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J T Epplen

Publications and source records attributed to J T Epplen.

At least 217 records · Page 12Linked to original sources
PubMed1994

Characterization of a human chromosome 4 flow-sorted cosmid library.

A cosmid library has been constructed from a hamster x human hybrid cell line and gridded into 270 microtiter plates containing a total of 25,920 single colonies. Approximately 84% of the recombinants contain human material, with an average length of 29 kb. This library represents a nearly three-fold coverage of human chromosome 4. We investigated this library for presumptive genes, using a set of oligonucleotides detecting SpI and splice-site consensus sequences. The presence of simple repeat motifs was investigated in the cosmids using the oligonucleotides (GGATTT)3, (GGAT)4, (CAC)5, (GCC)5, (AGC)5, (GATA)4, (GACA)4, and (CA)8 as hybridization probes.

Animals↗
PubMed1994

A multilocus DNA fingerprint with built-in security devices.

An unusual case of paternity testing is reported in which determination of paternity was an essential part of a genetic diagnosis. A Y-chromosomal abnormality, observed in a 33-year-old male whose wife had experienced a series of spontaneous abortions, was not found in his alleged father. DNA fingerprinting with the oligonucleotide multilocus probe (CAC)5 yielded two aberrant bands for the proband, i.e. bands exhibited by neither parent. This finding resulted in a comparatively low paternity probability of 0.02934 which is suggestive of, but does not unequivocally prove, false paternity. Subsequent analysis with other multi- and single-locus systems, however, failed to confirm this preliminary result. The paternity probability computed on the basis of the single-locus systems was 0.99997, providing compelling evidence in favour of true paternity. The present case thus demonstrates that even when two mutations turn up in a DNA fingerprint, these may be readily recognized as such.

Abortion, Spontaneous↗
PubMed1994

Exploiting the informativity of 'meaningless' simple repetitive DNA from indirect gene diagnosis to multilocus genome scanning.

Most eukaryotic genomes are characterized by excessively large amounts of non-coding DNA sequences among which redundant (repetitive) elements constitute a sizable portion. The functional role of an abundant subclass of repetitive sequences--simple, tandemly arranged repeats--remained mysterious so far. Even the biological meaning of most of these elements appears quite refractory to present-day techniques in molecular genetics. Notwithstanding simple repetitive sequences have been developed into superb tools for various aspects of eukaryotic genome research: Using oligonucleotide probes carrying simple repeat motifs multilocus DNA fingerprinting can be applied for individual identification and genetic relationship analyses in plants, animals and humans. Microsatellite analyses via polymerase chain reaction of simple repeat blocks allow for efficient investigations of such divers subject matters as criminal stains, detailed genome maps and indirect gene diagnoses.

Communicable Diseases↗
PubMed1994

Molecular biology techniques in the diagnosis of monogenic diseases.

Monogenic diseases are defined by their patterns of inheritance: autosomal dominant, autosomal recessive, or X-linked. This article examines the molecular biology techniques available for their study and detection. These techniques comprise direct detection of mutations (including trinucleotide repeats) and linkage analysis. The usefulness of these techniques is compared to conventional biochemical tests for the diagnosis of various inherited diseases. The authors stress the need for caution in the interpretation of test results and the importance of genetic counseling.

Forecasting↗
PubMed1993

Reproductive failure of dominant males in the poeciliid fish Limia perugiae determined by DNA fingerprinting.

Hierarchical structures among male individuals in a population are frequently reflected in differences in aggressive and reproductive behavior and access to the females. In general, social dominance requires large investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesis has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Burke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poeciliid fish Limia perugiae, in small mating groups the dominant male has a mating success of 100%, whereas in larger groups its contribution to the offspring unexpectedly drops to zero.

Animals↗
PubMed1993

Primitive sex chromosomes in poeciliid fishes harbor simple repetitive DNA sequences.

The demonstration of the chromosomal mode of sex determination via genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the most primitive form of sex chromosomes. In many different taxa the evolutionary process involved in the differentiation of advanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hybridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparous fishes of the family Poeciliidae. The hybridization pattern together with specific staining of the constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as well as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.

