Gene, 120 (1992) 75-83 0 1992 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

15

0378-l 119/92/$05.00

06667

The nitrate reductase-encoding sequence and induction kinetics (Algae; domain

structure;

induction;

nucleotide

gene

sequence;

carteri:

of Volvox

protozoa;

repression;

restriction-fragment

map

location,

length polymorphism

mapping)

Heribert Gruber”, u Lehrstuhlftir St.

Susan D. Goetinckb,

Genetik, Universitiit Regensburg,

David L. Kirkb and Rtidiger Schmitt

WD-8400 Regensburg,

Germany.

Tel. (49-941)943-3162;

a

and ’ Department ofBio1og.v. Washington University.

Louis. MO 63130, USA

Received

by J.R. Kinghorn:

1 February

1992; Revised/Accepted:

21 May/25

May 1992; Received

at publishers:

12 June 1992

SUMMARY

The nitrate reductase (NR) structural gene (nitA) of V&ox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 CM from the two /I-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period. The potential utility of the cloned nitA gene as an aid to molecular genetic studies of V. carteri development is briefly discussed.

Nitrate reductase (NR) catalyzes the first step of nitrate assimilation: the reduction of nitrate to nitrite. The enzyme

has been characterized in Solomonson and Barber, luf, 1981; Kinghorn and (Zeiler and Solomonson,

Correspondence

carteri strains

INTRODUCTION

to: Dr. D.L. Kirk, Department

of Biology,

Washington

University, St. Louis, MO 63130, USA. Tel. (314)935-6812; Fax (314)935-4432. Abbreviations: A., Aspergillus; aa, amino acid(s); bp, base pair(s); C., Chlamydomonas; chlR, gene encoding chlorate resistance; CM, centimorgans; ECM, extracellular matrix; FAD, flavin adenine dinucleotide; I, V.

certain higher plants (Wray, 1988; 1990), fungi (Cove, 1979; MarzCampbell, 1989) and green algae 1989; Fernandez and Cardenas,

derived from strains

isolated

in India: J, V. carteri strains

derived from strains isolated in Japan; kb, kilobase or 1000 bp; MoCo, molybdenum-pterin cofactor; N., Neurospora; nitA, gene encoding NR; N-free, medium lacking any utilizable nitrogen; NR, nitrate reductase; nt, nucleotide(s); RFLP, restriction-fragment length polymorphism; tsp, transcription type.

start point(s);

UTR, untranslated

region;

V., Volvox;

