for the following: BglI, BglII, BstEII, EcoRI, EcoRII, HpaI, HphII, KpnI, SacI, SalI, SmaI, SstI, SstII, and XhoI. Five plasmid specific proteins have been identified by DNA directed in vitro protein synthesis (43K, 41K, 30K, 16K and 14K). The location on the physical map of the coding regions for each of these proteins has been determined by the following methods: using plasmid DNA restricted by various enzymes in an in vitro protein synthesis system; identifying promoter-containing regions by digesting plasmid DNA with DdeI, adding RNA polymerase and then determining which fragments are retained by nitrocellulose. This plasmid contains both parental phenotypes in that it encodes penicillin resistance and possesses the sequence necessary for uptake in the gonococcus. Transformation data indicate that this plasmid can function in both E. coli and N. gonorrhoeae and that growth in E. coli has no effect on the plasmid's ability to transform the gonococcus.

Bacterial Proteins↗
PubMed1983

Identification of three homology classes of small, cryptic plasmids in intestinal Bacteroides species.

A systematic analysis of the plasmid content of intestinal Bacteroides spp. was made. Eight of fifteen clinical isolates and seven of nineteen normal rectal flora isolates examined contained small plasmids (less than 5 Mda). The majority of these small plasmids could be assigned to three homology classes by Southern hybridization. Further investigation demonstrated the presence of subclasses within one of the homologous classes. Class I plasmids were 1.8 Mda, Class III were 3.7 Mda, and Class II plasmids were of three different molecular weights: 2.6 Mda (IIA), 3.2 Mda (IIB), and 4.0 Mda (IIC). Among representative plasmids from each class there was remarkable sequence similarity based upon digestion patterns obtained using the restriction endonucleases, AluI and DdeI. In addition, similar polypeptide products were observed using purified Class I plasmids as template in an Escherichia coli in vitro coupled transcription-translation system. Small plasmids were found in seven of the ten recognized species of intestinal bacteroides, indicating that no species barrier exists for these plasmids. In addition, since plasmids from all three classes were found together in a single isolate, it was concluded that these plasmids are not incompatible.

Bacteroides↗
PubMed1983

Construction and characterization of a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae.

In vitro recombination techniques were used to construct a bifunctional shuttle vector capable of functioning in Neisseria gonorrhoeae and Escherichia coli. This 6-kb plasmid contains a selectable phenotype, beta-lactamase production, which functions in both organisms. It also contains the lac region from pUC9 that allows for the direct selection of hybrid plasmids in the appropriate E. coli hosts by disruption of beta-galactosidase alpha complementation. The lac region contains several unique restriction sites useful for cloning: EcoRI, SmaI, BamHI and SalI.

DNA Transposable Elements↗
PubMed1982

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli.

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.

Cloning, Molecular↗
PubMed1982

Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain.

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VIII.

Biological Assay↗
PubMed1982

High correlation of the presence of methyladenine in Neisseria gonorrhoeae DNA with the AHU auxotype.

Strains of Neisseria gonorrhoeae were tested for the presence of methyladenine in the DNA sequence GATC by using the site-specific restriction endonucleases MboI and DpnI. It was found that 43 of 83 strains tested contained methylated DNA. When methylation was compared with the auxotype of the organism, 35 of 35 strains with the AHU (arginine-, hypoxanthine-, and uracil-requiring) auxotype and 8 of 48 strains with other auxotypes contained methyladenine. When the incidence of methylation in strains isolated from patients suffering from disseminated gonococcal infection was compared with that in strains isolated from patients suffering from uncomplicated gonococcal infection, no correlation with methylation and disseminated gonococcal infection was observed.

Adenine↗
PubMed1982

Evidence for the secretion of soluble peptidoglycans by clinical isolates of Staphylococcus aureus.

