Characterization, chromosomal mapping, and ...

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Characterization, chromosomal mapping, and expression of different ubiquitin fusion protein genes in tissues from control and heatshocked maize seedlings1 Ling Liu, Daniel S. Maillet, J. Roger H. Frappier, Karen d9Ailly, David B. Walden, and Burr G. Atkinson

Abstract: Organisms possess at least two multigene families of ubiquitins: the polyubiquitins, with few to several repeat units, which encode a ubiquitin monomer, and the ubiquitin fusion (or extension) protein genes, which encode a single ubiquitin monomer and a specific protein. This report provides details about two ubiquitin fusion protein genes in maize referred to as MubG7 (uwo 1) and MubGlO (uwo 2). Each has one nearly identical ubiquitin coding unit fused without an intervening nucleotide to an unrelated, 237-nucleotide sequence that encodes for a 79 amino acid protein. The derived amino acid sequences of the two fusion proteins show that they differ by five amino acids (substitution by either a serine or threonine). MubG7 maps to chromosome 8L162 and MubGlO maps to chromosome 1L131. Analyses of the role(s) of these genes in response to heat shock (1 h at 42.5"C) reveal that the level of these fusion protein mRNAs in the radicles or plumules from 2-day-old seedlings does not change; however, heat shock does cause a marked reduction in the accumulation of these same gene-specific mRNAs in the radicles and plumules of 5-day-old seedlings. These data c o n f m the suggestion from our earlier work that there is precise modulation, in a gene-specific manner, of the response to developmental as well as environmental signals. Key words:ubiquitin, ubiquitin extension (or fusion) protein, maize, heat shock, heat shock proteins, gene expression, chromosome map.

R6sum6 : Les organismes ont au moins deux familles multigkniques d'ubiquitines : les gknes des polyubiquitines ayant de quelques-unes h plusieurs skquences rkptttes codant un monomtre d'ubiquitine et les gknes de prottines fusionnkes codant un seul monomkre d'ubiquitine et une protkine spkcifique. Dans cet article, nous donnons des dttails sur deux gknes de protkines fusionntes avec l'ubiquitine du mais, MubG7 (uwo 1) et MubGlO (uwo 2). Ces gknes ont une shuence codant l'ubiquitine presque identique, fusionnke directement B une sequence non apparentke de 237 nuclkotides codant une protkine constituQ de 79 acides aminks. Les skquences d'acides aminks de ces deux protkines fusionnkes, dtrivkes des stquences nuclhtidiques, indiquent que cinq de leurs acides aminks different (substitution par une skrine ou une thrkonine). Le gkne MubG7 se trouve au locus chromosomique 8L162 et le gkne MubGlO au locus chromosomique 1L131. L'ktude du r6le de ces gtnes dans la rkponse au choc thermique (1 h h 42,5"C) rkvble que le taux des ARNm de ces protkines fusionnkes ne change pas dans les radicules ou gemmules de semis de 2 jours; cependant, le choc thermique entraine une important rauction de l'accumulation de ces ARNm dans les radicules et gemmules de semis de 5 jours. Ces rksultats confirment ce que suggkraient nos travaux antkrieurs :il y a une modulation prkcise de gknes spkcifiques au cours de la rkponse h des signaux de dkveloppement ou environnementaux. Mots c l b : ubiquitine, protkine fusionnke avec l'ubiquitine, mays, choc thermique, protkines de choc thermique, expression gknique, carte chromosomique.

[Traduit par la rkdaction]

Received March 28,1995. Accepted August 19, 1995.

Abbreviations:nt, nucleotide(s); UTR, untranslated region(s); SDS, sodium dodecyl sulfate; kb, kilobase(s); bp, base pair@); kDa, kilodalton(s); HSWO, heat shock protein 70; HSP18, heat shock protein 18; CPM, counts per minute; SDP, strain distribution pattern. L. Liu, J.R.H. Frappier, K. d9Ailly,and B.G. ~ t k i n s o nMolecular .~ Genetics Unit, Western Science Centre, Department of Zoology, The University of Western Ontario, London, ON N6A 5B7, Canada. D.S. Maillet and D.B. Walden. Department of Plant Sciences, The University of Western Ontario, London, ON N6A 5B7, Canada. The nucleotide sequences reported in this paper have been submitted to the Gene BankTM/EMBLData Banks with accession numbers U29161 and U29160 for the MubG7 and MubGlO genes, respectively. Accession numbers for the maize polyubiquitin sequences reported in an earlier paper (Liu et al. 1995) are U29158 (MubCS), U29159 (MubGl), and U29162 (MubG9). Author to whom all correspondence should be addressed. Biochem. Cell Biol. 74: 9-19 (1996). Printed in Canada I Imprim6 au Canada

