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Biomedical subjects

T L Boehm

Publications and source records attributed to T L Boehm.

At least 19 recordsLinked to original sources
PubMed1988

DNA methylation levels in acute human leukemia.

We have studied the overall 5-methylcytosine content and the percentage of methylated CpG-dinucleotides in 25 cases of untreated human acute leukemias. For comparison, normal leucocyte subpopulations were similarly analyzed. The methylation levels in normal white blood cell DNA varied in the same range as those in leukemia cells with no apparent hypomethylation in tumor cell DNA. Such hypomethylation, however, was found in a patient studied in first and second relapses of the disease. These data suggest that genome-wide demethylation, a characteristic of other tumors, does not accompany leukemic transformation.

5-Methylcytosine↗
PubMed1987

[New possibilities of heterozygote detection of hemophilia A].

Hemophilia A is the most common inherited bleeding disorder in man. The recent isolation of the hemophilia gene has led to the identification of an intragenic restriction fragment length polymorphism (RFLP) which can be used for segregation analysis in families at risk for carrying the disease. In addition, a tightly linked extragenic RFLP can also be used for these analyses. In this paper, we exemplify the usefulness of DNA analysis in genetic counseling of families at risk for hemophilia A. Although DNA analysis allows carrier detection in the majority of families, bioassays are still required for accurate diagnosis when DNA analysis is not informative.

DNA↗
PubMed1987

T cell receptor gamma chain variable gene rearrangements in acute lymphoblastic leukemias of T and B lineage.

The status of immunoglobulin and T cell receptor genes in acute lymphoblastic leukemia (ALL) of T and B lineage has been studied. Our data indicate that illegitimate gene rearrangements at immunoglobulin heavy chain (in T cell ALL), and T cell receptor beta chain (in pre-B ALL) genes are only rarely found (2 out of 30 patients). In contrast, T cell receptor gamma chain gene rearrangements, characteristically found in T-ALL, are also present in 7 of 18 patients with pre-B ALL. Several features distinguish these illegitimate T cell receptor gamma chain gene rearrangements from those in normal and leukemic T cells. V gamma genes located far upstream of the J gamma/C gamma complexes (V gamma 2, V gamma 3, V gamma 4, V gamma 5) appear to be preferentially used in normal adult peripheral blood T cells. In contrast, V gamma genes located immediately 5' to the J gamma/C gamma complexes (V gamma 8, V gamma 9, V gamma 10, V gamma 11) predominate in V gamma -J gamma recombinations observed in T-ALL and pre-B ALL. Whereas the J gamma 2 region is primarily used in T cell receptor gamma gene rearrangements observed in T-ALL, those in pre-B ALL are confined mostly to the J gamma 1 region. These data suggest a limited accessibility of the T cell receptor gamma chain gene locus for recombination processes in early stages of B cell differentiation.

B-Lymphocytes↗
PubMed1987

Application of a bcr-specific probe in the classification of human leukaemia.

A genomic probe derived from the breakpoint cluster region (bcr) on chromosome 22q11 was used to assess whether Philadelphia (Ph) chromosome positive chronic myelogenous leukaemia patients have unique patterns of bcr rearrangements and whether this pattern is modified as the disease progresses from stable phase to blast crisis. The data indicated that bcr rearrangements are fairly unique to each patient and are not subject to additional modifications during the course of the disease. We have also found bcr rearrangements in acute lymphocytic leukaemia (ALL) patients, usually of the cALL phenotype. For the majority of Ph+ ALL patients, the breakpoint on 22q11 was in bcr. However, we describe a case of Ph+ ALL without bcr rearrangement, indicating heterogeneity of Ph chromosomes in ALL at the molecular level. Contrary to previous reports, a bcr rearrangement was also identified in a childhood cALL.

Aged↗
PubMed1987

In vitro differentiation of a null-acute leukemia: T-lymphoid surface antigen expression associated with rearrangement in T-cell receptor beta chain variable genes.

