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S Scherer

Publications and source records attributed to S Scherer.

At least 109 records · Page 6Linked to original sources
PubMed1995

A new procedure for efficient recovery of DNA, RNA, and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin.

A method for the rapid lysis of Listeria cells, employing a recombinant Listeria bacteriophage A118 lytic enzyme (PLY118), is described. The procedure can be used with all listerial species. It enables fast, efficient, and gentle recovery of DNA, RNA, or native cellular proteins from small-scale (2- to 5-ml) cultures. Moreover, this approach should be very useful in analytical detection and differentiation of Listeria strains when the release of native nucleic acids or proteins is required.

Bacterial Proteins↗
PubMed1995

Organization and transcriptional analysis of the Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh.

A511 is a broad-host-range, virulent myovirus for Listeria monocytogenes. The genes encoding major structural proteins of the capsid (cps) and tail sheath (tsh) were mapped to a 10.15-kb late gene fragment. We have determined the complete nucleotide sequence of this region and confirmed the identities of Cps (48.7 kDa) and Tsh (61.3 kDa) by N-terminal amino acid sequencing of both proteins. In addition, nine other open reading frames were identified. On the basis of amino acid sequence homologies to known phage-encoded proteins, some putative functions and locations could be assigned to some of the deduced gene products. We present evidence that the cps product is proteolytically cleaved between Lys-23 and Ser-24 to yield the 444-residue polypeptide found in the mature viral capsid. We also found that the N-terminal methionine is absent from the mature tail sheath protein. cps and tsh are late genes; mRNAs first appear 15 to 20 min after infection of L. monocytogenes. Northern (RNA) hybridizations of total late mRNA with specific oligonucleotide probes were used to determine the sizes of respective transcripts. Primer extension analyses enabled the positive identification of six late promoters, which were found to differ from those identified in the chromosome of Listeria spp. The bulk of transcripts from cps and tsh arise from two phage promoters with identical 13-nucleotide sequences (TGCTAGATTATAG [core region underlined]) in the -10 region which we speculate determines specific and timed expression of these genes. A 123-nucleotide leader sequence at the 5' end of the cps transcript was predicted to form a strong secondary structure (deltaG=-40.7 kcal [-170.3 kJ]/mol). Out results show that the strongly expressed A511 cps and tsh genes are included in two separate gene clusters and are independently regulated at the transcriptional level.

Amino Acid Sequence↗
PubMed1995

Stability of karyotype in serial isolates of Candida albicans from neutropenic patients.

Serial isolates of Candida albicans were obtained from 20 patients with leukemia over periods of up to 8 months. The fingerprinting of these isolates by interrepeat PCR and random amplified polymorphic DNA PCR has been described previously (A. van Belkum, W. Melchers, B. E. de Pauw, S. Scherer, W. Quint, and J. F. Meis, J. Infect. Dis. 169:1062-1070, 1994). Contour-clamped homogeneous electric field gel electrophoresis was used to examine the chromosomes of these isolates. When changes in the karyotype were seen in a series of isolates, additional interrepeat PCR and Southern blotting with a repeat DNA sequence from the 27A family were performed. These two genotyping tools were used to determine if karyotypic changes seen in a series of isolates were due to chromosome rearrangements in a single strain or due to colonization with more than one strain. It was determined that changes in karyotype in a series of strains indicated infection by a new strain.

Candida albicans↗
PubMed1994

Atypical regions in large genomic DNA sequences.

Large genomic DNA sequences contain regions with distinctive patterns of sequence organization. We describe a method using logarithms of probabilities based on seventh-order Markov chains to rapidly identify genomic sequences that do not resemble models of genome organization built from compilations of octanucleotide usage. Data bases have been constructed from Escherichia coli and Saccharomyces cerevisiae DNA sequences of > 1000 nt and human sequences of > 10,000 nt. Atypical genes and clusters of genes have been located in bacteriophage, yeast, and primate DNA sequences. We consider criteria for statistical significance of the results, offer possible explanations for the observed variation in genome organization, and give additional applications of these methods in DNA sequence analysis.

Bacteriophage lambda↗
PubMed1994

The ARG4 gene of Candida albicans.