Biochem. Cell Biol. Vol. 74. 1996


Introduction Genes and (or) cDNA sequences encoding ubiquitin fusion or extension proteins have been described in such diverse organisms as man (Lund et al. 1985; Salversen et al. 1987), yeast (Ozkaynak 1987), Dictyostelium discoideum (Westphal et al. 1986), Trypanosoma cruzi (Swindle et al. 1988), and Arabidopsis (Callis et al. 1990). The ubiquitin fusion protein genes typically encode a ubiquitin monomer (trypanosomes are an exception (Swindle et al. 1988)) followed by one of two unrelated proteins, the so-called fusion or extension proteins (Callis and Vierstra 1989; Schlesinger and Bond 1987). One of the fusion proteins is 52 amino acids in length and the other varies in length from 76 amino acids in yeast (Ozkaynak et al. 1987) to 81 amino acids in Arabidopsis (Callis et al. 1990). Both fusion proteins are highly basic, have a region of similarity to the consensus sequence for nuclear localization (Dingwall and Laskey 1986), and contain a 4-cysteine residue sequence in consensus with the zn2+finger domain present in nucleic acid binding proteins (Berg 1986; Klug and Rhodes 1987). The ubiquitin fusion protein genes are members of a multigene family. Both ubiquitin and the fusion proteins encoded by these genes are highly conserved among different organisms. The identification of these fusion proteins as ribosomal proteins (Finley et al. 1989; Redman and Rechsteiner 1989; MiillerTanbenberger et al. 1989) or as ribosome-associated proteins (Callis et al. 1990) helps explain the conservation of these fusion proteins and suggests a similar biological function for them. Studies, focused on the expression of the fusion protein genes in plants, have correlated their expression with ribosomal development and biogenesis (Genschik et al. 1992). Indeed, fusion protein encoding transcripts appear to be most abundant in light-grown and young plant tissues (Callis et al. 1990; Hoffmann et al. 1991) and to accumulate in plant tissues undergoing rapid cell growth (Binet et al. 1991; Genschik et al. 1992; Ozkaynaket al. 1987).While stress, such as heat shock, does not appear to affect the steady-state level of the fusion protein encoding transcripts in Arabidopsis and Nicotiana sylvestris (Callis et al. 1990;Genschiket al. 1992),it does cause adecrease from the steady-state level in Chlamydomonas reinhardtii (Pollmann et al. 1991) and in tomatoes (Hoffmann et al. 1991). Since we have been investigating the response of maize tissues to heat shock, particularly the expression of the genes encoding the maize small heat shock proteins (Goping et al. 1991; Atkinson et al. 1989, 1993; Bouchard et al. 1993) and maize polyubiquitins (Atkinson et al. 1989; Bouchard et al. 1993; Liu 1992; Liu et al. 1995), we decided to assess the effect of heat shock on the expression of the genes encoding some of the maize ubiquitin fusion proteins. Herein, we characterize the nucleotide sequences of genes encoding two different ubiquitin fusion protein transcripts in a maize inbred (Oh43) and use gene-specific DNA fragments from them to ( i ) map the chromosomal localization of the maize genes encoding these transcripts and (ii) demonstrate, in tissues of 2- and 5-day-old maize seedlings, the effect(s) of heat shock on the expression of each of these genes.

were germinated in the dark on moistened filter paper at 25°C for 2 or 5 days. Specific attention was directed to the developmental differences that may occur in the radicles and plumules of 2-day germinates (before cell division is initiated) and 5-day germinates (3-5 nuclear cycles after imbibition). The intact, etiolated seedlings were maintained at 25°C (control) or subjected to a 1-h incubation at 42.5"C (heat shock). This time and temperature regime was previously shown (Atkinson et al. 1989) to represent one at which radicles from 5-day-old maize seedlings exhibited maximal changes in the levels of mRNAs encoded from the ubiquitin, HSP70, and HSP18 gene families.

Screening of a maize (cv. Oh43) genomic library A maize genornic library, derived from 10-20 kb DNA fragments isolated (Dellaporta et al. 1983) from the radicles of 5day-old maize seedlings (inbred Oh43), was constructed in the Xho I site of Lambda FIX I1 (Stratagene, La Jolla, Calif.). The host bacterium, E. coli P2CPLK (Stratagene), was grown on NZY plates. The genomic library was screened with a 3 2 ~ labelled 0.471 kb Xho IISac I DNA fragment from a maize cDNA clone, cMubC1, containing two maize ubiquitin coding repeats, as described by Liu et al. (1995). Phage DNA was isolated from two positive plaques, designated as MubG7 and MubG10, and the inserted DNAs were released by digestion with Not I and sized by agarose gel electrophoresis. The DNA inserts from these clones (MubG7, -13 kb; MubGlO, -17 kb) were digested with Eco RI, electrophoresed on 0.6% agarose gel, and transferred to a Zeta-probe membrane (BioRad Laboratories, Hercules, Calif.) by capillary transfer (Southern 1975). The transferred DNA was UV cross-linked to the membrane by a UV Crosslinker (Stratagene) and hybridized with the 0.471 kb DNA fragment used to screen the library. The 32~-labelled probe recognized a 6.8 kb Eco RI DNA fragment excised from MubG7 and a 6.2 kb Eco RI DNA fragment excised from MubG10. Each of these fragments was ligated into the Eco RI cloning site of a pBluescript I1 SK- vector (Stratagene) and was designated as MubG7-1 and MubG10-1, respectively.

Materials and Methods

Subcloning and sequencing of the relevant portions in MubG7-1 and MubG10-1 A 3 kb fragment of MubG7-1 and a 1.4 kb fragment of MubG10-1 were completely sequenced in both directions by the dideoxy chain termination procedure (Sanger et al. 1977) using [ 3 5 ~(600 ] Cilmmol(1 ~ ~ ~ Ci = 37 GBq); Dupont Canada, Mississauga, Ont.) and a Sequenase Version 2 Sequencing Kit (United States Biochemical Corp., Cleveland, Ohio). Universal T3, SK, KS and (or) T, primers (Stratagene) were used initially to sequence from the pBluescript vector into either end of the inserted DNA. For complete sequencing of the defined areas of MubG7-1 and MubG10-1 in both directions, DNA fragments were generated by restriction enzyme digestions of the cloned DNA and the excised fragments were subcloned into the appropriate cloning sites in a pBluescript I1 SK- vector. T3, SK, KS and (or) T, primers (depending on the orientation of the inserted DNA fragment) and internal primers (prepared by VetroGen Corp., London, Ont.) were used to sequence the DNA fragments in the subclones.

Growth and treatment of maize (inbred Oh43) seedlings Dry seeds of Zea mays L. (inbred Oh43) were treated with a fungicide (Vitaflo; Uniroyal Chemical, Aylmer, Ont.) and

Preparation of gene-specificprobes for MubG7, MubG10, and the fusion protein sequence of MubG7 or MubGlO Since the DNA nucleotide sequence of MubG7 and MubGlO


were quite different from each other upstream from the putative ATG start codon, DNA fragments specific for each gene were isolated and used for Southern-blot hybridization analyses (Southern 1975). A 0.248 kb restriction fragment was excised from MubG7-1, using Apa I and Bgl I1 (MubG7-1-1), and a 0.264 kb restriction fragment was excised from MubG10- 1, using Acc I1 and Bgl I1 (MubG10-1-1), for use as gene-specific probes. To obtain a DNA probe containing both the sequence for ubiquitin and the fusion protein, a 0.545 kb DNA fragment was excised from MubG7-1 (or MubG10-1) with Bgl 11; this DNA fragment (MubG7-1-2) also contains 80 base pairs downstream from the TAA stop codon (see Results). Finally, to obtain a DNA probe containing the fusion protein sequence but lacking the ubiquitin sequence, a 0.323 kb DNA fragment was excised from MubG7- 1 (or MubG10-1) using Nar I and Bgl 11; this DNA fragment (MubG7-1-3) also contains 80 base pairs downstream from the TAA stop codon (see Results). In each case, the excised DNA fragments were separated by agarose gel electrophoresis, collected on a DEAE membrane (Dretzen et al. 1981), and quantitated by ethidium bromide fluorescence (Maniatis et al. 1982).

