Proc. Natl. Acad. Sci. USAVol. 90, pp. 8043-8047, September 1993Genetics

Sodium azide mutagenesis: Preferential generation of AT -> GCtransitions in the barley Antl8 gene

(anthocyanin/complementation/dihydroflavonol 4-reductase/microprojectile bombardment/RNA splicing)

OLE OLSEN, XINGZHI WANG, AND DITER VON WETTSTEINDepartment of Physiology, Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500 Copenhagen, Denmark

Contributed by Diter von Wettstein, May 24, 1993

ABSTRACT The molecular basis for the absence of an-thocyanins and proanthocyanidins in four independent sodiumazide-induced antl8 mutants of barley was examined by se-quencing the gene encoding dihydroflavonol 4-reductase inthese mutants. Sodium azide generated 21 base substitutions,which corresponds to 0.17% of the 12,704 nucleotides se-quenced. Of the substitutions, 86% were nucleotide transi-tions, and 14% were transversions. AT - G-C base pairtransitions were about 3 times more frequent than G-C -) ATtransitions. No deletions or mutation hot spots were found. Theabsence of dihydroflavonol 4-reductase activity in antl8-159,antl8-162, and antl8-164 plants is caused by missense muta-tions in the respective genes. By using microprojectile bom-bardment, a plasmid harboring the wild-type Antl8 gene wasintroduced into antl8-161 mutant cells and resulted in thedevelopment of anthocyanin pigmentation, which demon-strates that the mutation is corrected by expression of theintroduced gene. On the other hand, a plasmid derivative withthe two antl8-161-specific base transitions at the 5' splice siteof intron 3 prevented complementation. It is concluded that theabsence of detectable mRNA for dihydroflavonol 4-reductasein antl8-161 cells is due to the mutations in the pre-mRNAsplice donor site.

Sodium azide (NaN3) is the most efficient mutagen in barleyand has become the agent of choice for the induction ofmutants in this organism due to the ease with which themutagen is applied to dry and presoaked grains. Unlike otherphysical or chemical mutagens, it induces few, if any, chro-mosome aberrations (1-3). Up to 78% of chlorophyll-deficient mutant seedlings per M1 spike progeny can bescored. In Salmonella typhimurium sodium azide revertsonly base substitution mutants; the mutation process isfacilitated in strains deficient in excision-repair mechanisms(4). The first step in the azide mutagenic pathway in barleyand Salmonella has been elucidated: O-acetylserine (thiol)-lyase converts O-acetylserine with azide to ,-azidoalanine;azide substitutes for the natural sulfide substrate in thereaction (3, 5). Future experiments will have to show whetherthe f-azidoalanine reacts directly with the DNA bases caus-ing mispairing or whether further metabolic conversions (to,e.g., azidopyruvate) (6) are required for the interaction of theazide group with the DNA bases. While sodium azide is apotent mutagen in several other higher plants, Escherichiacoli, yeast, and the housefly, it fails to increase mutationfrequencies in Arabidopsis, Drosophila, and Neurospora andis only weakly mutagenic in mammalian cells (3).To date, the nucleotide changes induced by sodium azide

have not been investigated, although its high specificity hasbeen noted in Salmonella and E. coli (3, 4). Thus, only onemissense his mutation (G-46) of Salmonella could be reverted

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with high frequency, whereas several ochre, amber, andother missense mutations did not revert. We have thereforechosen the barley Antl8 gene and four of its azide-inducedmutant alleles to determine by nucleotide sequencing thebase changes elicited by this mutagen.The single-copy Antl8 gene encodes dihydroflavonol 4-re-

ductase (DFR), which catalyzes the conversion ofdihydrofla-vonoles into flavan-3,4-diols (7-9), the last common step inthe flavonoid biosynthetic pathway leading to anthocyaninsand proanthocyanidins (10, 11). Delivery of the structuralgene for DFR into mutant antl8-162 leaf sheath tissue bymicroprojectile bombardment gives rise to the synthesis ofanthocyanin in individual cells (12).Of the four mutant antl8 genes sequenced and compared

to their wild-type progenitor gene in the pure line Gula, threeare transcribed into normal-sized DFR mRNA, while thefourth (antl8-161) prevents formation of detectable amountsofDFRmRNA (9). The first analysis ofsodium azide-inducedmutant genes of barley revealed 21 individual base substitu-tions but no frameshift mutations. Microprojectile bombard-ment with chimeric plasmids containing either wild-type orantl8-161 DNA fragments to assay for anthocyanin pigmen-tation in antl8-161 mutant tissue identified two base transi-tions in the 5' splice site of intron 3 to be responsible for thelack of complementation.

