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Biomedical subjects

J Schmidtke

Publications and source records attributed to J Schmidtke.

At least 37 records · Page 2Linked to original sources
PubMed2000

Immortalization of murine male germ cells at a discrete stage of differentiation by a novel directed promoter-based selection strategy.

We developed a novel promoter-based selection strategy that could be used to produce cell lines representing sequential stages of spermatogenesis. The method is based on immortalization and subsequent targeted selection by using differentiation-specific promoter regions. As an example for this approach, a new murine germ cell line (GC-4spc) was established using a vector construct that contains the SV40 large T antigen and the neomycin phosphotransferase II gene under the control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptotene and early pachytene spermatocytes. Transcription of three germ cell-specific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the GC-4spc cell line using reverse transcription-polymerase chain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed different transcriptional activities in the GC-4spc cell line compared with the spermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing production of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.

Animals↗
PubMed2000

TSPY variants in six loci on the human Y chromosome.

We have studied the structure, organization, and evolution of the human TSPY gene family by mapping three sequence variants identified through RT-PCR analysis onto genomic clones derived from two different YAC contigs. TSPY gene family members occur in at least six locations on the human Y chromosome, and each cluster contains a unique combination of variants. Our data further suggest that an 18-bp tandem duplication found in TSPY exon 1 originated from an unequal sister chromatid exchange between two tandemly arranged TSPY clusters.

Cell Cycle Proteins↗
PubMed2000

Molecular evolution of the murine tspy genes.

Molecular aspects of murine evolution were studied by sequencing, and subsequently comparing, introns of the Y-chromosomal tspy genes from Apodemus agrarius, A. sylvaticus, A. flavicollis, Mus platythrix (subgenus Pyromys), M. booduga (subgenus Leggada), and from species of the subgenus Mus, including M. cervicolor, M. macedonicus and M. spretus. Estimates of nucleotide substitution rates in these lineages were in perfect agreement with phylogenetic data previously published by She et al. (1990), Catzeflis et al. (1992; 1993), and Lyon et al. (1996). The only exception was provided by a comparatively late divergence of M. spretus and M. macedonicus. Our data also suggest that M. booduga diverged from the subgenus Mus about 3 Myr ago.

Animals↗
PubMed1999

Identification of 9 novel FBN1 mutations in German patients with Marfan syndrome.

We report 9 new mutations in German patients presenting with classical Marfan syndrome. All mutations occur in exons with calcium-binding (cb) epidermal growth factor-like (EGF) domains. Five mutations are missense involving exons 12, 27, 30, 44, and 52 with the resultant substitution of cysteine by phenylalanine (C504F), cysteine by tyrosine (C1129Y), tyrosine by cysteine (Y1261C), cysteine by serine (C1833S), and cysteine by tyrosine (C2142Y), respectively. The other four mutations are single base deletions in exons 39, 43, 48, and 58, at nucleotide A4826, C5311, T6018, and A7291, respectively, each resulting in frameshift with premature termination. Four mutations were detected in sporadic cases and are likely to be de novo.

Adult↗
PubMed1999

Peutz-Jeghers syndrome: four novel inactivating germline mutations in the STK11 gene. Mutations in brief no. 227. Online.

The diagnosis of Peutz-Jeghers syndrome is based on the occurrence of hamartomatous gastrointestinal polyps and perioral pigment spots. In view of the development of hamartomatous polyps in several syndromes and the variability of pigment spots in Peutz-Jeghers patients, identification of affected individuals is difficult. Recently, germline mutations in the STK11 gene have been reported as a molecular cause of Peutz-Jeghers syndrome. We present four novel inactivating mutations identified by direct sequencing of all 9 exons of the STK11 gene in 4 patients suggestive of Peutz-Jeghers syndrome: three frameshift mutations (125-137del; 474-480del; 516-517insT) and one nonsense mutation (Q220X). Our data obtained in these patients and in those reported previously emphasize the diagnostic value of histological discrimination between different types of hamartomatous polyps and of molecular analysis, particularly in cases with no family history of the disease.

AMP-Activated Protein Kinase Kinases↗
PubMed1999

DNA sequence polymorphisms in genes involved in the regulation of dopamine and serotonin metabolism in rhesus macaques.