and a DNA fragment specific for the fusion protein sequence of both MubG7-1 and MubG10-1 (MubG7-1-3). Other DNA probes used in this report for Northern- and dot-blot hybridizations include DNA fragments specific for each of three different maize polyubiquitin genes (MubG1, MubG5, and MubG9) as described in Liu et al. (1995), a 0.471 kb DNA fragment containing two maize ubiquitin coding repeats as described in Liu et al. (1995), a 0.342 kb Pst IIBgl 11 DNA fragment from the open reading frame of a maize 18 kDa heat shock protein (HSP) as described in Goping et al. (1991), a 9.0 kb Eco R1 DNA fragment from a maize ribosomal genomic sequence as described in McMullen et al. (1986), and a 4.0 kb Eco RIJBam HI DNA fragment from a maize HSP70 genomic sequence as described in Shah et al. (1985). The DNA inserted fragments were excised with the appropriate enzymes, random prime labelled (BRL Life Technologies) with I X - [ ~ ~ P ] ~ C T P (Dupont Canada; specific activity -1 x lo9 cmplmg DNA), and hybridized to the RNA blots as described previously (Liu et al. 1995). In some cases, the labelled probe was removed from the blots (by boiling three times in 0.1 x SSC containing 0.5% SDS) and the blots were hybridized to other probes. Relative quantitation of the RNA dot blots was obtained by scanning autoradiograms of the dot blots with a laser densitometer (LKB Instruments Inc., Rockville, Md.).

Southern blots and hybridizations DNA was isolated from the plumules of 5-day-old maize (inbred Oh43) seedlings according to the method of Dellaporta et al. (1983). Isolated DNA was digested overnight with Barn Chromosomal mapping of maize (inbred Oh43) ubiquitin HI, Hind 111, or Eco RI and electrophoretically separated on fusion protein sequences 1% agarose gels. DNA standards (a lambda DNA-Hind III/ We used the recombinant inbred (RI) method (Burr et al. 1988; @X-174 DNA-Hae I11 digest, Pharmacia Fine Chemicals, PisBurr and Burr 1991) to map the ubiquitin fusion protein genes. cataway, N.J.) were coelectrophoresed with the DNA samples. Our execution of this method and analysis of the data are idenAfter electrophoresis, the gels were photographed using a UV tical with the description elsewhere (Liu et al. 1995).The DNA fragment used to map the chromosomal localization of MubG7 transilluminator, the DNA was transferred to a Zeta probe membrane (Bio Rad Laboratories) by capillary transfer was an Eco RVXba I restriction fragment. This DNA fragment (nt 1-1486) begins 452 bp upstream from the ATG start codon (Southern 1975), and the DNA was cross-linked to the memof MubG7 (see Fig. 1). The DNA fragment used to map the brane with a UV Crosslinker (Stratagene). Blots of the transchromosomal localization of MubGlO was a Xba UBgZ I1 ferred DNA were hybridized with a random prime labelled (BRL Life Technologies Inc., Burlington, Ont.) I X - [ ~ ~ P ] ~ C T Prestriction fragment (nt 1-736), which contains the first 10 bp from the open reading frame of MubGlO and 0.726 kb of DNA (Dupont; specific activity, -1 X lo9 cpmlmg DNA) DNA fragment specific for MubG7 (MubG7-1- 1), a DNA fragment upstream from the ATG start codon (see Fig. 2). specific for MubGlO (MubGlO-1-1), and a DNA fragment from MubG7 (MubG7-1-3), which is specific for the fusion Computer analysis of the nucleotide and deduced amino protein sequence in both genes. After hybridization, the DNA acid sequences blots were washed four times at room temperature in 2,2,0.5, Nucleotide sequences were analyzed and the deduced protein and 0.1 X SSC (15 mM Nacl and 1.5 rnM sodium citrate), consequences were determined by using DNA Inspector II+(Textaining 0.1% sodium dodecyl sulfate (SDS; 15 min per wash), tco, West Lebanon, N.H.). The EMBL and GenBank databases and subsequently washed at 55°C in 0.1 x SSC with 1% SDS were searched and alignments of various sequences were carfor 30 min before being exposed to Kodak X-Omat RP film ried out using a sequence analysis software package, version (Eastman Kodak Company, Rochester, N.Y.) with an intensi6.1, from the Genetics Computer Group (Devereux et al. 1984). fying screen at -70°C. For rehybridization of the Southern blots, the labelled probe was removed from the blots by boilResults ing them 3 times in 0.1 x SSC containing 0.5% SDS. Isolationand sequence analyses of genomic DNAs encoding two different maize ubiquitin fusion protein genes Isolation of total RNA and polyribosome-associated RNA Two maize genomic clones, termed MubG7 and MubG10, and Northern- and dot-blot hybridizations each containing a putative ubiquitin fusion protein gene, were Radicles and plumules were excised from control and heatisolated from our Lambda FIX I1 maize genomic library using shocked 5-day-old seedlings, and total RNA and polyriboa ubiquitin coding region fragment from cMubCl (Liu et al. somes were isolated from them and used for Northern- and 1995) as a probe. A portion of the nucleotide sequence in these dot-blot hybridizations as described elsewhere (Liu et al. clones (delineated by Eco RI and Sac II restriction enzyme 1995). The DNA fragments used as hybridization probes sites in MubG7-1 and by Xba I and Taq I restriction enzyme included a DNA fragment from MubG7- 1, which contains sites in MubG10-1) is shown in Figs. 1 and 2. The results show both the ubiquitin and fusion protein sequences (MubG7-1-2),

Biochem. Cell Biol. Vol. 74, 1996


Fig. 1. Restriction map and nucleotide sequence of a portion of the sense strand of maize (inbred Oh43) MubG7. (A) A limited restriction map of MubG7 is depicted and the following restriction enzymes are shown: Eco RI (W), Apa I (*),Pst I (a),Bgl I1 (0), Kpn I (V),and Sac I1 (U). The hatched bar represents the proteinencoding region of the gene. (B) The nucleotide sequence of an Eco RIISac I1 DNA fragment from MubG7 is shown. The portion of this gene encoding the ubiquitin fusion protein is in uppercase letters, and putative TATA and CAAT boxes are underlined. The sequence of MubG7, which encodes a ubiquitin monomer beginning with ATG, is separated by a double-headed arrow from the portion of the MubG7 sequence encoding the fusion protein. The boxed, shaded area denotes the sequence used as a probe to map the chromosomal localization of MubG7 (nt 1-1486).

