PubMed Health⌕ Search

Biomedical subjects

C M Pusch

Publications and source records attributed to C M Pusch.

29 records · Page 2Linked to original sources
PubMed2000

Voltage-induced release of nucleic acids from palaeontological samples.

Most of the protocols for the recovery of ancient DNA from palaeontological specimens are time-consuming and tend to yield inconsistent polymerase chain reaction (PCR) results. "Voltage-induced release" is a novel and rapid approach for the extraction of ancient DNA. Nucleic acids are directly electrophoresed out of powder derived from hard and soft tissues. This technique is much faster than other methods in which pulverized tissue conventionally undergoes time-consuming crude lysis steps. The total preparation time is 5-6 h. The reliability of the voltage-induced release method was validated by (i) measuring the ratio of D-to L-enantiomers of the amino acids aspartic acid, alanine, and leucine, and (ii) by specific PCR amplification of four single-copy markers of human chromosome 17 and 18. We compare voltage-induced release to a frequently used silica-based protocol. DNA extracted employing voltage-induced release was more effective in PCR amplifications, which may be attributed to the effective removal of PCR inhibitors.

DNA↗
PubMed2000

Physical mapping and exclusion of GPR34 as the causative gene for congenital stationary night blindness type 1.

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by impaired night vision, variably involving high myopia, nystagmus, decreased visual acuity, and strabismus. Linkage studies have identified two distinct loci for X-linked CSNB1 and CSNB2 on the short arm of chromosome X. The gene mutated in families displaying the "incomplete phenotype" of CSNB (i.e., CSNB2) has recently been identified. To identify novel candidate genes for the "complete form" of CSNB (i.e., CSNB1) we screened the physically vast region Xp11.3-Xp11.4 for cDNA sequences. This led us to identify and map the G protein coupled receptor (GPCR) gene GPR34 to Xp11.4 within 650 kb of the marker DXS993. Deletion screening via Southern blotting and direct sequencing of GPR34 revealed no mutations in 19 unrelated men with CSNB1, excluding a causal role in the disease. However, because of its expression in retinal and neural tissue and the involvement of GPCRs in transmembrane signal transduction, GPR34 remains a putative candidate gene for a number of ocular diseases which also map to the Xp11.4 region.

Blotting, Southern↗
PubMed2000

The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein.

X-linked congenital stationary night blindness (XLCSNB) is characterized by impaired scotopic vision with associated ocular symptoms such as myopia, hyperopia, nystagmus and reduced visual acuity. Genetic mapping in families with XLCSNB revealed two different loci on the proximal short arm of the X chromosome. These two genetic subtypes can be distinguished on the basis of electroretinogram (ERG) responses and psychophysical testing as a complete (CSNB1) and an incomplete (CSNB2) form. The CSNB1 locus has been mapped to a 5-cM linkage interval in Xp11.4 (refs 2,5-7). Here we construct and analyse a contig between the markers DXS993 and DXS228, leading to the identification of a new gene mutated in CSNB1 patients. It is partially deleted in 3 families and mutation analysis in a further 21 families detected another 13 different mutations. This gene, designated NYX, encodes a protein of 481 amino acids (nyctalopin) and is expressed at low levels in tissues including retina, brain, testis and muscle. The predicted polypeptide is a glycosylphosphatidylinositol (GPI)-anchored extracellular protein with 11 typical and 2 cysteine-rich, leucine-rich repeats (LRRs). This motif is important for protein-protein interactions and members of the LRR superfamily are involved in cell adhesion and axon guidance. Future functional analysis of nyctalopin might therefore give insight into the fine-regulation of cell-cell contacts in the retina.

Amino Acid Motifs↗
PubMed2000

[Molecular genetic analysis for taxonomic determination of fossil skull fragments of Warendorf-Neuwarendorf].

The isolation and examination DNA segments from prehistoric and fossil bone samples has become one of the biggest challenges in anthropology within the past years. By using specially developed and/or adapted genetic methods, it is possible under laboratory conditions to amplify portions of DNA from bone remains in states of good preservation by the polymerase chain reaction (PCR). DNA sequence data can provide far more specific answers to palaeanthropological questions than one would expect solely by morphologic comparison. Here we introduce an alternative approach for the classification of total ancient DNA by means of Southern hybridisation techniques.