The DNA sequence of a Candida albicans genomic fragment known to complement the arginine mutation designated arg57 in strain 1006 contains an ORF of 1404 nucleotides (nt) predicting a protein of 468 amino acids (aa). Database searches indicated that the deduced protein shares 75% identity and 85% similarity with the ARG4 protein of Saccharomyces cerevisiae. Analysis of the percent aa identity between C. albicans and S. cerevisiae sequences included in available databases suggested these values are within the range expected for biosynthetic enzymes from the two organisms which share similar function. Experiments to isolate C. albicans ARG4 by complementation in an arg4 strain of S. cerevisiae yielded a plasmid (pARG4-1) with a restriction map identical to that of the sequenced clone. From these data, we conclude that the gene previously designated ARG57 is in fact ARG4 encoding the enzyme argininosuccinate lyase (ASL). These results were unexpected, since ARG57 had been localized to chromosome 7, while a mutation causing an ASL deficiency had been linked to ade1, which is on chromosome R. Transformation of C. albicans strains with pARG4-1 indicated it complemented the arginine auxotrophy in strains TMSU221 and 1435, a derivative of 1006. Examination of commonly utilized C. albicans arginine auxotrophs by spheroplast fusion analysis indicated these strains comprise two complementation groups: one consisting of 1006 and TMSU221, which are arg4, and the other of A642, hOG318, hOG357, FC18-6 and WC-5-4, which possess an undefined defect in the arginine biosynthetic pathway which we designate arg100.

Amino Acid Sequence↗
PubMed1994

Water stress proteins of Nostoc commune (Cyanobacteria) are secreted with UV-A/B-absorbing pigments and associate with 1,4-beta-D-xylanxylanohydrolase activity.

Acidic water stress polypeptides (Wsp) with molecular masses of 33, 37 and 39 kDa are the most abundant soluble proteins in the cyanobacterium Nostoc commune. Wsp polypeptides and UV-A/B-absorbing pigments are secreted by cells, accumulate in the extracellular glycan sheath, and are released from desiccated colonies upon rehydration. No evidence was obtained for either glycosylation, phosphorylation, or acylation of Wsp polypeptides. NH2-terminal amino acid sequences of the 33-, 37-, and 39-kDa polypeptides were identical: Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu-Gln-X-Ile-Gln-Asn-Pro-Ser-Asn- Pro-Ser-Asn-Gly-Lys-Gln. This consensus NH2-terminal sequence and an internal sequence (Glu-Ala-Arg-Val-Thr-Gly-Pro-Thr-Thr-Pro-Ile-Asp) showed homologies with the sequences of carbohydrate-modifying enzymes. Purified Wsp polypeptides associate with a 1,4-beta-D-xylanxylanohydrolase activity that was inhibited specifically by Wsp antiserum. In the absence of salt, Wsp polypeptides, and the water-soluble UV-A/B-absorbing pigments, form multimeric complexes through strong ionic interactions. A possible role is suggested for Wsp polypeptides in the synthesis and/or modification of a xylose-containing UV-A/B-absorbing pigment.

Amino Acid Sequence↗
PubMed1994

Genotypic characterization of sequential Candida albicans isolates from fluconazole-treated neutropenic patients.

A polymerase chain reaction (PCR)-mediated genotyping assay for Candida albicans has been developed. By amplification of genomic regions bordered by eukaryotic or prokaryotic repeat-like motifs, differences between C. albicans isolates can be determined. The resolution of this typing procedure is at least as good as that of other genotypic assays. To ascertain the epidemiologic and clinical usefulness of this PCR genotyping, a retrospective analysis of serial C. albicans isolates from neutropenic adults treated with fluconazole was done. By PCR genotyping, 40 strains were detected in 24 patients. Eighteen C. albicans strains were found on multiple samplings in individual patients. It appears that most patients remain colonized with a C. albicans strain of constant genotypic characteristics. However, exceptions were observed. In 7 (29%) of 24 patients, strains deviating from the most frequently encountered type could be identified. All but 1 strain remained susceptible to fluconazole in vitro after treatment in vivo. It was not possible to demonstrate a relation of genotypic variation and antifungal susceptibility changes.

Adult↗
PubMed1994

Structural proteins and DNA characteristics of 14 Listeria typing bacteriophages.

