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L M Fisher

Publications and source records attributed to L M Fisher.

At least 37 records · Page 2Linked to original sources
PubMed1998

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues.

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.

Antibiotics, Antineoplastic↗
PubMed1998

DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae.

We examined the response of Streptococcus pneumoniae 7785 to clinafloxacin, a novel C-8-substituted fluoroquinolone which is being developed as an antipneumococcal agent. Clinafloxacin was highly active against S. pneumoniae 7785 (MIC, 0.125 microg/ml), and neither gyrA nor parC quinolone resistance mutations alone had much effect on this activity. A combination of both mutations was needed to register resistance, suggesting that both gyrase and topoisomerase IV are clinafloxacin targets in vivo. The sparfloxacin and ciprofloxacin MICs for the parC-gyrA mutants were 16 to 32 and 32 to 64 microg/ml, respectively, but the clinafloxacin MIC was 1 microg/ml, i.e., within clinafloxacin levels achievable in human serum. S. pneumoniae 7785 mutants could be selected stepwise with clinafloxacin at a low frequency, yielding first-, second-, third-, and fourth-step mutants for which clinafloxacin MICs were 0.25, 1, 6, and 32 to 64 microg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of the gyrA, parC, gyrB, and parE genes by PCR, HinfI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in gyrA or gyrB, in parC, in gyrA, and finally in parC or parE. No evidence was found for other resistance mechanisms. The gyrA mutations in first- and third-step mutants altered GyrA hot spots Ser-83 to Phe or Tyr (Escherichia coli coordinates) and Glu-87 to Gln or Lys; second- and fourth-step parC mutations changed equivalent hot spots Ser-79 to Phe or Tyr and Asp-83 to Ala. gyrB and parE changes produced novel alterations of GyrB Glu-474 to Lys and of Pro-454 to Ser in the ParE PLRGK motif. Difficulty in selecting first-step gyrase mutants (isolated with 0.125 [but not 0.25] microg of clinafloxacin per ml at a frequency of 5.0 x 10(-10) to 8.5 x 10(-10)) accompanied by the small (twofold) MIC increase suggested only a modest drug preference for gyrase. Given the susceptibility of defined gyrA or parC mutants, the results suggested that clinafloxacin displays comparable if unequal targeting of gyrase and topoisomerase IV. Dual targeting and the intrinsic potency of clinafloxacin against S. pneumoniae and its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.

Anti-Infective Agents↗
PubMed1997

Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast.

Candida albicans topoisomerase II, encoded by the TOP2 gene, mediates chromosome segregation by a double-strand DNA break mechanism and is a potential target for anti-fungal therapy. In this paper, we report the characterization of the C. albicans TOP2 gene and its use to develop a yeast system that allows the identification and study of anti-fungal topoisomerase II inhibitors in vivo. The gene, specifying a 1461-residue polypeptide with only 40% identity with human topoisomerase IIalpha and beta isoforms, was isolated from C. albicans on a 6.3 kb EcoRI fragment that mapped to chromosome 4. It was used to construct a plasmid in which TOP2 expresses a recombinant enzyme (residues 57-1461 of C. albicans topoisomerase II fused to the first five residues of Saccharomyces cerevisiae topoisomerase II) under the control of a galactose-inducible promoter. The plasmid rescued the lethal phenotype of a temperature-sensitive S. cerevisiae DNA topoisomerase II mutant allowing growth at 35 degrees C. Yeast cells, bearing ISE2 permeability and rad52 double-strand-break-repair mutations the growth of which at 35 degrees C was dependent on C. albicans topoisomerase II, were killed by the known topoisomerase II inhibitors amsacrine and doxorubicin. Parallel experiments in yeast expressing human topoisomerase IIalpha allowed the relative sensitivities of the fungal and host topoisomerases to be examined in the same genetic background. To compare the killing in vivo with drug inhibition in vitro, the recombinant C. albicans topoisomerase II protein was expressed and purified to near-homogeneity from S. cerevisiae yielding a 160 kDa polypeptide that displayed the expected ATP-dependent DNA-relaxation and DNA-decatenation activities. The enzyme, whether examined in vitro or complementing in S. cerevisiae, was comparably sensitive to amsacrine and doxorubicin. Our results suggest that potential topoisomerase II-targeting anti-fungal inhibitors can be identified and studied in S. cerevisiae.

Amino Acid Sequence↗
PubMed1997

Detection of a common mutation in factor V gene responsible for resistance to activate protein C causing predisposition to thrombosis.