A systematic search was performed for DNA sequence variation in genes regulating neurotransmitter metabolism in rhesus macaque (Macaca mulatta). These genes included dopamine and serotonin receptors and transporters, and tyrosine hydroxylase. A total of 13 single nucleotide polymorphisms in five different genes were identified, namely: DRD1 (-244T->G), q = 0.45; DRD1 (-179C->T), q = 0.19; DRD1 (-127G->A), q = 0.25; DRD1(-11T->G), q = 0.08; DRD1(-81C->T), q = 0.19; DRD3 (248G->A), q= 0.08; DRD3(341G->C), q = 0.11; DRD3(377A->G), q = 0.19; DRD3 (403C->T; A59V), q= 0.11; DRD4(2608G->A), q= 0.48; HTR1D(-506G->T), q = 0.47; HTR1D(-173C->T), q = 0.47; and HTT(340G->A), q = 0.39. The nucleotide positions listed correspond to the human homologs.

Alleles↗
PubMed1999

A search for chromosome 22q11.2 deletions in a series of 176 consecutively catheterized patients with congenital heart disease: no evidence for deletions in non-syndromic patients.

Microdeletions in chromosome 22q11.2 are associated with DiGeorge syndrome (DGS), velo-cardio-facial syndrome (VCFS), and several other syndromes, collectively referred to as DG/VCF. Non-dysmorphic patients with cardiac defects have also been attributed to deletions in this chromosomal region. In this study 157 consecutively catheterized patients with isolated, non-syndromic cardiac defects, and 25 patients with cardiac defects and additional stigmata (10 of whom were clinically diagnosed as DG/VCF cases prior to chromosome analysis) were analysed by fluorescence in situ hybridization with the DGS-specific probe D0832. Chromosome 22q11.2 deletions were observed only in the ten patients with the clinical diagnosis of DG/VCF. Conclusion In a large unselected cohort of patients with congenital heart disease no association between isolated or non-syndromic heart defects and the 22q11.2 microdeletion was observed. One can conclude that testing for the 22q11.2 microdeletion is clearly indicated in cases when even mild extracardiac abnormalities are present, particularly in very young infants.

Adolescent↗
PubMed1998

Prenatal diagnosis of congenital alveolar proteinosis (surfactant protein B deficiency).

We report on the DNA-based prenatal diagnosis of congenital pulmonary alveolar proteinosis in a family in which alveolar proteinosis was associated with surfactant protein B (SP-B) deficiency. The parents had lost an eight-week-old female child due to this fatal disorder. The affected child was homozygous and both parents were heterozygous for a frame-shift mutation in codon 121 of the surfactant protein B gene (SFTP3-gene). Chorionic villus sampling (CVS) was performed in two subsequent pregnancies. DNA analysis revealed homozygosity for the codon 121 mutation in the first fetus, and the pregnancy was terminated. Homozygosity for the parental wild-type alleles was detected in the following prenatal diagnosis, and a healthy child has been born. DNA-based prenatal diagnosis of congenital alveolar proteinosis is simple, fast and reliable, and can be performed much earlier in pregnancy than any other method, e.g. the direct measurement of SP-B in amniotic fluid. In families with a term infant who dies of unexplained respiratory failure, genetic testing of the parents should be evaluated, since the presence of the codon 121 mutation enables prenatal diagnosis in later pregnancies.

Amniotic Fluid↗
PubMed1998

Paternity assessment in rhesus macaques (Macaca mulatta): multilocus DNA fingerprinting and PCR marker typing.

Establishing kinship relations in primates using modern molecular genetic techniques has enhanced the ability to scrutinize a number of fundamental biological issues. We screened 51 human short tandem repeats (STRs) for cross-species PCR amplification in rhesus macaques (Macaca mulatta) and identified 11 polymorphic loci with heterozygosity rates of at least 0.6. These markers were used for paternity testing in three social groups (M, R, and S) of rhesus macaques from Cayo Santiago, Puerto Rico. Several consecutive birth cohorts were analyzed in which approximately 200 males were tested for paternity against more than 100 mother/ infant pairs. Despite a combined exclusion rate of more than 99.9% in all three groups, some cases could not be solved unequivocally with the STR markers and additional testing of the MHC-associated DQB1 polymorphism. A final decision became possible through multilocus DNA fingerprinting with one or more of the oligonucleotide probes (GATA)4, (CA)8, and (CAC)5. Paternity assessment by multilocus DNA analysis with probe (CAC)5 alone was found to have limitations in rhesus macaques as regards the number of potential sires which might be involved in a given case. Multilocus DNA fingerprinting requires large amounts of DNA, and the ensuing autoradiographic patterns present difficulties in comparisons across gels and even within the same gel across remote lanes. Computer-assisted image analysis was incapable of eliminating this problem. Therefore, a dual approach to DNA typing has been adopted, using STR markers to reduce the number of potential sires to a level where all remaining candidates can be tested by multilocus DNA fingerprinting on a single gel, preferably in lanes adjacent to the mother/infant pair.

