Search PubMed⌕ Search

Biomedical subjects

H Zischler

Publications and source records attributed to H Zischler.

29 records · Page 2Linked to original sources
PubMed1989

DNA fingerprinting with the oligonucleotide probe (CAC)5/(GTG)5: somatic stability and germline mutations.

DNA fingerprints were generated from various human somatic tissues and from peripheral blood of 179 children and their 80 parents using (CAC)5/(GTG)5 oligonucleotide probes. Whereas somatic stability of the fingerprint patterns was demonstrated, the average rate for germline mutations was estimated to be approximately 0.001 per DNA locus and gamete, with the three different restriction enzymes used. Seven out of eight mutations observed appeared to be of paternal origin.

Base Sequence↗
PubMed1989

Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ.

A fast, reproducible and non-hazardous technique for non-isotopic DNA fingerprinting is presented. The method is based on digoxigenated oligonucleotides, which are specific for simple repetitive DNA sequences. The use of digoxigenin/anti-digoxigenin detection avoids many drawbacks inherent in e.g. the biotin/streptavidin system which often causes a poor signal-to-background ratio. Synthesis and purification of digoxigenated oligonucleotides and their use in filter hybridization are described in detail. Hybridization patterns obtained with four different radioactively labeled oligonucleotides have been compared with those of the respective digoxigenated probes. When slightly less stringent hybridization conditions are applied for digoxigenated oligonucleotides than for those labeled with 32P, the signal intensities are satisfying but additional minor bands occur as a result of the reduced stringency. With one explainable exception, these bands increase the information content of the fingerprint. In addition, hybridization of the digoxigenated (CAC)5 probe has been performed in situ with human metaphase chromosomes. The hybridization patterns in many mitoses resemble R-bands.

DNA↗
PubMed1989

Electrophoretic and isoelectric focusing studies in Brazilian Indians: data on four systems.

Three-hundred ninety-nine individuals living in seven populations of two Brazilian Indian tribes (Macushi and Içana River Indians) were tested for the phosphoglucomutase 1 (PGM1), properdin factor B (BF), haptoglobin (HP), and alpha-1-antitrypsin (PI) systems. We observed significant internal heterogeneity in the two tribes for the PGM1 alleles and in the Macushi for the HP markers. Frequencies in three of the four systems (the exception being BF) also show clear differences in the Macushi and Içana River Indians. Compared with other ethnic groups, South American Indians generally present high frequencies of PGM1*1B, BF*S, HP*1S, and PI*M3. On the other hand, PGM1*1A, PI*M1, and PI*M2 are reduced, and HP*1F is absent or rare. This is the first report about HP subtypes among American Indians.

Brazil↗
PubMed1988

Optimized oligonucleotide probes for DNA fingerprinting.

The three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 x 10(-8). The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e.g. in paternity testing and in forensic applications. In most of these experiments 32P-labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P-labelled probe (GACA)4.

Blotting, Southern↗
PubMed1988

Generation of mature CD3+ and T cell receptor (TCR) + T cells from a leukemic analogue of the putative human stem cell by T cell conditioned medium containing IL-3, IL-2, and GM-CSF.

Malignant cells of a patient with acute leukemia expressed hematopoietic stem cell antigens such as CD34 and HLA-class II but lacked lineage specific differentiation markers. The leukemic blasts differentiated into mature T cells within 14 days in the presence of a T cell conditioned medium or with a mixture of highly purified interleukin-2 (IL-2) plus recombinant interleukin-3 (IL-3) and recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF). Phenotypically, the maturing cells acquired the T cell-specific differentiation antigens CD2, CD3, and CD8, whereas immature differentiation antigens such as CD34 and Leu19 as well as HLA-class II and the IL-2 receptor CD25 were concomitantly down-regulated within 14 days of in vitro culture. This in vitro maturation involved two to three synchronized cell divisions. Beyond 10 days of culture the leukemic cells produced mRNA specific for the T cell receptor beta and alpha chain, but at no time transcription of T cell receptor gamma chain-specific message was detectable. To our knowledge, these data represent the first in vitro model demonstrating the differentiation of phenotypically mature T cells from immature leukemic cells induced by the combined activities of IL-2 plus IL-3 and GM-CSF.

Antigens, CD19↗
PubMedDate not supplied

Oligonucleotide fingerprinting with (CAC)5: nonradioactive in-gel hybridization and isolation of individual hypervariable loci.

