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PubMed1993

A 31P NMR spectroscopy study of Xenopus laevis heart perfused in vitro with creatinol-O-phosphate, phosphocreatine, adenosine triphosphate, fructose diphosphate and ouabain.

Xenopus laevis heart was studied by 31P NMR using a 200 MHz proton spectrometer; hearts were perfused, at pH 7.35 and room temperature, with normal oxygenated or K(+)-enriched Ringer. Solution was later added with creatinol-O-phosphate (COP), phosphocreatine (PCr), adenosine triphosphate (ATP), fructose-1,6-diphosphate (FDP) and ouabain. NMR spectra of the heart show organic phosphomono- and phosphodi-esters, inorganic phosphate, PCr, overlapping alpha-ATP/ADP and gamma-ATP/beta-ADP, and beta-ATP signals. Their chemical shift positions and areas showed no significant changes in the course of 1.5 h perfusions with either solution, except in a few preparations, whether the heart was beating or reversibly arrested. While COP reduced the signals in beating hearts, the same spectra exhibited no consistent, substantial changes under PCr, ATP and FDP 1 to 10 mM, pH 7.35 perfusion with either solution, nor when ouabain mumol was added. The spectra are briefly discussed in comparison with those observed in the perfused heart of mammals (mostly rat), and particularly with those obtained in the frog (Rana temporaria) heart, both by analysing the bioenergetic equilibria on the basis of total tissue substrate levels measured in extracts of freeze-clamped tissue, and by evaluating cytochrome-b, flavin and pyridine nucleotide in vitro oxido-reduction read-outs in separate, similar experimental settings.

Adenosine Triphosphate↗
PubMed2002

[Clonging, expression and activity determination of the recombinant human soluble B lymphocyte stimulator and its two mutants].

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily that functionally involved in B cell proliferation. Here, the full length cDNA of human soluble BlyS (hsBLyS) was amplified by reverse transcription-PCR from total RNA of human peripheral blood lymphocytes and sequenced. The cloned cDNA fragments was inserted into pBV220, a thermo-sensitive expression plasmid, forming recombinant plasmid pBV220/hsBLyS. The two mutants of hsBLyS: hsBY-A (Cys(146) right curved arrow Ala(146), TGC right curved arrow GCT) and hsBY-V (Cys(146) right curved arrow Val(146), TGC right curved arrow GTT), were amplified by overlap PCR from pBV220/hsBLyS and inserted into the pBV220 to form pBV220/hsBY-A and pBV220/hsBY-V expression plasmids as the method above. The three expression plasmids were transformed into E.coli DH5alpha and it was found that the recombinant proteins were highly expressed as inclusion bodies. Pure recombinant proteins were obtained by Sephacryl-200 gel filtration and renaturation. The experimental results demonstrated that the sequences of the PCR products were identical with the published hsBLyS cDNA sequence or expected mutant sequence and the purity of the recombinant proteins obtained was relatively high. The activity of the purified recombinant proteins was very significant in the B cell survival and proliferation assay, and statistical tests indicated that the mutant rhsBY-V promoted proliferation of B lymphocyte cell remarkably better than the wild type.

B-Cell Activating Factor↗
PubMed1996

Fast amplification of the low density lipoprotein receptor gene and detection of a large deletion by means of long polymerase chain reaction.

To demonstrate the usefulness of Long PCR in analyzing gene structures and large deletions we have developed a method to amplify the entire LDL receptor gene, including the promoter region and intron 1. This opens new ways for studies of the gene and allows the detection of certain LDL receptor-specific deletions. For the amplification of the LDL receptor gene, spanning approximately 45.5 x 10(3) bases and divided into 18 exons and 17 introns, we have designed overlapping PCR products (ranging from 4 to 16 x 10(3) bases in length), which can be amplified simultaneously overnight for fast results. It was possible to positively identify two samples from heterozygote carriers of the "5 kB French-Canadian" deletion using this method. As a side result the length of intron 1 of the LDL receptor gene could be established to be approximately 9.5 x 10(3) bases. The method is sensitive enough to detect deletions in 1:10 mixes of positive control with wildtype DNA.

Gene Deletion↗
PubMed1996

A novel A-->G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency.

