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T Kieser

Publications and source records attributed to T Kieser.

At least 37 records · Page 2Linked to original sources
PubMed1990

Pleural multicystic mesothelial proliferation. The so-called multicystic mesothelioma.

We report on the clinical and pathological features of a hitherto unrecognized multicystic and multifocal mesothelial lesion arising in the pleural cavity of a 37-year-old Caucasian woman. The lesions consisted of clusters of thin-walled cysts separated by connective tissue and lined by a single layer of flattened and cuboidal mesothelium. Mucin stains, immunohistochemistry, and electron microscopy were consistent with a mesothelial origin. The pathological features are identical to those of the previously reported multicystic mesotheliomas of the peritoneum. Although these multicystic peritoneal mesothelial lesions have been regarded as neoplasms, absent stromal extension, lack of mitotic activity, and (in this case) continuity with morphologically normal surrounding mesothelium are suggestive of a reactive process. The term "multicystic mesothelial proliferation" may therefore be more appropriate. Because these lesions may be detected as discrete pleural based masses on chest radiograph and CT scan, they may be submitted for frozen section during operative resection. It is therefore important to be aware of their existence, morphology, and differential diagnosis.

Adult↗
PubMed1989

Development of genetic systems for the mycobacteria.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system which allows for the transfer, mutation, and expression of specific genes. Genetic analysis of mycobacteria has been exceedingly difficult since the mycobacteria grow slowly and no natural efficient method of gene transfer within the pathogenic has thus far been found. Using a molecular genetic approach, we have developed both the vectors and the methodology for efficient gene transfer in the mycobacteria. Initially, a novel of type of mycobacteriophage vector was developed, termed a shuttle phasmid. This hybrid shuttle vector replicates in Escherichia coli as a plasmid and in mycobacteria as a phage, capable of introducing foreign DNA into a wide variety of mycobacterial species. A set of shuttle phasmids, constructed from a temperate mycobacteriophage, retained their ability to lysogenize their mycobacterial hosts and could thus introduce foreign DNA stably into mycobacterial cells. An E. coli gene conferring kanamycin-resistance was cloned into these vectors and shown to express in the mycobacteria, thus providing the first selectable marker gene for subsequent genetic studies. Using kanamycin-resistance gene as a selection, the M. fortuitum plasmid pAL5000 replicon, and electroporation; a plasmid transformation system has been developed for both M. smegmatis and BCG. We now plan to use these phage and plasmid systems to analyze, genetically, the virulence attributes of the pathogenic mycobacteria. In addition, by introducing and expressing foreign antigens in BCG, we hope to develop a novel recombinant multi-vaccine vehicle capable of conferring immunity to a variety of bacterial, viral, and parasitic pathogens.

BCG Vaccine↗
PubMed1988

Site-specific degradation of Streptomyces lividans DNA during electrophoresis in buffers contaminated with ferrous iron.

Streptomyces lividans DNA contains a modification which makes it susceptible to double-strand cleavage during electrophoresis in buffers contaminated with ferrous iron (which may be present in some batches of EDTA). The cleavage of the DNA is site-specific and the average fragment size resulting from limit digestion of total S. lividans DNA is about 6kb. DNA from Streptomyces coelicolor A3(2) and several other Streptomyces strains, and from E. coli, is not cleaved under the same conditions. A S. lividans mutant has been isolated which lacks the DNA modification. We suspect that many reports of "poor" preparations of S. lividans plasmids may be due to the above effect.

Buffers↗
PubMed1988

Plasmid pIJ699, a multi-copy positive-selection vector for Streptomyces.