The major structural proteins of 13 temperate and one virulent Listeria typing bacteriophages were analysed and compared using isoelectric focusing in immobilized pH gradients (IPG), ultrathin-layer two-dimensional electrophoresis, amino acid analysis and N-terminal amino acid sequences of selected proteins. Isoelectric points for major capsid and tail proteins of the 12 members of the siphoviridae family included in this study ranged from 4.70 to 5.92, whereas one of the two myoviridae investigated (B054) showed structural proteins in the 6.1 to 6.3 range. In comparison to protein profiles from one-dimensional SDS gels, the IPG technique gave better resolution and improved discrimination of phage proteins. Combination of this technique and SDS gel electrophoresis made it possible to correlate M(r) and isoelectric points of major structural proteins. Tail polypeptides of all siphoviridae are smaller and, with one exception, more acidic than their corresponding capsid counterparts. We also determined the amino acid composition of capsid and tail proteins. When compared with an average protein, they were found to be fairly rich in acidic and short-chain hydrophobic amino acids, as well as in lysine. In addition, the N-terminal amino acid sequences of major capsid and tail proteins of four representative listeriaphages were compared. The base composition of listeriaphage DNAs was between 37% and 39% G + C, reflecting that of their bacterial hosts. Each phage had a distinct restriction endonuclease pattern, and genome sizes ranged from 35 to 116 kb. DNA-DNA hybridization permitted the identification of five DNA homology groups. The two myoviruses studied (A511 and B054) showed no DNA homology to other phages, confirming their unique nature. The 12 siphoviruses were classified into three DNA homology groups with little cross-homology. Furthermore, phage A006 was found to share little DNA homology with the other investigated members of species 2671. Therefore, a new species (A006) is proposed. With respect to phage classification and taxonomy, a good correlation between the various approaches was observed, mostly corresponding to particle morphology.

Amino Acid Sequence↗
PubMed1994

HLA class II associations with idiopathic nephrotic syndrome in children.

The occasional familial occurrence of idiopathic nephrotic syndrome (NS) points to a genetic predisposition. Reports on associations with certain HLA class II antigens support this hypothesis. In order to define the immunogenetic background of NS more precisely, HLA class II allele frequencies in 161 children with NS were studied by restriction fragment length polymorphism (RFLP) typing. The patient cohorts consisted of 87 children from Southwest-France and 74 from Southwest-Germany. The control group consisted of 118 French and 101 German unrelated individuals from the same geographical areas. HLA alleles were defined in patients with steroid-sensitive (SS) and steroid-resistant (SR) NS and in controls. RFLP typing revealed that the previously reported association between SSNS and HLA-DR7 is confined to the RFLP split 7.1 (DRB1*07) with a combined relative risk (RRcomb) of 6.2. HLA-DQB typing showed an increased frequency of the allele DQB2b (DQB1*0201) (RRcomb = 7.8). HLA-DQA typing showed an association of SSNS with DQA3 (DQA1*0201,0301,0302) (RRcomb = 4.1). The highest RR (16.5) for SSNS was found in German patients who carried the two DRB1 specificities 17.1 (DRB1*0301) and 7.1 (DRB1*07). All associations were stronger in SS patients with frequent relapses or steroid dependency than in non- or infrequent relapsers. SR patients exhibited no significant associations with HLA class II alleles.

Alleles↗
PubMed1994

A method for HLA-DQA typing by the PCR-SSP technique.

There is preliminary evidence that matching for HLA-DQ is important for kidney graft survival. We developed a method for HLA-DQA typing based on the PCR-SSP principle. The procedure consisted of three steps: DNA isolation, PCR amplification and visualization of the PCR product under UV light. For the identification of all currently known DQA1 alleles, we designed 18 different primers that allowed typing for the specificities DQA1*0101, *1012, *0103, *0104, *0201, *03, *0401, *0501 and DQA1*0601. For the typing of a single individual, 12 PCR mixes were needed, each containing a primer pair specific for a certain allele group, and a pair of control primers that amplified a non-polymorphic region. The time required for this procedure was approximately 3 h from the time of blood collection. Comparison of this method with DQA typing by the RFLP method in 151 individuals revealed only a single discrepancy. The method can be easily applied for prospective cadaver donor typing.

DNA Primers↗
PubMed1994

Comparison of serological and DNA HLA-DR typing results for transplantation in Western Europe, Eastern Europe, North America and South America.

