Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Amplification and molecular cloning of murine adenosine deaminase gene sequences

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.     

Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver.

The complexity of rat liver poly (A)+ messenger RNA (mRNA) has been measured by analysis of the kinetics of hydridization with both complementary DNA (cDNA) and single copy DNA. The complementary DNA-poly(A)+ mRNA hybridization reaction demonstrates the existence of three abundance classes representing 18, 37, and 45% of the cDNA and 4, 290, and 24 000 different 1800-nucleotide sequences respectively. The poly(A)+ mRNA driven single copy DNA hybridization reaction reveals a single major transition accounting for 1.9% of the haploid rat genome. The kinetics of the poly(A)+ mRNA driven single copy DNA reaction suggest that approximately 45% of the mass of the mRNA population contains over 95% of the complexity. Although higher than previous estimates, the base sequence complexities of rat liver poly(A)+ mRNA measured in these two ways are in good agreement, suggesting that the technique of poly(A)+ mRNA-cDNA hybridization may be used in approximating the complexity as well as abundance of a messenger RNA population. DNA-driven cDNA reactions reveal that about 10% of rat liver poly(A)+ mRNA is transcribed from repetitive sequences in the rat genome.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Maize phosphoenolpyruvate carboxylase. Cloning and characterization of mRNAs encoding isozymic forms

The isozymic forms of maize phosphoenolpyruvate carboxylase (P-enolpyruvate carboxylase) involved in photosynthetic CO2 fixation were shown by protein gel blot analysis to consist of 100-kDa subunits. The nonautotrophic isoform found in roots is comprised of 96-kDa subunits and is about 50-100-fold less prevalent. Further analysis of P-enolpyruvate carboxylase isoforms made use of cloned cDNA probes. Two cDNA clones were isolated from a library constructed from maize leaf poly(A) RNA. The largest clone was complementary to about 25% of P-enolpyruvate carboxylase mRNA, which is 3.4 kilobases in length. The quantity of P-enolpyruvate carboxylase mRNA in green, mature leaf tissue was estimated to be 0.20% of poly(A) RNA, whereas P-enolpyruvate carboxylase mRNA in roots was about 100-fold less prevalent. We used thermal denaturation of a P-enolpyruvate carboxylase cDNA probe hybridized to RNA gel blots to estimate the degree of sequence difference between mRNAs encoding different P-enolpyruvate carboxylase isoforms. There appear to be at least two prevalent P-enolpyruvate carboxylase mRNAs in green leaves which are significantly different in sequence, as are P-enolpyruvate carboxylase mRNAs in roots and shoots. The hybridization pattern of maize genomic DNA Southern blots indicates that P-enolpyruvate carboxylase is encoded by a small gene family.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.     

The messenger RNA sequences in human fibroblast cells induced with poly rI.rC to produce interferon

Induction of human fibroblast cells with poly rI.rC induces interferon mRNA which can be translated into interferon precursor in wheat germ cell free system or in Xenopus oocytes into biologically active interferon. The extent of gene expression in the poly rI.rC induced cells was compared to that of the uninduced cells by hybridization of the mRNA to complementary DNA. Homologous template driven hybridization of cDNA revealed the presence of two clearly defined transitions in the total poly A RNA from the induced cells; abundant class and a scarce class comprising approximately 37,000 diverse species of RNA. Heterologus hybridization of the cDNA with total uninduced mRNA showed that the majority of the mRNA sequences are the same in both the induced and uninduced cells. The results of the hybridization using cDNA prepared to the fraction enriched for interferon mRNA, however, showed that about 4% of the sequences present in the interferon enriched fraction are not present in the uninduced cells. These differences may result from the poly rI.rC induced alterations in gene expression.     

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.     

Cloning of the human oestrogen receptor cDNA

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.     

Sequence complexity of cDNA transcribed from a diverse mRNA population

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Identification of HCV-1b by Low-Density cDNA Microarray-Based Assay

A rapid and sensitive microarray assay for the detection of HCV-1b was developed in our laboratory and a cDNA fragment library for HCV-1b cDNA microarray probes was constructed. The full-length cDNAs of HCV-1b were digested with restriction endonuclease Sau3A I and the fragments were cloned with the pMD18-T vectors. Positive clones were isolated and identified by sequencing. The cDNA microarray was prepared by spotting the gene fragment on the surface of an amido-modified glass slide using the robotics system and samples were fluorescent labeled by the restriction display PCR (RD-PCR) technique, In the present study, modified protocols were used for probe selection and hybridization temperature. The detection of a microarray was validated by the hybridization and the sequence analysis. A total of 22 different specific gene fragments of HCV-1b ranging from 250 to 750 bp were isolated and sequenced, and these fragments were further used as probes in the microarray preparation. The diagnostic validity of the microarray method was evaluated after the washing and scanning process. The results of hybridization and sequence data analysis showed a significant specificity and sensitivity in the detection of HCV-1b RNA. The method of preparing microarray probes by construction of cDNA fragments library was effective, rapid, and simple; the optimized microarray was sensitive in the clinical detection of HCV-1b. The RD-PCR technique for the sample labeling was useful for significantly increasing the sensitivity of the assay. The cDNA microarray assay can be widely used in the clinical diagnosis of HCV-1b.     

Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia.

Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Expression of catalase and myeloperoxidase genes in hydrogen peroxide-resistant HL-60 cells.

We studied the expression of catalase and myeloperoxidase genes in the hydrogen peroxide-resistant variants of human myeloid leukemia HL-60 cells HP50-2 and HP100-1. Southern blot hybridization with catalase and myeloperoxidase cDNA probes indicated that the copy number of the catalase gene in HP50-2 and HP100-1 cells was two and eight times, respectively, higher than that in HL-60 cells, whereas the copy number of the myeloperoxidase gene was the same. The amplified catalase and c-myc genes in HP100-1 cells were not decreased by treatment of the cells with inhibitors of poly(ADP-Ribose) polymerase, such as nicotinamide and benzamide. RNA blot hybridization with cDNA probes indicated that the content of catalase mRNA in HP50-2 and HP100-1 cells was four and 16 times higher, respectively, than that in HL-60 cells. By contrast, the content of myeloperoxidase mRNA in HP50-2 and HP100-1 cells was only a few percent of that in HL-60 cells. Furthermore, fluorescent in situ hybridization of a catalase cDNA probe to chromosomes indicated that the catalase gene in HP100-1 was amplified in the p13 region of a derivative chromosome 11. These results indicate that the increased synthesis of catalase in these resistant cells is mainly due to increased expression of the catalase gene, and that the lack of myeloperoxidase synthesis in these cells is due to the absence of its mRNA.