EXPERIMENTAL PROCEDURESMutagenesis, Plant Material, and Growth Conditions. Bar-

ley (Hordeum vulgare L.) cv. Gula grains were presoaked for15 h in distilled water at 5°C and then treated in an oxygenatedsolution of 1 mM NaN3 at pH 3.0 for 2 h (13). The grains wererinsed and planted in the field. The M2 progenies were plantedthe following year (1978), and the homozygous mutantsantl8-159, antl8-161, antl8-162, and antl8-164 were se-lected.For the present analyses, grains were planted in potting

compost and grown in a growth chamber under controlledconditions for 6 days at 22°C in continuous light followed by9 days of incubation with a 16-h photoperiod at 15°C and 8 hin the dark at 10°C.For microprojectile bombardment, 2-cm-long pieces of

stem tissue surrounded by leaf sheath but without coleoptilewere used. Ten tissue segments were placed in one 5-cm Petridish and treated as described (12).

Cloning of Genomic DNA. Barley genomic DNA was iso-lated from 0.1 g ofwild-type or mutant leaftissue as described(14) and dissolved in 400 ,ul of water. An aliquot of 70 ,ul wasdigested with 200 units of HindIII for 15 min before desaltingand gel chromatography using Croma Spin-1000 columns(Clontech). Twenty microliters of purified DNA was used asa template for PCR. By using phosphorylated oligonucleotideprimers (30-mers) identical to the Antl8 gene sequence, four

Abbreviation: DFR, dihydroflavonol 4-reductase.

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partially overlapping fragments spanning 3176 bp of thechromosome segment were amplified from each mutant andwild-type gene. According to the published sequence (9), theproducts contain the following endpoints: -1283 to -606(fragment 1), -723 to 145 (fragment 2), -243 to 919 (fragment3), and 853 to 1893 (fragment 4) as illustrated in Fig. 1. Theamplification reactions were carried out for 25 cycles usingdenaturation at 94°C for 1 min, annealing at 60°C for 2 min,and primer extension at 72°C for 2 min before separation ofthe amplified products by agarose gel electrophoresis. DNAfragments of interest were purified from the gel by usingPrep-A-Gene matrix (Bio-Rad), ligated into pUC18 linearizedwith Sma I (Pharmacia), and cloned in E. coli DH5a cells(GIBCO/BRL). Plasmid DNA for sequencing was purifiedwith Qiagen columns.

Nucleotide Sequence Determination. Double-stranded plas-mids containing cloned gene fragments were sequenced withgene-specific oligonucleotide primers using a model 373ADNA sequencer (Applied Biosystems) according to the man-ufacturer's recommendations.

Construction of Chimeric Genes. Plasmid pgDFR, whichharbors the genomic 3188-bp Antl8 Kpn 1-HindIII fragment(9), was digested with Nco I, and the small fragment wasreplaced with the corresponding part ofPCR fragment 3 fromthe antl8-161 gene, giving plasmid pgDFR[NcoI-161],thereby introducing the mutated 5' splice site of intron 3derived from antl8-161 into the Antl8 sequence.PCR fragments 1, 2, and 3 (Fig. 1) were combined, and the

fragment spanning the region -1283 to 145 was amplifiedusing the outermost primers (15). Similarly, the resultingproduct was combined with fragment 4 (Fig. 1) to yield thefull-length antl8-161 gene, which was cloned in pUC18 givingpgDFR-161. For introduction of the wild-type 5' splice site of

Ant 18

ant 1 8-1 59

ant 18-1 61

ant 18-162

ant 18-164

TATATA AATAAA

ATG TAA 3188

A A T G A

G G C A G

T A GT

C G AC

T T T A G

A C C T A

AA GT C T AUWII I'H

G GAA CtAA T C G

2 4

1 3A A

N N

FIG. 1. (Upper) Diagram of the barley wild-type Antl8 gene (9)and localization of mutations identified in azide-induced antl8 al-leles. Only specific bases of the upper strand of the DNA sequenceare shown; these include the TATA and polyadenylylation signals aswell as start (ATG) and stop (TAA) codons for the gene productDFR. The transcribed region includes three introns, which areillustrated as white boxes. Vertical arrows denote the position ofazide-induced sequence alterations in the mutant genes, with thewild-type base above the mutated base. Except for these bases, thegene sequences are identical. (Lower) Location of PCR fragments1-4 generated with gene-specific oligonucleotide primers (horizontalarrows). Arrowheads show the position ofNco I (N) restriction sitesused for the construction of chimeric plasmids as detailed in the text.

intron 3 into the antl8-161 gene, the small Nco I fragment ofplasmids pgDFR-161 was exchanged with the similar frag-ment of pgDFR, giving plasmid pgDFR-161[NcoI-wt].

