Proc. Natl. Acad. Sci. USAVol. 84, pp. 3753-3757, June 1987Genetics

Complementation of areA- regulatory gene mutations of Aspergillusnidulans by the heterologous regulatory gene nit-2 ofNeurospora crassa

(gene regulation/nitrogen-metabolite repression/transformation)

MERYL A. DAVIS AND MICHAEL J. HYNESDepartment of Genetics, University of Melbourne, Parkville, Victoria 3052, Australia

Communicated by Norman H. Giles, January 28, 1987

ABSTRACT Loss-of-function mutations in the regulatorygene areA of Aspergillus nidulans prevent the utilization of awide variety of nitrogen sources. The phenotypes of nit-2mutants of Neurospora crassa suggest that this gene may beanalogous to the areA gene. Transformation has been used tointroduce a plasmid containing the nit-2 gene into A. nidulans.The nit-2 gene of Neurospora complemented mutations in theareA gene, restoring the ability to use a variety of nitrogensources. This indicated that the activator function of nit-2 andareA gene products was retained across these two fungalspecies. Southern blot analysis revealed both single-copy andmulticopy integrations and, in at least one case, integrationappeared to generate a nit-2 mutation. Integration of thetransforming plasmid appeared to be by nonhomologous eventsat a number of different sites in the Aspergillus genome. Thetransformants were less sensitive to nitrogen-metabolite re-pression of extracellular protease activity and nitrate reductase(EC 1.6.6.3) than were wild-type A. nidulans. This indicatedthat nitrogen control was not completely normal in the nit-2transformants.

Gene transfer techniques have been used recently to inves-tigate the regulation of heterologous genes in eukaryotes. Insome cases evidence for essentially normal regulation hasbeen found, for example, in transgenic mice and tobacco(1-3). This implies that regulatory genes and their sites ofaction may be conserved during evolution. Here we showdirectly that a regulatory gene from one fungal species canfunction in another species.

Nitrogen-metabolite repression is a widespread regulatoryphenomenon in microorganisms. Studies with fungi haverevealed a complex situation in which mutations in a varietyof genes alter the response of the cell to changes in nitrogenstatus (4, 5). In Aspergillus nidulans, previous work hassuggested that the areA gene plays a central role in nitrogencontrol. The areA gene product has been shown to be aprotein (6), and mutants are available that result in loss offunction, altered activator specificities, or derepression(7-9). Loss-of-function (areA-) mutants have lost (partiallyor totally) the ability to use nitrogen sources other thanammonium, whereas mutations that affect areA specificitiesresult in allele-specific alterations of growth responses on avariety of nitrogen sources. The properties of these mutantsindicate that the areA protein controls the expression ofunlinked structural genes by binding at a recognition site toactivate their expression. The derepressed class of areAmutants comprises two mutations generated by chromosomalrearrangements (5), and, therefore, their status in relation toareA function is unclear.

Studies in Neurospora crassa have identified the nit-2gene, which is postulated to work in an analogous fashion tothe areA gene. The nit-2 gene has been characterized byloss-of-function mutations, and although derepressed alleleshave been sought, none have been isolated (4). The isolationof nonsense suppressible nit-2 mutations indicates that thenit-2 gene product is a protein (10). Studies in both organismshave pointed to glutamine as the key effector of nitrogen-metabolite repression (4, 5).Given the apparent functional similarity between the areA

gene of A. nidulans and the nit-2 gene of N. crassa, we haveused transformation studies to determine whether the nit-2gene product could replace the function of the areA gene inareA- mutants ofA. nidulans. Our results show that the nit-2gene complements areA- mutations allowing transformantcolonies to utilize a variety of nitrogen sources. Furtheranalysis has shown that nitrogen-metabolite repression in thetransformants differs from nitrogen control in the wild-typestrain.

MATERIALS AND METHODSStrains, Media, and Genetic Methods. The areA- strains

used in transformation experiments were yAl areA217prnA309 riboB2 or yAl areA19 pyroA4 nicB8. The areA217and areA19 mutations have been described previously (8, 11).Transformants were crossed to the wild-type strain biAlniiA4 and to a biAl areA102 niiA4 strain. The wild-type strainused for nitrate reductase assays was biAl. For haploidiza-tion analysis, a pabAl galAl pyroA4 facA303 nicB8 prnC6Jstrain was used. All markers have been described previously(12). Genetic analysis was by standard techniques (13), andbasic media have been described (14).