We describe a human acute unclassified leukemia with a unique t(4;17) translocation that coexpresses T-lymphoid and myeloid surface antigens after in vitro culture in the presence of the tumor promoter, TPA. Under these conditions, the joining regions of the immunoglobulin heavy chain and T-cell receptor gamma and beta chain complexes remained in germ line configuration. A T-cell receptor beta chain variable gene probe, however, revealed the presence of rearrangements in the V beta M3-2 gene region after bilineage differentiation. These results may be pertinent to the interrelationship of T-cell receptor gene rearrangements and the control of T-cell antigen surface expression.

Acute Disease↗
PubMed1987

Oncogene amplifications, rearrangements, and restriction fragment length polymorphisms in human leukaemia.

We have determined the prevalence of amplification and rearrangements for c-myc, c-myb, c-mos, bcr, c-abl, c-Ha-ras-1, c-N-ras, and c-K-ras-2 in a total of 51 cases of human leukaemia (19 patients with AML, 13 cases with CML, 14 cases with ALL, and 5 cases with CLL). Amplifications at a level of more than 2 two copies per haploid genome are apparently very rare and were found only once for c-myb in a c-ALL patient. Oncogene rearrangements were not found except for bcr, which was rearranged in all cases of CML, and 5 cases of ALL studied. Restriction fragment lengths polymorphisms (RFLPs) were also analysed. A previously described rare 5 kb EcoRI allele at the c-mos locus was absent in our patients. Rare alleles at the c-Ha-ras-1 locus were found to be significantly more prevalent in our patients than in a control group. Transfection experiments revealed no dominant transforming oncogenes in the tumour DNA of 3 patients carrying such rare alleles.

Alleles↗
PubMed1987

A rapid and efficient screening method for DNA restriction fragment length polymorphisms.

Genomic loci displaying DNA sequence polymorphisms represent useful landmarks on the genetic linkage map. We describe an integrated experimental approach to facilitate the detection of DNA restriction fragment length polymorphisms (RFLP) with useful allelic frequencies at loci covered by cloned DNA sequences. The essential feature of the screening method presented is the pooling of DNA from unrelated individuals for Southern blot hybridization analyses using non-repetitive DNA sequences identified in and preparatively isolated from genomic lambda phage clones. This procedure results in the detection of RFLP with maximal values of heterozygosity while counterselecting for RFLP with unfavourable allelic frequencies. The described experimental protocol should therefore facilitate the identification and characterization of polymorphic loci with frequent heterozygosity.

DNA, Recombinant↗
PubMed1987

Immunoglobulin heavy chain and T-cell receptor gamma and beta chain gene rearrangements in acute myeloid leukemias.

Somatic rearrangements of immunoglobulin (Ig) and T-cell receptor (TCR) genes are the basis for the production of receptors for antigen in B-cells and T-cells, respectively. Here, we have studied the extent and pattern of rearrangements at Ig and TCR loci in 17 patients presenting with acute myeloid leukemia (AML). Our data demonstrate illegitimate clonal rearrangements at Ig heavy and/or TCR beta chain genes in 5 of 17 AML patients. In four of these five patients, rearrangements at the TCR beta chain gene locus were also observed. Seven patients displayed clonal TCR gamma chain gene rearrangements as the only abnormality. Rearrangements at Ig light chain and TCR alpha chain gene loci were not detected. Illegitimate TCR gamma chain gene rearrangements in AML involve recombinations of only a subset of V gamma genes, predominantly with the J gamma 1 region. Rearrangements at the TCR beta chain gene locus are characterized by both D-J and V-D-J recombinations, with predominant use of the J beta 1 region. The absence or presence of illegitimate antigen receptor gene rearrangements in AML may constitute a prognostic marker. In addition, these alterations can be used to establish clonality in AML with direct applications in the monitoring of disease. Finally, the present data relate to the problem of lineage assignment of acute leukemias based on Ig and TCR gene rearrangements and strongly suggest that the latter cannot be regarded as unequivocal evidence for the B-cell or T-cell lineage, respectively.

Adult↗
PubMed1986

Use of a biotinylated DNA probe specific for the human Y chromosome for rapid antenatal sex determination.

