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GABA (γ-aminobutyric acid) is a major inhibitory neurotransmitter in the central nervous system (CNS) which activates both ionotropic (GABA[sub A] /GABA[sub C] ) and metabotropic (GABA[sub B] ) receptor systems. We identified two alternatively spliced cDNA variants of the murine GABA[sub B] receptor 1 that are predominantly expressed in the CNS. Deduced protein structures are highly homologous to the previously characterized rat and human receptors. Comparison of the genomic structures of mouse and human revealed that alternative splicing occurred at the same position, whereas the mouse gene has an additional 5′ exon. Radiation hybrid mapping, combined with database searches, indicated that the GABA[sub B] receptor gene (Gabbr1) is located on mouse chromosome 17, adjacent to the marker D17Mit24 in a region homologous to human chromosome 6p21.3. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Females with XY gonadal dysgenesis are sterile, due to degeneration of the initially present ovaries into nonfunctional streak gonads. Some of these sex-reversal cases can be attributed to mutation or deletion of the SRY gene. We now describe an SRY-deleted 47,XXY female who has one son and two daughters, and one of her daughters has the same 47,XXY karyotype. PCR and FISH analysis revealed that the mother carries a structurally altered Y chromosome that most likely resulted from an aberrant X-Y interchange between the closely related genomic regions surrounding the gene pair PRKX and PRKY on Xp22.3 and Yp11.2, respectively. As a consequence, Yp material, including SRY, has been replaced by terminal Xp sequences up to the PRKX gene. The fertility of the XXY mother can be attributed to the presence of the additional X chromosome that is missing in XY gonadal dysgenesis females. To our knowledge, this is the first human XXY female described who is fertile. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The evolution, inheritance and recombination rate of genes located in the pseudoautosomal region 1 (PAR1) is exceptional within the human genome. Pseudoautosomal genes are identical on X and Y chromosomes and are not inherited in a sex linked manner. Due to an obligatory recombination event in male meiosis, pseudoautosomal genes are exchanged frequently between X and Y chromosomes. During the isolation, characterization and sequencing of a novel gene PPP2R3L, which was classified by sequence homology as a novel member of the protein phosphatase regulatory subunit families, it became apparent that cosmids of different origin harboring this gene are highly polymorphic between individuals, both at the nucleotide level and in the number and sequence of tandem repeats. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objectives: Left ventricular hypertrabeculation/noncompaction(LVHT) is characterized by extensive trabeculations. LVHT has been reported to be associated with stroke or embolism(S/E). Aim of the study was to compare characteristics and prognosis of LVHT-patients with and without S/E to identify potential risk factors for S/E., Materials and Methods: Retrospectively included were consecutive patients with echocardiographically diagnosed LVHT in a cardiologic department in 1995-2020. Baseline characteristics and follow-up data were collected. The etiology of S/E was assessed by applying the Trial of Org 10172 in Acute Stroke Treatment(TOAST) criteria., Results: The follow-up of 319 patients, mean age 53 years, 30% females, was 7.4 ± 6 years. In 49 patients(15%), either stroke(n = 44), peripheric embolism(n = 3) or both(n = 2) occurred. The etiology of S/E was cardioembolic(n = 32), atherothrombotic(n = 12), undetermined(n = 4) and intracerebral hemorrhage(n = 1). S/E occurred in 31 patients before, in 15 patients after and in 3 patients before as well as after the diagnosis of LVHT. Patients with S/E were older, suffered more frequently from arterial hypertension, diabetes mellitus, atrial fibrillation, neuromuscular disorders and heart transplantation than patients without S/E. Of the patients with S/E, only 8% were without risk factors for S/E. For the patients in whom S/E occurred after the diagnosis of LVHT, the rate of S/E was 0.74%/year. The death rate was 4.17%/year in patients with and without S/E., Conclusion: Cardiovascular risk factors are more prevalent in LVHT-patients with than without S/E. S/E in LVHT-patients is not always cardioembolic why investigations for etiology are useful. LVHT by itself seems to be only a minor risk factor for S/E., Competing Interests: Declaration of competing interest None (all authors)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