A plasmid vector, pIJ699, which provides positive selection for cloned DNA, was constructed using the replication functions of the Streptomyces wide-host-range multi-copy plasmid pIJ101. The selection for inserts is based on the principle that plasmids with long uninterrupted perfect palindromes (inverted repeats) are 'not viable' in bacteria. For cloning, pIJ699 is digested with BglII. This produces two fragments, one of which is the linearized vector, with two arms of the palindrome at its ends, and the other is a 'spacer' which is needed to keep the inverted repeat sequences apart. The vector fragment is separated from the 'spacer' fragment and ligated with the DNA to be cloned. Plasmids with a fragment of cloned DNA, but not the circularized vector, give rise to thiostrepton-resistant transformants in Streptomyces lividans. The inverted repeat sequences contain a strong transcription terminator which reduces transcriptional read-through both in and out of the cloned fragment. This improves the stability of many hybrid plasmids and facilitates the study of the regulation of cloned genes.

Cloning, Molecular↗
PubMed1988

"Strong incompatibility" between derivatives of the Streptomyces multi-copy plasmid pIJ101.

Some derivatives of pIJ101, a 8.9 kb Streptomyces multi-copy plasmid, can co-exist with each other at similar copy numbers but others are strongly incompatible. The DNA sequence, sti, which causes this "strong incompatibility" was localised on a DNA segment of about 200 bp which is not part of the essential replication region of pIJ101. The sti function is active only when the DNA fragment carrying it is present in the natural orientation with respect to the basic replicon region of pIJ101. Pairs of plasmids which either both possess sti in the correct orientation (Sti+) or both lack sti or carry it in reverse orientation (Sti-) can co-exist, but Sti+ and Sti- plasmids cannot; in this case the Sti+ plasmid is retained and the Sti- plasmid is lost. This phenomenon is called strong incompatibility to distinguish it from classical incompatibility where identical or related plasmids are incompatible and dissimilar plasmids are compatible. pIJ101 probably replicates via a single-stranded intermediate; sti would be a site where the synthesis of the second (lagging) DNA strand is initiated because Sti- plasmids accumulate more single-stranded plasmid DNA than Sti+ plasmids. The copy number of pIJ101 and its derivatives is influenced by sti and by an additional trans-acting function (cop).

Chromosome Deletion↗
PubMed1988

Lysogeny and transformation in mycobacteria: stable expression of foreign genes.

Requisite to a detailed understanding of the molecular basis of bacterial pathogenesis is a genetic system that allows for the transfer, mutation, and expression of specific genes. Because of the continuing importance of tuberculosis and leprosy worldwide, we initiated studies to develop a genetic system in mycobacteria and here report the use of two complementary strategies to introduce and express selectable genetic markers. First, an Escherichia coli cosmid was inserted into the temperate mycobacteriophage L1, generating shuttle phasmids replicating as plasmids in E. coli and phage capable of lysogenizing the mycobacterial host. These temperate shuttle phasmids form turbid plaques on Mycobacterium smegmatis and, upon lysogenization, confer resistance to superinfection and integrate within the mycobacterial chromosome. When an L1 shuttle phasmid containing a cloned gene conferring kanamycin resistance in E. coli was introduced into M. smegmatis, stable kanamycin-resistant colonies--i.e., lysogens--were obtained. Second, to develop a plasmid transformation system in mycobacteria, M. fortuitum/E. coli hybrid plasmids containing mycobacterial and E. coli replicons and a kanamycin-resistance gene were constructed. When introduced into M. smegmatis or BCG (Mycobacterium tuberculosis typus bovinus var. Bacille-Calmette-Guérin) by electroporation, these shuttle plasmids conferred stable kanamycin resistance upon transformants. These systems should facilitate genetic analyses of mycobacterial pathogenesis and the development of recombinant mycobacterial vaccines.

Cloning, Molecular↗
PubMed1987

Activity of a Streptomyces transcriptional terminator in Escherichia coli.

A 205bp DNA fragment from the Streptomyces multi-copy plasmid pIJ101 has in vivo terminator activity both in Streptomyces lividans and in Escherichia coli. Termination of RNA synthesis, detected by high-resolution S1 nuclease mapping, occurs at precisely the same nucleotides in both organisms. This suggests that the E. coli RNA polymerase recognizes the same sequence elements and chooses the point(s) of termination in the same way as the corresponding S. lividans enzyme.