Transformation. Protoplasts were prepared and trans-formed as described by Tilburn and coworkers (15). Trans-formed protoplasts were plated on selective protoplast me-dium containing the appropriate nitrogen source as describedunder Results.

Nitrate Reductase Assay and Protein Determination. Nitratereductase (EC 1.6.6.3) was assayed as described (14), withmodified controls (16). Results are expressed as milliunits permilligram of soluble protein, where a unit is defined as theamount of enzyme catalyzing the reduction of 1 ,umol ofnitrate to nitrite per minute. Soluble protein was estimated byusing the Bradford procedure (17) and commercially avail-able reagents (Bio-Rad).

Molecular Methods. Isolation of Aspergillus DNA andSouthern blot analysis were as described (18), except that theprehybridization solution contained 50% deionized formam-ide, 4 mM EDTA, 32 mM NaOH, 40 mM NaH2PO4, 0.72 MNaCl, 1% NaDodSO4, 0.5% skim milk powder, and 0.05 mgof denatured herring sperm DNA per ml. Hybridizationsolution contained 47% deionized formamide, 3 mM EDTA,24 mM NaOH, 30 mM NaH2PO4, 0.54 M NaCl, 1% NaDod-

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S04, 0.5% skim milk powder, and 10% dextran sulfate.Nitrocellulose filters were hybridized with 32P-labeled nick-translated pNIT2 plasmid for 18 hr at 42°C and were washedat 65°C under stringent conditions (18).

RESULTS

Transformation of areA- Mutants. The nit2+ gene of N.crassa was cloned on a cosmid that carried an insert of at least40 kilobases (kb) of Neurospora DNA (19). An EcoRIfragment of -6 kb was subcloned into the EcoRI site ofplasmid pUC8 to make pNIT2 (G. Marzluf, personal com-munication). pNIT2 was transformed into several differentareA- mutants of A. nidulans. The Neurospora gene wasfound to complement the areA217 mutation (8) and theareA19 mutation (11), both ofwhich prevent the utilization ofmost nitrogen sources by A. nidulans. Transformants wereobtained by direct selection for growth on either nitrate orglutamate as sole nitrogen sources. In addition, the pNIT2plasmid was introduced into an areA217 strain by cotrans-formation with a second selectable plasmid. The cotrans-forming plasmids used were pAN222 (20), containing thegenes of the prn cluster, which allowed selection oftransformants on proline as a sole nitrogen source in theareA217 prnA309 strain or p3SR2 (18), containing the amdSgene, which allowed selection of cotransformants on aceta-mide as a nitrogen source. Transformants obtained by prolineor acetamide selection grew on a variety of nitrogen sources.Control transformation experiments in which areA217 strainswere transformed with either pAN222 or p3SR2 alone yieldedtransformants able to grow only on proline or acetamide,respectively. These transformants retained an areA217 phe-notype on all other media.Growth Properties of pNIT2 Transformants. A more de-

tailed analysis was undertaken on pNIT2 transformants of anareA2J7 strain obtained by direct selection for growth onnitrate as a sole nitrogen source. The twelve transformantschosen were representative of the phenotypic classes ob-tained in the transformation experiment. The growth prop-erties of these strains on a variety of media are given in Table1, and examples are shown in Fig. 1. In the majority of cases,the nit-2-containing transformants showed growth properties

resembling those of the wild type (areA+) such as stronggrowth on nitrate, glutamate, and uric acid and poor growthon nitrogen sources such as histidine and acetamide, whichare used poorly by wild type A. nidulans. Slight differencesin growth or colony morphology distinguished the transform-ants from the wild-type strain. By contrast, three transform-ants, TNIT7, TNIT20, and TNIT22 showed a restoration ofgrowth only on particular nitrogen sources in addition tonitrate (Table 1). The TNIT22 strain was the only one that didnot grow as strongly as the wild type on nitrate as a nitrogensource. Additional growth tests showed that TNIT1 andTNIT22 were temperature sensitive for nitrate utilization at420C.