With the decipherment of the human genome, new questions have moved into the focus of today's research. One key aspect represents the discovery of DNA variations capable to influence gene transcription, RNA splicing, or regulating processes, and their link to pathology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a powerful tool for the qualitative investigation and relative quantification of variations like single nucleotide polymorphisms, DNA methylation, microsatellite instability, or loss of heterozygosity. After its introduction into proteomics, efforts were made to adopt this technique to DNA analysis. Initially intended for peptide/protein analysis, it held several difficulties for application to nucleic acids. Today, MALDI-TOF-MS has reached worldwide acceptance and application in nucleic acid research, with a wide spectrum of methods being available. One of the most versatile approaches relies on primer extension to genotype single alleles, microsatellite repeat lengths or the methylation status of a given cytosine. Optimized methods comprising intelligent primer design and proper nucleotide selection for primer extension enabled multiplexing of reactions, rendering the analysis more economic due to parallel genotyping of several alleles in a single experiment. Laboratories equipped with MALDI-TOF-MS possess a universal technical platform for the analysis of a large variety of different molecules.

Advanced glycation end-products (AGEs) of DNA are formed spontaneously by the reaction of carbonyl compounds such as sugars, methylglyoxal or dihydroxyacetone in vitro and in vivo. Little is known, however, about the biological consequences of DNA AGEs. In this study, a method was developed to determine the parameters that promote DNA glycation in cultured cells. For this purpose, the formation rate of N2-carboxyethyl-2'-deoxyguanosine (CEdG), a major DNA AGE, was measured in cultured hepatic stellate cells by liquid chromatography (LC)-MS/MS. In resting cells, a 1.7-fold increase of CEdG formation rate was observed during 14 days of incubation. To obtain insights into the functional consequences of DNA glycation, CEdG was introduced into a luciferase reporter gene vector and transfected into human embryonic kidney (HEK 293 T) cells. Gene activity was determined by chemiluminescence of the luciferase. Thus, CEdG adducts led to a dose-dependent and highly significant decrease in protein activity, which is caused by loss of functionality of the luciferase in addition to reduced transcription of the gene. When the CEdG-modified vector was transformed into Escherichia coli, a loss of ampicillin resistance was observed in comparison to transformation with the unmodified plasmid. These results indicate that CEdG accumulates in the genomic DNA of resting cells, which could lead to diminished protein activity.

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that can occur spontaneously or can be caused by infection or mutations within the prion protein gene PRNP. Nonsynonymous DNA polymorphisms within the PRNP gene have been shown to influence susceptibility/resistance to infection in sheep and humans. Analysis of DNA polymorphisms within the core promoter region of the PRNP gene in four major German bovine breeds resulted in the identification of both SNPs and insertion/deletion (indel) polymorphisms. Comparative genotyping of both controls and animals that tested positive for bovine spongiform encephalopathy (BSE) revealed a significantly different distribution of two indel polymorphisms and two SNPs within Braunvieh animals, suggesting an association of these polymorphisms with BSE susceptibility. The functional relevance of these polymorphisms was analyzed using reporter gene constructs in neuronal cells. A specific haplotype near exon 1 was identified that exhibited a significantly lower expression level. Genotyping of nine polymorphisms within the promoter region and haplotype calculation revealed that the haplotype associated with the lowest expression level was underrepresented in the BSE group of all breeds compared to control animals, indicating a correlation of reduced PRNP expression and increased resistance to BSE.

Background: Non-synonymous polymorphisms within the prion protein gene (PRNP) influence the susceptibility and incubation time for transmissible spongiform encephalopathies (TSE) in some species such as sheep and humans. In cattle, none of the known polymorphisms within the PRNP coding region has a major influence on susceptibility to bovine spongiform encephalopathy (BSE). Recently, however, we demonstrated an association between susceptibility to BSE and a 23 bp insertion/deletion (indel) polymorphism and a 12 bp indel polymorphism within the putative PRNP promoter region using 43 German BSE cases and 48 German control cattle. The objective of this study was to extend this work by including a larger number of BSE cases and control cattle of German and Swiss origin., Results: Allele, genotype and haplotype frequencies of the two indel polymorphisms were determined in 449 BSE cattle and 431 unaffected cattle from Switzerland and Germany including all 43 German BSE and 16 German control animals from the original study. When breeds with similar allele and genotype distributions were compared, the 23 bp indel polymorphism again showed a significant association with susceptibility to BSE. However, some additional breed-specific allele and genotype distributions were identified, mainly related to the Brown breeds., Conclusion: Our study corroborated earlier findings that polymorphisms in the PRNP promoter region have an influence on susceptibility to BSE. However, breed-specific differences exist that need to be accounted for when analyzing such data.

