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R Schaffrath

Publications and source records attributed to R Schaffrath.

11 recordsLinked to original sources
PubMed1999

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Biological Evolution↗
PubMed1997

Microbial linear plasmids.

While plasmids were originally considered to be generally circular until almost two decades ago, linear elements were reported to exist as well. They are now known to be common genetic elements in both, pro- and eukaryotes. Two types of linear plasmids exist, the so-called hairpin plasmids with covalently closed ends and those with proteins bound to their 5' termini. Hairpin plasmids are common in human-pathogenic Borrelia spirochetes, in which they are instrumental in escape from the immunological response; cryptic hairpin elements are present in mitochondria of the plant pathogenic fungus Rhizoctonia solani. Plasmids with 5' attached proteins constitute the largest group. In actinomycetous bacteria they are conjugative and usually confer advantageous phenotypes, e.g. formation of antibiotics, degradation of xenobiotics, heavy-metal resistance and growth on hydrogen as the sole energy source. In contrast, the majority of linear plasmids from eukaryotes are cryptic, with only a few exceptions. In some yeasts a killer phenotype may be associated, the most thoroughly investigated elements being those from Kluyveromyces lactis killer strains. In Neurospora spp. and in Podospora anserina, senescence and longevity respectively are correlated with linear plasmids. This review focuses on the biology of linear plasmids, their environmental significance and their use as tools in molecular and applied microbiology.

Bacteria↗
PubMed1997

ORF7 of yeast plasmid pGKL2: analysis of gene expression in vivo.

ORF7 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a putative RNA polymerase subunit of 16 kDa. RNA analysis detected a single, plasmid-dependent ORF7 transcript of 550 nt indicating that the gene is transcribed mono-cistronically. Attempted one-step gene disruption of ORF7 resulted in chromosomal integration of the marker gene rather than the formation of stable recombinant k2ORF7(0) deletion plasmids. Thus, ORF7 appears to be a potential cis-dominant locus the integrity of which is indispensable for plasmid stability. The ORF7 gene product was over-produced as a c-myc-tagged fusion protein in Escherichia coli. Western-blot analysis of total yeast protein extracts using an antibody against this Orf7-c-myc fusion product identified a protein band with an apparent molecular weight of 17 kDa. This protein corresponds in size to the predicted product and is only detectable in plasmid-carrying killer yeasts.

Blotting, Northern↗
PubMed1996

Yeast killer plasmid pGKL2: molecular analysis of UCS5, a cytoplasmic promoter element essential for ORF5 gene function.

A k2/k1 plasmid gene shuffle system has been used to investigate linear plasmid promoter function in Kluyveromyces lactis. By transplacing various ORF5 deletion constructs from the larger plasmid k2 onto k1, and analysing trans-complementation of an ORF5(0) deletion on k2, a 40 bp k2 fragment, including the UCS motif of ORF5 (UCS5), has been identified as a cis-acting promoter element essential for ORF5 gene function. Qualitative and quantitative transcript analyses of a UCS5-ScLEU2 fusion gene using Northern blot analysis and phosphor image technology revealed a plasmid-dependent LEU2 transcript distinct in size (1.55 kb) and regulation from its nuclear counterpart (1.35 kb): cytoplasmic, UCS5-driven expression of the marker gene was non-repressible by leucine and reduced five- to eight-fold compared to fully derepressed nuclear K1LEU2 mRNA levels. Thus, the killer plasmids k2 and k1 appear to express low levels of transcript overall, when relative gene copy numbers (one for the nuclear allele versus 50-100 copies for the plasmid-borne LEU2 gene) are taken into account.

Base Sequence↗
PubMed1996

A cytoplasmic gene-shuffle system in Kluyveromyces lactis: use of epitope tagging to detect a killer plasmid-encoded gene product.

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis. By transplacing k2ORF5 from larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF5(0) deletion on k2 in trans, giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.

Amino Acid Sequence↗
PubMed1995

Kluyveromyces lactis killer plasmid pGKL2: molecular analysis of an essential gene, ORF5.

The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.

Amino Acid Sequence↗
PubMed1995

The DNA and RNA polymerase genes of yeast plasmid pGKL2 are essential loci for plasmid integrity and maintenance.

Novel recombinant plasmids derived from the Kluyveromyces lactis killer plasmid k2 have been constructed to study plasmid biology and gene function. In vivo recombination between native resident k2 and suitable disruption vectors, employing the KITRP1 gene fused to a plasmid promoter as selection marker, yielded ORF2 and ORF6 deletion plasmids at high frequencies. As judged from Southern hybridization and plasmid restriction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6, respectively, carry deletions in their putative DNA (ORF2) and RNA (ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for TRP1 over 350 generations of growth did not favour maintenance of hybrids over wild-type k2. Thus, neither rk2/2 nor rk2/6 was fully functional and able to displace parental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy number relative to k2 with rk2/2 more drastically affected than rk2/6 implying a direct involvement of the ORF2 product in plasmid replication and an indirect maintenance function for the ORF6 gene product.