Southern Blot Analysis. Each of the trarisformants pro-duced a unique Southern blot pattern in EcoRI and Pvu IIdigests of genomic DNA probed with nick-translated pNIT2plasmid (Fig. 2A and B). A preliminary restriction map ofthepNIT2 plasmid (G. Marzluf, personal communication) isshown in Fig. 2C. Evidence from Southern analysis indicatedthat the pNIT2 plasmid was present either in a single copy(TNIT1, 7, 30, and 32) or in tandem arrays, with or withoutadditional rearrangements in multicopy transformants(TNIT3, 5, 6, 19, 20, 27, and 28). Multicopy tandem integra-tions were shown by the presence of plasmid-sized bands inbQth digests. The TNIT22 transformant appeared to containmulticopies of a rearranged plasmid. In single-copy trans-formants, a plasmid-sized band is replaced by different bandsgenerated by integration into the Aspergillus genome. Fromthe EcoRI digests, integration was found to be via the insertsequences of the pNIT2 plasmid in TNIT1, TNIT7, andTNIT32 and via vector sequences in TNIT30. Pvu II digestsshowed that in TNIT30, integration was adjacent to theinsert. Preliminary data (not shown) suggest that the TNIT7transformant was generated by an integration event in thesmall Pst I/Pst I fragment ofthe pNIT2 plasmid (see Fig. 2C).The unusual phenotype of this transformant suggested thatthe nit-2 gene extends into this region and that the integrationevent has generated a novel nit-2 "allele," possibly with analtered 3' sequence. It is likely that the rearrangement of theplasmid in TNIT22 also generated an altered nit-2 sequenQe.

Genetic Analysis. The pattern of integration of the pNIT2plasmid into the Aspergillus genome in the transformants was

Table 1. Growth properties of transformants

MediumMilk + Milk +

Uric Milk ammonium glutamineStrain Nitrate Glutamate acid Formamide Histidine Acetamide A B A B A B

WT +++ +++ +++ +++ + + +++ 1 +++ 0 +++ 0areA217 - - - - - _ _ 0 +++ 0 ++ 0TNIT1 +++ ++ ++ +++ + + +++ 3 +++ 2 +++ 1TNIT3 +++ +++ +++ +++ + + +++ 8 +++ 6 +++ 2TNIT5 +++ +++ + + + + + +++ 2 +++ 2 +++ 1TNIT6 +++ +++ +++ + + +++ 7 +++ 4 +++ 1TNIT7 +++ + + + - - +++ 1 +++ 1 +++ 1TNIT19 +++ +++ +++ +++ + + +++ 4 +++ 2 +++ 1TNIT20 +++ + + + - - +++ 4 +++ 2 +++ 1TNIT22 + - + + - - ++ 1 +++ 1 +++ 0TNIT27 +++ ++ ++ +++ + + +++ 3 +++ 2 +++ 1TNIT28 +++ +++ +++ +++ + + +++ 5 +++ 1 +++ 0TNIT30 +++ +++ +++ +++ + + +++ 8 +++ 6 +++ 2TNIT32 +++ ++ +++ ++ + + +++ 6 +++ 3 +++ 2

Growth was scored relative to the wild-type strain on each medium. The symbols used range from + + + (strong growth) to - (negligiblegrowth). Scoring across media is not strictly comparable. Milk clearing was scored on a numerical system from 0 (no detectable clearing) to8. Plates containing skim milk protein were scored both for growth (A) and milk clearing (B). A halo of milk clearing was used as an indicatorof extracellular proteise levels (9). All growth tests were carried out at 370C on glucose minimal media with the exception of acetamide platesthat contained sucrose as the carbon source. Nitrogen sources were used at a concentration of 10 mM except skim milk protein, which was addedat a final conceptration of 1%.

