PU.1 is a transcription factor present in B-cells and macrophages. Here, we report our studies on the role of PU.1 in myelopoiesis using human neutrophil elastase (HNE) as a model. HNE, a component of the primary granules of mature granulocytes, is a serine protease which is transcriptionally restricted to the late promyelocytic stage of granulocytic maturation. The first 200 bp of the HNE promoter directs myeloid specific expression of a reporter gene and a 30-bp element within this region was been identified as the major determinant of myeloid specific expression [S. Srikanth, T. Rado, A 30-bp element is responsible for the myeloid specific activity of the human neutrophil elastase promoter, J. Biol. Chem. 269 (1994) 32626-32632.]. We now show that the B-cell and macrophage specific transcription factor, PU.1, binds to the PU.1 consensus site within the 30-bp element to activate transcription. Substitution mutations within this recognition sequence results in the loss of PU.1 binding and in a 90% decrease in promoter activity in myeloid cells. Cotransfection of PU.1 and a reporter gene controlled by the HNE promoter into non-myeloid HeLa cells resulted in activation of reporter gene transcription.

A 42-year-old Caucasian male with sporadic primary polycythemia has been followed by us for 13 years. During the time of observation, his hemoglobin had been stable, and he has never had an elevated white count or platelet count or any other stigmata of polycythemia vera (PV). Both of his parents, his three children, and all siblings have been hematologically normal. The in vitro culture of erythroid progenitors revealed an absence of autonomous erythropoietin (Epo)-independent erythroid colonies but demonstrated a marked increase in the sensitivity of erythroid progenitors to Epo. We have undertaken a study designed to determine whether a mutation in the Epo receptor (Epo-R) gene could cause the polycythemia phenotype seen in either dominant or recessive primary polycythemia described by us and others, or in polycythemia vera. We have sequenced the cytoplasmic positive and negative regulatory domains of the Epo-R genomic DNA, and a transversion of C to T in nucleotide 6148 was found in one of the patient's chromosomes. This mutation is located in the negative regulatory domain and results in a change from proline to serine (P488S). We have subsequently analyzed more than 40 chromosomes from unrelated normal subjects, as well as autosomal dominant, recessive, and sporadic primary polycythemia and polycythemia vera subjects. In no instance was the same or any other mutation in the Epo-R found. To determine if this Epo-R mutation is a cause of increased sensitivity of erythroid progenitors to erythropoietin, Ba/F3 cells (interleukin-3-dependent murine lymphoid line) were transfected with normal and mutated Epo-R cDNA, rendering the transfected cells viable and able to proliferate in Epo. Transfectants with wild-type and mutant Epo-R cDNA exhibited no difference in the presence of Epo. More recently, we were able to obtain DNA from the seven family members of the propositus and found that the nonpolycythemic mother and one of the siblings have the same Epo-R mutation. We conclude that this first described mutation of Epo-R encountered in humans does not appear on its own to explain the polycythemia phenotype; however, the possibility that it may interact with some other acquired or congenital abnormality in generating the polycythemia phenotype cannot be excluded.

Human neutrophil elastase (HNE), a serine protease, is expressed only in the promyelocytic stages of granulocyte maturation. We examined several regions of the promoter for transcriptional activity and report that a 30-base pair (bp) element located between -76 and -106 in the 5'-flanking region of HNE is sufficient for myeloid-specific expression of HNE. Gel shift assays using nuclear extracts from myeloid and non-myeloid cells reveal several myeloid-specific complexes binding to the 30-bp element. Examination of DNA-protein interactions shows that at least two myeloid-specific proteins of 38 and 55 kDa bind to this element. DNase I protection analysis reveals two distinct footprints between -80 to -91 and -94 to -104 within this element. Transient expression studies using deletion constructs of the HNE 5'-flanking region show that the 30-bp element is active in myeloid cells K 562 and U 937 but not in HeLa cells. Internal deletion of this element results in a 60-85% loss of promoter activity in myeloid cells. Additional functional studies also show that a 19-bp region between -112 and -131 contributes to transcriptional activity of the elastase promoter as well.

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl- permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a ''CF phenotype.'' These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are responsible for cystic fibrosis. The CFTR gene has recently been identified and encodes a 6.5-kb mRNA transcript. Recent observations showing that CFTR expression increases during differentiation of epithelial cells suggested that CFTR may also be regulated in the liver in response to partial hepatectomy (PH). We studied the expression of CFTR in rat regenerating liver and investigated the mechanisms that regulate CFTR RNA levels during a 120-h period after PH. Northern and slot-blot analysis revealed a liver-specific biphasic increase of CFTR mRNA levels, which peaks at 2 and 24 h post-PH. In contrast to these findings, the mode of regulation of the homologous gene MDR-1 showed a clearly different pattern. Nuclear run-on analysis demonstrated increased levels of CFTR transcription corresponding to the time points where an increase in CFTR message was observed. Similarly, the beta-actin gene, which increases transiently during liver regeneration, showed increased nuclear run-on activity 4 h posthepatectomy, indicating that the nuclei were functional. No increase of MDR-1 gene transcription was detected, confirming the previous finding that the increase in MDR-1 mRNA level in regenerating liver results from a post-transcriptional event such as message stabilization. This study indicates that expression of the CFTR gene is regulated during the regenerative process of the liver. The data also suggest that the increase in CFTR and MDR expression levels result from two distinct regulatory mechanisms.

We complemented the Cl‐ conductance defect in cystic fibrosis lymphocytes by transfection with wild‐type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non‐CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector‐specific sequences. Both fluorescence and patch‐clamp assays showed that transfectants expressing wild‐type CFTR acquired properties previously associated with Cl‐ conductance (GCl) regulation in non‐CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist‐induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild‐type CFTR corrects the defects in Cl‐ conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR‐mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23-2q24 by chromosome microdissection-PCR (CMPCR). The distribution of HBSC I and II MRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain, and that the relative ratio of the two forms can vary as much as 7-fold between different regions.

We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (alpha subunit) gene sequence to chromosome 2q22-q23. When DNA from single, dissected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-specific 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome microdissection-polymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer significant advantages for other applications, such as cancer cytogenetics and linkage analysis.

Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin.

Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability ( P Cl ) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for P Cl increased specifically during the G 1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3′,5′-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of P Cl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

Cystic fibrosis (CF) is the most common disease caused by a single gene abnormality within the caucasian population. Its severity of expression in homozygotes varies widely, and the disease involves multiple organ systems. During the past few years, major advances in CF research have been made. These advances have occurred primarily in the fields of physiology and molecular genetics. As a result of these advances, it is now generally accepted that the basic defect in CF is the inability of an epithelial chloride channel to respond to adrenergic stimulation in affected organs. The recent major breakthrough in CF research is the localization of the CF gene and identification of the mutation responsible for the majority of cases of CF. In this article, the evidence which has led to this conclusion, as well as possible mechanisms by which a mutation in a single codon can produce the CF defects are reviewed. Finally, new approaches to the characterization of the CF gene by complementation of the defect in immortal cell lines displaying the transport phenotype associated with CF are discussed.