The first topic to be treated in this paper is the nonradioactive DNA fingerprinting by means of in-gel hybridization with digoxigenated (CAC)5. Besides the fact that time-consuming Southern blotting can be avoided, the dried agarose is an excellent matrix to produce background-free nonradioactive DNA fingerprints. There is no tendency of either the oligonucleotide probe or the antibody towards unspecific binding to the dried agarose. Prehybridization and blocking steps are therefore superfluous. Furthermore, we will discuss what effect the degree of crosslinking of the antibody-enzyme conjugates has. The second topic concerns the isolation and characterization of locus-specific probes from a human (CAC)5 fingerprint. The isolation and characterization of one variable probe, by screening complete genomic libraries, is described and discussed. This probe is compared to a hypervariable single-copy probe, isolated from a size-enriched genomic library. The sequence of the repeat flanking locus-specific probe is presented and a semi-specific, adaptor-mediated polymerase chain reaction was designed to amplify (CAC)n/(GTG)n flanking sequences.

Base Sequence↗
PubMedDate not supplied

Highly instable (GATA)n-containing sequences of the mouse during the cloning process.

Mouse DNA fingerprints were obtained by HaeIII digestion of genomic DNA and in-gel hybridization with the (GATA)4 oligonucleotide probe. In order to obtain locus-specific probes that hybridize with only one fragment of the (GATA)4 DNA fingerprint, a genomic library of size-selected inserts was constructed using a system of direct subcloning from the phage clones. During the cloning procedure, the phage as well as the plasmid insert DNAs changed primarily within their repetitive DNA but also within adjacent nonrepetitive sequences, as was demonstrated for several clones by in-gel hybridization with the (GATA)4 probe as well as by sequence analysis. Isolated subclones varied within their (GATA)n repeats, resulting in different insert lengths. Several "metastable" as well as stable (GATA)4-positive subclones could be isolated. Also, vector sequences were affected by alterations during the cloning process. These phenomena are discussed within the context of possible mechanisms for cloning artifacts.

Animals↗
PubMedDate not supplied

Chromosomal organization of simple repeated DNA sequences used for DNA fingerprinting.

Stretches of short, simple DNA sequences are widespread in all eukaryote genomes studied so far. Simple sequences are thought to undergo frequent expansion and deletion due to intrinsic genomic mechanisms. Some of the simple sequences were used successfully to detect hypervariable loci in various genomes. Hybridization experiments using synthetic probes not only revealed the informative simple repeats suitable for DNA fingerprinting in a particular species, but also reflected the wide range of distribution of the simple sequences among eukaryotes. The organization of these simple repetitive sequences at the chromosomal loci was investigated using in situ hybridization with chemically synthesized, pure oligonucleotide probes. Both biotin- and digoxigenin-attached probes detected specific chromosomal sites that are enriched in the respective simple-repeat blocks. Depending on the organism and probe used, accumulation of simple DNA sequences at individual or multiple sites on the chromosomes of different vertebrates could be demonstrated. The simple repetitive DNA sequences are located in different chromosomal regions (e.g., heterochromatin on the sex chromosomes, nucleolus organizer regions, and R-band sites), which are constrained considerably during evolution.

Amphibians↗
PubMedDate not supplied

Characterization of hypervariable locus-specific probes derived from a (CAC)5/(GTG)5 multilocus fingerprint in various Eurasian populations.

Population genetic studies were performed using oligonucleotide probes (Hz1103, Hz4103, and Hz4201) that recognize three hypervariable loci (D11S859, D9S128 and D22S265) in the human genome. DNA from 17 Eurasian population samples including 37 monozygotic twin pairs were digested with HinfI and hybridized with Hz4103. Allele frequency distribution profiles and high degrees of heterozygosity were similar in each ethnic group. Among 804 unrelated individuals tested, we detected one case of mosaicism caused by a somatic recombination event in a monozygotic twin. In addition, samples of DNA from three ethnic groups (Germans, Assamese Hindus and Thais) and from German and Thai families were restricted with MboI and probed with Hz1103, Hz4103, and Hz4201. The results showed considerable degrees of heterozygosity and locus-specific allele distribution profiles, rather than interpopulation differences. Among 262 meioses (12 three-generation families with a total of 131 children) analyzed, a single recombination event was observed following hybridization with the DNA probe Hz4201.

Alleles↗