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Biomedical subjects

H Luthman

Publications and source records attributed to H Luthman.

At least 91 records · Page 5Linked to original sources
PubMed1990

TaqI HLA-B and -DRB RFLP analysis can predict disease in siblings of affected children with 21-hydroxylase deficiency.

The possibility of using TaqI restriction fragment length polymorphism (RFLP) analysis of the HLA-B locus and the HLA-DR-DQ subregions, flanking the 21-hydroxylase genes, for predicting disease in siblings of children with 21-hydroxylase deficiency was analyzed in 12 nuclear families with at least one affected child and a total of 18 at-risk off-spring. As part of the study allelic TaqI HLA-B RFLP patterns were determined in homozygous cell lines and families. The frequencies of individuals homozygous for TaqI allelic patterns of the different investigated HLA loci, each locus alone and in various combinations, were determined in 100 random controls. In all 12 families it was possible to make correct genetic diagnosis by the use of only one restriction enzyme, TaqI, and two locus-specific HLA cDNA probes, HLA-B and -DRB. In all families four haplotypes were obtained. Thus, affected siblings as well as carriers could be identified. Seven of the eight sibling pairs concordant for 21-hydroxylase deficiency had pairwise identical TaqI HLA-B-DRB-DQA-DQB haplotypes. The last disease-concordant sibling pair had inherited different haplotypes from their mother, who had nonclassical 21-hydroxylase deficiency. None of the ten healthy children shared both haplotypes with their affected sibling(s). Early prenatal suppression of the fetal adrenal cortex with fluorinated corticosteroids can prevent virilization of female fetuses with 21-hydroxylase deficiency. In most cases RFLP analysis of the 21-hydroxylase genes is not informative enough for prenatal diagnosis. Our results from the present retrospective family study indicate that TaqI HLA-B and -DRB RFP analysis will be a valuable tool for first trimester assessment of 21-hydroxylase deficiency. TagI HLA-B and -DRB RFLP analysis can be performed on DNA from chorionic villi biopsies obtained in the 8th week of pregnancy. Supplemented with sex determination, early withdrawal of prophylactic steroid therapy will thus be feasible when the mother carries a male or an unaffected female fetus.

Adrenal Hyperplasia, Congenital↗
PubMed1990

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells.

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other "housekeeping genes," thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3-0.8 pg/micrograms DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8-1.2 pg/microgram DNA, which corresponds to 6-10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5-10 h before the onset of DNA synthesis, and a steady-state level was reached after 2-3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (Lesch-Nyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.

Amnion↗
PubMed1990

The gene for insulin-like growth factor-binding protein-1 is localized to human chromosomal region 7p14-p12.

Insulin-like growth factors (IGF) I and II are bound to high-affinity binding proteins in the blood circulation and other body fluids. These IGF-binding proteins are expressed at different concentrations in different tissues and are thought to regulate the activity of IGF I and II. Cloned cDNA for IGF-binding protein-1 (IGFBP1) has been used to verify the location of its gene to human chromosome 7 by Southern blotting to DNA from a human-mouse hybrid cell line. Further, by in situ hybridization the gene was regionally localized to 7p14-p12, and a Mendelian-inherited two-allele BglII restriction enzyme length polymorphism was identified, with the most frequent allele occurring in 53% of the chromosomes.

Carrier Proteins↗
PubMed1990

21-hydroxylase deficiency: disease-causing mutations categorized by densitometry of 21-hydroxylase-specific deoxyribonucleic acid fragments.

The types of disease-causing mutations were studied in 43 unrelated patients with 21-hydroxylase deficiency. Densitometry of Southern blots after cleavage with the restriction enzymes TaqI, PvuII, and BglII was used to measure the ratio of the copy-number of the 21-hydroxylase gene (CYP21) to the copy-number of its pseudogene (CYP21P). DNA from 16 unrelated patients showed equal hybridization intensities of the 2 genes, indicating that point mutations caused the enzyme deficiency. One of the 2 haplotypes in 7 patients showed evidence of a large gene conversion between the CYP21 and the CYP21P gene without loss of the total number of 21-hydroxylase genes. Deletion of at least 1 21-hydroxylase gene was found in 11 patients. DNA from 8 of these patients had relative hybridization intensities compatible with a deletion of the active 21-hydroxylase gene, CYP21. Two patients with the salt-wasting form of the disease showed homozygous loss of DNA fragments that are specific for the 5' end of the active 21-hydroxylase gene. Nine patients showed relative 21-hydroxylase hybridization intensities compatible with duplication of the gene in 1 or both haplotypes. In conclusion, point mutations, gene conversions, or CYP21 gene deletions are the typical mutations in patients with the simple virilizing and salt-wasting forms of the disease, while duplications of the locus are overrepresented in patients with nonclassical 21-hydroxylase deficiency.