Restriction mapping and nucleotide sequence analysis con-firmed the structures of the chimeric genes.

Microprojectile Bombardment. Plasmid DNA for transferinto cells was purified with Qiagen columns. The DNA wasintroduced into intact barley antl8-161 leaf sheath tissue byhigh velocity microprojectiles using the delivery system PDS1000/He (DuPont) as described (12). After 5 days of incuba-tion at 15°C with illumination for 16 h alternating with 8 h inthe dark at 10°C in a growth chamber, tissues were visuallyanalyzed for transformed cells containing red anthocyaninpigmentation. Plasmid pEmuGN (16), which directs high-level expression of 3-glucuronidase in monocots, was used asan internal control to monitor the efficiency of gene transfer.The substrate 5-bromo-4-chloro-3-indolyl glucuronide wasused for the histochemical analysis of transfected tissue. Thisresults in a blue staining of cells expressing the 3-glucuroni-dase enzyme (17).

RESULTSAbsence of Base Deletions and DNA Rearrangements in the

ant18 Genes. The PCR products derived from wild-type andmutant plants were ligated into vector pUC18, and thenucleotide sequences were determined. To rule out thatmutations had been introduced during the amplifications,several control experiments were required. First, the ob-tained wild-type Antl8 gene sequence should be identical tothat previously obtained (9). Second, identification of amutation should be confirmed by sequencing of both DNAstrands in a region surrounding the mutation, as well as beingidentified either in an independent PCR product derived froman additional amplification or in an overlapping PCR frag-ment. The mutations described here fulfill these criteria,except for a single base pair that was absent in both thewild-type and the mutant gene promoters at position -386with respect to the transcription initiation site. Accordingly,the base is considered the only error in the original sequence.DNA sequencing uncovered no base deletions in the

azide-induced alleles of Antl8, which is consistent with theobservation that sodium azide, 3-azidoalanine, 3-azidoglyc-erol, azidoalcohols, and 6-deoxy-6-azidohexoses producebase substitutions in the S. typhimurium base pair substitu-tion mutation tester strains TA100 and TA1535 with a similarefficiency, but are nonmutagenic in the frameshift mutationtester strain TA98 (6). The results are also consistent with theabsence of chromosome aberrations in azide-mutated barley(3, 18).Base Transitions Predominate in the antl8 Genes. DNA

sequence analysis of the 3176-bp-long mutant genes revealedfive, four, five, and seven base substitutions in the antl8-159,antl8-161, antl8-162, and antl8-164 genes, respectively;thus, a total of 21 mutations were identified, which gives amutation frequency of 0.17% (Fig. 1 and Table 1). Mutantgene antl8-161 contained two contiguous base changes,whereas the remaining substitutions were scattered randomlythroughout the genes, which demonstrates that azide-induced mutagenesis is neither locally nor regionally targetedwithin the gene.

Table 1. Types of base substitutions among four 3176-bp-longantl8 genes

Base Transition Transversionsubstitutions A-T -- G'C G-C -- A-T A-T -- T-A

No. identified 13 5 3Percent of total 62 24 14

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Table 1 summarizes the distribution of the mutationsaccording to the type ofbase changes observed. The majorityof substitutions, 18 out of 21, were transitions. Both types oftransitions and only one type of transversion had occurred.Three times as many base substitutional events occurred atA-T as at G-C sites. Preferential modifications of adenine orthymine by organic azide may thus be the cause for mispair-ing.