Animals↗
PubMed1998

A murine TSPY.

Sequences homologous to human and bovine TSPY were isolated from M. musculus testicular cDNA, and a nearly full-length gene was polymerase chain reaction (PCR) amplified from mouse genomic DNA. This gene is apparently non-functional. Contrary to the situation encountered in species along the primate and artiodactyl lineages, in which TSPY is moderately repetitive, murine Tspy appears to be single copy. Murine Tspy is located on Yp, i.e. in the same syntenic group as in man. Sequence comparisons of murine, human and bovine TSPY exons suggest that TSPY became non-functional during rodent evolution.

Animals↗
PubMed1998

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Molecular cloning and characterization of P47, a novel boar sperm-associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins.

P47, a peripherally associated 47-kDa protein of porcine spermatozoa, was identified by affinity chromatography in the fraction of solubilized plasma membrane proteins bound to immobilized porcine zona pellucida glycoproteins. N-terminal and internal amino acid sequences revealed structural similarity between P47 and rat O-acetyl ganglioside synthase, bovine mammary gland protein (MGP)57/53 and mouse milk fat globule protein E8-polypeptides of unknown function secreted by mammary gland epithelial cells in both species. A polyclonal antibody directed against bovine MGP57/53 displayed cross-reactivity with P47. Indirect immunofluorescence analysis located porcine P47 on the acrosomal cap of testicular sperm and on sperm recovered along different sections of the ductus epididymidis, as well as on swim-up and in vitro-capacitated sperm. Porcine P47 was demonstrated on sperm bound to the zona pellucida of a homologous oocyte. Western blot analysis identified P47 (or MGP57/53) homologous proteins in porcine and human milk. Like the sperm-associated protein, porcine milk P47 possesses affinity for isolated, biotinylated sow oocyte zona pellucida glycoproteins. Reverse transcription-polymerase chain reaction was used to isolate P47 homologous cDNAs from porcine testis and mammary gland tissues as well as from bovine, mouse, and human testis. P47 proteins deduced from these cDNA sequences showed 60-100% amino acid sequence identity. These proteins display a mosaic structure organized into two N-terminal, tandemly arranged epidermal growth factor (EGF)-like domains followed by a region with similarity to C1 and C2 domains found in blood clotting factors V and VII. The second EGF-like domain contains an arginine-glycine-aspartic acid sequence, a motif often found in integrin receptor ligands. P47-like proteins are not expressed solely in testicular and mammary gland tissues. Northern blot analysis showed that P47 mRNA is transcribed in several porcine and bovine tissues. These data indicate a potential role for boar sperm-associated P47 in membrane remodeling and/or as a zona pellucida binding protein.

Amino Acid Sequence↗
PubMed1998

Murine and human TSPYL genes: novel members of the TSPY-SET-NAP1L1 family.

Using expressed sequence tag (EST) sequence information, novel genes related to the Y chromosome gene family TSPY were isolated and characterized. Because of a significant homology to TSPY, the novel genes were designated TSPY-Like (TSPYL) and were assigned as new members of the TSPY-SET-NAP1L1 family. A human TSPYL gene was localized on chromosome 6 and a murine Tspyl gene was localized on chromosome 10. Expression of TSPYL was observed in all tissues investigated, as well as early onset during development. Both the human and murine Tspyl homologs lack introns over the entire region so far investigated and are thought to have arisen by an ancient retroposition event. Retroposition of Tspyl genes is supported by the isolation of a murine Tspyl pseudogene on chromosome 12 which also lacks intronic sequences, and by its observed proximity to an R element, a family of dispersed repetitive DNA.

Animals↗