Anthropology, Physical↗
PubMed2000

[Contamination through preparation: risk of molecular genetic studies by using biological preservatives for museum collections].

In paleogenetic science, artifacts (i.e. non-authentic DNA sequences) are mainly produced by cryptic contamination with (i) edaphon DNA sequences and/or (ii) human biomolecules derived from the involved researchers and the laboratory equipment. A third, and yet underestimated source of contamination with exogenous nucleic acids is provided by (iii) conservation practices applied to old material. Bone glue has been successfully used from the beginning of the 19th century up to the middle of this century, and comprises a rich source of non-authentic nucleic acids. An unequivocal identification of treated samples remains difficult since bone and the glue used for conservatory purposes bear similar chemical properties. Since the majority of agents used for the preservation of museum collections are of biological origin, the differentiation between contaminated and non-treated samples is required.

Artifacts↗
PubMed2000

Cremation practices and the survival of ancient DNA: burnt bone analyses via RAPD-mediated PCR.

Numerous burial rites have been developed in different time periods of human cultural evolution. One of the most interesting burial practices was the ritual cremation of human bodies. Due to the respective cultural and religious background, brand graves are known where human remains had been buried together with burnt bones of animal origin. To date, burnt bone samples have been refractory to PCR-mediated amplification. D/L values of aspartic acid far greater than 80 x 10(-3) were measured in the samples thus indicating the presence of severely nicked and fragmented nucleic acids. In order to differentiate between burial gift of animal origin and burnt human specimens we established a highly sensitive protocol that addresses all the shortcomings connected to degraded ancient DNA. With the novel procedure it was possible to classify 4 specimens ranging from 2,000-5,000 BP on the basis of mitochondrial DNA sequences.

Age Determination by Skeleton↗
PubMed2000

Adaptor-mediated amplification of minute amounts of severely fragmented ancient nucleic acids.

It has been repeatedly shown that high copy number mitochondrial DNA sequences can be recovered from ancient samples. A significant increase in the volume of information available to researchers will be observed when the amplification of nuclear DNA becomes commonplace and reproducible. To this end we established a modification of the Rapid Amplification of cDNA Ends (RACE) procedure normally used for the generation of cDNA ends from adaptor-ligated expressed sequence tag libraries. The modifications were designed to specifically address the problems associated with the highly damaged nucleic acids extracted from palaeontological specimens. For this study we used 6 human samples dating to 450 AD and approximately 6.500 BP that were refractory to reliable amplification of single copy loci by PCR. Racemate contents (ratio of D/L enantiomers) of aspartic acid, alanine, and leucine also indicated that no amplifiable DNA is present in 5 of the 6 samples. The proposed technique allowed us (i) to amplify four X-chromosomal loci from 5 human specimens, and (ii) to correct allelic drop-out phenomena at the amelogenin locus in one individual; thus showing that the threshold of 80 x 10-3 for D/Lasp as a borderline for the presence/absence of amplifiable aDNA requires reassessment. Reliability of the proposed technique (i.e. amplification of DNA sequences endogenous to the find) was validated by the application of "ancient RACE" (aRACE) to prehistoric animal samples.

Journal Article↗
PubMed1998

Repair of degraded duplex DNA from prehistoric samples using Escherichia coli DNA polymerase I and T4 DNA ligase.

The most notable feature of DNA extracted from prehistoric material is that it is of poor quality. Amplification of PCR products from such DNA is consequently an exception. Here we present a simple method for the repair of degraded duplex DNA using the enzymes Escherichia coli DNA polymerase I and T4 DNA ligase. Adjacent sequences separated by nicks do not split up into intact strands during the denaturation step of PCR. Thus the target DNA is refractory to amplification. The proposed repair of nicked, fragmented ancient DNA results in an increase of amplification efficiency, such that the correct base order of the respective nuclear DNA segment can be obtained.

Antigens, Neoplasm↗