Structure of the antl8-159, antl8-162, and antl8-164 Afleles.Mutant plants containing these alleles are without DFRactivity, anthocyanin pigmentation, and proanthocyanidins,although the levels of DFR-specific transcripts are similar tothat of wild-type plants (9). Since the wild-type codingsequence for DFR complements the antl8-162 mutations(12), it seems reasonable to suppose that the effect of theantl8 mutations on synthesis ofactive enzyme is restricted tothose aberrations identified in the DFR coding region (Figs.1 and 2). Given below is a summary of the base substitutionswith base number 1 corresponding to position -1283 of thepublished sequence (9).Three substitutions, A-203 -- G, A-596 -- G, and T-931

C, were identified in the 5' end of the promoter sequence ofantl8-1S9, and two transitions were identified in the protein-coding region. One, G-2163 -- A, introduces a basic Lysresidue for an acidic Glu at position 176 where heterogeneityexists among plant DFR enzymes (Fig. 2). The other muta-tion, A-2292 -* G, substitutes Ala for the conserved Thr-214.Mutations T-234 -* A and T-459 -- C were identified in thepromoter region of the mutant antl8-162 gene, and G-2947 -+A was identified in the 3' noncoding region. It also carries twomutations in the DFR coding region, T-1434 -* C and A-1849-* T, that alter conserved residues-namely, Val-32 -+ Alaand Lys-101 -+ Met, respectively. Two of the transitions inthe protein-coding region of antl8-164, C-1696 -- T andT-1940 C, are silent, whereas two base substitutions,G-1374 A and T-1386 -) A, change Gly-12 -) Glu andPhe-16--+ Tyr in the translated product. It seems unlikely thatthe mutations A-69 -* G and A-180 -+ G at the 5' end of thepromoter, or A-2080 -- G in the center of intron 3, cause the

absence of DFR activity because message was identified inantl8-164 plants.

Intron Mutations Affect Splicing of DFR mRNA in theantl8-161 Mutant. Analysis ofgenomic DNA from antl8-161plants identified four base substitutions. One, T-70 -+ C,located 1214 bases upstream of the transcription start point,seems unlikely to explain the absence of detectable DFRmessage (9), whereas the transition A-1406 -- G, whichsubstitutes Val for Met-23, is a potential missense mutation.In the intron 3 sequence, however, two neighboring substi-tutions, G-2031 -- A and T-2032 -- C, were identified at the5' splice site of intron 3 (Fig. 1). Monocot genes invariablycontain the sequence GT in these positions (22).To establish whether the splice site mutations are respon-

sible for the absence of normal-sized transcript, severalchimeric plasmids were constructed. Plasmid pgDFR[NcoI-161] carries the two splice site base substitutions, whereasplasmid pgDFR-161 harbors the full-length antl8-161 se-quence and pgDFR-161[NcoI-wt] contains the wild-type 5'splice site of intron 3 in the antl8-161 sequence. Eachconstruct was separately introduced into antl8-161 cells bymicroprojectile bombardment, and complementation wasvisualized by the appearance of cells containing red antho-cyanin pigmentation derived from transcription/translationof the chimeric gene (Fig. 3). Complementation was obtainedwhen mutant tissue was transfected with plasmids pgDFRand pgDFR-161[NcoI-wt], but transfection with plasmidspgDFR-161 and pgDFR[NcoI-161] did not restore DFR ac-tivity in the mutant cells (Table 2). The results identify the 5'splice site mutations ofthe antl8-161 gene as the cause for theabsence of anthocyanin production in antl8-161 plants. Asnonfunctional mRNA is a frequent cause of message insta-bility (23, 24), it seems likely that the absence of detectablepolyadenylylated message for DFR in antl8-161 plants is dueto degradation of alternatively processed or unspliced DFRpre-mRNA. The results reinforce the importance of a properdonor site for the splicing of nuclear pre-mRNA into maturecytoplasmic mRNA.

E Y V A1 MDGNKGPVVVTGASGFVGSWLVMKLLQAGYTVRATVRDPANVEKTKPLLELPGAKERLSIWKADLSED1 VSQ ET C L R ER F G LK VOM D N TO T E1 MEGGAGASE T L A G MD T A E1 MPLHLRCSAT C A R ER N H E KK V H K DTN TLL TVE1 MSPTSLNTSSETAPPSSTT C A R ER G MK V H K DTN TL MTVE

M69 GSFNEAIAGCTGVFHVATPMDFDSQDPENEVIKPTVEGMLSIMRACKEAGTVKRIVFTSSAGSVNIEERPRPAYDQDNWS69 YDD N D E K N G K VK K FFF T V HQKNV END74 HD R L K R T 0 0 V EES T72 D 0 0 E K R IEA AK N L TLDVQ QOKLF TS81 D 0 E L S E V ID N IKS VO K KFI T G T V HOK V ETDS

