Gilberto Hintermann

Simple procedure for distinguishing CCC, OC, and L forms of plasmid DNA by agarose gel electrophoresis.

Abstract A simple and rapid method discriminating between covalently closed circular (CCC), open circular (OC), and linear (L) forms of plasmid DNA is presented. Two consecutive steps of agarose gel electrophoresis with a single DNA sample are used; use is made between the steps of ultraviolet light irradiation which introduces single-strand nicks in ethidium bromide-stained DNA, converting CCC into OC forms. Unambiguous assignments of OC and CCC forms are possible in samples containing several plasmids.

Cloning and expression of the genetically unstable tyrosinase structural gene from Streptomyces glaucescens

SummaryThe gene from Streptomyces glaucescens coding for an inducible tyrosinase was cloned using the low copy vector pIJ41 and the melanin-negative strain Streptomyces lividans TK23 as host. Hybridisation experiments as well as complementation studies showed that melC mutant strains carry large deletions of more than 10.5 kb, comprising the structural gene for tyrosinase, while melA and melB strains carry mutations in genes involved in the expression of tyrosinase activity. Strong DNA homology was found between the Streptomyces antibioticus and the S. glaucescens tyrosinase structural genes and both genes showed a similar regulation when introduced into melanin-negative hosts. While both tyrosinases exhibited clear induction in S. glaucescens, constitutive expression was observed in S. lividans. Northern blot experiments showed that tyrosinase expression is regulated at the transcriptional level and that the gene (822 bp) is part of a 2.3 kb transcript. The main start of the mRNA at about 475 bp upstream from the tyrosinase N-terminus was located by S1-mapping experiments.

Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.

Isolation and characterization of an extrachromosomal element from Nocardia mediterranei

Strain LBG A3136 of Nocardia mediterranei (ETH Collection) was found to contain a low-copy-number covalently closed circular extrachromosomal element, pMEA 100, which could only be isolated from mycelium grown on agar plates. pMEA 100 could not be isolated from the closely related strain ATCC 13685. Hybridization experiments showed that pMEA 100 is present in strain LBG A3136 in the free as well as in the integrated form whereas in strain ATCC 13685 only an integrated form was detected. Excision and reintegration in strain LBG A3136 seemed to be site specific. pMEA100 was found to be self-transmissible, eliciting the lethal zygosis phenotype, and is possibly involved in fertility in N. mediterranei.

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

SummaryThe wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.

Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions

SummaryStreptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in lenght. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements.

Deoxyribonucleic Acid Restriction Endonuclease Fingerprint Characterization of Actinomycete Strains

Restriction endonuclease digestion of total purified genomic deoxyribonucleic acid gives rise to deoxyribonucleic acid fragments of discrete sizes. These can be separated by one-dimensional agarose gel electrophoresis. A unique banding pattern (fingerprint) is obtained for each actinomycete wild-type strain tested. These fingerprints are useful for recognizing a given wild-type strain and its derivatives, but cannot be used for species characterization.

A technique for the detection of large DNA alterations in complex genomes

Abstract As part of our effort to understand the chromosomal organization of streptomycetes, we have developed a method for detecting large alterations in the DNA, in particular to visualize insertions, transpositions, and deletions. The method involves the labeling of the DNA of two strains with [3H]thymidine or [14C]thymidine, extraction and purification of the DNA, digestion with restriction endonucleases, and one-dimensional agarose gel electrophoresis of the two samples in the same slot. Following electrophoresis, the gel is cut into thin slices, and the 14 C 3 H ratio is measured in each slice. Deviations from the average standard ratio are caused by differences in the restriction site arrangement in the DNA of the two strains, which may be caused by rearrangements in the DNA. The method has a high resolution of one restriction fragment change.

Genetic Instability in Streptomycetes

Streptomycetes and other actinomycetes can exhibit a remarkable degree of phenotypic variability, which is frequently due to genetic variability. This behaviour attracted interest, because it affects not only morphological characters but also various enzymatic activities, antibiotic resistance and the commercially important antibiotic production. The problem has already been dealt with extensively in various review articles. Therefore only a short summary with a number of recent relevant references will be given, focusing on the role of plasmids and on chromosomal instability and without any claim of completeness.