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Biomedical subjects

J De Ley

Publications and source records attributed to J De Ley.

At least 37 records · Page 2Linked to original sources
PubMed1975

Improvements of the membrane filter method for DNA:rRNA hybridization.

We describe and recommend the following improvements of DNA:rRNA membrane filter hybridization methods. One of our aims was to avoid DNA release from filter discs during hybridization. 1. Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr. 2. Duplexing is over in 8-10 hr. 3. Formamide has to be very pure (O.D. less than or equal to 0.2/cm light path at 270 nm). 4. RNAase treatment: 250 mug/5 ml 2 SSC/filter at 37 C for 1 hr. 5. Our conditions for stepwise thermal denaturation are: 5 degrees C steps from 50 C to 90 C in 1.5 SSC in 20% formamide. 6. Single-stranded DNA, fixed on membrane filters, and stored in vacuo at 4C can be used reliably for hybridization for up to 20 months. 7. Concentrated DNA in 0.1 SSC, quick-frozen at -50 C and stored at -90 C for up to 2 years can be used for hybridization without much change. 8. A CsCl gradient purification step yields much purer DNA, but increases the release of DNA from filters by about 20%. Filters with 20% more DAN is a compensation. 9. rRNA can be stored for 20 months in SSC or 2 SSC at -12 C without changing the hybridization results.

Arthrobacter↗
PubMed1975

Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns.

Improved methods for the identification and grouping of bacteria by polyacrylamide gel electrophoresis of soluble proteins are described. Electrophoretic protein patterns were obtained in rigorously standardized comditions. The results were much more reproducible than any described previously. Some of the factors affecting reproducibility were; growth conditions, time and speed of centrifugation of extracts, and conditions of gel electrophoresis. Protein patterns were compared by computing correlation coefficients from normalized densitometric tracings and clustering the strains by the unweighted average pair group method. As model systems, both Agrobacterium and Zymomonas were used because of differences in the sharpness of the peaks. The methodwas applied to 42 Agrobacterium strains. The agreement with the results of clustering by either phenotypic tests or DNA:DNA hybridization was excellent. Computerized comparisons of electrophoretic protein patterns can be a fast, easy and powerful tool for classification and identification of bacteria.

Bacteria↗
PubMed1975

Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.

Amino Acid Sequence↗
PubMed1975

Particulate cytochrome c in Agrobacterium tumefaciens.

In Agrobacterium tumefaciens the main part of c-type cytochromes is tightly bound to the bacterial cell envelope structures. Several techniques were attempted to solubilize these cytochromes. The highest yield of cytochromes released is obtained by treatment of particle suspensions with 5% Triton X-100. Further purification confirms that the proteins are not really solubilized, but still aggregated in small heterogeneous complexes. Chromatography on a CM-cellulose column demonstrates that at least three different c-type cytochromes are present: cyt c-550, cyt c-552 and cyt c-556.

Cytochrome c Group↗