Hereditary predisposition to thrombosis due to activated protein C resistance (APCR) has been attributed to a missense mutation in the factor V gene at nucleotide 1691 (G to A), causing replacement of arginine at codon 506 with glutamine. Using an RFLP-PCR assay to detect this mutation, we measured a prevalence of 3.3% in healthy Caucasians and 1.25% in healthy African-Americans. In addition, we evaluated a total of 90 consecutive specimens submitted to the coagulation laboratory at the Medical College of Virginia for the presence of this mutation. We compared our results for 78 of these specimens with the values measured by a modified partial thromboplastin assay, the COATEST. Twelve of the 90 samples could not be tested using the COATEST because the patients were undergoing anticoagulant therapy. One of the latter 12 specimens was positive by the RFLP-PCR test. Using the genetic test as the definitive assay and the cutoff value established for distinguishing between normal and abnormal results by the COATEST, the COATEST had a sensitivity of 50% and specificity of 93% for the detection of factor V mutation. Analysis of the 90 samples stratified by ethnic groups revealed a frequency of mutation of 13.3% for Caucasians and 6.88% for African-Americans, although with the present sample size, the difference was not statistically significant. Although the COATEST is technically simpler to perform than the genetic test for diagnosing the presence of the factor V mutation, its use for this purpose is limited due to low sensitivity. Thus where this disorder is clinically suspected, submission of the specimen directly for genetic testing by RFLP-PCR or equivalent assay should be considered.

Black People↗
PubMed1997

Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2 beta construct allows the study of topoisomerase II beta inhibitors in yeast.

We show herein that human DNA topoisomerase II beta is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants. A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta.

Amsacrine↗
PubMed1997

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Alleles↗
PubMed1997

Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones.

gyrA and parC mutations have been identified inn Streptococcus pneumoniae mutants stepwise selected for resistance to sparfloxacin, an antipneumococcal fluoroquinolone. GyrA mutations (at the position equivalent to resistance hot spot Ser-83 in Escherichia coli GyrA) were found in all 17 first-step mutants examined and preceded DNA topoisomerase IV parC mutations (at Ser-79 or Glu-83), which appeared only in second-step mutants. The targeting of gyrase by sparfloxacin in S. pneumoniae but of topoisomerase IV by ciprofloxacin indicates that target preference can be altered by changes in quinolone structure.

Anti-Infective Agents↗
PubMed1996

Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta.

Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for alpha. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase II beta (overexpressed and purified from yeast) and a direct comparison with the recombinant alpha isoform. DNA cleavage in plasmid pBR322 and SV40 DNA was induced by alpha or beta in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human alpha promoted DNA breakage at sites akin to those of beta, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for beta and 70 sites for alpha revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1. The result for recombinant alpha isoform is in agreement with previous studies using alpha purified from human cell lines. Thus, alpha and beta proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II alpha; the results presented here demonstrate that beta is an in vitro target with similar site determinants, strongly suggesting that beta should also be considered a target of mAMSA in vivo.

Amino Acid Sequence↗
PubMed1996

Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

Amino Acid Sequence↗
PubMed1996

Mode of action of GR122222X, a novel inhibitor of bacterial DNA gyrase.

GR122222X is a potent inhibitor of the supercoiling reaction of bacterial DNA gyrase. We show that this compound binds stoichiometrically to inactivate the ATPase activity of a 43-kDa N-terminal fragment of the B subunit and competitively inhibits the binding of a radiolabelled coumarin drug to N-terminal fragments of GyrB. These and other data suggest that GR122222X has a mode of action similar, but not identical, to that of coumarin antibiotics.

DNA Topoisomerases, Type II↗
PubMed1996

NorA plasmid resistance to fluoroquinolones: role of copy number and norA frameshift mutations.

Staphylococcus aureus NorA protein is a transmembrane multidrug efflux pump that confers low-level resistance to hydrophilic fluoroquinolones. The norA gene promoter is active in Escherichia coli HB101. We have examined the genetic basis of norA-mediated resistance in E. coli by introducing a wild-type norA gene into HB101 in plasmid pCL1921, pBR322, or pUC18 exhibiting copy numbers that spanned a 22-fold range. Increased ciprofloxacin resistance correlated with norA transcript levels seen by Northern (RNA) analysis. Thus, contrary to some reports, a wild-type norA gene confers fluoroquinolone resistance in E. coli in a copy-number-dependent fashion and does not require mutational activation. Interestingly, a multicopy pUC19norA derivative gave transformants exhibiting a range of resistance phenotypes. The norA gene of one transformant carried a single base deletion (ATACAAT to AACAAT; the deleted base is underlined) in the putative--10 Pribnow box resulting in a promoter down-regulatory mutation; a second plasmid had acquired a frameshift producing a null mutation at codon 112. These mutations override the dual resistance-growth-inhibitory phenotype of high-copy-number norA plasmids. The results have implications for using the standard E. coli HB101 system to assess NorA function and potentially for plasmid-borne transmission of norA-mediated drug resistance.