1

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1 $ d h t t cugtf geccl Ccatc ~ g a gt ta t ct Zf gagtot c t rrgcact t c t e g t ~ e g ct ta a c c t c c c t g c c 71 cgaggcatca gcacat p a c t c a a t e g t c c g gt ecgat gca t ccagct t gg t ggeggct ~g agggcatgt c I41 t t g c i a g g r a a g t c a t cgga oggcccotag agcgt t t c t a c g c t tcoacc caaggt tagc ctccgacgag 211 t c g t g a c t g t a t c i t c t g c t cgaggttggc ctcaggcgga cacccagagt t c g c c t c g g g agagacgaaa 281 c u g t t t e a c c cgftcgagga t g g c c t t a g g cgagacagaa c c g c g t c t c o cgeecugugc t g g c c t c a g g 351 caocacggga c t g c a t c t c c cgtccgaggf i g a c c t c a g a cgagatggaa t c g i g t c t c c I g c c t a g a g c 421 tagcctcgag cgagacaaaa c t g c a t c t c t c g t c c g o g g i t g g c c t c a o g cgagtcgaaa g t g c g t t t a t 491 g t g c l t a t a u ggacccgagu g a t a t c t a t e tcccacaagc c c c g a g c t t c g g g t t g t t i t gagaaacaat 561 c a a c t t g a a g g t c c a a a a t c t g c t t t c a g a a t g c t i g g c c i t g t a g t c t t c g a a a a a t t t c t g a t g a t a t 631 e c t e c u i g t t t t g a g g a t c a a g c a a c t t a i Zcctecgugt ggtcttggcg aggtggacaa caacotccgu . 701 gocgacgagc g t a g t g t a g c c t a a c a t a a l cgacgaagca gcaagtcggc gaggcaggcg a t g g c c t u g c 771 ccctaogccg gtgaugcagt aagccgocga gcgagcgatg g c c t a g t c t c igctgtcggtg aagcaggaat . 841 ctgcgaggcg caogacggtg tagcccccga gceaoigaag cagcgagtcg gegoggcaga c u a t g t c c c a 911 accccceaga cggtgaagco acaagtcagi gaggeacaig gcggtgcagc c e t c a a g g t g acgaagcagt 981 gastcggcga ggtgggtgac gaactagccc cclagccagu gaagcaacga ggggctaggc ggacoacacc 1051 tagcceccga gccgatgaag t a g t g a t t e g gcgaggcaga caacatccca gecatcgagc t g t c o a a g t a 1121 gtcactcggc aaggcuggtg tagtccagcc ttgogcltgct g a a g c a g c t i gtaagcaaag tggacgatgg 1 191 t c t agccccc gagcggtcaa agf agt g a c t cogcgaggca ggcgat ~ U E Et a g c c c c t ga g t cggt gaag 1261 cagcgagtcg gcgaagtgga ggatggggga g f c c c t a a g c t g g t g a t t g c gactcgatgg t a t g t g c g g a 1331 t g i a c g t eag atcacacaog g c t t t a i a c a g g t t c g g g c i g a t a t a a l g s g t a g g t c c c t acticctgtc 1401 g a c g t t g a a t a t t t c a a t a t cactaaoatt c a r q q u t i a c a t a t a a q c t a t q ~ u c u a t a ao a o a t a a a t t 1471-ctccaqteaa t c i u a d g g t g g t g t g g t g g c tgaaacggag g t g g t a t t t t a a t t g c c t t t t a c a a a t c a t 1541 c t tgaaaata g t a t t t t a a t t gcct t t t a c aaaacatct t gaaaataagt actgcacaag gcagcaagca 1611 g c t c a t c g t g a a t c t g a t c t t g a c a t c t c t g t g t t c a a g c t c a t a t g c a c gccggcgtca g t c c a t t t a g 1681 t a a c c t a t g g gcccacgaga cgaagcagcccagcc catcgaacac c a a c t a c t g c gagatccagc 1751 c g t c a g a t c c t g a a a c c t t t agatccaaca g t c c a c g t c g t c t g c a t t c t c a a u t c aaacacagcc 1821 gcgaacacaa a c c c t a g c c t ccacccctca cccgccgcag c c a a t c c c t c cccagcagcc g t c g t c g c c a 1891 ccgctgcagc gcggaggcga ccccccatcg ccgccaagflT GCAGATCTTC GTGAAGACCC TGACGGGGAA 1961 AACGATCACG TTGGRGGTGG AGTCCTCGGA CACCATCGAC AACGTGAAGG CCARGRTACR GGACARGGAG 2031 GGCATCCCGC CGGACCAGCA GCGGCTAATC TTCGCCGGCA AGCAGCTCGA GGACGGC ACCCTCGCCG 2101 ACTACAACAT CCAGAAGGAG TCCACCCTCC ACCTGGTGCT CCGCCTCCGA GGT G A AGAAGCGCAA 2171 GAAGAAGACG TACACCAAGC CCAAGAAGAT CAAGCACAAG CACAAGAAGG TA GCTCGC AGTGCTGCAG 2241 TTCTACRRGG TGGACGACGC CACCGGCAAG GTGRCCCGCC TCCGCAAGGA GTGCCCCAAC ACCGRGTGCG 2311 GTGCGGGTGT CTTCATGGCC AACCACTTCG ACCGCCACTA CTGCGGCRRG TGCGGCCTCA CCTACGTCTA 2381 CRATCAGAAG GCGTflfltccc atgcgccgct t t g c t t c a c c c g c c t a c c t a t c a a g c a t t c a c c t g t t g g t 2451 t a c t c g a t t t g a a t a t c a t t t a g a t c t g t t t g a g g t t g g a acataaatgc a g c a g t a g t t t c t g t c t t a t 2521 t t a t a t t g g t a c c t c t g t t a c t g a g t t g a t g t g g t c c t g g t t c c t g a g c t t a a t t t t g t t t t c g t g t t c t 2591 a t c c t t a g a g a c g a t t g t a c c t t t t g g c c g t t t c a c t t t c a t t c a t a c a t a g g t a g a t t a t g g t a g t a t t 2661 g c a t g c t t a g a t t a a t t g c g t t c c t t a g c t a t t g c t a t t g c t t g a t t a g c c t t g a g g t g g caactgacaa 2731 gggaaaatca tcactggaaa cctggaatgc a t g a a t t g g a a t g t ataaat agt g t a a g t a t c a t g c a t a a 2801 a c a t a a t c a t cgatggcata aacat t g a t t ggacagt t c a tcgcggaggc tgataccaag g t c g c t a g a t 2871 caaaggacgc cgcctaaggc accggattgg aggagttggt cgtcgaagga cgctagagcc a c c c a t g c c t 2941 c a c g t g g c t g a c a t c g t t a g agacggtcgt gcctcgcgac cgcgg