wt, wild

76 1989) and an understanding of the structure and regulation of the enzyme has been aided by analysis of NR-deficient mutants (Caboche and Rouze, 1990). Each NR subunit contains three redox prosthetic groups, FAD, heme and MoCo, that act (in that order) to transfer two electrons from NAD(P)H to nitrate (Campbell and Kinghorn, 1990). Typically, both the synthesis and the activity of NR are highly regulated by factors such as light and the available nitrogen source(s). The NR genes of several higher plants, fungi and algae have been cloned in recent years (Cheng et al., 1986; Crawford et al., 1986; 1988; Fu and Marzluf, 1987; Vaucheret et al., 1989; Daniel-Vedele et al., 1989; Fernandez et al., 1989; Kinghorn and Campbell, 1989; Johnstone et al., 1990; Okamoto et al., 1991; Unkles et al., 1992); such cloned genes have been used to show that NR transcription generally is both induced by nitrate and repressed by ammonium and glutamine (reviewed in Solomonson and Barber, 1990), and in some cases is also subject to negative autoregulation (Fu and Marzluf, 1988). l’. carteri is a multicellular green alga with many features that recommend it as an object for molecular genetic analysis of development (reviewed in Kirk and Harper, 1986; Schmitt et al., 1992). It exhibits simple and regular cellular differentiation, and mutants with developmental defects are readily recovered and analyzed genetically (Starr, 1970; Huskey et al., 1979a; Adams et al., 1990; Kirk et al., 1991). Although Volvox has proven quite amenable to certain types of molecular genetic analysis (Harper and Mages, 1988; Mages et al., 1988a,b; Miiller and Schmitt, 1988; Rausch et al., 1989; Cresnar et al., 1990; Miiller et al., 1990; Tam and Kirk, 1991a,b; Tam et al., 1991; Larson et al., 1992) others have been precluded by the absence of a transformation system. A transformation system has been developed for A. niger (Unkles et al., 1989) and more recently (Fernandez et al., 1989; Kindle, 1990) for C. reinhardtii, a close unicellular relative of Volvox, by the use of cloned, homologous NR-encoding genes as selectable markers. Its potential utility as a homologous transformation marker was one reason that we were motivated to clone and characterize the gene encoding the Volvox NR. A second was that cloning of this locus would open up the possibility of using it as a ‘transposon trap’ (as was done in Nicotiana; Grandbastien et al., 1989), in order to identify transposable elements that might be useful for tagging and cloning of various loci of developmental interest. The usefulness of the NR-encoding gene for such studies is based on the fact that it is the only Volvox gene for which methods are presently available for positive selection of mutations in both directions: NR- mutants can be selected on the basis of their resistance to chlorate (Huskey et al., 1979b), whereas back mutations to NR’ confer ability to grow on nitrate as a nitrogen source. Both transformation and transposon trapping have now been accomplished in our laboratories

by use of the gene described here; these advances the subject of future communications.

RESULTS

will be

AND DISCUSSION

(a) Isolation of genomic clones and determination number

of copy

Genomic clones containing the V. carteri gene that encodes NR were isolated by sequential screening of two different genomic libraries: clones hybridizing to probes representing two adjacent portions of the coding region of the C. reinhardtii NR-encoding gene (Fernandez et al., 1989) were first isolated from a Lambda DashTM library of V. carteri DNA. When two of these clones that had been partially sequenced and found to contain a portion of the NR coding region were used to screen a AEMBL3 library of V. carteri DNA (Mages et al., 1988b), /ZNR102, a clone containing the 3’-terminal half of the gene, and ANR107, a partially overlapping clone that contains the 5’ portion of the gene, were recovered (Fig. 1A). The presence of NRencoding sequences in 3,NRl02 and 1NR107 was confirmed by showing that both hybridized to a 3.6-kb transcript that was present in Volvox grown on nitrate - but not ammonium - as a sole nitrogen source (similar results shown in Fig. 6A). A plasmid, pVcNR1, that contains the entire region diagrammed in Fig. 1B was constructed by recombining three restriction fragments derived from INR107 and ANR102 (Fig. 1A); this plasmid was used as the hybridization probe for several studies reported below. Southern hybridization of pVcNR1 to genomic DNA digested with SalI, EcoRI, and Hind111 revealed a set of fragments (Fig. 2A), all of which are consistent with the physical map of the cloned element that is shown in Fig. 1B. This indicates that sequences homologous to the cloned probe are present only once in the genome, and that the restriction maps of the cloned fragment and its genomic equivalent are colinear. (b) Linkage mapping RFLPs were detected when either SalI-, EcoRI- or HindIII-digested DNAs from J strains (originating from Japan; Starr, 1969) and I strains (originating from India; Adams et al., 1990) were compared on Southern blots hybridized with pVcNR1 (Fig. 3B). When progeny derived from a cross between strain HBllA (a chlR, reducednitrogen-requiring mutant J female) and PM1 (an I male) were analyzed with respect to their Sal1 and Hind111 restriction patterns and their chlorate sensitivity, 87/88 exhibited a parental combination of traits: either the J restriction pattern and chlorate resistance or the I restriction pattern and chlorate sensitivity. This near-perfect cosegregation indicates that the NR structural gene locus is syn-

77 XNRlO2

A

SSHSSSB

EH

INR107

SB

SE

ES

S

__--

HEBB

*.