Animals↗
PubMed1993

Human T cell autoimmunity against myelin basic protein: CD4+ cells recognizing epitopes of the T cell receptor beta chain from a myelin basic protein-specific T cell clone.

We have investigated whether the normal immune system contains T cells that are able to recognize T cell receptor (TcR) determinants of autologous autoantigen-specific T cells. The T cell clone HW.BP3, specific for myelin basic protein (MBP) was isolated from a healthy donor. HW.BP3 is restricted by HLA-DR2a, and reacts to human MBP 139-153. The expressed alpha beta TcR genes of HW.BP3 were cloned and sequenced, and the sequences analyzed for potential T cell epitopes. Two synthetic peptides, one from the VDJ beta junctional (beta 1) and one from the V beta region (beta 2) of the TcR of HW.BP3, were used to select four TcR peptide-specific T cell lines from the donor of HW.BP3. All anti-TcR lines had the phenotype CD3+/CD4+/HLA-DR+/CD25+/CD45RO+, and recognized the antigen in the context of HLA-DR. Three anti-TcR lines, which had been selected for reactivity to peptide beta 1, recognized exclusively this peptide restricted by HLA-DR2b. One anti-TcR line, selected for peptide beta 2, responded to both peptides beta 1 and beta 2 when presented by autologous blood mononuclear cells, but not by HLA-DR2a- or HLA-DR2b-transfected L cells. All TcR peptide-specific T cell lines were efficiently cytotoxic. They specifically lysed autologous macrophages or HW.BP3 line cells in the presence of exogenous peptide antigen. In contrast, HW.BP3 did not present endogenous TcR peptides to the anti-TcR lines. The results demonstrate that the normal human immune system contains not only autoantigen-specific T cells, but also T cells that recognize antigenic determinants of autologous autoreactive TcR.

Amino Acid Sequence↗
PubMed1993

Simple repetitive (GAA)n loci in the human genome.

In order to investigate the organization (and inheritance) of simple tandem (GAA)n repeats in the human genome, different restriction enzymes were employed for DNA digestion followed by separation of the resulting fragments by agarose gel electrophoresis. Frequently cutting enzymes (4 bp recognition sites) revealed highly complex multilocus banding patterns after conventional horizontal submarine gels. Fragments larger than 25 kb, resulting from digestions with rarely cutting enzymes (6 bp recognition sites), were separated by pulsed-field gel electrophoresis (PFGE). Hybridizations were carried out directly in the gel matrix. Nearly all of the enzymes produce at least one predominant signal band after hybridization with (GAA)6. The frequency of (GAA)n stretches was estimated on human chromosome 4 by probing a respective cosmid library. In comparison with other simple di-, tri- and tetranucleotide repeats, (GAA)n stretches appear underrepresented. Hybridization of a fetal human brain cDNA library indicated very few expressed (GAA)n repeats. These data are discussed with particular reference to genomic organization and other simple repetitive trinucleotide stretches.

Chromosome Mapping↗
PubMed1993

Calculating paternity probabilities from DNA multilocus fingerprints in some cases of deficiency.

Paternity determination via DNA multilocus fingerprints in normal trio cases, namely when the fingerprints of the trio mother, child and putative father are available, is performed on a routine basis. But in cases of deficiency, where the DNA fingerprint of mother or putative father is missing, there exists no systematic, analytical way for paternity calculations, even in cases where DNA fingerprints of other persons related to the missing individual are available. The aim of this paper is to establish a mathematical, analytical background, that enables paternity calculations in cases of deficiency if complementary information can be obtained from the DNA fingerprints of at least one of the parents of the missing individual. Using the basic outline of our previously described procedure, the formulas for paternity calculations also allow mutations.

Chromosome Mapping↗
PubMed1993

Detection of somatic changes in human renal cell carcinomas with oligonucleotide probes specific for simple repeat motifs.