K A149 DIDYCRRVKMTGWMYFVSKALAEKAAMEYASENGLDFISIIPTLVVGPFLSAGMPPSLVTALALITGNEAHYSILKQVQL149 LEFIMSK S DF E K ITTT SP R IR GGY154 V F T LA A H LVT S S AP152 L FIYAK A E KKKNI P ITPTF S C G Y161 M FINSK 6 A K N P IMPTF SP C Y

229 VHLDDLCDAMTFLFEHPEANGRUICSSHDATIHGLARMLQDRFPEYDIPQKFAGVDDNLQPIHFSSKKLLDHGFSFRY-T229 N HI Y OAAAK LTISKF RPKY NV ST E E KS E TEM N K -S234 El N AA V V A R Y V RP IO D VR Q L T K232 E HI Y K D F H YDV K VREKW YV TE K KD PVVS T M 0 K -T241 EGHI Y K E YDI KLITENW H DE E KDIPVVS MIGM K -

308 TEDMFDAAIHTCRDKGLIPLGDVPAPAAGGKLGAL--AAGEGOA-IGAET4308 L E IES E 0 FL VSLSYQSISEI VPTKNEII-EVKT-GDGL DGMKPVNKTETGVTGERTDAPMLAOQMCA4314 L R GE ---- T DGF SVR P TE T 4311 L YKG D a 0 L FSTRS EDN HNRE IAIS ONYASGKENAPVANHTEMLSNVEV4320 L VRG D E ML YSTKNNKGDEKEPILNSLENNYNIODKELFPISEEKHINGOENALLSNTODKELLPTSEEK

400 RVNGLESALLSKI0DKEVLPTSGVKHAKGQENALLPDIANDHTDGRII

HvAtZmPhAm

HvAtZmPhAm

HvAtZmPhAm

HvAtZmPhAm

HvAtZmPhAm

Am

FIG. 2. Structural alignment of the DFR amino acid sequences (single-letter code) from H. vulgare [Hv (9)], Arabidopsis thaliana [At (19)],Zea mays [Zm (20)], Petunia hybrida [Ph (21)], and Antirrhinum majus [Am (21)]. Only differences from the barley enzyme are shown; emptyspaces denote identities. The C-terminal residue ofeach enzyme is followed by an arrowhead. Dashes are used for optimal alignment. The derivedamino acid sequence from antl8-159 includes the marked substitutions Glu-176 -. Lys and Thr-214 -. Ala; for antl8-161, Met-23 -+ Val; forantl8-162, Met-23 -. Val, Val-32 -- Ala, and Lys-101 -- Met; for antl8-164 Gly-12 -- Glu and Phe-16 -* Tyr.

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FIG. 3. Leaf sheath tissue of the barley mutant antl8-161 wastransfected with expression plasmid pgDFR-161[NcoI-wt]. Comple-mentation of the mutation in the gene encoding DFR is visualized byanthocyanin pigmentation in single cells.

A larger number of colored cells was obtained from trans-fections using plasmid pEmuGN compared with DFR ex-pression plasmids (Table 2). Whether this reflects a differ-ence in the sensitivity of the assays used or instead is aconsequence of their expression (such as message stability)remains to be established.

DISCUSSIONA total of 451 sodium azide-induced barley mutants blockedin the synthesis of anthocyanins and proanthocyanidins-i.e., the flavonoid pathway prior to the (+)-2,3-trans-3,4-cis-leucocyanidin intermediate-have been analyzed by dialleliccrosses. These mutants have important implications forbreeding malting barley varieties lacking testa-localizedproanthocyanidins in order to avoid chemical stabilization ofbeer against permanent haze and chill haze (25, 26). Themutants were found to belong to five complementationgroups (11). Among these, Antl3 with 134 investigated allelesencodes a transcription factor apparently required for thetranscription of a majority, if not all, of the genes operatingin the flavonoid pathway (27). Ant2l represented by 7 NaN3-induced mutants is possibly another regulatory gene. GenesAnti7 with 172 known mutants and Ant22 with 5 knownmutants are candidates for the gene of flavanone 3-hydrox-ylase (11, 27), whereas gene AntJ8 (133 mutants induced withNaN3) encodes DFR (9, 12).That the four analyzed antl8 mutant genes contained 21

base substitutions is a surprise and implies a high number ofother base changes in the mutant plant genomes. For exam-ple, antl3 mutants display small yield depressions, whichhave not yet been overcome by recombination breeding toprovide commercial varieties, although significant progresshas been achieved toward this aim. Many antl7 mutant plantsprovide excellent yield but are often inferior in maltingquality, reflecting the absence of uniform secretion of amy-

