Immunological detection of pro-corticotropin releasing factor (CRF) in rat hypothalamus and pancreatic extracts. Evidence for in vitro conversion into CRF

Extracts of both rat hypothalamus and pancreas were analyzed for their corticotropin releasing factor (CRF)-like immunoreactivity by radioimmunoassay (RIA). In the case of the hypothalamus, besides the rat CRF, further identified by high-pressure liquid chromatography (HPLC), two peptide components, a 20-kDa and a 10-kDa species were detected. The 20-kDa component was stable under acidic pH conditions and was further purified by reverse-phase HPLC. When exposed to proteolytic activities coeluting with 'high-molecular-mass CRF' at pH 6, processing was observed and the CRF generated was identified both by RIA, molecular sieve filtration and HPLC under different experimental conditions. It is concluded that this 20-kDa CRF may represent the CRF precursor and that hypothalamic extracts may contain processing enzymes involved in its selective post-translational cleavage. In the pancreatic extract two immunoreactive forms of CRF were detected, the smaller coeluting with the rat CRF and the other corresponding to the intermediate 10-kDa component detected in the hypothalamus. Pancreatic rat CRF, analyzed using RIA both by molecular sieve filtration and HPLC, was indistinguishable from the hypothalamic rat CRF.     

Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.     

Posttranslational processing of concanavalin A precursors in jackbean cotyledons

Metabolic labeling of immature jackbean cotyledons with 14C-amino acids was used to determine the processing steps involved in the assembly of concanavalin A. Pulse-chase experiments and analyses of immunoprecipitated lectin forms indicated a complex series of events involving seven distinct species. The structural relatedness of all of the intermediate species was confirmed by two-dimensional mapping of 125I-tryptic peptides. An initial glycosylated precursor was deglycosylated and cleaved into smaller polypeptides, which subsequently reannealed over a period of 10-27 h. NH2-terminal sequencing of the abundant precursors confirmed that the intact subunit of concanavalin A was formed by the reannealing of two fragments, since the alignment of residues 1-118 and 119-237 was reversed in the final form of the lectin identified in the chase and the precursor first labeled. When the tissue was pulse-chased in the presence of monensin, processing of the glycosylated precursor was inhibited. The weak bases NH4Cl and chloroquine were without effect. Immunocytochemical studies showed that monensin treatment caused the accumulation of immunoreactive material at the cell surface and indicated that the ionophore had induced the secretion of a component normally destined for deposition within the protein bodies. Consideration of the tertiary structure of the glycosylated precursor and mature lectin showed that the entire series of processing events could occur without significant refolding of the initial translational product. Proteolytic events included removal of a peptide from the surface of the precursor molecule that connected the NH2- and COOH-termini of the mature protein. This processing activated the carbohydrate-binding activity of the lectin. The chase data suggest the occurrence of a simultaneous cleavage and formation of a peptide bond, raising the possibility that annealment of the fragments to give rise to the mature subunit involves a transpeptidation event rather than cleavage and subsequent religation.     

Characterization of endozepine-related peptides in the central nervous system and in peripheral tissues of the rat

Endozepines represent a novel family of regulatory peptides that have been isolated by their ability to displace benzodiazepines from their binding sites. All endozepines derive from an 86 amino acid precursor polypeptide called diazepam binding inhibitor (DBI), which generates, through proteolytic cleavage, several biologically active endozepines. The aim of the present study was to compare the molecular forms of endozepines present in different regions of the rat brain and in various peripheral organs using an antiserum raised against the central (biologically active) region of DBI. Combination of HPLC analysis and RIA detection revealed the existence of two major forms (peaks I and II) of endozepine-immunoreactive peptides. The retention times of the two peaks (36 and 39 min, respectively) were identical in all tissues or organs tested. Western blotting analysis of cerebral cortex extracts confirmed the existence of two immunoreactive species with apparent molecular weights 4000 and 6000 Da, which respectively correspond to peaks I and II. Tryptic digestion of peaks I and II generated a single immunoreactive peptide that coeluted with the synthetic octadecaneuropeptide ODN [DBI(33–50)]. These results show that, in different parts of the brain and in various peripheral organs, DBI is rapidly processed to generate two peptides of apparent molecular weight of 4000 and 6000 Da, which both possess the biologically active determinant of endozepines.     