fl


Fig. 2. Restriction map and nucleotide sequence of a portion of the sense strand of maize (inbred Oh43) MubG10. (A) A limited restriction map of MubGlO is depicted and the following restriction Acc I1 (O),Nar I (O), Bgl I1 (O), and Tag I (0).The enzymes are shown: Eco RI (M), Xba I (e), hatched bar represents the protein-encoding region of the gene. (B) The nucleotide sequence of a Xba IITaq I DNA fragment from MubGlO is shown. The portion of this gene encoding the ubiquitin fusion protein is in uppercase letters, and putative TATA and CAAT boxes are underlined. The sequence of MubG10, which encodes a ubiquitin monomer beginning with ATG, is separated by a double-headed arrow from the portion of the MubGlO sequence encoding the fusion protein. The boxed, shaded area denotes the sequence used as a probe to map the chromosomal localization of MublO (nt 1-736).

RCGGGGAAGA C C A T C A C T T T GGAGGTGGAG T C C T C G G A C A CCATCGACAA TGTGAAGGCC AAGATCCAGG ACAAGGAGGG CATCCCCCCG GACCAGCAGC G G C T C A T C T T CGCCGGCAAG CAGCTCGAGG ACGGTCGCAC CCTCGCCGAC TACARCRTCC C ~ A R RGCACAAGCA GACGACRCCA RGAGTGCGGC GCGGCARGTG gccccgattt

AGAAGGAGTC RAGCGTARGR G CAAGRRGGTG CTAGCAAGGT GCGGGCGTCT CGGCCTCACC gctttatccg

CRCCCTCCAC RGAAGACGTR RAGCTCTCCG GACCCGCCTC TCATGGCCAA TATGTCTACA cctaccctat

CTGGTGCTGC CRCCRRGCCC TGCTGCRGTT CGCAAGGAGT CCACTTCGAC ACCAGAAGGC cgagcatcca

GTCTTCGCGG AAGRRGRCTA CTATAAGGTG GCCCCAACTC CGCCACTACT GTAflttgctt gctgttggtt

actcgaactg t a t c t c a t g t a g a t c t g t c t gaggttgaaa cttaaatgga gcagtagtat c t g a c a t a t t ggtacccctg t t a c t g a a t t gacgtgatgc t g a g t t t a a t t t t g t t t t c g t a t c t a t c c t tacagaccat t g c a c t c t t t t c c c g t t t c a c c a t g a t t t t t t t g a t t c a t aggtcga

14


Fig. 3. A comparison of the nucleotide sequence of MubGlO (written in full) with MubG7. Dashes indicate identity and dots indicate insertions or deletions to allow for maximal alignment (Devereux et al. 1984).The ATG and TAA codons are boxed, and the sequences in these genes that encode the ubiquitin monomers are separated by a double-headed arrow from those that encode the fusion proteins. Numbers on either side of these sequences correspond with those assigned to the sequences in Figs. 1 and 2.


1385 GTCCCTRCTTCCTGTCGRCtTT...... GRRTRTTTCARTRTCGCTGGRGTTCACGGRTTACRTRTGRGCTRTGGGCGGTGGRGRGTGRGTTCTCCRGTC 1478

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I I Ill

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I

200 RRRCRTRTRTTCRCRTG~CRTTGTTRGRGGRG~TTGRRGRRGRTRTGTRCRGTTTGRRGCRCCTGTGGRRCRRGTRCCTCTTCCGGGTRRRCRTTTTTCR 299

1579 T T G R R R f l T R R G T R C T G C ~ C R R G G C R G C R R G C R G C C . . . . . .

I

I

I I

1 1 1 1

1

I

. . .TCATCGTGRRTCTGRTCTTGRCATCTCTGTGTTCRRGCTCRTRTGCRCGCCGGCG IIIIII

I

I

II I I I

II II

II

1667

II I I I

400 G G R f l G G G R C C G G C G G G G R T T C G T T R G G R G T C T C C ~ T C C G C C A A C G C G G C G G C G R R G G T C C C G G C G C G T C R G R T C G G T G G C T G G C G G R C G C 499 1668 TC~GTCCRTTTRGTRRCCTRTGGGCCCRCGRGRCG~RGCRRRTCRGCCCRGCCCRTCGRRCRCCRRCTRCTGCGRGRTCCRGCCGTCRGRTCCTGRR.RC I766