We have identified the underlying molecular defect in a patient with hepatic lipase (HL) deficiency presenting with hypertriglyceridemia and premature cardiovascular disease. DNA sequencing of polymerase chain reaction (PCR) amplified DNA and digestion with BsrI established homozygosity for an A-->G mutation in intron I of the patient's hepatic lipase gene. This mutation introduces an additional AG motif within a potential branch lariat signal located 13 bp upstream of the native 3' splice site. Two minigene constructs (normal and mutant) consisting of exons 1 and 2 as well as 192 bp of intron I of HL were generated by the overlap PCR extension method and transfected in human 293 cells. Sequence analysis of reverse transcribed, amplified cDNA generated from total RNA isolated from transfected cells demonstrated the presence of abnormally spliced products containing 13 and 78 additional bases as well as the accumulation of unspliced mRNA. No normally spliced mRNA was identified. Thus, the A-->G mutation disrupts normal splicing of intron I and generates a new AG site that is utilized as an alternative 3' splice signal leading to the most prominent RT-PCR product in vitro. Translation of these alternatively spliced products leads to premature termination resulting in the synthesis of a truncated, non-functional enzyme. The absence of normal HL protein in post heparin plasma of this patient was confirmed by Western blotting. DNA restriction analysis demonstrated that all four of the proband's children, who exhibit HL activity levels between those of the HL-deficient father and the mother with normal HL activity, are heterozygotes for the splice site mutation. Thus, our studies establish the functional significance of a novel mutation in the HL gene of a patient presenting with HL deficiency.

Adenine↗
PubMed2007

Separation of near full-length hepatitis C virus quasispecies variants from a complex population.

A long RT-PCR (LRP) protocol was developed recently for robust amplification of a near full-length HCV genomic sequence from clinical samples, followed by efficient cloning [Fan, X., Xu, Y., Di Biceglie, A.M., 2006. Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples. Biochem. Biophys. Res. Commun. 346, 1163-1172]. In the present study, the LRP protocol has been estimated for its error rate and the validation by sequencing fully the near full-length HCV inserts from six recombinant clones derived from a patient sample with complex viral diversity. These sequences were compared with the near full-length HCV sequence that was generated by direct sequencing of multiple overlapped PCR products from the same sample, referred to as the population sequence. Comparative analysis confirmed the artificial nature of the PCR-assembled population sequence and identified potential domains for linked viral mutations. The data also suggested that the hypervariable region 1 (HVR1) may be a biological marker for the phenotype at the quasispecies level. These observations emphasize the significance of the use of near full-length genomic sequences for HCV genetic studies and for reverse genetic analysis using authentic quasispecies variants.

Cloning, Molecular↗
PubMed1999

[Growing divergence inside primate T cell lymphotropic viruses].

During our studies on the evolution of the PTLVs, we have isolated and genomically characterized two divergent simian T-lymphotropic viruses, not belonging to the previously well-established PTLV lineages. STLV-PH969 has been isolated from an Eritrean sacred baboon (Papio hamadryas), with an HTLV-like indeterminate serological profile. The entire 8916 nt long genomic sequence, was obtained from a cDNA library of PH969 mRNA, in combination with a PCR based strategy. All open reading frames (ORF) common to all HTLV related viruses could be identified without important deletions or insertions. Sequence comparison of the STLV-PH969 genomic sequence with the HTLV-I and -II prototype sequences revealed that this virus is equidistantly related to HTLV-I and -II. A phylogenetic analysis on the envelope and regulatory Tax proteins conclusively proved the ancient separation of HTLV-I, -II and STLV-PH969. Together these data provided enough evidence to justify the classification of this virus as a new type of primate T-lymphotropic virus, designated PTLV-L. We isolated a second highly divergent virus from pygmy chimpanzees (bonobo's, Pan paniscus) housed in the Antwerp Zoo. The animals infected with this virus showed an aberrant HTLV-I like serological profile. The entire 8855 nt long genomic sequence of the STLV-PP1664 provirus was obtained by sequencing of overlapping PCR fragments. On this sequence, all ORFs common to all HTLV related viruses could be identified without any important deletions or insertions. Sequence comparison showed that the STLV-PP1664 proviral sequence was related to HTLV-II and the virus is called STLV-II. However, in all genomic regions, STLV-IIPP1664 was much more divergent from any of the HTLV-II subtypes than these are from each other. In order to further investigate the genomic relationship of STLV-LPH969 and STLV-IIPP1664 with HTLV-I and -II, we also analyzed the genomic organization of the pX region, which differs between HTLV-I and HTLV-II. In the STLV-LPH969 and STLV-IIPP1664 producing cell lines, 2 and 5 viral messengers could be detected respectively, potentially expressing 3 and 5 different accessory proteins respectively. The splicing pattern and the resulting proteins differ from those of HTLV-I and HTLV-II. In the light of the growing interest for xenotransplantation, the prevalence of distinct PTLVs in the wild and their capacity to cross the species barrier deserves further attention.