Anti-Infective Agents↗
PubMed1996

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.

Amino Acid Sequence↗
PubMed1996

Quantitative evaluation of dizziness characteristics and impact on quality of life.

Patients attending an outpatient clinic with the complaint of dizziness were given a set of five items to rank the magnitude of the frequency and severity of their dizziness and the impact of dizziness on their quality of life. They were given instruments to evaluate their degree of anxiety and depression. The impact of frequency and severity on the patients' daily activities, on the quality of life, and on the fear of becoming dizzy was self-rated by the patient. The score on quality of life was compared with that provided by the physician in a blind questionnaire. The items that were developed addressed the physical, functional, and emotional impact of dizziness and can be considered promising for the evaluation of the patients' degree of overall impairment.

Adolescent↗
PubMed1996

Drug sensitivity and sequence specificity of human recombinant DNA topoisomerases IIalpha (p170) and IIbeta (p180).

Effective anticancer agents, such as epipodophyllotoxins and anthracyclines, exert their antitumor activity through stabilization of cleavable topoisomerase II/DNA complexes, which may result in DNA breakage on detergent addition. Two isozymes (alpha and beta) of DNA topoisomerase II are present in human cells; however, their roles as drug targets have not been completely defined. We determined the in vitro isoenzyme sensitivities to VM-26 (teniposide) and 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (an anthracycline analog) and established the sequence selectivity of isoenzyme-mediated DNA cleavage. Human topoisomerases IIalpha and IIbeta were purified from yeast cells overexpressing the corresponding plasmid-borne cDNA. Enzyme sensitivities to drugs were measured by a DNA cleavage assay using 32P-labeled simian virus 40 DNA fragments, and cleavage sites were mapped using agarose and sequencing gels. Both isozymes were sensitive to the studied poisons. They stimulated similar cleavage intensity patterns in agarose and sequencing gels; however, minor differences could be detected. The results showed that local base preferences for DNA cleavage without drugs were different at positions -2 and -1. On the other hand, sequence specificities of VM-26 and 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin were identical for both isozymes and corresponded to those of the native murine enzyme. The identical drug sequence specificities suggested that molecular interactions of the tested drugs in the ternary complex are likely similar between the two isozymes. The current findings indicate that both topoisomerase IIalpha and IIbeta may be in vivo targets of antitumor poisons.

Animals↗
PubMed1995

Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta.

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants.

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions (alpha to theta) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.

Amino Acid Sequence↗
PubMed1995

Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters.

Human eosinophil major basic protein (MBP) is one of the principal mediators of injury to parasites and tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil action. Previous studies have identified an MBP gene promoter that generates a 1.0 kb mRNA transcript encoding MBP preproprotein which undergoes processing to the mature storage form. To investigate how the MBP gene is regulated, we have examined the identity and levels of the MBP transcripts both in precursor cells and in blood eosinophils. It was found that the gene was expressed from two upstream promoters, a distal promoter P1 in addition to the previously described promoter P2. Evidence for the second promoter was initially provided by isolation from a human HL-60 leukaemic cell cDNA library of a novel 1.6 kb MBP cDNA that was distinct from the known 1.0 kb cDNA. The complete nucleotide sequence of the 1.6 kb cDNA was determined, and showed that the two cDNAs had identical coding and 3' untranslated regions but differed in their 5' sequences. By isolating and sequencing MBP genomic clones from an arrayed chromosome 11 library, it was demonstrated that the MBP gene is composed of nine upstream exons and five coding exons. The 1.6 and 1.0 kb cDNAs arise by differential splicing of alternate MBP transcripts from promoters P1 and P2 respectively, located 32 kb apart in the genomic DNA. Primer extension analysis identified two transcription start sites at P1, neither associated with a typical TATA box motif. Northern blotting and reverse-transcription PCR analysis showed that the 1.0 kb mRNA was present at higher levels than the 1.6 kb species in immature cells including HL-60 and bone-marrow cells. By contrast, low levels of 1.6 kb mRNA transcripts predominated in differentiated blood eosinophils. The results are compatible with differential use of P1 and P2 promoters as a mechanism for regulation of MBP expression during eosinophil maturation.

Alternative Splicing↗