SHS

‘kb

-.

*.

. .

. .

---Y-+T+

*.

-.

__--

-.

__--

. .

*,

-.

_.-*

*.

,_--

B

E

S

HE

B

B

SH

I

I

II

I

I

I

Se

SC

I

I

I

s

s

s

I

. . B

I I

I

C 1 kb

Fig. 1. Physical

maps of two overlapping

NR genomic

the nitA gene (C). Clones bearing a portion EVE; Harper

et al., 1987) in Lambda

gene (Fernandez

clones, I.NRIO2

and I.NR107

of the gene were first identified by screening

DashTM (Stratagene,

Starr,

1969) in IEMBL3.

coding region plus upstream

The 7.6-kb region (shaded

and downstream

UTR s has been sequenced

et al., 1977). The three lines labeled with roman tor PBS + to generate

the plasmid,

pVcNR1,

numerals

a genomic

La Jolla, CA) with two cloned fragments

et al., 1989). These Vdvox clones were then used to identify additional

from strain HKlO;

(A); sequencing

strategy

(I, II, III) in A indicate

by arrows)

three restriction

that was used as a probe in several studies reported

structure

of

(B6a-2 and B6a-3) of the C. reinhardtii NR-encoding

clones, including

INR102

in A and shown on an expanded (as indicated

(B); and derived exon-intron

library (derived from Volvox carteri f. nagariensis, strain and INR107,

in a second library (derived

scale in B) that contains

using the dideoxy fragments

the entire Volvos NR

chain-termination

that were recombined

below. The stylized exon-intron

method

(Sanger

in the plasmid

vec-

(thick and thin lines,

respectively) structure diagrammed in C was derived by comparison of gcnomic and cDNA sequences. B, BarnHI; E. EcoRI; H, HindIII; S, SalI; SC, ScaI (sites linking insert and vector DNA are given in bold face; the phage i DNA is shown in the reverse of the conventional orientation in order to display

the coding

sequence

of the insert in the 5’-3’, left-to-right

orientation).

onymous with the chlR locus on linkage group IX that had been defined earlier by analysis of progeny from the same type of cross (Adams et al., 1990) and hence also corresponds to the nitA locus defined by Huskey et al. (1979a,b). These data place the nitA gene 20-30 CM from the two /I-tubulin-encoding genes of Volvox, and indicate that existing mutants with lesions at the nitA locus are candidates for transformation with the cloned gene. (c) Nucleotide sequence analysis Partially overlapping fragments from INR102 and 3,NRl07 (Fig. 1A) were used to determine the complete 5871-nt sequence of the V. carteri nitA coding region plus its 5’ and 3’ UTRs (Fig. 1B). All regions were sequenced in both directions, Exon-intron organization was deduced by alignment with higher-plant NR peptide sequences, and by comparison of genomic and cDNA sequences. These assignments were supported by accordance of all presumptive intron-exon boundaries with splice-site-consensus sequences (Breathnach and Chambon, 1981). Fig. 1C summarizes the relative locations of the eleven exons and ten introns, the tsp and translation start sites, and the presumed polyadenylation signal of the nifA gene. The 2595-nt coding region, upstream and downstream UTRs, and the deduced aa sequence of the the NR polypeptide are given in Fig. 4. The sequences of the ten