Animals↗
PubMed1996

Systematic screening for mutations in the 5'-regulatory region of the human dopamine D1 receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder.

A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5'-flanking region of the dopamine D1 receptor (DRD1) gene. This region has previously been shown to contain a functional promoter [Minowa et al., 1992: Proc Natl Acad Sci 89:3045-3049; Minowa et al., 1993: J Biol Chem 268:23544-23551]. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5'-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder.

Alleles↗
PubMed1997

Metachromatic leukodystrophy: identification of the first deletion in exon 1 and of nine novel point mutations in the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459 + 1G-->A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459 + 1G-->A, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene.

Alleles↗
PubMed1995

Systematic screening for mutations in the promoter and the coding region of the 5-HT1A gene.

In the present study we sought to identify genetic variation in the 5-HT1A receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette's syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5' untranslated region of the 5-HT1A gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A-->G) in nucleotide position 82 which leads to an amino acid exchange (Ile-->Val) in position 28 of the receptor protein and a silent mutation (C-->T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT1A gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette's syndrome.

Base Sequence↗
PubMed2004

General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.

Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.

Animals↗
PubMed1994

The seventh transmembrane domain of gastrin/CCK receptors contributes to non-peptide antagonist binding.

The related rat cholecystokinin (CCK)-A and gastrin/CCK-B receptors can be selectively blocked by the antagonists L364718 and L365260, respectively. In order to determine receptor domains which are important in conferring specificity for L365260 and L364718 we constructed by overlap-PCR a rat gastrin/CCK-B receptor chimaera which contained the seventh transmembrane domain of the rat CCK-A receptor. Receptor binding assays on transiently transfected COS cells demonstrated a selective reduction in the affinity of the chimaeric receptor for L364718, so that the L365260 and L364718 affinities were of a similar order. Since the chimaera differs from the wild-type gastrin/CCK-B receptor by only six amino acids we conclude that one or more of these six amino acids contributes to L364718 binding and that the affinity determinants of L365260 and L364718 must, at least in part, be different. Furthermore, the affinity of the chimaera for gastrin is essentially the same as the gastrin/CCK-B receptor, indicating that the six different amino acids probably do not contribute to peptide agonist binding.

Animals↗
PubMed1994

Identification of genetic variation in the human serotonin 1D beta receptor gene.

Disturbances of serotonergic pathways have been implicated in a wide variety of neuropsychiatric disorders such as depression, anxiety, migraine, and substance abuse. Genetic variation in genes coding for serotonin receptor proteins might well be involved in the genetic predisposition to these diseases and/or of pharmacogenetic relevance. Genomic samples from 46 unrelated healthy subjects were investigated by single-strand conformation analysis (SSCA) to screen for genetic variation in the human serotonin 1D beta (5-HT1D beta) receptor gene. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as 5' untranslated regions of the 5-HT1D beta gene. Four nucleotide sequence variants were found: a coding mutation in nucleotide position 371 which leads to an amino acid exchange (Phe-->Cys) in position 124 of the receptor protein and three mutations in the 5' flanking region. For all mutations specific PCR-based assays were developed which allow rapid genotyping in populations and families. To our knowledge, the Phe-124-Cys substitution is the first natural occurring molecular variant which has been identified for the 5-HT1D beta receptor so far.

Alleles↗
PubMed2001

A humanized monoclonal antibody constructed from intronless expression vectors targets human hepatocellular carcinoma cells.