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4'

4T 0

~~ NJJ

4c

N.0

4'/

4)

areA217

TNIT 1

TNIT3

TNIT7

TNIT 19

TNIT22

WT

FIG. 1. Growth properties of transformants. Growth tests weredone on glucose minimal media to which the appropriate nitrogensource was added to a final concentration of 10 mM. Plates wereincubated at 370C for 2 days.

investigated by genetic analysis. The phenotypic propertiesof the transformants (Table 1) were found to segregatetogether in the progeny of outcrosses to a wild-type strain.The appearance of areA217-type progeny in the outcrossessuggested that the plasmid had not integrated into the areAgene in any of the transformed strains. However, in themajority of cases where the growth properties of the

A

"I 'V 4, t A ) A, R) 0

e' e AeZ e A~ A,$ Ate ,QC

transformants were similar to those of the wild-type strain, itwas not possible to reliably identify areA' nit-2 recombinantprogeny from the parental areA217 nit-2 types. Therefore,plasmid instability through meiosis generating the areA-phenotype could not be excluded in these crosses. Mitoticstability was found to be high for the transformants, althoughmulticopy transformants occasionally produced areA- sec-tors under nonselective conditions. Haploidization analysisconfirmed the- nonhomologous integration sites for some ofthe transformants; TNIT1, TNIT7, and TNIT19-chromo-some VII, TNIT20-chromosome VIII, TNIT32-chromo-some IV. Only TNIT22 was found to carry an integration onthe same chromosome as the areA gene (III); howeverrecombinational studies showed that the integration wasunlinked to the areA locus.The progeny ofthe cross between TNIT1 and the wild-type

strain further demonstrated the complementarity betweenthe nit-2 and areA gene products. The temperature sensitivityof the TNIT1 "allele" meant that at 370C, but not at 420C, thenit-2 gene product allowed areA strains to utilize nitrate. Inrecombinant progeny containing the areA' allele, the defi-ciency in the TNIT1 nit-2 product at 420C was, in turn,compensated for by a functional areA product, allowinggrowth on nitrate. Crosses were also made between sometransformant strains and strains carrying another areA mu-tation, areA102 (21). The areA102 mutation results in an areAproduct with altered specificities leading to increased growthon acetamide and acrylamide and reduced growth on uricacid as sole nitrogen sources compared with the wild-typestrain. In progeny of cross between TNIT1, TNIT3, TNIT7,or TNIT19 and areA102 strains, the double mutants hadregained the ability to use uric acid but were slightly weakeron acetamide or acrylamide than the areA102 strain alone. Itis possible that the phenotype of these double mutants isindicative of an interaction between the areA and nit-2

B

fA, 0,Ae&e A,4.4.4.4. 4. 4 4. '~~~~~~ ~ 4. 4.

- aom -. am_~~~~~oi- 23.1 - _-

m- 9.42 - - _

~ 6.56- 4.37

- 2.32 -o 2.03

"Wom -

a---._

- 23.1_ 9.42- 6.56

_4a3aO 4.37

- o 2.32O 2.03

0 0.56

0.56

EcoRI Pvu 11

C

Pst Pvu 11EcoRl PVU 11

81 1-- t~~~~I

PUC8

Pst Pst EcoRII II

nit-2

FIG. 2. Southern blot analysis oftransformants. Autoradiographs of (A) EcoRI digests and (B) Pvu II digests of transformants and the pNIT2plasmid. Southern blots were probed with 32P-labeled nick-translated pNIT2 plasmid. The standard used was end-labeled X cut with HindIll,and fragment sizes are given in kb. (C) Restriction map of the pNIT2 plasmid containing Neurospora crassa DNA inserted at the EcoRI siteof pUC8. The plasmid is -9 kb in length, and the direction of transcription of the nit-2 gene is shown (G. Marzluf, personal communication).

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products or competition at their site of action. In all crosses,the milk clearing phenotype of the transformant strains wasretained independent of the status of the areA gene (are217,areA', or areA102).

Nitrogen Metabolite Repression Control. As evidenced bythe growth properties of most of the transformants, the nit-2gene product of N. crassa has similar affinities for a varietyof Aspergillus structural genes as does the areA product.However, one distinguishing property of many of thetransformants was an increased level of extracellular prote-ase production compared to wild-type A. nidulans (Table 1).Plate tests also indicated that these transformants producedsubstantial, although reduced, levels of milk clearing in thepresence of either ammonium or glutamine, conditions underwhich extracellular protease activity is not detectable in thewild type. This is illustrated in Fig. 3. Extracellular proteaseproduction is under areA control in Aspergillus and isabolished in areA- mutants. The phenotypes of thetransformants suggested that the nit-2 gene product maypromote a greater level of expression than the areA geneproduct and, more significantly, may not be as sensitive tothe signals for repression.To further quantify differences between the areA and nit-2