introns (3276 nt in all) are included in the sequence deposited in the GenBank database, but they have been omitted from Fig. 4 for conciseness. However, their locations have been indicated within the coding sequence by arrows, and their individual lengths are tabulated at the bottom of the figure. The elevated A+T content that typically precedes the 3’-splice site of higher plant introns (Wiebauer et al., 1988) is not observed here; in Vofvox, the prevalent 3’ consensus sequence is GCAGG (Cresnar et al., 1990). In contrast to the ten introns in the VoIvox nitA gene, higher plant genes encoding NR are interrupted by only three introns; introns 3 and 4 of Volvox are at positions congruent with introns 1 and 2 of higher plants, but intron 6 of Volvox is shifted by 26 nt from the location of intron 3 of higher plants. The A. nidulans and A. niger niaD (NR-encoding) genes contain six introns at identical positions (Kinghorn and Campbell, 1989; Johnstone et al., 1990; Unkles et al., 1992), one of which is in the same location as the single intron of the N. crassa gene (Okamoto et al., 1991) but all of which are at different locations from the Vo/vox introns. These relationships are indicated at the bottom of Fig. 5. Primer extension was used to identify two tsp, 439 and 452 nt upstream from the start codon (Fig. 3). Repeated studies, using primer-extension and S 1-nuclease mapping, failed to reveal any tsp signals in more proximal regions of the 5’ UTR. A hexanucleotide, UGUAA, resembling the

78 DNA --

ACGTVY

mRNA

G

kb

T

- 23

5

-9.4

_/ /

-4.4

T-_ A C

A +l

T” T c T T T A

A A G A A A u A IJ A A C

U G U

T A T+ T G

- 2.3

(+I)

A

‘T

-2.0 Fig. 3. Dcternlination

of the f.qi by primer-extension

were carried out using a commercial

analysis.

prinlcr-extension

ison, WI) according

to the vendor’s

recommendations,

annealing

were modified

(9fl min at 42’C);

conditions

Reactions

kit (Promega, except

Madthat the

the region of the

5’ UTR complementary to the primer used for the analysis. pnr40, is indicated in Fig. 4. The reaction products obtained using 10 ng samples of f/oiw~s RNA (lane V) or control plates were precipitated,

A

acrylamide

Fig. 2. Southern

hybridization

with the plasmid

pVcNR1.

tions of theHind

fragments

0.7.kb SalI fragment wise nvercxposed,

analysis ofgenomic Numbers

between

is readily detected on autoradiograms

indicated

by numbers

fragments

that contain

and arrows flanking

to termini of the pVcNRl chlorate

and PM1 (a wt l-male RFLPs EcoRI:

Estimated

indicate

strain) digested

arc

gcnomic

to regions homologous

insert. (Panel B) DNA samples

from HBl IA

.I-fcmalc strain derived from EVE) with three enzymes.

generated

the corresponding indicated

portion

transcript

by + I and

lane V) as tcm-

and clcctrophoresed

on a h”, poly-

get next to the sequence

by use of the same primer.

is shown the relevant

RNA (yeast tRNA,

ladders (lanes A, 0. G, T)

To the right of the autoradiogram

of the nitA genomic

scqucncc

(DNA) and

(mRNA), with the two 5’ ends of the mRNA

(+ 1).

reproduced

wt J strain of E;. carteri,

to the left; asterisks

and the methods

A predicted

sizes of all gcnomic fragments

DNA in addition

resistant

the posi-

that arc other-

but is not visible on the autoradi(?~~~ls

digested with three enzymes.

tion of these strains

indicate

of 1DNA used as size markers.

here. (Panel A) DNA from EVE, the standard

(a multiply marked,

DNA samples probed

panels

sequencing

redissolved,

The deriva-

used for linkage analysis

of such

were previously described (Adams et al., 1990). Abbreviations: E, H, NindIII; I, DNA from PMI; J, DNA from FIB1 IA; S, Sir/I.

putative polyadeny~atioll signals of other volvocalean genes (Cresnar et al., 1990) was identified 355 nt downstream from the UAG stop codon (Fig. 4), and sequencing of the 3’-end of the cDNA revealed the poly(A) start 24 nt downstream from this UGUAA. From these data, the length of the mature n&4 mRNA was calculated to be 3413-3426 nt, not including the poly(A) tail. This calculated size is consistent with the apparent size of the n&I transcript detected on Northern blots, namely approx. 3.6 kb (Fig. 6). (d) Amino acid sequence comparisons and domain structure