An anti-human hepatocellular carcinoma (HCC) monoclonal antibody, hHP-1, was genetically humanized from a murine monoclonal antibody. In this study, a concept of positional template approach was applied to design the amino acid sequence of hHP-1's variable region, and synthetic DNA fragments for protein expression were produced through overlapping PCR from single strand oligonucleotides. Synthetic DNA fragments and human antibody constant region cDNA were used to construct two CMV promotor-based expression vectors for the antibody light and heavy chains, in which the variable region was connected directly to the constant region without an intron sequence. Completely assembled humanized antibody was successfully expressed in mammalian cells as IgG1 kappa molecules and purified using protein A affinity column. The immunogenicity of the hHP1 was estimated by the amino acid sequence and determined through a HAMA (human anti-murine antibody) serum reaction assay. Results indicated that the immunogenicity of hHP-1 was significantly reduced. In vitro binding activity assay showed that the hHP-1 had retained its binding function to a human HCC SMMC-7721 cell-line, without cross binding to other human normal tissues. Immunofluorescence staining showed that hHP-1 had a strong binding activity to SMMC cells. A competitive binding assay showed that the relative binding activity of hHP-1 was approximately 25% binding activity of the original murine antibody. Our results indicate that a humanized antibody could be produced using intronless vectors and expressed as a complete IgG1 kappa antibody. Hence we believe that hHP-1 could be a potential candidate for HCC treatment.

Amino Acid Sequence↗
PubMed1999

alphaSU2, an epithelial integrin that binds laminin in the sea urchin embryo.

At gastrulation in the sea urchin embryo dramatic cell adhesion changes contribute to primary mesenchyme cell ingression movements and to cell rearrangements during archenteron invagination. At ingression, quantitative adhesion assays demonstrated previously that primary mesenchyme cells (PMCs) change their affinity for neighboring cells, for a fibronectin-like substrate, and for the hyaline layer. To investigate the molecular basis for these and other differential cell affinities at gastrulation, we have identified an integrin that appears to be responsible for specific alterations in cell-substrate adhesion to laminin. During early cleavage stages blastomeres adhere poorly to laminin substrates. Around hatching there is a large increase in the ability of blastomeres to bind to laminin and this increase correlates temporally with the expression of an integrin on the basal surface all blastomeres. PMCs, after undergoing their epithelial-mesenchymal transition, have a strongly reduced affinity for laminin relative to ectoderm cells and, correspondingly, do not stain for the presence of the integrin. We identified the alpha integrin cDNA from Lytechinus variegatus by RT-PCR. Overlapping clones were obtained from a midgastrula cDNA library to provide a complete sequence for the integrin. The composite cDNA encoded a protein that was most similar to the alpha5 subgroup of vertebrate integrins, but there was not a definitive vertebrate integrin homolog. Northern blots and Western immunoblots showed that the sea urchin integrin, which we have named alphaSU2, is present in eggs and during all stages of development. Immunolocalization with specific polyclonal antibodies showed that alphaSU2 first appears on the basal cell surface of epithelia at the midblastula stage, at a time correlating with the increase in adhesive affinity for laminin. The protein remains at high levels on the basal surface of ectoderm cells but is temporarily reduced or eliminated from endoderm cells during their convergent-extension movements. To confirm integrin binding specificity, alphaSU2 was transfected into an alpha-integrin-deficient CHO cell line. alphaSU2-expressing CHO cells bound well to isolated sea urchin basal lamina and to purified laminin. The transfected cells bound weakly or not at all to fibronectin, type I collagen, and type IV collagen. This is consistent with the hypothesis that alphaSU2 integrin functions by binding epithelial cells to laminin in the basal lamina. In vivo, modulation of alphaSU2 integrin expression correlates with critical adhesive changes during cleavage and gastrulation. Thus, this protein appears to be an important contributor to the morphogenetic rearrangements that characterize gastrulation in the sea urchin embryo.

Amino Acid Sequence↗
PubMed1995

Sequence-based analysis of properdin deficiency: identification of point mutations in two phenotypic forms of an X-linked immunodeficiency.