gene products, the nitrate reductase enzyme (EC 1.6.6.3) wasassayed in the transformants under repressed and dere-pressed conditions (Table 2). The results indicated clearlythat all nit-2-containing strains were partially derepressed forthe synthesis of the nitrate reductase enzyme in the presenceof either ammonium or glutamine. Furthermore, the inducedlevels were also found to be higher (up to 2-fold) in thetransformants than in the wild-type strain. The high nitratereductase levels of the TNIT22 strain suggested that thepoorer growth of this strain on nitrate must be due to effectsat another step of nitrate utilization or other unknownmetabolic defects. The elevated levels in the transformants

v.

areA217

TNIT 1

TNIT3

TNIT7

TNIT19

TNIT22

WT

FIG. 3. Growth and extracellular protease activities of transform-ants on milk protein media. Skim milk protein was added to glucoseminimal medium at a concentration of 1%. Ammonium tartrate andglutamine were used at a final concentration of 10 mM. Plates wereincubated at 370C for 2 days. A halo of milk clearing around thecolony was used as an indicator of extracellular protease activity (9).

Table 2. Nitrate reductase activity of transformants

Medium

Nitrate + Nitrate +Strain Nitrate ammonium glutamine Alanine

WT 25.9 0.9 0.8 0.4areA217 <0.1 <0.1 <0.1 NDTNIT1 40.8 36.8 28.5 0.7TNIT3 32.0 17.1 22.4 0.6TNIT5 40.9 37.6 30.5 NDTNIT6 41.6 22.2 30.0 NDTNIT7 29.5 13.0 17.5 NDTNIT19 48.6 32.7 35.2 0.8TNIT20 31.1 29.8 27.2 NDTNIT22 43.2 12.5 27.1 0.8TNIT27 47.1 17.3 22.5 NDTNIT28 56.7 34.8 29.5 0.5TNIT30 42.6 30.4 33.4 NDTNIT32 59.8 43.7 33.6 ND

Nitrate reductase activities were determined at least in duplicate.Mycelia were grown at 370C for 16 hr in glucose minimal media with10 mM ammonium before transfer to media containing the appro-priate nitrogen source(s) at a concentration of 10 mM for 4 hr. ND,not determined.

were not due to altered inducibility. The uninduced nitratereductase levels, as measured in alanine-grown mycelia,were comparable to the wild-type levels in all transformantstested (Table 2). Nitrate reductase levels were also assayedin derivatives of the TNIT1 and TNIT3 strains containing theareA+ allele. The properties of these strains were indistin-guishable from those of the original strains carrying theareA217 mutation (data not shown). The level of expressiontherefore appears to be a property of the nit-2 allele carriedby the strain rather than any interaction with the residentareA allele.

DISCUSSIONTransformation of areA- strains with the pNIT2 plasmidcarrying the nit-2 gene of N. crassa showed that theheterologous regulatory gene can be expressed in A. nidulansand that the regulatory protein functions to activate expres-sion of nitrogen-regulated genes in this organism. The growthproperties of the transformants suggest that the nit-2 geneproduct recognizes the appropriate regulatory sequencesadjacent to the various structural genes in a way comparableto the native areA protein. The functional similarity betweenthe regulatory products was also apparent where one productsubstituted for deficiencies in the other when present in thesame nucleus. An example of this was described above wherea functional areA product restored the ability of the temper-ature-sensitive TNIT1 strain to utilize nitrate at 420C. Clearlythere are differences in the apparent efficiencies with whichthe nit-2 product and areA products activate expression ofmany, if not all, nitrogen-regulated genes. However, relativeaffinities appear to be similar for both regulatory geneproducts.

It appears that the similarities between these products canbe disturbed if the integration event involves the integrity ofthe nit-2-coding sequence. In these instances, the transfor-mation event would appear to be generating nit-2 mutationsin vivo. In this context, it is interesting to note that Perrineand Marzluf (10) were able to generate a nonsense mutationin the nit-2 gene that resulted in a truncated protein withaltered affinity for a variety of N. crassa structural genes.The ability of the nit-2 gene to complement areA mutants

suggests that there has been a strong conservation of con-formation and functionality between these two regulatoryproteins despite the evolutionary distance between the two

-A: :;

k:- :: :iAnnnnHnrnAn_Day.s...........,...