Previous studies have used regions exhibiiing strong sequence similarities to other enzymes of analogous catalytic

fullction, plus regions of proteoiytic sensitivity, to subdivide the primary structure of higher-plant NRs into a total of six regions: MoCo-, heme- and FAD-binding regions have been defined on the basis of sequence similarities to the corresponding cofactor-binding sites of sulfite oxidase, cytochrome b5, and cytochronle-~j reductase, respectively, and a variable N-terminal domain and two ‘hinge regions’ flanking the heme-binding site have been identified (Crawford et al., 1988; Daniel-Vedele et al., 1989). When the deduced 864-aa sequence of Volvos NR was aligned and compared to the 917-aa NR sequence of Arabido~s~~, an overall sequence-identity value of 48.9 y,, and a sequence-similarity value of 69.1 Y; (taking conservative interchanges into consideration) were observed; in a VolvoxJA. nidulnns comparison, the corresponding values obtained were 38.2 and 60.8%. respectively. More detailed comparisons revealed, however, that the sequence is much more strongly conserved within each of the cofactor binding domains than it is at the termini and in the inter-domain regions (Fig. 5). When these comparisons were extended to include the NR sequences of tomato (911 aa) and tobacco (904 aa), the following noteworthy relationships were confirmed (H.G., unpubIished): (i) All previously defined fimctional domains are present, and highly conserved, in the aa

TMEEVAAHNTEESCWFVHGGKVYDATPYLDEHPG

ALAAVKEGATAAAAPAAPPPVVAAAANGGPRQY

CItXTXZX?A DGVMGLRFQHPAPVELGERGNMGWREEDNLVAQ

DETMNTQPAIITWN"MGMMNNCYYRIKIHPQ'JDS

PNEYGKYWCWVHWSLDVMTFDFLNAKEVLLRAW

Ql YAYAGGGRKIIRCEVSLDDGKTWRLGDIQRFEK

LsATcA?Ti-p-m EFIINDLNINSAVARPWHDEVVRLDANKPYTMRG

"ESQNFYHFMDNRVLPSHVDEALAKEEGWWYKP

WLTPDHGFP"RMIIPGFIGGR""KWLSEIT"TE

clWi-TTALPRGSDGSYGTSLTYAKAMDPSSDVIIAYKQNHR

WTGVRLRDLLLLAGIKSPEQGANFVCFRGPKGE

CTLVCAGNRRKEENMLKKSIGFNWGPCATSTTY

ATpcccxxzxxTpGcAT(xipAloQIAATcAAaxxxlpco IPWAEHRIEINGLVDKPLFLTMDELVALPSITFP

PLNCEPPMDVLMEYGFITPPAVHFVRNHGAAPR

P-

“H”PAAA”DKKDQDTPDNW”RRDPRlLRLTGRH

A MTIPELPLGGI”SQA”ELGAPYEPPLTPDHPEWK

c-?Ql HRIGLP"GKHVFTYATINGEN""RAYTPISGDE

1

482

1548

2

313

respectively.

Intron

5

365

2113

6

7

144

2562

S

368

2778

525

Sequence

analysis

(Fig. 3).

complemen-

signal (TGTAA)

under

sequence,

are given by Database of the sequence used for primer-extension

the portion

polyadenylation

Nucleotide tsp and the presumptive

in the GenBank

but their positions

in the margin refers to

and downstream

181

3389

10

Tvrrx

mrpAGaRlcc

of the figure. The complete

are not shown,

(lower lines). Numbering

nt in the 5’ UTR indicate

9 3030

region plus the upstream

183

2273

at the bottom

oligodeoxyribonucleotide

the underlined

No. X64136. The two potential

has been deposited accession

are bold-faced;

336

sequences are tabulated the introns,

and their lengths

tary to pnr40, the synthetic

4 2014

aa sequence

including

arrowheads,

nt and aa residues,

3 1882

of the 2595-m NR-encoding

379

1700

UTRs (upper lines), and the deduced

sequence

mpj

pas.