1

111 1 1 1 1

11

I

I1111111 1 1 I I I I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

11111111 1111111 111111111 II II

I

500 TGCTCCCAGTCRGTCGCCCCT..CCGCRCGAGRTGRGGCRRRTCCGCCCRGCGCRTGGRACRCTGRCTRCTGCRRGRTCCRRCCGTCRGRTTCTCRRGCC

597

1767 CTTTRGRTCCRRCRGTCC~CGTCGTCTGCRTTCTCRRTRTRRTCRRRCRCRGCCGCGRCRCRRCCCTRGCCTCCRCCCCTCRCCCGCCGCGCCRRTC

1866

1

111111111111 111 1111111

I Ill 11111

I

11111 II 1 1 1 1 1 1 1 11 I1111111111

598 CCCTRGRTCCARCRGCCCRTGTCGTCTTTCTCCTCTATRTRGGCGCRCRCRCCCRCRRRCRCRGRCRCCRGCCTCCRCC.

................... 676

0

1 RGRTCTTCGTGRAGACCCTCACGGGGARRRCGRT 1966 1111 1111 11 IIIIIIIIIIIIIII 11111111 11111111111 II II

1867 CCTCCCCRGCRGCCGTCGTCGCCRCCGCTGCRGCGCGGRGGCGCCCCCCRTCGCCGCC

II II

677

I1 111

111 I I

11

. . . . . . . . . . . .CCRTCRACCCTRCCTRGGCGCCGCCGCRGGCGGCCCCGRTCGTCGGCRR

f

AGATCTTTGTGARGRCTCTGRCGGGGRRGRCCRT 764

1967 CRCGTTGGRGGTGGAGTCCTCGGRCRCCRTCGRCRRCGTGRRGGCCRRGRTRCRGGRCRRGGRGGGCRTCCCGCCGGRCCRGCRGCGGCTRRTCTTCGCC

2066

111 11111111111111111111111111111111 IIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIII IIIIIllllllllllll l l l l l l l l l 765 CRCTTTGGRGGTGGRGTCCTCGGRCRCCRTCGRCRRTGTGRRGGCCRRGRTCCRGGRCRRGGRGGGCRTCCCCCCGGRCCRGCRGCGGCTCRTCTTCGCC 864

d

2067 GGCRRGCRGCTCGAGGRCGGCCGCRCCCTCGCCGACTRCRRCRTCCRGRRGGRGTCCRCCCTCCRCCTGGTGCTCCGCCTCCGAGGT

GCGCCRRGRAGC 2166

I I I I I I I I I I I I I I I I I I I I 1111111111111111111IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I1 I1 I1 I1 l l l l l l l l l l l l l 865 GGCRRGCAGCTCGRGGACGGTCGCRCCCTCGCCGACTACRRCRTCCRGRRGGRGTCCRCCCTCCRCCTGGTGCTGCGTCTTCGCGG

GCGCCRRGRRGC 964

2167 GCRRG~~GRRGRCGT~CRCCRRGCCC~RG~RGRTCRRGCRCRRGCRCRRGRRGGTRRRGCTCGCRGTGCTGCRGTTCTRCRRGGTGGRCGRCGCCRCCGG 2266

1 1111111111111111111111111111111

11111111111111111111 IIIIII 1 11111111111111 111111111111 1111

I

965 G T R R G R R G R R G ~ C G T ~ C R C C R R G C C C ~ ~ G ~ R G R C T R R G C R C R R G C R C R R G R R G G T G R R G C T C T C C G T G C T G C R G T T C T R T G G T G G C G C C C I064 TRG 2267 C~RGGTGRCCCGCCTCCGCRRGGAGTGCCCCRRCRCCG~GTGCGGTGCGGGTGTCTTCRTGGCCRRCCRCTTCGRCCGCCRCTRCTGCGGCRRGTGCGGC2366

IIIII11TtllllllllllIIIIlIIIIIIIIII I IIIIIIII 1 1 1 1 1 IIIIIIIIIIIIIIIIIIIIIIIIIIllllllllllllllllllllll 1065 CRRGGTGACCCGCCTCCGCRRGGRGTGCCCCRRCTCRGRGTGCGGCGCGGGCGTCTTCRTGGCCRRCCRCTTCGRCCGCCRCTRCTGCGGCRRGTGCGGC

I164

CCCRTGCGCCGCTTTGCTTCRCCCGCCTRACCTRTCRRGCRTTCRCCTGTTGGTTRCTCGRTTTGRATR2465

1 1 1 1 111 1 1 1 1 1 1 1 1 IIIIIII 111111 11111 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

11 II

TGCTTGCCCCGATTTGCTTTRTCCGCCTRCCCTRTCGRGCRTCCRGCTGTTGGTTRCTCGRRCTGTRTC

1264

2466 TCRTTTRGATCTGTTTGRGGTTGGRRCRTAAATGCAGCRGTRGTTTCTGTCTTRTTTRTRTTGGTRCCTCTGTTRCTGRGTTGflTGTGGTCCTGGTTCCT 2565

IIII IIIIIIIII IIIIIIII I l l IIIIII IIIIIIIII Ill1 I

IIIIIIIIIII IIIIIIIIII IIII II

I I II

1265 T C R T G T R G R T C T G T C T G R G G T T G R R ~ C T T ~ R R T G G R G C R G T R G T f l T C T G R C . . . . . R T R T T G G T R C C C C T G T T ~ C T G R ~ T T G R C G T T . . . . . . G ~1351 TGCT 2566 GRGCTTRflTTTTGTTTTCGTGTTCTRTCCTTRGRGRCtATTGTRCCTTTTGGCCGTTTCRC....TTTCRTTCRTRCRTRGGTAGA

111 1111111111111111

1111111111 1111 1111 11

111

111111111

111

1352 GRGTTTflRTTTTGTTTTCGT.RTCTRTCCTTRCRGRCCRTTGCRCTCTTTTCCCGTTTCRCCRTGRTTTTTTTGRTTCRTRGGTCGfl

that the ORF of each gene consists of one ubiquitin coding unit fused to an unrelated 237 nucleotide sequence that encodes a protein containing 79 amino acids. There is no intervening sequence between the ubiquitin sequence and the fusion protein sequence in either gene. Nucleotide identity (Fig. 3) between the coding regions of these genes (95%) is slightly higher than that between any two ubiquitin repeats (84-94%) in some maize polyubiquitin genes (Liu et al. 1995). The identity drops sharply to 45% in the 318