Table 2. Number of red and blue cells per 10 antl8-161 stemsegments after three independent transfections withexpression plasmids

Cell count,Plasmid mean + SD

pgDFR 7.5 ± 3.1pgDFR-161 0pgDFR-161[NcoI-wt] 9.3 ± 3.2pgDFR[NcoI-161] 0pEmuGN 47.0 ± 6.1*

*Blue cells.

lase from the aleurone tissue to the endosperm during ger-mination. Breeding by genome fitting has not even beenattempted for antl8 mutants, since they produce shriveledgrains. That this is primarily the result of biochemical pleiot-ropy of eliminating DFR activity is indicated by the recenttesting of mutants blocked in the branch of the pathwayspecific to the conversion of leucocyanidin to catechin andproanthocyanidins (28). NaN3-induced mutants in the genesAntl9, Ant25, Ant26, and Ant27 seem to produce normalyield, have good disease resistance, and have acceptablemalting quality.

Plants with mutations in the AntJ3, Antl7, andAntl8 genesarise after NaN3 mutagenesis with a frequency of 0.003% inthe M2 generation (26). From this, it can be estimated that the451 mutants were screened from 15 million M2 plants, firstselected as anthocyanin-free plants in the field and subse-quently analyzed for the absence of proanthocyanidin-freegrains with the vanillin test (29). When the mutants werestudied in the following generations, 79% of the antl3 mu-tants, 69% ofthe antl7 mutants, and 72% ofthe antl8 mutantssegregated for chlorophyll deficiencies, morphological vari-ants, or partial sterility. In cases where an analysis wascarried out, these characteristics were due to independentmutational events as is expected with the high mutationfrequencies elicited by NaN3.The absence of GC -- C*G transversions in the analyzed

barley mutants is by itself an important finding. Since GiC-CG transversions induced by 6Co y-rays or Fe2+ in DNA insolutions are due to the action ofoxygen free radicals (30, 31),their absence rules out that oxygen free radicals from azide-derived peroxide accumulation are the mutagens for the basesubstitutions identified. Except for the data presented, noinformation is available for barley with respect to natural andmutagen-induced sequence alterations. However, the pref-erential induction of transitions seen here is similar to thespectrum of spontaneous and UV-induced mutations arisingduring passage ofthe DNA through mammalian cells (32) andin systems using the E. coli lacI or rpsL gene (33, 34) butdifferent from spontaneous mutations in yeast (35) and the E.coli supF gene (36) where transversions predominate.

It has been demonstrated by the transfection experimentsthat the mutation in the promoter mutation in combinationwith the mutation in the first exon of antl8-161 giving thechange Met-23 -- Val in the DFR enzyme is silent, whereasthe two base changes at the 5' splice site of the third intronare responsible for the block in the synthesis of DFR. Mostlikely, similar tests will reveal that several of the pointmutations in the other three mutant antl8 genes are silent.Domains determining the function of DFR enzymes are atpresent poorly understood except that circumstantial evi-dence indicates that a 13-residue-long sequence spanningVal-132 to Asn-144 determines substrate specificity (21). Nomutations were identified in this region. It is interesting tonote that most of the mutations identified are located atpositions with high homology among the DFR enzymes (Fig.2), but it remains to be established whether these mutationsindividually or in combination inactivate DFR. Since the genesegments corresponding to the amino- and carboxyl-terminalparts ofDFR are devoid of mutations, heterogeneity in theseregions is apparently not deleterious to enzymic activity.For functional characterization of induced mutants in

barley, the combination of sequencing genomic DNA andcomplementation tests with individual base changes by mi-croprojectile gene delivery appears to be a useful technique.It will permit exploration by gene walking away from Antl8if sodium azide at the doses employed causes base transitionsin a larger chromosome segment than that analyzed here.

We are indebted to Dr. Barbro Jende-Strid for the information onthe behavior of progeny tests with ant mutants. Eva Gertman is

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thanked for technical assistance. This work was in part supported byEUREKA Grant Eu270, by a grant from the Danish BiotechnologyProgram 1991-1995, and by the Danish Program for Food Technol-ogy, Grant 6028.

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