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J T Epplen

Publications and source records attributed to J T Epplen.

340 records · Page 19Linked to original sources
PubMed1975

DNA replication patterns of human chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique.

A technique is described for visualizing late-replicating regions by a Hoechst 33258-Geimsa-staining procedure combining the techniques of Latt (1973) and of Perry and Wolff (1974). The advantages are two-fold: distinct bands are obtained and many possible mistakes and interpretation difficulties with autoradiography are avoided. The time sequence of late-replication patterns (excepting C-group chromosomes) has been established in human fibroblasts of adults, and these results have been compared with three of four different cell types from amniotic fluid. No significant differences in late-replication patterns and time sequence of the different cells could be discovered. As expected, the replication patterns are in good concordance with the patterns of G- and Q-bands. Some exceptions are described.

Amniotic Fluid↗
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On paternity determination from multilocus DNA profiles.

Various approaches have been worked out to calculate the probability of paternity from DNA multilocus profiles. Despite promising results, the application and practicability of these approaches are often restricted (due to, e.g., the equality assumption of the a priori probabilities by using Bayes' theorem, no allowance of mutations, etc). Here we present two methods for paternity determination, one of which is an extension of the well-known procedure of Evett et al. J. Forens. Sci. Soc. 1988, 28, 249-254. These methods depend on fewer assumptions and therefore may be applied to a wide spectrum of disputed paternity cases.

DNA↗
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A gel retardation assay system for studying protein binding to simple repetitive DNA sequences.

Simple repetitive DNA sequences have been regarded as mere "junk" present in all eukaryotic genomes. In fact, mixed simple repeat (gt)n(ga)m sequences are present in major histocompatibility complex MHC-DRB genes for long evolutionary times, including such distant animals as artiodactyla and man. We describe herein an unsophisticated method which reveals that at least certain simple repetitive (gt)n(ga)m sequences bind nuclear proteins and show characteristics of a specific DNA-protein interaction via gel retardation.

Animals↗
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PCR amplification products are of limited use for the study of DNA/protein interaction.

Conventional methods for labeling double-stranded DNA lead to high specific activity. Yet they often alter the target DNA sequence to such an extent as to prevent a meaningful protein/DNA interaction analysis. Therefore we tried to establish a polymerase chain reaction (PCR)-based method which allows radiolabeling to high specific activity and should maintain the protein binding capability of small double stranded DNA fragments. By using PCR it is possible to label double stranded DNA to high specificity, but the protein binding capability of such DNA is drastically reduced.

Base Sequence↗
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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↗
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Preferential expression of a defined T-cell receptor beta-chain gene in hapten-specific cytotoxic T-cell clones.

A multitude of different antigens can be recognized by T cells through specific receptors. Both the alpha- and beta-chains of the T-cell receptor contribute to the antigen recognition portion. The repertoire of beta-chain variable region (V beta) gene segments is limited to some 20 elements which seem to be used randomly in different T cells. Diversity at the beta-chain level can be created in several ways: a multiplicity of germline gene segments; combinatorial diversity by rearranging different V, diversity (D), joining (J) and constant (C) region elements; junctional diversity by joining gene segments at different sites; N-region diversity, that is, insertion of random nucleotides at junctional sites; and somatic mutation. However, the major sources and the extent of diversity of the T-cell receptor are unclear. To address this issue, 42 H-2Kb-restricted, 2,4,6-trinitrophenyl (TNP)-specific cytotoxic T-cell (Tc) clones from C57BL/6 mice were characterized with respect to expression of different beta-chain gene segments in messenger RNA using specific oligonucleotide probes. We report here that nearly half of the Tc clones use identical elements for productive beta-chain gene rearrangement. Thus, there is a restriction in the use of beta-chain gene segments in this panel of Tc clones which favours a particular V beta--D beta--J beta--C beta combination with a defined D beta element.

Animals↗
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Dominance of one T-cell receptor in the H-2Kb/TNP response.

T-cell receptors (TCRs) recognize foreign antigens in the context of major histocompatibility complex (MHC)-encoded cell surface proteins. These receptors are heterogeneous, dimeric glycoproteins composed of disulphide linked alpha- and beta-chains. We analysed the diversity of TCRs in a collection of H-2Kb-restricted, 2,4,6-trinitrophenyl (TNP)-specific (H-2Kb/TNP) cytotoxic T-cell (Tc) clones from C57BL/6 mice. Investigation of the beta-chain messenger RNAs revealed that nearly half of these independent clones expressed an identical beta-chain gene. We show here that almost all the Tc clones expressing the predominant beta-chain gene also express an identical alpha-chain gene. These results show that a strong selective pressure acted on the Tc population, resulting in a skewing of the TCR repertoire for H-2Kb/TNP and in the dominant expression of one TCR with this specificity. Possible explanations for this skewing include antigen-driven clonal expansion and network interactions.

Animals↗
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On simple repeated GATCA sequences in animal genomes: a critical reappraisal.