2647

1 1 II IIIIIII II 1437

nt upstream from the ATG start site. In the 3'-UTRs, the identity is 83% for the 248 nt following the TAA stop codon. A putative TATA sequence is located 125 nt upstream from the ATG start codon in MubG7-1 and 94 nt upstream from the ATG start codon in MubG10-1, and CAAT box-like sequences (5'-CAAAT) are located 98 nt upstream from the putative TATA sequence in MubG7-1 and 97 nt upstream from the putative TATA sequence in MubG10-1. Unlike the maize polyubiquitin genes (Liu et al. 1995), neither the typical

Liu et al

Table 1. Map position of maize ubiquitin fusion protein genes. Designation on maize mapa


uwol uw02

Designation this text

Fragment used to produce S D P ~

MubG7 MubGlO

5'-UTR (Fig. 1) 5'-UTR (Fig. 2)

Chromosome

arm assignmentC 8L 162 1L131

aPublished annually in the Maize Genetics Cooperation Newsletter. 'SDP, strain distribution pattern (Burr et al. 1988). CChromosomes 1-10 (Rhoades 1950); S, short arm; L, long arm; position 000, telomere of short arm;numbering proceeds sequentially to telomere of long arm; units are estimates of centiMorgans.

Fig. 4. A comparison of the amino acid sequences in the fusion proteins derived from two maize genes, MubG7 and MubG10, with the long extension-protein - fusion protein sequences (7680 amino acids) reported in barley (Gausing and Jensen 1990), Arabidopsis (Callis et al. 1990), yeast (Ozkaynak et al. 1987), and humans (Lund et al. 1985). Identical amino acid residues are denoted by dashes, and asterisks symbolize gaps introduced to maximize the alignment. The nucleotide sequence similar to a motif required for protein localization to a nucleus is boxed, and the four cysteine residues with similar arrangement to that found in zinc figure structures are underlined. i7Al ZE H u b t - 7 M R l Z E nubG-lO BRRLEY

YT KPKKIKHKHK KUKLAUCOFY RUOOATGKUT ----T--------s--------f-$---

------------ ----Q----- ---------- ---------- -I-C$*---0 ---GS*--- 0 -- T------------------ ---RIEL---- f---N---R------KY--Eln--IS

YERST HUnRn

79

41 I l R l Z E IlubG-7 n A l Z E nubG-10 BRRLEY RRRBIDOPSIS ~ ~ #Rf)B/DOPS/S UB06 YEAST HUnAN

RLRKECPNTE --------S--------AD --------AT 0 5 --------AT K--R--S-pT

CGAGUFnANH FDRHYCGKCG LTYUYMQKA

---R---sRD

-----R--s-

-------------T--------T---s----T---S-

-------------------

------I----

K--L-----H

---------c

--------------------+KEG~ -----QKEGR SU-KU-A ---CF-KPED

ER QE K

animal-type polyadenylation signal, AATAAA, nor a partial polyadenylation consensus sequence, AATAA, is evident in the 3'-UTRs of these ubiquitin fusion protein genes.

Derived amino acid sequences of the ubiquitin and fusion proteins in MubG7 and MubGlO The derived amino acid sequences of the ubiquitin moieties in MubG7 and MubGlO are identical with those established for the ubiquitin coded for from maize polyubiquitin genes (Liu et al. 1995) and other plant ubiquitin sequences (Callis and Vierstra 1989). The derived amino acid sequences of the two fusion protein moieties are aligned in Fig. 4 and this comparison shows that only five amino acids differ between the fusion proteins encoded by MubG7 and MubG10. Interestingly, the amino acid differences between these fusion proteins involve the substitution of either a serine or threonine for another amino acid. Each of these fusion proteins has a stretch of seven highly basic amino acid residues at their amino-terminal end, which is similar to a sequence motif required for protein local-

ization to the nucleus (Dingwall and Laskey 1986), and contain four cysteine residues (underlined in Fig. 4) capable of forming a zinc-finger structure similar to that found in nucleic acid binding proteins (Klug and Rhodes 1987). A comparison of the amino acid sequences of these maize ubiquitin fusion proteins to barley, Arabidopsis, yeast, and human ubiquitin fusion proteins is also shown in Fig. 4. The two maize fusion proteins share -90% identity with the sequences of two different barley 79 amino acid fusion proteins (Mubl and Mub2; Gausing and Jensen 1990), -80% identity with two different 81 amino acid fusion proteins in Arabidopsis (UBQ5 and UBQ6; Callis et al. 1990), 76% identity with a yeast 76 amino acid fusion protein sequence (Ub13; Ozkaynak et al. 1987), and 72% identity with a human 80 amino acid fusion protein sequence (Lund et al. 1985).

Genomic Southern-blothybridization analyses Southern-blot hybridization analyses of Eco RI, Barn HI, or Hind 111digests of maize (inbred Oh43) genomic DNA with a DNA fragment from a maize ubiquitin fusion protein gene (either MubG7- 1-3 or MubG10- 1-3), which lacks the ubiquitin sequence but contains the fusion protein sequence, reveal that this probe hybridizes with at least three different DNA fragments from each restriction enzyme digest (Fig. 5A). Since no Eco RI, Barn HI, or Hind 111enzyme restriction sites were found in the MubG7-1 or MubG10-1 sequences, we assumed that the maize ubiquitin fusion protein genes arise from a multigene family consisting of at least three different genes. Southern-blot hybridization analyses, using DNA fragments from the noncoding 5' regions of MubG7-1 (Fig. 5B) and MubG10-1 (Fig. 5C), confirmed our assumptions. As shown in Fig. 5, MubG7- and MubG10-specific probes hybridize with a single restriction fragment in each digestion, and each hybridizing DNA fragment corresponds to only one of the three restriction fragments appearing when the common fusion protein coding region is used as a probe. Since all of the DNA fragments hybridizing with the fusion protein coding region (Fig. 5A) are not accounted for by sequences in MubG7-1 or MubG10-1, at least one additional ubiquitin fusion protein gene is probably present in the maize genome. Genes encoding different maize ubiquitin fusion protein mRNAs map to different chromosomes The results of the recombinant inbred mapping strategy are listed in Table 1. In as much as the mapping of MubG7 was our first attempt to map a DNA sequence, Maillet (1993) confirmed the chromosome 8 assignment using the monosomic method of analysis in maize (data not shown). Northern-blothybridization analyses of the ubiquitin fusion protein mRNAs associated with polyribosomes in tissue from control and heat-shocked 2- and 5-day-old maize seedlings Figure 6 presents the results from Northem-blot hybridization analyses in which DNA fragments containing only ubiquitin, a gene-specific polyubiquitin, ubiquitin, and fusion protein, or fusion protein sequences were hybridized with polyribosomeassociated rnRNAs from radicles (R) and plumules (P) of control (25°C) and heat-shocked (42.5"C for 1 h) 2- and 5 4 a y old maize (inbred Oh43) seedlings. The results shown in Fig. 6A, using a DNA fragment from the ubiquitin coding region