The purpose of our study was to detect somatic changes in renal cell carcinoma by multilocus fingerprinting. DNA fingerprints were generated from the DNA of normal and malignant renal tissue samples of 29 patients with nonhereditary kidney carcinoma by using oligonucleotide probes specific for simple repeat motifs such as (GTG)5, (CA)8, (GACA)4, or (TTAGGG)3. Each probe rendered a typical fingerprint pattern, because it is specific with respect to the target regions recognized in the genome. The restriction enzymes used were HinfI and HaeIII. Changed banding patterns were detected by using (GTG)5 in 20% of the tumors, in 20% for (CA)8 after HinfI digestion, and in 10% after HaeIII digestion. Even more informative probes were (GACA)4, showing 70% changes after HaeIII digestion, and (TTAGGG)3, with 80% changes after digestion with either enzyme. Since the simple repeat motifs recognized by (GACA)4 are localized on the short arms of the acrocentric chromosomes (13, 14, 15, 21, and 22), it is possible that sequences important for renal carcinogenesis are present in these regions. The observation of changes in regions to which (TTAGGG)3 hybridizes points to an involvement of DNA elements in telomeric sequence related regions in human kidney tumor formation.

Adult↗
PubMed1993

Microsatellite and HLA class II oligonucleotide typing in a population of Yanomami Indians.

We have used three different microsatellites (on chromosome 12 and Y) together with HLA class II oligonucleotide typing (DQA and DQB) to analyze families of Yanomami indians settling in villages in Southern Venezuela. There exist complex networks of biological relationship between villages as a result of wife exchange, village fissioning and changing patterns of alliances associated with inter-village warfare. Social status in this society is largely determined by the kinship system. Polygyny is common, especially among headmen, with additional wives, frequently being chosen among the sisters of the first wife. Our preliminary results mainly obtained from inhabitants of the village HAP show the expected allele distribution in populations with a high degree of consanguinity: (i) deficiency of observed heterozygotes at the autosomal loci and (ii) almost all men carry the same Y chromosomal allele. Nevertheless in the Yanomami village two thirds of the described autosomal microsatellite alleles were identified. Several paternities were clarified.

Alleles↗
PubMed1993

Testing deficiency paternity cases with a Y-linked tetranucleotide repeat polymorphism.

Because a son's genotype at a Y-linked locus uniquely specifies his father's genotype at that locus irrespective of the maternal contribution, Y-linked polymorphisms show increased exclusion power over autosomal polymorphisms in paternity cases involving a male child. This advantage is even more obvious when the alleged father is deceased or otherwise unavailable for testing. In this situation, any individual connected by patrilineage to the alleged father may be tested in his place. The usefulness of the Y-linked tetranucleotide repeat locus Y-27H39 in deficiency cases was evaluated in a set of 41 families that had a deceased alleged father and that had been tested at GENE/MG with multilocal and unilocal DNA fingerprinting probes. In sixteen of these cases the proband (child tested) was male and there were male relatives. In the thirteen cases in which paternity was included, Y-27H39 would contribute significantly to the paternity index. In one of three cases in which there was exclusion by fingerprinting probes there was also exclusion by Y-27H39. Thus, Y-27H39 is useful in deficiency paternity cases and will be specially valuable in situations where autosomal polymorphisms have limited power, such as when there is only one male living relative of the deceased father. However, our experience is that Y-linked probes can only be applied in approximately 40% of cases.

Base Sequence↗
PubMed1993

On the essence of "meaningless" simple repetitive DNA in eukaryote genomes.

Various kinds of simple tandemly repetitive DNA sequences are abundantly interspersed in the genomes of practically all eukaryotic species studied. The comparatively elevated mutation rates of simple repeat blocks result in highly polymorphic and therefore extremely informative investigation systems for studies on forensic, ecological and genetic relationship questions. Recently the techniques for analyzing simple repeats have achieved great effectivity and simplicity. Beyond their utility as tools for differentiation and individualization, certain of these repeated elements harbor quite unexpected qualities which may be discussed in the context of their biological meaning. i) A specific subset of simple (cac)n or (gtg)n repeats is expressed in mature mRNA and total cellular RNA. ii) Despite the apparently high mutation rate certain (gt)n or mixed (gt)n/(ga)m stretches of intronic simple repeats are preserved in immunologically relevant genes for at least 70 x 10(6) years and they bind nuclear protein molecules with high affinities. Consequently in addition to their tool character, the biological aspects of simple repeated DNA should be taken into consideration.