..

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species. However, probing oftotal A. nidulans genomic DNAwith the pNIT2 plasmid has revealed only weak homologythat has been insufficient for use in screening an Aspergillusgene library. The possibility remains that there are smallareas of sequence conservation that would be detected indirect hybridization between cloned areA and nit-2 genes ashas been reported for the gdhA and am genes of A. nidulansand N. crassa (22).

Plate tests and enzyme assays have provided direct evi-dence that, at least for those systems studied, the nit-2 geneproduct leads to partial derepression of enzyme synthesis inA. nidulans, irrespective ofwhether a wild-type areA productis simultaneously produced. This is suggestive that the areAproduct does not assume a repressor function under repress-ing conditions. The interpretation of the nit-2 result isdifficult, as the mechanism by which repression is achievedhas not been established. In the presence of metabolitesderived from growth on ammonium or glutamine, the areAproduct may assume a conformation incompatible withactivator function, or, alternatively, the synthesis of the areAproduct itself may be curtailed by a means of autoregulationor the intervention of a second regulatory product thatcontrols areA synthesis. Given this level of uncertainty, anumber of possibilities exist. The nit-2 product may notregister the signal for the onset of repression conditions. Ifthis signal is received via direct interaction between the nit-2or areA product and a low molecular weight effector, there isno reason to assume that this signal would not be recognizedin Aspergillus as it is in Neurospora, unless the nit-2 productitself affects the distribution of this metabolite. Similarly, ifthe nit-2 gene is autoregulated, there is no obvious reasonwhy such control should not be retained when the intact gene(see below) is expressed in Aspergillus. The involvement ofan additional hierarchy of regulatory gene(s) controlling thesynthesis of nit-2 product could therefore be suggested. Thiswould imply that such regulatory genes are either absent fromAspergillus or do not interact correctly with the nit-2 gene ornit-2 gene product. Mutations in the Neurospora gene definedby the nmr-J and ms-S mutations may identify such ahigher-order regulatory locus (ref. 23; G. Marzluf, personalcommunication). A further alternative is that the partialderepression of enzyme synthesis in the transformants mayindicate that the pNIT2 plasmid does not contain the entirenit-2 gene and that regions adjacent to the structural gene arenecessary for the regulated expression of the nit-2 gene.The finding that the heterologous regulatory gene can be

expressed and can function in A. nidulans has opened up newpossibilities of studying the activation and repression ofenzyme synthesis. The properties of the pNIT2 transform-ants may allow these aspects to be considered separately, anapproach that is not easily taken with the currently availableareA mutants in which derepression is associated with grosschromosomal rearrangements. The xprDI mutation, for ex-ample, is thought to have arisen by an inversion event whichhas placed the areA gene under the control of a new promoter(24). Such gross alterations make the function of the areAgene in nitrogen metabolite repression more difficult todissect. Clearly much remains to be understood about howcells register their nitrogen status and how this is translatedinto effective activation or repression of a variety of unlinkedstructural genes subject to nitrogen control. The finding thatthe cloned nit-2 gene of N. crassa can complement mutations

in the areA gene of A. nidulans will provide an additionalapproach to gaining this understanding.

Transformation in Aspergillus, as in Neurospora andmammalian systems, is integrative and is not strongly de-pendent on homology (25). Although the transformantsdescribed here appear to be generated by such nonhomolo-gous events, it is possible that transformants could beobtained in which the integration event generates a hybridareA-nit-2 gene. Furthermore, the areA gene has recentlybeen cloned (26), allowing a detailed comparison of thesegenes to be undertaken as well as reciprocal experiments tothose reported here in which expression of the areA gene inN. crassa is studied. Such approaches will provide valuableinformation about the conservation of regulatory sites andindicate sequences of functional importance to nitrogencontrol of gene expression by trans-acting regulatory mole-cules.

We are indebted to George Marzluf for providing us with thepNIT2 plasmid. We also thank Margaret Delbridge and NicoleTurner for their technical assistance. Support for this work wasprovided by the Australian Research Grants Scheme and a RowdenWhite Fellowship to M.A.D.

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