Biomedical subjects

K Sperling

Publications and source records attributed to K Sperling.

At least 73 records · Page 4Linked to original sources

PubMed1990

The rapid molecular genetic diagnosis of cystic fibrosis by polymerase chain reaction: an experience report.

The authors report their experience with about two thousand DNA amplifications by polymerase chain reaction (PCR) in prenatal diagnosis of cystic fibrosis. The method is demonstrated on examples of diagnostic informativity and prenatal diagnosis examination in a family at 1 in 4 risk of the disease using closely CF-linked diagnostic polymorphisms: J3.11/MspI, MetH/MspI, CS7/HhaI, KM19/PstI, Mp6-d9/MspI and XV2c/TaqI, PCR methodology and safety precautions are discussed.

Cystic Fibrosis↗

PubMed1990

[Sex determination using the polymerase chain reaction method for amplification of DNA segments on the X and Y chromosomes].

The authors tested the rapid and accurate prenatal and postnatal diagnosis of sex by the method of amplification of specific portions of DNA of sex chromosomes. To ensure a maximum reliability of the diagnosis the authors recommend combined examination of specific sequences from the heterochromatin region of the long arm of the Y chromosome (Yq) and from alphoid satellite sequences of DNA from the pericentromeric portion of chromosome X and Y (Xc and Yc). For further improvement of the reliability the authors recommend to digest the Yc amplification product obtained by means of restriction endonucleases Msp I, Eco Ri and Hini I. Correct assessment of the diagnosis was confirmed in all examined subjects (12) from DNA and chorium.

Female↗

PubMed1989

DNA fingerprinting with the oligonucleotide probe (CAC)5/(GTG)5: somatic stability and germline mutations.

DNA fingerprints were generated from various human somatic tissues and from peripheral blood of 179 children and their 80 parents using (CAC)5/(GTG)5 oligonucleotide probes. Whereas somatic stability of the fingerprint patterns was demonstrated, the average rate for germline mutations was estimated to be approximately 0.001 per DNA locus and gamete, with the three different restriction enzymes used. Seven out of eight mutations observed appeared to be of paternal origin.

Base Sequence↗

PubMed1989

Identification of a HeLa mRNA fraction which can correct the DNA-repair defect in Fanconi anaemia fibroblasts.

Injection of Fanconi anaemia (complementation group A) fibroblasts with HeLa mRNA is shown to correct their abnormal response to a psoralen cross-linking challenge, namely permanent repression of DNA synthesis. Injection of gradient-fractionated mRNA led to identification of a single fraction, containing mRNA of approximately 650 bases, which is responsible for this effect. This finding suggests that Fanconi anaemia (group A) cells are deficient in a small protein, up to 20 kDa in size, which is involved in the cellular response to DNA interstrand cross-links.

Adenine↗

PubMed1989

[Introduction to molecular genetic diagnosis].

A short review is given of the scientific basis and practical application of molecular approaches to the direct and indirect diagnosis of heritable disease and the forsensic identification of individuals.

Child↗

PubMed1988

Trisomy 14 mosaicism leading to cytogenetic discrepancies in chorionic villi sampled at different times.

Short- and long-term cultures of chorionic villi obtained in the 11th week of pregnancy revealed trisomy 14. After induced abortion trisomy 14 mosaicism was established in fetal skin and umbilical cord tissue while a second long-term culture of chorionic villi exhibited a normal karyotype. The results of the pathological investigations are discussed with respect to the cytogenetic findings.

Abortion, Induced↗

PubMed1988

Fanconi's anaemia: correlation of genetic complementation group with psoralen/UVA response.

The correlation found by Moustacchi (1987) between cellular response to a crosslinking challenge and genetic heterogeneity in Fanconi's anaemia is confirmed for an earlier set of complementation groups (Zakrzewski and Sperling 1980). This allows the matching of the two independently established complementation groupings and better characterization of their DNA repair-related biochemical properties.

Anemia, Aplastic↗

PubMed1988

Simple GATCA repeats characterize the X chromosomal heterochromatin of Microtus agrestis, European field vole (Rodentia, Cricetidae).

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GATCA probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.

Animals↗

PubMed1988

DNase I sensitivity of Microtus agrestis active, inactive and reactivated X chromosomes in mouse-Microtus cell hybrids.

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique--the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.

Animals↗

PubMed1988

A new chromosomal instability disorder confirmed by complementation studies.

Two sisters with a complex clinical pattern, including microcephaly, microgenia, defects of skin pigmentation, anal stenosis/atresia, and combined immunodeficiency together with spontaneous chromosomal instability and cellular hypersensitivity to X-rays and bleomycin are described. Complementation studies on heterokaryons proved that the underlying genetic defect is non-allelic with that of patients with ataxia telangiectasia (complementation groups AB-E) and the Nijmegen breakage syndrome, but identical with the case described by Conley et al. (1986).

Ataxia Telangiectasia↗

PubMed1987

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication.

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbilllus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially "reinactivated", suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.

Animals↗

PubMed1987

Transcriptional activity of constitutive heterochromatin in the mammal Microtus agrestis (Rodentia, Cricetidae).