Properdin deficiency is an inherited X-linked disorder causing increased susceptibility to meningococcal disease. Here, underlying genetic defects in the properdin gene were identified for the first time. Samples from individuals with type I deficiency, defined as complete absence of properdin in serum, and individuals with type II deficiency, characterized by low concentrations of properdin in serum, were analyzed by direct chromosome sequencing of overlapping PCR products. The complete gene, including 10 exons and 9 introns, covering 6460 bases of the region Xp11, was investigated by direct solid-phase sequencing. In the related individuals with type I deficiency a C to T mutation in exon 5 was identified, which gives rise to a stop codon TGA and thus a truncated gene product. In addition, point mutations were found in 4 introns and a silent mutation in exon 10. In the properdin gene from related individuals with type II deficiency two point mutations were found, one in intron 3 and one in exon 4. The latter mutation yields a substitution of arginine to tryptophan, which may affect folding, secretion, and/or turnover of the protein. The genetic and biochemical implications of these mutations are discussed.

Base Sequence↗
PubMed1997

Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins.

Ovalbumin is a non-inhibitory serpin which lacks the ability to undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition of uPA, SDS-PAGE stable complex formation with uPA and increased instability on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may have a key role in serpin beta-sheet opening and inhibitory function.

Amino Acid Sequence↗
PubMed2002

DNA-based diagnosis of isolated sulfite oxidase deficiency by denaturing high-performance liquid chromatography.

Isolated sulfite oxidase deficiency is a rare autosomal recessive disease, characterized by severe neurological abnormalities, seizures, mental retardation, and dislocation of the ocular lenses, that often leads to death in infancy. There is a special demand for prenatal diagnosis, since no effective treatment is available for isolated sulfite oxidase deficiency. Until now, the cDNA sequence of the sulfite oxidase (SUOX) gene has been available, but the genomic sequence of the SUOX gene has not been published. In this study, we have performed a DNA-based diagnosis of isolated sulfite oxidase deficiency in a Chinese patient. To do so, we designed oligonucleotide primers for amplification of the predicted exons and intron-exon boundaries of the SUOX gene obtained from the completed draft version of the human genome. Using overlapping PCR products, we confirmed the flanking intronic sequences of the coding exons and that the entire 466-residue mature peptide is encoded by the last exon of the gene. We then performed mutation detection using denaturing high-performance liquid chromatography (DHPLC). The DHPLC chromatogram of exon 2b showed the presence of heteroduplex peaks only after mixing of the mutant DNA with the wild-type DNA, indicating the presence of a homozygous mutation. Direct DNA sequencing showed a homozygous base substitution at codon 160, changing the codon from CGG to CAG, which changes the amino acid from arginine to glutamine, i.e., R160Q. The DNA-based diagnosis of isolated sulfite oxidase deficiency will enable us to make an accurate determination of carrier status and to perform prenatal diagnosis of this disease. The availability of the genomic sequences of human genes from the completed draft human genome sequence will simplify the development of molecular genetic diagnoses of human diseases from peripheral blood DNA.

Child, Preschool↗
PubMed1999

A mutation in the promoter of the multidrug resistance gene (MDR1) in human hematological malignancies may contribute to the pathogenesis of resistant disease.

The overexpression of the multidrug resistance gene MDR1 has been found to be associated with therapy-resistance in hematological malignancies. Yet the cellular mechanisms underlying this increased expression are completely unknown. Point mutations in the MDR1 promoter have been found in osteogenic sarcoma (Stein et al., Eur J of Cancer, 30A: 1541-1545, 1994). We therefore analyzed DNA from hematological malignancies for MDR1 promoter point mutations. Two pairs of overlapping PCR primers were designed which did not amplify the MDR3 gene. Amplified DNA was screened using single strand conformation polymorphism (SSCP). 139 patients and 93 normal controls were studied. Fifteen patients (11%) were found to have abnormal bands on the SSCP analysis. Of these, 9 had acute myeloid leukemia (AML), 4 chronic lymphocytic leukemia (CLL), 1 acute lymphocytic leukemia (ALL), and 1 nonHodgkin's lymphoma (NHL). Sequence analysis revealed that all patients were heterozygous for a point mutation in the promoter (T-C transition at +8). Four normals (4%) were found to be heterozygous for the mutation. Confirmation of the mutation was performed by oligonucleotide probe hybridization. All but two of the AML patients have died due to chemoresistant disease (one is lost to followup). Of the CLL patients, one is alive with progressive disease, and the others have died. Further studies will assess the effect of this mutation on MDR1 gene transcription.

ATP Binding Cassette Transporter, Subfamily B↗