In a previous study, DNA typing revealed that 25% of serological HLA-DR typings of kidney transplants were incorrect. In the current study, we analyzed whether this error rate had improved in recent years, and whether there were differences according to geographical region. From 1988 to 1991 the error rate of serological typing improved slightly in Western Europe from 19% to 16%, and in North America, from 21% to 16%. In Eastern Europe, the error rate decreased from 49% to 33% in 1991, whereas the rate remained high in South America at 60% in 1988 and 72% in 1991. The high error rates in South America and Eastern Europe reflected a lack of good quality serological typing reagents. The 16% typing errors in Western Europe and North America demonstrated the current limit of serological techniques for cadaver donor typing and underlined the need for prospective DNA typing.

DNA↗
PubMed1994

Supplementary Listeria-typing with defective Listeria phage particles (monocins).

The potential use of monocins for listeria typing was investigated. Monocins are defective phage particles still capable of lysing listerial cells. They were induced by u.v.-irradiation, precipitated with polyethylene glycol and purified by density gradient centrifugation. Using 26 monocins, it was possible to type 48% of Listeria monocytogenes strains, 92% of L. innocua strains and 94% of L. ivanovii strains. Overall typability of 480 strains was 68%. None of the monocins was able to lyse L. grayi. Monocin typing was found to be a valuable supplementary tool for typing strains which were non-typable by the Weihenstephan phage typing set. A combination of phage typing and monocin typing increased overall typability of Listeria strains to 95%.

Bacteriophage Typing↗
PubMed1994

Isolation and characterization of Linocin M18, a bacteriocin produced by Brevibacterium linens.

Brevibacterium linens M18, isolated from red smear cheese, produces a substance that inhibits the growth of Listeria spp. and several coryneform and other gram-positive bacteria. No gram-negative bacteria were inhibited. The substance is heat labile, sensitive to proteolytic enzymes, and stable between pH 3 and 12. High levels of this bacteriocin, named Linocin M18, were obtained in the stationary growth phase. Linocin M18 was purified by ultrafiltration, ultracentrifugation, and gel filtration chromatography. In its native form, it is a proteinaceous aggregate with a high molecular weight. Fractions with Linocin M18 activity contained particles of 20 to 30 nm in diameter. The bacteriocin consists of a single protein subunit with a molecular mass of 31 kDa and an isoelectric point of 4.5 N-terminal sequence analysis yielded Met-Asn-Asn-Leu-Tyr-Arg-Glu-Leu-Ala-Pro-Ile-Pro-Gly-Pro-Ala-Ala-Ala-Glu- Ile. Significant homology with published sequences was lacking.

Amino Acid Sequence↗
PubMed1994

Induced chromosome rearrangements and morphologic variation in Candida albicans.

We have isolated a mutant of Candida albicans that switches between colony morphologies at high frequencies in a strain with several genetic markers. This strain, 1183, has an altered karyotype with two extra chromosomes. The 1183 karyotype is unstable upon passage. Using DNA transformation with the URA3 gene flanked by sequences from the C. albicans repeat sequence 27A, we have marked individual chromosomes of 1183 and 1161, a related smooth, stable strain. Many transformants contained one or more extra chromosomes, ranging in size from 150 kb to 2.1 Mb. Most were less than 800 kb and appeared to be fragments of a single chromosome. All fragments tested derive from one of the two smallest chromosomes. Six of 13 fragments contained the URA3 gene. In some cases, URA3 was located at the end of a fragment with adjacent telomere repeats. The integrated copy of URA3 was unstable in some 1183 transformants. Our results suggest that 1183 has a mutation affecting genomic stability. A connection between karyotypic changes and morphologic variation has been suggested from studies of several C. albicans strains; however, we find that gross karyotypic and morphological changes are separable processes.

Blotting, Southern↗
PubMed1994

Taxonomical classification of 20 newly isolated Listeria bacteriophages by electron microscopy and protein analysis.

A set of 20 newly isolated temperate bacteriophages for phage typing of the pathogen Listeria monocytogenes has been investigated by means of electron microscopy and electrophoretic analysis of phage structural proteins. All phages had isometric capsids (60-64 nm diameter), and long contractile or non-contractile tails of 170-320 nm length. They could be classified into 2 morphotypes (A1 and B1) and were assigned to 3 listeriaphage species (4211, 2671, and 2389). Individual protein profiles were generated by SDS-PAGE of viral polypeptides, as well as isoelectric focusing of solubilized phage proteins in immobilized pH gradient gels. The major structural proteins ranged in size from approximately 15 to 38 kD, and showed isoelectric points from pI 4.3 to 6.2. Protein compositions permitted the differentiation of individual phages as well as the recognition and grouping of similar viruses.

Bacteriophage Typing↗