Animals↗
PubMed1993

DNA fingerprinting of trait-selected mouse lines and linkage analysis in reference families.

The first aim of the study was the molecular genetic characterization of long-term trait-selected (growth, adaptability and fertility) lines of mice using multilocus DNA fingerprinting with the simple tandem repetitive oligonucleotide probes (GAA)6 and (GACA)4. Secondly polymorphic markers were screened for association with growth performance based on DNA fingerprint analysis in reference families. Pooled DNA samples of ten unrelated mice of trait-selected mouse lines (over 40 generations) and the unselected control lines were analyzed. Resulting differences in band patterns were reanalyzed comparing individual fingerprints of the animals included in the pooled DNA samples of the different lines. Between the mouse lines about 30% of analyzed fingerprint bands were polymorphic. Individuals of long-term selected mouse lines show only a few individual-specific bands. Most polymorphic bands observed in DNA mixes appear in all animals included in the DNA mix of the corresponding line. Line-specific DNA fingerprint bands were analyzed for their inheritance and linkage with growth performance in reference families using animals with extreme growth performance of the first backcross after crossing of growth-selected with unselected mice. Scanning the distribution of line-specific bands in the reference panel few bands were identified which are associated with growth performance. They appear as useful markers for growth selection. Nevertheless most line-specific bands result from genetic drift rather than from selection.

Adaptation, Physiological↗
PubMed1993

Oligonucleotide fingerprinting of free-ranging and captive rhesus macaques from Cayo Santiago: paternity assignment and comparison of heterozygosity.

Multilocus DNA fingerprinting with oligonucleotide probes (GTG)5, (GATA)4, and (CA)8 was applied in order to determine paternity in one birth cohort (15 infants) of social group (S) from the free-ranging colony of rhesus macaques (Macaca mulatta) on Cayo Santiago. While sires could be identified in 11 cases, all males tested (N = 19) could be excluded from paternity for the remaining four infants. Data revealed marked discrepancies between actual paternity and paternity as inferred from the observation of copulation behavior. Thus, a dominant social rank does not appear to be strongly associated with reproductive success. Furthermore, alternative reproductive strategies were found to yield comparable net benefits in reproduction. A second group of animals (M) was translocated from Cayo Santiago to the Sabana Seca Field Station in 1984. They have continuously resided together in a large outdoor enclosure since then. Here paternity assessment was seriously impeded by a reduced number of discriminating bands, i.e. offspring bands which were unequivocally derived from the sires. This was initially held to be indicative of a smaller degree of heterozygosity in Group M, and was attributed to inbreeding due to a lack of male immigration or extra-group fertilizations. However, a comparison of the DNA fingerprint patterns obtained in Group S and Group M lends only partial support to this idea.

Animals↗
PubMed1993

Towards covering immunological genes with highly informative markers: a trans-species approach.

To establish a highly informative screening system for immunologically relevant genes ("immunoprinting") we co-amplified via polymerase chain reaction (PCR) polymorphic exons plus adjacent intronic simple repetitive dinucleotide stretches in the T-cell receptor (Tcr) Vb6 and Major Histocompatibility Complex (MHC)-DRB loci in man and several ungulate species. In both gene families the basic structure of the simple repeat was found to be preserved for more than 70 x 10(6) years in all investigated species. The simple repeats exhibit extensive length variability. Distinct exon sequences are correlated with a defined repeat length and substructure. In addition, PCR and the oligonucleotides for typing were applicable to a broad range of species from different mammalian orders. Multiplex PCR of different members of the Tcr Vb6 family and MHC-DRB resulted in a complex pattern similar to an oligolocus fingerprint. Hence immunoprinting can be employed for searching for associations of immunologically relevant genes with diseases even across species barriers.

Animals↗