Fig. 4. Nucleotide

length

seq.

intron X

CXXX_M-mm LMCGPPAMLEHCCVPFLESMGYSKEQMIHF*

SQTNSSDWKFSTGRVTLEMFKQHLFACSGPECLA

PTA--% "FANNTEED~LLREELDELANNHPDRFHLWHTV

HMSFVAGGTGITPCYAVIKAALRDPEDKTQISL

CIXAC4-----

RIGDTVEFKGPLGHFVYDGRGSYTLNGKLHKHAT

a

ELGRLDMLIKVYFANEHPAFPDGGKMSQHFESL

-’

""LNPRQK"KLPLIERIELNRNTRIFRFGLPSPQ

IGDLVASKPAAAGATVPEPQPVASTSSPAVDPL

GAESILIVAGADATDEFNSIHSSKAKAMLAQYY

-m

80 Heme MO-CO ~CI______-_H___-______--_, ,

=QT

excms

12131415161

1

Sf F

F

+

ff$f$

F

FP

Fig. 5. A histogram cations

P

indicating

Is

7

indicate (Crawford

/halima

(Crawford

the approximate

I9

F

help of the GCG were calculated

of regions

GAP

program

dow size of 40 aa residues the histogram

indicate

lo-

of V. curteri and

that have been defined domain

(by rcfcrence

(Devereux

(by reference to human had been aligned with the

et al., 1984) identity

regions of the two sequences,

and a step size of 20 residues.

regions of the aa sequence

eleven exons of the V. carteri gene. Arrows

to

domain (by reference to bovine-

After the sequences

for successive

Ill

identity values at successive

b5) and the FAD-binding domain

reductasc).

10

et al., 1988). The lines above the histogram

boundaries

rat-liver sulfite oxidase), the hcme-binding cytochrome-b5

1

F’

P

sequence

et al., 1988) as the MoCo-binding

liver cytochrome

I

+

along the derived and aligned NR aa sequences

Arabidopsis

FADINADH

encoded

values

using a winBoxes below by each of the

below these boxes indicate the

locations of introns in the NR-encoding gcncs of higher plants (P) and fungi (F, specifically A. n&w and A. nidulms, except that the asterisk identifies

a position

occupied

also by the only intron

of the Neurmporu

gene).

sequence of Volvox NR. (ii) Relative to the higher plant NRs in particular, the I’&ox NR exhibits a gap in the highly variable N-terminal domain. (iii) Two additional gaps are located internally, in the ‘hinge’ regions flanking the heme-binding domain, and the aa sequences surrounding these internal gaps differ substantially from those in corresponding regions of the higher plant and fungal genes. (e) Induction kinetics

Both the synthesis and the activity of NR are known to be regulated in many higher plants and algae in a complex manner, by factors including light and the available nitrogen sources (Guerrero et al., 1981; Franc0 et al., 1988; Galangau et al., 1988). The availability of a genomic clone allowed us to examine at the transcriptional level the regulatory effects of reduced and oxidized nitrogen sources that had previously been studied in Volvox only at the level of NR enzymatic activity (Huskey et al., 1979b). We first performed quantitative Northern blot analysis of RNA samples harvested at intervals after a transfer of a culture from ammonium to nitrate as a nitrogen source. An approx. 3.6-kb transcript hybridizing to pVcNR1 appeared within 10 min, reached peak abundance at about 30 min, and then declined over the next 3.5 h, to establish a plateau level at about one-third of the peak value (Fig. 6A and B). Similar observations have been reported for nitratestarved plants of Arubidopsis (Crawford et al., 1988), tomato and tobacco (Galangau et al., 1988): leaves of such plants accumulate NR mRNA very rapidly following ni-