Biosynthesis and processing of renal mitochondrial glutaminase in cultured proximal tubular epithelial cells and in isolated mitochondria

Primary cultures of rat renal proximal tubular epithelial cells were used to characterize the biosynthesis and processing of the mitochondrial glutaminase. When the cells were labeled with [35S]methionine in the presence of 20 microM carbonylcyanide m-chlorophenylhydrazone, only a 72-kDa peptide, which co-migrates with the primary translation product of the glutaminase mRNA, was immunoprecipitated. At lower concentrations of carbonylcyanide m-chlorophenylhydrazone, the 68- and 65-kDa peptides that are characteristic of the mature glutaminase and a 71-kDa peptide were synthesized. Pulse-chase experiments suggest that the 72-kDa cytosolic precursor could be quantitatively chased to generate the mature mitochondrial species. The observed kinetics indicate that the 71-kDa species is an intermediate in the import pathway. In addition, the 65-kDa glutaminase peptide was synthesized more rapidly than the 68-kDa peptide, and the two peptides were produced in a final ratio of 3:1, respectively. These results suggest that one subunit of the tetrameric glutaminase may be subject to covalent modification. In vitro processing was also characterized by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in vitro translation of acidotic rat renal poly(A+) RNA. This system produced an identical sequence of processing reactions. The in vitro formation of the 71-kDa intermediate required a transmembrane potential. Both the intermediate and the mature forms of the glutaminase were recovered in the mitochondria and were resistant to trypsin digestion. Thus, the glutaminase precursor is rapidly translocated across the inner mitochondrial membrane and initially processed to yield an intermediate. The intermediate is subsequently processed to yield the two peptides that constitute the mature enzyme.     

Identification of the released form of atrial natriuretic factor by the perfused rat heart

Atrial natriuretic factor (ANF), released by the isolated perfused rat heart, was extracted from the perfusates by C18 Sep-Pak cartridges and then isolated by immunoaffinity chromatography and by reverse phase HPLC. About 500 ng of immunoreactive material were so obtained and submitted to amino acid sequencing. The C-terminal Tyr was detected by radiolabelling. Identification of these residues indicated that the primary structure corresponds to ANF (Ser 99-Tyr 126) which is identical to the circulating form in the rat. These results indicate that the ANF released by the atria corresponds to a short peptide. Therefore, its maturation process may therefore take place either intracellularly or during secretion and implicates a tryptic-like cleavage after a single Arg residue in position 98.     

Biosynthesis and processing of rat alpha 1-antitrypsin

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.     

Sequence of the precursor to rat bone gamma-carboxyglutamic acid protein that accumulates in warfarin-treated osteosarcoma cells

A biosynthetic precursor to rat bone gamma-carboxyglutamic acid protein (BGP) was isolated from warfarin-treated ROS 17/2 osteosarcoma cells by antibody affinity chromatography followed by reverse phase high performance liquid chromatography. Thirty-two residues of its NH2-terminal sequence were determined by gas-phase protein sequence analysis. Comparison of this sequence with the known structure of rat BGP established that the intracellular precursor is a 76-residue molecule of Mr = 9120 that differs from 6000-Da bone BGP in having an NH2-terminal extension of 26 residues. This precursor appears to be generated from the primary translation product by cleavage of a hydrophobic signal peptide and is the probable substrate for gamma-carboxylation by virtue of its accumulation in the presence of warfarin. The putative targeting region for gamma-carboxylation previously identified in the leader sequences of vitamin K-dependent proteins is found in the propeptide portion of the precursor. Since the immunoreactive component secreted by warfarin-treated cells is identical in sequence to the 6000-Da BGP from bone, propeptide cleavage from the precursor is independent of gamma-carboxylation and precedes secretion of BGP from the cell.     

Identification of an urotensin I-like peptide in the pituitary of the lungfish Protopterus annectens: immunocytochemical localization and biochemical characterization