Simple tandemly organized GATCA sequences occurred in all eukaryotic genomes investigated. The amount and organization of individual GATCA sequences or derivatives thereof vary considerably in animal DNAs and can be assessed by simple but specific hybridization procedures with chemically pure oligonucleotide probes. In several animal species, including humans, GATCA sequences show extensive polymorphism, thus allowing individual-specific "DNA fingerprints." In selected rodents the sex-chromosomal organization of GATCA sequences is being studied extensively, revealing rapid evolutionary changes. In addition, insight can be expected into the sequences involved in obligatory meiotic crossing over between the X and Y chromosomes, into unequal crossing-over events, and into the linkage of GATCA elements to male-specific as well as to male-determining genes on the Y chromosome. The exact provenance of GATCA sequences in present-day eukaryotes cannot be pinpointed, but evolutionary conservation and several modes of de novo generation are discussed. Among these are unequal recombination, slipped strand mispairing, and other unspecified mechanisms. The latter include inherent properties that are responsible for the "selfish" or "ignorant" nature of simple repeats. Expression, if any, of GATCA sequences is critical to the overall significance of these ubiquitously interspersed simple repeats.

Animals↗
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Rapid prenatal diagnosis of aneuploidies in uncultured amniocytes by fluorescence in situ hybridization. Evaluation of >3,000 cases.

OBJECTIVE: A new method in prenatal diagnostics allows to demonstrate certain numeric chromosomal aneuploidies in amniotic cells within 24 h in contrast to conventional methods which take 1-3 weeks. MATERIALS: The experience with this rapid fluorescence in situ hybridization (FISH) method is compared to standard karyotyping and its clinical relevance is described in a large clinical pilot study. FISH on uncultured amniocytes has been performed from 12 weeks of gestation to the third trimester using commercially available chromosome-specific DNA probes for chromosomes 13, 18, 21, X and Y. RESULTS: FISH was performed successfully in 3,150 prenatal cases. All trisomies 13, 18 and 21 and all cases with gonosomal aberrations were detected by FISH analysis. Neither false-positive nor false-negative results were obtained using FISH. For all analyzable disorders the FISH results were in complete agreement with standard cytogenetics. CONCLUSIONS: In our experience, FISH is a valuable and reliable method for rapid diagnosis of numeric chromosomal aneuploidies.

Adult↗
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Extensive gene flow in human populations as revealed by protein and microsatellite DNA markers.

Population genetic studies are mainly based on the description of genetic variability and on interpopulational comparisons using genetic distance measures. The evolutionary dynamics of the populations are inferred from these parameters and accurate estimates of gene flow may be critical. The present study reevaluates the role of gene flow in human populations by different statistical methods from a number of microsatellite and protein polymorphism data. The estimated number of individuals exchanged per generation (Nm) was greater than 1 in all data sets with all statistical methods. The correlation between geographic and genetic distances suggests a pattern of isolation by distance, characteristic of demographic and genetic equilibrium conditions among populations worldwide. Thus the high values of Nm may be interpreted as a reflection of high gene flow between geographically close populations. As expected, gene flow appears to exert a pivotal role in the genetic history of humans.

Gene Frequency↗
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[Multi-locus DNA fingerprints using oligonucleotide probe (CAC)5/(GTG)5 in the Chinese population].

In order to test the practical applicability of oligonucleotide fingerprinting in China we have investigated unrelated individuals, family members and a pair of twins from the Beijing area using the probe (CAC)5/(GTG)5. Except for the monozygotic twins highly variable banding patterns were demonstrated as expected for the randomly selected individuals but also for the relatives. On the basis of an initial survey of 50 unrelated individuals the calculated probability for obtaining by chance two identical multilocus patterns is very small (less than 1.93 x 10(-13). Therefore it seems reasonable to conclude that like in caucasians, (CAC)5/(GTG)5 fingerprints are completely individual-specific also in this population. Therefore they have already been used successfully for identification purposes and paternity tests in many actual cases.

China↗
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[Oligonucleotide fingerprinting using (GTG)5 and (GACA)4 probes for the differentiation of body fragments].

For the detection of postmortem stability of DNA and for the identification of parts of dead bodies of unknown origin the oligonucleotide probes (GTG)5 and (GACA)4 can be used. (GTG)5 is a highly discriminating probe which allows to differentiate in the 4 to 25 kilobase range of DNA fragments. DNA fingerprints obtained by (GACA)4 show useful results especially in the short fragment range. The (GACA)4 probe can therefore be used to analyze partially degraded DNA.

DNA Fingerprinting↗
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[Identification of blood stains and fetal tissue using genetic fingerprinting].

Two cases from practical forensic serological investigations which could not be clarified by means of conventional serological methods, but could be resolved on the basis of the DNA finger print technique with oligonucleotide probes are presented. Both the comparison of the identity of the blood sample which had been stored for a long time and the determination of paternity in fetal material in a case of incest demonstrate the enormous possibilities of this method. However, some problems which still have not been resolved at least at present with regard to the evaluation and appraisal of the band patterns are also discussed.

Accidents, Traffic↗
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[Typing of stored, hemolyzed blood samples by DNA fingerprinting].

DNA fingerprints were established from 3 months old hemolysed blood samples. In spite of partial degradation of the DNA, the hybridization with the oligonucleotide probe (GTG)5 allowed unequivocal identification of the source of the blood.

Accidents, Traffic↗