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Structural abnormalities of chromosome 18p mainly consist of isochromosomes of the short arm, which result in tetrasomy 18p. Trisomy 18p is much rarer, and less well characterized. We report on a 12-year-old girl with minor facial anomalies, delayed development, abnormal hands, atopic dermatitis, and hearing loss. She was mosaic for two abnormal cell lines in peripheral blood. In 90% of cells, a dicentric chromosome with duplication of the whole short arm of chromosome 18 resulted in trisomy 18p; 10% of cells had monosomy 18p, arising from a t(14;18)(p11;q11). FISH mapping, with multiple region specific and locus specific probes from the short and long arm of chromosome 18, showed that the structure of the dicentric chromosome 18 was 18pter-->18q23::18q11-->18pter. DNA polymorphisms for chromosome 18 showed that the abnormalities of chromosome 18 were paternal in origin. Combining all results, we could link the trisomy 18p and monosomy 18p to a common origin via a complex series of events in an early mitosis., (Copyright 2000 Wiley-Liss, Inc.)

The three-dimensional positioning of the X-chromosomal adenine nucleotide translocase genes, ANT2 and ANT3, were compared in the active and inactive X chromosome territories (Xa and Xi) of female human amniotic fluid cell nuclei. ANT2 is located in Xq24-q25 and is transcriptionally active on Xa, but inactive on Xi. ANT3 is located in the pseudoautosomal region Xp22.3 and escapes X-inactivation. Three-color fluorescence in situ hybridization, confocal laser scanning microscopy, and three-dimensional image analysis revealed that transcriptionally active ANT2 and ANT3 genes were positioned more peripheral within their chromosome territory than the inactive ANT2 gene. The position of the latter was significantly more interior in the Xi territory. Although the volumes of both X territories were similar, 3D distances between ANT2 and ANT3 were significantly smaller in Xi compared to Xa territories reflecting different territory shapes. Our data show a correlation between 3D positioning and transcriptional activity of these X-specific genes., (Copyright 1999 Academic Press.)

We have isolated the prosimian lemur homologues for STS and SRY. FISH unambiguously co-localized STS with SHOX, IL3RA, ANT3 and PRK into the meiotic X-Y pairing region (PAR) of lemurs. In contrast to the close proximity of SRY to the pseudoautosomal boundary (PAB) on the Y chromosome in simian primates, SRY maps distant from the PAR in lemurs. Most interestingly, we were able to determine a DNA sequence divergence of 12.5% between the human and lemur SRY HMG box. This divergence directs to a 52 million year period of separate evolution of human and lemur SRY genes. Phylogenetically, this time period falls in between the times that prosimians and New World monkeys branched from the human lineage. Thus, we conclude that approximately 52 million years ago a transposition of SRY into the ancestral eutherian PAR distal to STS and PRK defined a new PAB in a simian progenitor. By this event, STS and PRK, amongst other genes, were excluded from the X-Y crossover process and thus became susceptible to rearrangements and/or deterioration on the Y chromosome in simian primates.

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-beta (PKC-beta) and several stable PKC-beta transfectants, we found that PRKX gene expression is under control of PKC-beta; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.

Genes within the differential region of the human Y chromosome do not recombine, and therefore the determination of their location depends on physical mapping. Yeast artificial chromosome (YAC) contigs spanning the euchromatic region of the human Y have become a powerful tool for the generation of an overlapping clone map. With this approach, however, complete physical mapping is difficult in Y euchromatic regions that are rich in repetitive sequences. We have, therefore, made use of the fluorescence in situ hybridization technique as an alternative strategy for physically mapping the PRKY and AMELY genes as well as the TSPY, RBM and DAZ gene families to human Y chromosomes in prometaphase and to extended Y chromatin in interphase. From our results, the following order of gene sequences in interval 3 of the short arm of the human Y chromosome is suggested: TSPY major with few RBM sequences interspersed-PRKY-AMELY-TSPY minor with few RBM sequences interspersed-cen. On the long arm, RBM sequences appear to be distributed over wide regions of intervals 5 and 6 with few TSPY sequences interspersed. Distal to an RBM signal cluster, a large cluster of DAZ signals is located with only a few DAZ and RBM signals overlapping in between the two clusters.