Four isolates of Staphylococcus aureus from patients with endocarditis and bacteremia were capable of secreting high-molecular-weight soluble peptidoglycans when grown in a minimal cell wall medium containing penicillin G. Vancomycin was not able to substitute for penicillin G in triggering this secretion. Secretion reflected de novo synthesis of soluble peptidoglycan and was strongly dependent on time of incubation (30 to 60 min), and number of bacteria (2 X 10(8) to 5 X 10(8) colony-forming units per ml), but not on penicillin G concentration (10 to 250 micrograms/ml). The incorporation of alanine into the peptidoglycans secreted in vitro by these isolates incubated in the presence of penicillin G under optimal conditions was variable. The least incorporation of alanine into peptidoglycan occurred with an isolate from a patient treated with nafcillin who had no detectable antipeptidoglycan titer.

Alanine↗
PubMed1982

Anaerobic survival of clinical isolates and laboratory strains of Neisseria gonorrhoea: use in transfer and storage.

Eleven laboratory strains and 67 clinical isolates of Neisseria gonorrhoeae were tested for the ability to survive during anaerobic incubation. The survival of the laboratory strains was dependent on auxotype, temperature, and cell density on agar plates. For both the laboratory strains and the clinical isolates, anaerobic survival was better at lower temperatures. We concluded that anaerobic incubation, for as long as 7 days, is useful when transporting or storing N. gonorrhoeae.

Anaerobiosis↗
PubMed1982

Correlation of the Genetic Map and the Endonuclease Site Map of Bacillus subtilis Bacteriophage SP02.

By marker rescue of bacteriophage SP02 sus mutants with purified bacteriophage SP02 DNA fragments, 11 of the 17 known bacteriophage SP02 sus loci were assigned to discrete DNA fragments. The left-most genetic locus, susA, was found to reside near one bacteriophage SP02 terminus (EcoRI-C1 fragment), whereas the right-most genetic locus, susP, was found to reside near the other bacteriophage SP02 terminus (EcoRI-C2 fragment). The physical locations of the intervening genetic loci were found to be consistent with the previously determined genetic order. Evidence was also obtained which suggested that at least one end of a transforming DNA fragment is degraded during DNA uptake by the competent bacterium.

Journal Article↗
PubMed1981

Interaction with lectins and differential wheat germ agglutinin binding of pyocin 103-sensitive and -resistant Neisseria gonorrhoeae.

Strains of Neisseria gonorrhoeae were treated with pyocin 611 131 (pyocin 103) from Pseudomonas aeruginosa PA103, and isogenic resistant variants were isolated. The interaction of pyocin-sensitive and isogenic pyocin-resistant strains with wheat germ agglutinin (WGA) agglutinated all pyocin-sensitive, but not pyocin-resistant, strains. Binding of WGA to three pyocin-sensitive strains and their isogenic pyocin-resistant variants was examined quantitatively by using fluorescein-conjugated lectin. Pyocin-resistant strains maximally bound one-third to one-eighth the quantity of WGA bound by isogenic-sensitive strains. Linear Scatchard plots revealed homogeneous WGA-binding sites on three pyocin-sensitive and one pyocin-resistant strains. Biphasic Scatchard plots, obtained with two pyocin-resistant strains, show that WGA-binding sites in these strains are heterogeneous. The number of WGA-binding sites for pyocin-sensitive organisms ranged from 8 x 10(5) to 1 x 10(6) sites per coccus and from 1 x 10(5) to 3 x 10(5) sites per coccus for pyocin-resistant strains. The apparent association constant for WGA binding to pyocin-sensitive strains ranged from 3 x 10(6) to 6 x 10(6) liters/mol and from 6 x 10(6) to 1 x 10(7) liters/mol for pyocin-resistant strains. Gonococcal lipopolysaccharide was shown to serve as the pyocin 103 receptor by inhibition of pyocin activity. Lipopolysaccharide from a pyocin 103-resistant strain was not able to inhibit pyocin 103 activity. Pyocin 103 resistance was correlated with a structural alteration involving N-acetylglucosamine residues in gonococcal lipopolysaccharide. Based on interactions with wheat germ, soybean, and ricin lectins, a model of lipopolysaccharide structure in N. gonorrhoeae is presented.

Agglutination↗
PubMed1980

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli.

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.

Bacillus subtilis↗