trate replenishment, whereas accumulation of NR protein proceeds much more slowly. Next, transcript levels following 30 or 60 min exposure to different nitrogen sources were examined. Upon transfer from ammonium to N-free medium, NR transcript levels rose nearly tenfold; but upon transfer from ammonium to nitrate they rose by more than 35-fold (Fig. 6C). This indicates that in I’ofvox (as in many other organisms: Guerrero et al., 1981; Franc0 et al., 1988) NR is both repressed by ammonium and induced by nitrate. Urea represses less strongly than ammonium. This conclusion is supported both by the observation that NR transcripts accumulate significantly following a transfer from ammonium to urea medium, and by the observation that urea counteracts the inductive effect of nitrate somewhat less strongly than ammonium (Fig. 6C). Whether this repression is a direct effect of urea, or a result of intracellular ammonium that is produced by catabolism of urea, cannot be determined from these data. The transcript levels achieved within 30-60 min in the presence of an equimolar mixture of ammonium plus nitrate (Fig. 6C) are similar to the levels observed after 4 h exposure to nitrate alone (Fig. 6B). This suggests that the plateau value attained after 4 h in medium containing only nitrate may be a result of the combined effects of nitrate induction and feedback repression by the intracellular ammonium that is generated by NR activity. It cannot yet be ruled out, however, that nitA transcription in Volvox may also be subject to negative autoregulation by the NR polypeptide, as it is in Neurosporu (Fu and Marzluf, 1988). Finally, the effects of light were examined. In higher plants maintained on an illumination cycle similar to the one routinely used for Volvox cultures (8 h dark/l6 h light), it has been reported that the level to which NR-encoding transcripts are accumulated by nitrate-fed plants increases late in the dark period, reaches a maximum value at the beginning of the light period, and then declines fairly rapidly (Galangau et al., 1988). When such studies were performed with Volvox, a similar pattern was observed (Fig. 6D): following a 30- or 60-min exposure to nitrate as the sole nitrogen source, accumulation of nitA transcripts was substantial in cultures tested in the last hour of the dark period, but rose to a maximum in cultures tested during the first hour of illumination, and then declined sharply. Moreover, cultures exposed to light for 1 h and then exposed to nitrate in the dark accumulated only about half as much transcript as equivalent cultures that were exposed to nitrate in the light. This suggests that although the nitA gene can be induced in darkness, there may be two quantitative effects of light on inducibility: an immediate, stimulatory effect of illumination during the time of nitrate exposure, plus a more complex effect of the prior illumination history of the culture.

81

A

10

0

20

30

60

120 240 460 min 4.4 kb

nitA 26 S

,16 S

0

I

I

I 0

-1

I

I 1

Hours After Beginning

,1.35 kb

of nitA transcript containing

following

NH,Cl

The locations

transfer

to medium

I

I 2

of lllumlnatlon

of juvenile

containing

Period

spheroids

KNO,

in the (1 s’, agarose/2.2

occupied

I

plus the large and small subunits

rRNA,

on the right margin

are indicated

medium, sources.

in juvenile

or after transfer Juveniles

gel by

of cytoplasmic

of panel A. (C) Relative

spheroids

to media

source.

M formaldehyde)

two size standards,

dance of NR transcripts

from medium

as the nitrogen

abun-

grown in NH,-containing

containing

for the sample labeled NH,+

various

other

nitrogen

were removed

from the

culture at the time the remaining spheroids were harvested for transfer to other media. Samples were removed for Northern blot analysis after 30 and 60 min in the new media; because

these two time points gave qual-

itatively similar results, the figures plotted

are averages

of 30 min and 60

min values. (D) Relative abundance of nit.4 transcripts following transfer from NH., + medium to NO,- medium, for 30 or 60 min, at various times near the end of one dark period

and the beginning

of the next light pe-

riod. Open circles indicate cultures exposed to NO,-

medium in the light;

blackened

circles indicate cultures harvested in subdued light and then medium in complete darkness. Methods. Medium was exposed to NO, standard