Two cytological approaches were applied to the analysis of genetic activity in euchromatin and heterochromatin in the European field vole Microtus agrestis. The first is based on the transcriptional activity of prematurely condensed chromosomes, the second on in situ hybridization of labeled cellular RNA to metaphase chromosomes. The results show that in cultivated fibroblasts, the transcriptional activity of constitutive heterochromatin is in the same order of magnitude as that of euchromatin; its chromosomal pattern is nonrandom. It is concluded that--as has already been shown for insects and amphibians--also in mammals, constitutive heterochromatin is transcribed in its decondensed state but is genetically inactive when forming chromocenters at interphase.

Animals↗

PubMed1986

Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF).

During an in vitro fertilization (IVF) program 122 inseminated eggs showing polar body extrusion, but neither formation of pronuclei nor cell cleavage were analysed cytogenetically. Nine of these eggs showed prematurely condensed sperm chromosomes of the G1-phase (G1-PCC) besides the haploid set of maternal metaphase II chromosomes. This phenomenon can be explained by the permanent arrest of the oocytes at metaphase II after sperm penetration and hence the continuing presence of cytoplasmic chromosome condensing factors which lead to the induction of PCC in the sperm nucleus. The overall frequency of this aberrant type of fertilization was calculated to be in the order of 3-4% of all in vitro fertilized eggs.

Chromosome Banding↗

PubMed1986

Organization and chromosomal distribution of a novel repetitive DNA component from Muntiacus muntjak vaginalis with a repeat length of more than 40 kb.

The organization and chromosomal distribution of the repetitive DNA component IB from Muntiacus muntjak vaginalis (MMV) was investigated. DNA fragments of component IB were cloned in cosmids and their structure analysed using restriction nucleases and blot-hybridization experiments. Two cosmids were found to be practically identical by restriction enzyme mapping. The repeat unit of component IB DNA is more than 40 kb and contains the 11 and 18 kb Bam HI fragments, which have previously been shown to cross-hybridize with MMV satellite IA. In addition, the repeat unit contains long stretches of DNA sequences which are unique to component IB. In situ hybridization experiments showed that component IB has the properties characteristic of long interspersed repetitive DNA rather than tandemly repeated satellite DNA. Consistent with this conclusion, only a minor fraction of component IB is located on the X chromosome as demonstrated by the analysis of somatic cell hybrids. This is in marked contrast to satellite IA that is specific for the X chromosome. These results have interesting implications for the evolution of the component I DNA family of the MMV genome.

Animals↗

PubMed1985

DNase I sensitivity in facultative and constitutive heterochromatin.

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes in Microtus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. In Microtus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.

Animals↗

PubMed1984

Description of a chromosome replication unit in individual prematurely condensed human S-phase chromosomes.

Mammalian chromosome replication was studied by the aid of premature chromosome condensation (PCC). After induction of PCC the sites of DNA replication appear as "gaps" between condensed chromosomal regions. These condensed particles are unineme before and bineme after DNA replication. The two phases are due mainly to the unineme or bineme nature of the particles. During early S-phase almost all particles are unimene, during late S-phase they are bineme and there is only one transitory stage between these two main stages. Premature chromosome condensation was studied in detail on a specific human chromosome 22 which is marked by its heterochromatin constitution. This led to easy identification of these elements in S-phase PCC (S-PCC) preparations. For each stage of the S-phase there was a reproducible pattern of condensed chromosomal particles making up the whole chromosome. The number of these particles was rather limited and a complementary pattern was found in early versus late S-phase. The pattern of early S-PCC corresponded to the banding pattern of G-banded prometaphase chromosomes; the pattern of late S-PCC, to R-banded prometaphase chromosomes. Thus, "gaps" and condensed particles as observed after PCC induction are obviously homologous to chromosome replication units. Replication of constitutive heterochromatin occurred during the very late S-phase. During this stage PCC induction led to condensation of the heterochromatin into several small, highly fluorescent particles.

Cell Line↗

PubMed1984

Patterns of heterochromatin replication and condensation correlate in rat kangaroo PtK2 cells.

Chromosome replication in mammalian cells in an ordered phenomenon. This is true also for the condensation in G2 of the heterochromatic chromosomal regions in mouse cells. The generality of this phenomenon and its mechanism are not known, nor is it known whether the order of condensation of the heterochromatic chromosomal segments in G2 reflects the order of replication or is independent of it. We determined the order of replication during the S phase and of condensation in G2 of the short heterochromatic chromosomal regions in the rat kangaroo cell line PtK2. The kinetics of condensation of these regions in G2 was studied in cells treated with Hoechst 33258. Their order of replication was established with the use of a sensitive technique based on the treatment of living cells with 5-bromodeoxyuridine and Hoechst 33258. Our results show that these regions exhibit a similar pattern of replication in S and condensation in G2.

Animals↗

PubMed1983

[Genetic integrity following cryopreservation].

The use of human cryosperm in artificial insemination raises the question of its safety with respect to genetic damage. As outlined here, the rate of congenital malformations among those newborn cannot be regarded as a reliable indicator for any genetic hazard. We have therefore studied the effect of deep freezing on 1. the rate of chromosomal aberrations in human lymphocytes, and 2. the number of gene mutations at the HGPRT locus, and 3. the frequency of sister chromatid exchanges in Chinese hamster ovary cells. In all three test systems no evidence of induced genetic damage after cryoconservation was observed.

Animals↗