Adrenal Hyperplasia, Congenital↗
PubMed1989

Human insulin-like growth-factor-binding protein. Low-molecular-mass form: protein sequence and cDNA cloning.

Two different insulin-like growth-factor (IGF)-binding proteins have been found in human blood, one of high molecular mass and dependent on growth hormone for synthesis, the other of low molecular mass and independent of growth hormone. The small IGF-binding protein is abundant in human amniotic fluid. Its amino acid sequence has now been determined by direct analysis of the protein and its proteolytic fragments. Also, by immunoscreening a partial cDNA clone was isolated from a human hepatoma cell line. The mature protein consists of 234 amino acids and is coded for by an mRNA of approximately 1700 nucleotides in length. The primary structure of the protein reveals 18 Cys residues in N-terminal and C-terminal clusters and an Arg-Gly-Asp peptide sequence, common to extracellular proteins binding to receptors of the integrin family. A protein-sequence polymorphism was detected at position Ile/Met-228, indicating possible allelic variation. The 3'-untranslated mRNA sequence has a high A + T content and shows five copies of an ATTTA sequence, which has been shown to be involved in the regulation of the stability of certain mRNAs coding for growth-regulating proteins.

Amino Acid Sequence↗
PubMed1989

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells.

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.

Animals↗
PubMed1985

Cytochrome P-450-dependent fragmentation of DNA in reconstituted membranes.

Membrane vesicles containing various forms of rabbit liver microsomal cytochromes P-450 and NADPH-cytochrome P-450 reductase were found to degrade plasmid DNA in an NADPH-requiring reaction. When cytochrome P-450 was replaced by cytochrome b5, only a negligible extent of DNA disintegration occurred. The complete inhibition of the process by hydroxyl radical scavengers, superoxide dismutase and catalase, indicated an iron-catalyzed Haber-Weiss reaction for the generation of hydroxyl radicals that subsequently react with the nucleic acid.

Animals↗
PubMed1984

Inhibition of polyoma DNA synthesis by base pair substitutions at the replication origin.

The effect of base pair substitutions on the function of the polyoma virus origin of DNA replication was studied. The mutations were all C-G to T-A transitions, induced by bisulfite treatment of recombinant DNA molecules. The mutagenesis was directed to short single-stranded gaps in duplex DNA, or to loops in heteroduplex molecules. Modification of a 34 base pair sequence of dyad symmetry led to cis-acting inhibition of viral DNA synthesis, ranging from slight defects to total inactivation. One of the mutants was temperature sensitive. Mutants with base changes in an adjacent DNA segment, including an 18 base pair long purine-pyrimidine tract, had similar, but less severe, deficiences. In contrast to the effect of mutations in the homologous region of the simian virus 40 genome, there was no strict relationship between mutation of the putative large T-antigen-binding base sequence GPuGGC and defective viral DNA synthesis.

DNA Replication↗
PubMed1983

High efficiency polyoma DNA transfection of chloroquine treated cells.

Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods. Exposure to chloroquine increased the proportion of transfected mouse cells to approximately 40%. From a culture of one million such cells, microgram quantities of newly synthesized viral DNA could be isolated. Similarly, the transformation frequency of rat cells following polyoma DNA transfection was approximately 6-fold increased by chloroquine treatment. The effect of the compound was even more pronounced in transfections with linear forms of polyoma DNA, suggesting that chloroquine inhibits degradation of DNA absorbed by the cells.

Animals↗
PubMed1982

Non-contiguous segments of the polyoma genome required in cis for DNA replication.

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 basepairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A . T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions. The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.

Base Sequence↗
PubMed1982

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA.

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.

DNA Restriction Enzymes↗