V&ox medium (Kirk and Kirk, 1983) with Ca(NO,)z

by CaCI,,

urea excluded

(except where indicated),

ments were added to a final concentration I

4

liter. Dark samples

were incubated

replaced

and nitrogen

supple-

of lo- ’ g atom N (each) per

in foil-wrapped

flasks. Other culture

conditions were as previously described (Kirk and Kirk, 1983). To forestall the possibility of induction kinetics that were distorted by reutilizaHours After Transfer

From NH ; to NO j

tion of reduced spheroid,

nitrogen

analyses

from the glycoprotein-rich

were performed

ECM of the parental

with isolated juvenile

spheroids

at a

stage before they had accumulated any significant amount of ECM. To this end, ammonium-grown cleaving embryos were isolated from parental spheroids

and returned

to fresh ammonium-supplemented

medium

near the end of one light period (Kirk and Kirk, 1983); the next morning (one h after the lights came back on, except where indicated the juveniles

were harvested

by filtration,

dium, resuspended

in appropriately

to normal

conditions.

samples

culture

were harvested

phoresed,

transferred

rinsed

supplemented

medium

After the designated

for analysis to membranes

and RNA

incubation

and hybridized

NH;

Fig. 6. A representative normalized transcripts

N-tree

Wea

autoradiogram

NO;

of a Northern

NO; and

NH; and

NH:

Urea

blot (panel A) and

densitometry data (B, C, D) indicating the abundance of nitA detected in cultures exposed to various nitrogen sources under

defined conditions.

(Panel A and graph

B) Time course of accumulation

Research Labs) to estimate apparent blots were hybridized simultaneously

me-

and returned

was extracted,

scribed (Tam and Kirk, 1991a), using co-electrophoresed (Bethesda Northern

otherwise)

with nitrogen-free

periods, electro-

as previously

de-

RNA standards

sizes of transcripts. to pVcNR1 (n&4)

All and

C38 (a cDNA that detects a 0.8-kb transcript that is present in essentially constant abundance throughout the life cycle; Tam and Kirk, 1991a), densitometry

was performed

as previously

1991 a), and the data were normalized units of nit,4 transcript

described

by calculating

(Tam

and Kirk,

the number of density

per 100 density units of C38 transcript

For display purposes, normalized data have been converted maximum value obtained within that experiment.

in each lane. to % of the

82 (f) Conclusions (1) Vdvox NR is encoded

by a single locus, nitA, that is

located on linkage group IX, 20-30 CM from the two p-tubulin genes. (2) The nitA transcription unit is 6689-6702 nt long and comprises the following regions: a 5’ UTR 439-452 nt long with two potential tsp, a coding region 5871 nt long that consists of eleven exons and ten introns, and a 379-nt 3’ UTR containing a typical volvocalean polyadenylation signal (UGUAA) 24 nt upstream from the polyadenylation site.

cukaryotic split genes coding (1981) 349-383.

for proteins.

Caboche, M. and Rouz&, P.: Nitrate reductase: and cellular studies in higher plants. Trends W.H. and Kinghorn,

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Daniel-Vedcle,

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‘gaps’ and nonconservative interchanges in the N-terminal portion and two internal regions of the Volvox polypeptide may aid in defining the essential features and boundaries of NR domains. (4) An approx. 3.6-kb transcript of the nitA gene accumulates abundantly within 30 min after transfer from ammonium to nitrate as a nitrogen source; the level of the transcript then declines to reach a stable, intermediate level by 4 h. (5) Accumulation of the nitA transcript is both induced by nitrate and repressed by reduced nitrogen sources. (6) Two types of light-dark effects on nitA expression have been tentatively identified: an immediate stimulatory effect of light during the time of nitrate induction, and a more complex set of effects related to the illumination history of the culture.

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