Base Sequence↗
PubMed1992

Kluyveromyces lactis killer system: ORF1 of pGKL2 has no function in immunity expression and is dispensable for killer plasmid replication and maintenance.

To functionally characterize the genes encoded by the larger killer plasmid pGKL2 from Kluyveromyces lactis a previously developed in-vivo recombination system was exploited. An in-vitro modified version of the cytoplasmically expressible LEU2 gene cartridge (LEU2*) flanked by appropriate pGKL2 segments was used to replace the central part of the ORF1 region of pGKL2. Transformation of a Leu- killer strain resulted in the expected disruption of ORF1 in the resident pGKL2. The Leu+ transformants obtained can be assigned to three classes. Class I carries both killer plasmids, pGKL1/2, and the recombinant pGKL2 derivative termed pRKL2. Class II and III additionally harbor palindrome and hairpin-like plasmids, respectively. Upon subculturing of class I transformants under selective pressure, segregation of the native pGKL2 and the recombinant pRKL2 eventually occurs resulting in total loss of pGKL2. No differences concerning killer and immunity phenotype between a pRKL2-harboring strain and the native pGKL2-carrying recipient could be detected. Thus pGKL2 ORF1 is dispensable for both expression of killer/immunity phenotypes and for the replication and maintenance of the K. lactis killer plasmids.

Immunity↗
PubMed1989

[Hearing handicap in childhood--responsibilities of public health, early diagnosis].

Serious congenital hearing impairment occur in 1 per 1,000 people. Hereditary disease, perinatal complications, and postnatal disease present particular risk factors. Children, who do not hear or are extremely hard of hearing are slow in their language development and thus become retarded in their mental growth if not given appropriate stimulation. The Counseling Center for the hearing impaired was stablished as a multidisciplinary institution of the Health Office of Berlin-Neukölln for early recognition of and therapy for hearing impaired children. The average age at initial diagnosis of children with hearing impairment in the Federal Republic of Germany is between 3.3-3.4 years according to statistics from 1981. An effort should be made, however, to use the time before the completion of the first year of on's life for an optimal early therapy. Statistics from the Counseling Center for the hearing impaired indicate that at the present time all children with distinct hearing impairments, born in the FRG and West-Berlin, are diagnosed before they reach 32 months of age and are then assigned to special education programs. The average age of recognizing hearing impairments is 13.4 months. This favorable, however not optimal result can be attributed to the situation of a metropolis and its population informed by special professional events for target groups including employees of theYouth Health Services, established pediatricians and ENT specialists, teachers, social workers, educators, and also to the model of the "Berlin-Neukölln Counseling Center" which has proven successful over the past 30 years.

Child, Preschool↗
PubMed1987

Buccal versus sublingual nitroglycerin administration in the treatment of angina pectoris: a multicentre study.

Buccal (Suscard) was compared with sublingual nitroglycerin (Nitromex) in 126 patients with stable angina pectoris. Following a dose adjustment period they were randomized to cross-over treatment, two weeks on sublingual and buccal nitroglycerin, respectively. The dosage of buccal nitroglycerin was 2.5 mg in 49% and 5.0 mg in 51%. The total number of treated acute anginal attacks was 31% less during the buccal compared to the sublingual nitroglycerin period (P less than 0.001) despite a reported physical activity level which was significantly higher during the buccal nitroglycerin period. The use of sublingual nitroglycerin and buccal nitroglycerin was considered equally simple by 67%, sublingual nitroglycerin easier by 19% and buccal nitroglycerin easier by 14% of the patients. Sublingual nitroglycerin was used prophylactically on 806 occasions and buccal nitroglycerin on 929 occasions (P less than 0.05) with success in 66% of the sublingual nitroglycerin-attempts and 74% of the buccal nitroglycerin-attempts (P less than 0.05). Considering only prophylactic use, buccal nitroglycerin was preferred by 81% and sublingual nitroglycerin by 4% (P less than 0.05). When given the opportunity only to select one of the two nitroglycerin formulations, 65% (P less than 0.05) preferred buccal nitroglycerin and 19% sublingual nitroglycerin. In conclusion, buccal nitroglycerin seems to be more efficacious than sublingual nitroglycerin. The explanation is probably that the two formulations are comparable in the treatment of acute anginal attacks, while buccal nitroglycerin has a more pronounced prophylactic effect due to its longer duration of action.

Administration, Buccal↗