Chloramphenicol↗
PubMed1986

Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator.

Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

Cloning, Molecular↗
PubMed1986

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G.

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.

Cloning, Molecular↗
PubMed1986

Expression of a Streptomyces plasmid promoter in Escherichia coli.

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region.

Base Sequence↗
PubMed1986

Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans".

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.

Cloning, Molecular↗
PubMed1985

Cloning of a multicopy plasmid from the actinorhizad nitrogen-fixing bacterium Frankia sp. and determination of its restriction map.

An 8.3-kb multicopy plasmid, pFQ31, from the nitrogen-fixing Frankia sp. strain ArI3, was cloned into Escherichia coli plasmid vectors and analysed physically. pFQ31 has no detectable sequence homology with another Frankia plasmid, pFQ32, which is present in the same host. Derivatives of pFQ31 with an antibotic resistance marker were introduced into Streptomyces lividans, which is taxonomically related to Frankia, but no stable replication could be achieved.

Actinomycetales↗
PubMed1984

DNAGEL: a computer program for determining DNA fragment sizes using a small computer equipped with a graphics tablet.

The program DNAGEL is used to determine the size of DNA fragments run on agarose or polyacrylamide gels. The positions of the bands are read from gel photographs by means of a digitizer. Standard curves are calculated by the method of Southern (1979). The bands, as they are measured, are reproduced on the screen so that erroneous input can be recognized and corrected immediately. Similarly the estimated fragment sizes are printed in a table in the same relative positions as the bands on the gel. This makes it especially easy to relate fragment sizes with the bands on the gel picture. As an additional function the calculated positions of bands can be displayed on the screen. The program DNAGEL is written in APPLESOFT BASIC, suitable for APPLE II computers with 48K memory connected to a monitor, printer and a HOUSTON graphics tablet.

Base Sequence↗
PubMed1984

Integrated DNA sequences in three streptomycetes form related autonomous plasmids after transfer to Streptomyces lividans.

When Streptomyces parvulus ATCC 12434 was crossed with a plasmid-free S. lividans 66 derivative, some S. lividans exconjugants contained plasmid DNA, pIJ110 (13.6 kb). In a similar way, pIJ408 (15.05 kb) was found after mating S. glaucescens ETH 22794 with S. lividans. CCC DNA was not visualized in the donor strains. pIJ110 and pIJ408 each originates from a larger replicon, probably the chromosome, of S. parvulus or S. glaucescens. Restriction maps of pIJ110 and pIJ408, each for 10 enzymes, were derived. Derivatives of each plasmid were constructed carrying antibiotic-resistance markers (thiostrepton or viomycin) in a nonessential region and each plasmid was cloned into an Escherichia coli plasmid vector (pBR327 or pBR325). pIJ110 and pIJ408 resemble, in their origin, the previously known SLP1 plasmids (such as SLP1.2) which come from integrated sequences in the chromosome of S. coelicolor A3(2). pIJ110 and pIJ408, like SLP1.2, are self-transmissible, elicit the so-called lethal zygosis reaction (pock formation) and mobilize chromosomal markers. The three plasmids, in spite of their very different restriction maps, were found to be related: SLP1.2 and pIJ110 were strongly incompatible, showed complete resistance to each other's lethal zygosis reaction, and shared a segment of DNA with a considerable degree of cross-hybridization; pIJ110 and pIJ408 were weakly incompatible and showed partial resistance to lethal zygosis and a weak DNA cross-hybridization; pIJ408 and SLP1.2 were only distantly related on these criteria. pIJ110, pIJ408, and SLP1.2 hybridized with varying degrees of homology in Southern transfer experiments to DNA from 7 out of 13 of an arbitrary collection of wild-type streptomycetes. Integrated sequences capable of forming plasmids after transfer to S. lividans may therefore be widespread in the genus Streptomyces.

Base Sequence↗
PubMed1984

Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli.

Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.

Chloroform↗