Comparative in situ hybridization in various primate species has revealed a pseudoautosomal location for the human ANT3 gene and an X-specific location for the steroid sulfatase (STS) gene throughout the higher primate species up to the New World monkeys. However, ANT3 and STS map together on an autosome of two prosimian species of the genus Lemur and Eulemur. These results suggest an autosome-to-X/Y translocation after the simians radiated from the prosimians, resulting in a pseudoautosomal location of genes such as ANT3 and STS. In simian primates, STS then became X-specific by a pericentric inversion in the Y chromosome followed by mutational inactivation of the Y allele.

We report the cloning of a highly conserved pseudoautosomal gene on the human sex chromosomes. A cDNA clone was selected by crosshybridization with a microdissected clone from the chromosomal subregion Xp22.3. It encodes a previously characterized member of the ADP/ATP translocase family and plays a fundamental role in cellular energy metabolism. This gene, ANT3, is located approximately 1,300 kilobases from the telomere, proximal to the pseudoautosomal gene CSF2RA, and escapes X-inactivation. Interestingly, a homologue of ANT3, ANT2, maps to Xq and is subject to X-inactivation. These genes provide the first evidence of two closely related X-chromosomal genes, which show striking differences in their X-inactivation behaviour.

The nucleotide sequence of a cDNA coding for rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the cDNA are sufficient to encode the total protein as determined by comparison with the liver type of glycogen phosphorylase of man. Human and rat liver glycogen phosphorylase showed 86% homology at the DNA level whereas the deduced amino acid sequence has 93.5% identity.

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.

Restriction enzyme analysis and cloning of the 18S, 5.8S, and 25S ribosomal RNA genes (rDNA) of the mung bean (Vigna radiata = Phaseolus aureus) reveal length heterogeneity in the repeating units (10-11 kbp) localized within two different regions in the ribosomal spacer. The 1.5-2.0 kbp region flanking the 3' end of the 25S rRNA contains various numbers (8-10) of tandemly arranged 180 bp subrepeats. After DNA sequencing a complex organized length heterogeneous 5' external spacer built up of different numbers of 340 bp subrepeats, each flanked by 52 bp direct repeats, is detected and described for the first time for plant ribosomal DNA repeating units. Sequences occurring in front of and within this repeated structure (elements II-IV) can be combined with the motifs P1, P2, and P3. These exhibit a strong similarity to transcription initiation sites specific for RNA polymerase I described for other plant and animal rDNA investigated to date. Transcription products complementary to the complex repeated structures are detected by hybridization to total nuclear RNA. The 9 bp element V located in fron of the first 340 bp region appears in duplicated form as a direct repeat with sequence similarity to SV40 (or RNA polymerase II) enhancer sequences.

In mung bean (Vigna radiata, formerly Phaseolus aureus) one length heterogeneity in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) is due to a variable number of 175-bp subrepeats. This spacer region downstream of the 25S rRNA coding region was characterized by sequencing the 2.4 kb EcoRI/HindIII fragment of a 10.5 kb mung bean rDNA repeat. Within the 175-bp repetitive elements a sequence was detected showing strong similarity to the T2/T3-box (GACTTGC) found in Xenopus rDNA and involved in termination and enhancing transcription. In mung bean this sequence partly forms the stem of a possible stem-loop structure at the 3'end of each subrepeat. Nuclease mapping of transcription termination sites (TTS) results in two signals, 65 bp and 315 bp downstream of the 3'end of the 25S rRNA coding region. The longer transcript terminates 20 bp downstream of the stem-loop structure at the end of the first 175-bp subrepeat. A spacer model is proposed which allows "readthrough enhancement". No cross-hybridization was observed between the 180-bp subrepeats in pea (Pisum sativum) rDNA and the mung bean 175-bp subrepeat.