In the present study we have investigated the localization and biochemical characteristics of urotensin I (UI)-like and urotensin II (UII)-like immunoreactive peptides in the central nervous system (CNS) and pituitary of the lungfish, Protopterus annectens, by using antisera raised against UI from the white sucker Catostomus commersoni and against UII from the goby Gillichythys mirabilis. UI-like immunoreactive material was found within the melanotrope cells of the intermediate lobe of the pituitary. By contrast, no UI-immunoreactive structures were found in the brain. No UII-like peptides structurally similar to goby UII were found in the brain and pituitary of P. annectens. The UI-immunoreactive material localized in the pituitary was characterized by combining reversed-phase high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The UI-like immunoreactivity contained in a pituitary extract eluted as a single peak with a retention time intermediate between those of sucker UI and rat corticotropin-releasing factor (CRF). Control tests on adjacent sections of pituitary showed that the UI antiserum cross-reacted with the frog skin peptide sauvagine, but lungfish UI did not co-elute with synthetic sauvagine on HPLC. On the contrary, no cross-reaction was observed between the UI antiserum and CRF or alpha-melanocyte-stimulating hormone (alpha-MSH). The occurrence of an UI-like peptide in the intermediate lobe of the pituitary of P. annectens suggests that, in lungfish, this peptide may act as a classic pituitary hormone or may be involved in the control of melanotrope cell secretion.     

Identification of procalcitonin in a rat medullary thyroid carcinoma cell line

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.     

Atrial natriuretic peptide in lymphoid organs of various species

1. Evidence for the occurrence of atrial natriuretic peptide (ANP) in various lymphoid organs of different species (rat, mouse, pig, chicken) is provided. 2. ANP precursor material (1-126) as well the physiologically active ANP (99-126), were identified by chromatographic analysis and RIA in extracts of thymus, spleen and lymph nodes of rat, mouse and pig. 3. mRNA coding for ANP was demonstrated both in the thymus and in isolated thymocytes of these species. Furthermore, mRNA for ANP was detected in spleen and lymph nodes (rat and pig). 4. The bursa of Fabricius, thymus glands and spleen of chickens were also shown to express mRNA coding for ANP. 5. These findings provide a firm basis for a link of ANP to the immune system, a novel aspect of possible biological functions of this peptide.     

Rodent islet cell antigens recognized by antibodies in sera from diabetic patients

Rat islets, rat insulinoma cells and islets from three different mouse strains were labelled with 35S-cysteine and/or 35S-methionine. Detergent lysates of the cells were subjected to immunoprecipitation with sera from 5 newly diagnosed diabetic children and 5 control sera. The immunoprecipitates were analysed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography. One of the sera immunoprecipitated a protein of Mr 64K from lysates of rat islets, rat insulinoma cells, A. TH and NMRI but not CBA/H mouse islets. This protein was not consistently immunoprecipitated by the other diabetic sera, however, it was never found with control sera nor was it detected in rodent lymphocytes. Some proteins of lower molecular weight (59K, 57K, 40K, 29K) were specifically immunoprecipitated by one or more diabetic sera from some of the rodent islet cell preparations. It is concluded that rodent islet cells contain a protein of Mr 64K which may be antigenically related to a 64K protein previously detected in immunoprecipitates of human islet cells with the same diabetic sera. The variable results with rat and mouse islet cell material suggest that the level of cross-reactivity is low. Further studies are needed to clarify whether the lower molecular components detected in some immunoprecipitates represent other antigenic determinants or degradation products of the 64K protein.     

Synthesis and biological properties of ovine corticotropin-releasing factor (CRF)

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05-125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.     

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.     

Biosynthesis of an asparagine-linked oligosaccharide-containing calcitonin by a rat medullary thyroid carcinoma cell line

Calcitonin contains an amino acid sequence that provides a potential site for glycosylation of the peptide at the asparagine at position 3. Preliminary evidence has suggested that there are glycosylated forms of calcitonin and its precursor, procalcitonin. The CA-77 rat medullary thyroid carcinoma cell line, recently developed to study calcitonin biosynthesis, was used to demonstrate the synthesis of glycosylated forms of this hormone by intact cells. Cultures were incubated in medium containing either [3H]mannose or [35S]methionine. Two species incorporating both labels were specifically immunoprecipitated when cell extracts were treated with calcitonin antibodies. Gel filtration chromatography in 6 M guanidine hydrochloride indicated that one peptide had a molecular weight of 5500, approximately 2000 daltons larger than calcitonin, while the second peptide had a molecular weight of 14 400, the approximate size of procalcitonin. Treatment of the [3H]mannose-labeled cell extract with endo-beta-N-acetylglucosaminidase H before immunoprecipitation removed the labeled sugar from the calcitonin species. Microsequence analysis of the radiolabeled immunoreactive 5500-dalton calcitonin species showed methionine at cycle 8 and mannose at cycle 3, suggesting that this peptide is calcitonin containing an N-linked oligosaccharide at Asn-3. These results suggest that in this cell line a minor but significant biosynthetic pathway exists for the production of glycosylated calcitonin from glycosylated procalcitonin.     