We have cloned a male-specific 3.4 kb human DNA sequence which showed only little crosshybridisation to autosomal sequences. To further enhance the specificity of the probe, we subcloned an internal TaqI-fragment resulting in clone pH343T33. This clone was used to determine the presence of Y-chromosomal sequences in DNA extracted from amniotic cells and from chorionic villi by Southern and dot hybridisation assays, respectively. Using this clone, we correctly predicted fetal sex in all of 148 cases analysed. To facilitate the use of this clone in clinical practice, we simplified the dot hybridisation procedure so that it can be performed in less than 48 h. The procedure with 32P-labeled DNA probes requires less than 0.5 mL of amniotic fluid; when biotinylated DNA probes are used, 3-5 mL of amniotic fluid usually suffice. We have used this probe in genetic counseling of families at risk for X-linked disorders.

Biotin↗
PubMed1986

Classical and late-onset forms of congenital adrenal hyperplasia caused by 21-OH deficiency reveal different alterations in the C4/21-OH gene region.

Congenital adrenal hyperplasia (CAH) is due to defective adrenal cortisol biosynthesis. In most cases, deficiency of a P 450-C21 specific steroid hydroxylase impairing cortisol synthesis has been found. The disease is HLA-linked, and on clinical grounds it can be divided into two major forms, the classical and the non-classical type. Here, evidence is presented that the classical and the non-classical forms of CAH caused by 21-OH deficiency are due to different genetic alterations in the C4/21-OH gene region. In most cases of classical CAH associated with the HLA-Bw47 antigen, a specific and selective loss of the 21-OH B gene was observed with some interesting exceptions. Alterations in the 21-OH gene region in the non-classical forms of CAH, patients either HLA-B14; DR1 homo- or heterozygous, are different. Our data indicate the possibility of gene conversion events in this genomic region in non-classical CAH.

Adrenal Hyperplasia, Congenital↗
PubMed1985

Purification and characterization of mammalian DNA methyltransferases by use of monoclonal antibodies.

Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.

Animals↗
PubMed1985

Mouse DNA-cytosine-5-methyltransferase: sequence specificity of the methylation reaction and electron microscopy of enzyme-DNA complexes.

Monoclonal antibodies prepared against DNA methyltransferase from human placenta undergo immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells. One of these monoclonal antibodies, M2B10, was used for the immunoaffinity purification of this enzyme. Complexes of the immunoaffinity-purified mouse DNA methyltransferase with DNA were visualized by electron microscopy. DNA methyltransferase was found to be distributed along linearized plasmid DNA with a higher incidence of enzyme molecules at the terminal segments. This binding to strand ends was significantly increased after dG- or dGdC-tailing of the DNA, which is compatible with a preferred binding of the enzyme to single-stranded DNA. Sequence specificity analysis using methyl-sensitive restriction enzymes showed that the mouse DNA methyltransferase transferred methyl groups to the internal cytosines in 5'CCGG and 5'GCGC sequences, however, the external cytosine in 5'CCGG sequences was also methylated.

Animals↗
PubMed1984

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels.

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.

Animals↗
PubMed1983

Isolation and characterization of DNA cytosine 5-methyltransferase from human placenta.

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.

Animals↗
PubMed1983

Alteration of enzymatic DNA methylation by chemical carcinogens.

It is generally accepted that the DNA methylation pattern in a particular cell type is stable over the cell generations and is clonally inherited by a semiconservative mechanism in which the methylation of the progeny strand is determined by the 5-methylcytosine residue present in the parental strand. In our experiments, we have been able to show that the chemical carcinogens MNU, MNNG, and L-ethionine affect the methylation process either by modification of the DNA substrate or by interference at the cofactor level. The changes introduced into the cellular methylation pattern persist and are inherited in the progeny cells regardless of whether the carcinogens or their adducts in the DNA persist in the cell or not. The carcinogen-induced hypomethylation correlates with the increased transcriptional complexity of the nRNA of treated cells, indicating the initiation of transcription at sites previously inactive. This means that chemical carcinogens can cause, by interference with the process of DNA methylation, stable changes in the program of genetic expression; the possibility that such changes are related to the process of initiation of carcinogenesis should not be overlooked.

Animals↗