Granular and extra-granular forms of immunoreactive calcitonin in normal rat "C" cells

The intracellular localization of the intermediate calcitonin-related peptides produced during calcitonin maturation (CT, 3.4 Kd) from the polyprotein precursor (15 Kd) has been studied by means of subcellular fractionation of normal adult rat thyroid using a continuous hypertonic Percoll gradient. A fraction B (Bottom), containing the secretory granules, was separated from a fraction T (Top) constituted of rough and smooth membranes devoid of granules. Immunoreactive calcitonin extracted from both fractions was heterogeneous when analysed by gel electrophoresis followed by radioimmunoassay. In fraction T, the immunoreactive molecules assayed were associated with the membranes. Their apparent molecular weights (lower than 18 Kd) were compatible with the intermediates predicted by the aminoacid sequence of the precursor. However the maturation was not complete, as indicated by the absence of the monomer. The smallest molecular weight detected (4-4.5 Kd) was compatible with a calcitonin-katacalcin uncleaved molecule. These data strongly suggest the occurrence of a cleavage separating the N-terminal part of the molecule during the pre-granular step of calcitonin secretory pathway. In the granular fraction, the monomer constituted the granular matrix, representing 65% of the total immunoreactivity. The remainder was associated with the granules membranes, consisting of the 4-4.5 Kd precursor and some 43 Kd material which has already been demonstrated by others to be covalent polymers of calcitonin [17]. It is thus concluded that a second cleavage could take place in the granules where the major immunoreactive form is the processed calcitonin, either as free monomer or as covalent polymers associated with the membrane of the granules.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Differential brain expression of the Alzheimer''s amyloid precursor protein.

The expression of the Alzheimer amyloid protein precursor (AAPP) was examined in human, monkey, dog and rat brains. Two proteins, one identified as AAPP695 and the other as AAPP751, were immunoprecipitated from the in vitro translation of human, dog and rat brain polysomes. The AAPP751 to AAPP695 ratio was highest in human, intermediate in dog and lowest in rat brain polysomes. Human cerebral cortex contained higher levels of the AAPP751 mRNA than either dog or rat cortex. AAPP695 was detected in both cerebral cortex and cerebellum of all species examined. In contrast, AAPP751 was detected predominantly in the cortex of human, monkey and to a lesser extent dog brains while it was not detected in rat brain. These findings indicate that the amyloid precursors are differentially expressed in different mammalian brains and suggest that AAPP751 is mainly expressed in the brain regions involved in plaque formation.     

Concomitant changes of ACTH, β-endorphin and N-terminal portion of pro-opiomelanocortin in rats

Radioimmunoassay developed to measure N-terminal peptide of pro-opiomelanocortin isolated from porcine pituitaries was used to measure changes in the concentration of immunoreactive material in rat plasma. The N-terminal peptide immunoreactive material decreased in plasma after hypophysectomy of both female and male rats below the level of detectability and substantially increased after adrenalectomy as compared to normal control rats. The same changes were observed when β-endorphin and ACTH like immunoreactive material was measured. The primary culture of rat anterior pituitary cells released ACTH and N-terminal peptide-like immunoreactive material into the incubation medium. The results seem to indicate that the N-terminal immunoreactive material is a secretory product produced by the pituitary gland.     

Characterization of complementary DNA encoding the rat neuromedin U precursor.

Neuromedin U (NmU), a peptide originally isolated from porcine spinal cord, is known for its ability to stimulate uterine smooth muscle contraction and to cause selective vasoconstriction. It was subsequently isolated from a number of species. Among the species studied, the five amino acids at the C-terminus of the peptide are totally conserved, suggesting that this region is of major importance. We have cloned and sequenced the cDNA encoding the rat NmU precursor protein using the anchor polymerase chain reaction technique. Sequence analysis revealed that NmU is synthesized as a 174-amino acid precursor. Like the precursors of most other small regulatory peptides, it has a hydrophobic signal peptide and a number of paired dibasic amino acids, which may serve as signals for enzymatic cleavage, to release NmU and a series of other peptides. These predicted flanking peptides of NmU show no significant homology with entries in the protein databases searched, and the cDNA likewise shows no homology with entries in the GenBank database. Northern blot analysis using total RNA extracted from different rat tissues shows high levels of NmU mRNA in the ileum, thyroid, and anterior pituitary. Southern blot analysis of rat genomic DNA demonstrates that NmU is a single copy gene.