16



Fig. 5. Southern-blot hybridization analyses of homologous fusion protein sequences in maize DNA. DNA was isolated from maize, digested with Hind I11 (I), Barn HI (2), and Eco RI (3), electrophoretically separated on 1% agarose gel, and transferred to a Zeta-probe membrane. Autoradiograms from the hybridizations of maize DNA to (A) the fusion protein coding region, (B) a MubG7-1 gene specific probe, or (C) a MubG10-1 gene specific probe are shown. The relative mobility and size of coelectrophoresed DNA standards are indicated on the right side of Fig. 5C.

1 2 3

1 2 3

1 2 3

as a probe, demonstrate that at least five different size, ubiquitin-containing transcripts (-2.0, 1.7, 1.4, 1.1, and 0.7 kb) are associated with polyribosomes in maize radicles and plumules of both 2- and 5-day-old seedlings. Figures 6C and 6D show the same blots hybridized with sequences containing either the entire coding region for the ubiquitin fusion protein complex in MubG7-1 (Fig. 6C) or only the fusion protein coding region common to MubG7 and MubGlO (Fig. 6D). These results demonstrate that the ubiquitin fusion protein mRNA transcripts are about 0.7 kb in size and that heat shock does not affect the accumulation of these fusion protein mRNAs in the radicles or plumules of 2-day-old seedlings but does markedly decrease the accumulation of these transcripts on polyribosomes in tissues of 5-day-old seedlings. Hybridization of gene-specific DNA fragments from the 5'-UTRs (see Materials and Methods) of both MubG7 and MubGlO on these same blots (not shown) confirmed that the amount of mRNA transcripts encoded from each of these fusion protein genes is markedly decreased in radicles and plumules of 5-day-old heat-shocked seedlings. Hybridization of the same RNA blots with a DNA fragment specific for a maize polyubiquitin (Fig. 6B), one containing seven repeats of a ubiquitin encoding region (MubG1; see Liu et al. 1995), emphasizes the difference in the size of the polyubiquitin and ubiquitin fusion protein encoding transcripts and demonstrates that the amount of this polyubiquitin transcript, unlike the fusion protein transcript, markedly increases in polyribosomes in tissue of heatshocked 5-day-old seedlings. Finally, results from hybridizing these same blots with DNA sequences encoding a maize HSP18 gene sequence (Fig. 6D), a maize HSP70 gene sequence (Fig. 6E), and a DNA sequence encoding maize ribosomal RNA (Fig. 6G) validate the heat shock response by these tissues and, in the case of the rRNA hybridizations, assure that equal quantities of RNA were used in preparing these blots.

Discussion Results from our studies, using gene-specific oligonucleotide probes to appraise the expression of particular polyubiquitinencoding genes in tissues of heat-shocked maize seedlings (Liu et al. 1995), prompted us to assess the effect of heat shock on the expression of other ubiquitin encoding genes, the ubiquitin fusion proteins. In this study, we characterize the nucleotide sequence of two different ubiquitin fusion protein genes (MubG7 and MubGlO) in a maize inbred (Oh43), map the chromosomal localization of these genes, and demonstrate that heat shock affects the polyribosome-associated levels of the rnRNAs encoded from these particular genes. The nucleotide identity between the coding regions of the maize ubiquitin fusion protein genes that we isolated is 95%; however, the identity drops sharply to 45% in the first 318 nt upstream from their ATG start sites and to 83% for the first 248 nt after their TAA stop codons. One of the genes, MubG7, which we isolated from our maize inbred, Oh43, shares >90% identity with a ubiquitin fusion protein gene (UBF9), which Chen and Rubenstein (1991) isolated from another maize inbred, W22. The ORF of both MubG7 and MubG10 contains a sequence consisting of one ubiquitin coding unit fused to an unrelated 237-nucleotide sequence encoding a fusion protein 79 amino acids long. A comparison of the amino acid sequence of the fusion proteins encoded from MubG7 and MubGlO reveals that they differ from each other by five amino acid substitutions and that they share between 72 and 90% identity with the 79-81 amino acid ubiquitin fusion proteins reported for plants (Gausing and Jensen 1990; Callis et al. 1990), yeast (Ozkaynak 1987), and humans (Lund et al. 1985). Southern blots of restriction enzyme digested maize (Oh43) genomic DNA, hybridized with DNA fragments from the 5' noncoding regions of these genes, suggests that these are single-copy genes. However, since all of the DNA frag-

Liu et al.

17

Fig. 6. Northern-blot hybridization analyses of the relative changes in the amount of ubiquitin fusion protein mRNAs associated with polyribosomes in radicles (R) and plumules (P) of control (C; 25OC) and heat-shocked (H; 42.5"C for 1 h) 2-day-old (upper) and 5-day-old (lower) maize (inbred Oh43) seedlings. Northern blots of polyribosome-associated RNAs were hybridized to DNA fragments encoding (A) a maize ubiquitin coding region (Ub), (B) a sequence specific for a maize polyubiquitin gene (MubG1; Liu et al. 1995), (C) a maize sequence containing both ubiquitin and the fusion protein (MubG7-1-2), (D) a maize sequence specific for the fusion protein coding region (MubG7-1-3), (E) a maize 18 kDa HSP, (F) a maize 70 kDa HSP, and (G) a maize ribosomal RNA (rRNA). The mobility and size (kb, kilobases) of standards from an RNA ladder are shown on the left side of this figure.

Ub

MubGl

MubG7-1-2 MubG7-1-3 HSP 18


kb

n 0

n

Es e-: y

7 '

4.4- A 2.4-

HSP 70

rRNA

-

t=-

31

E

1.4-

I

CU

0.3 C H C H R P

C H C H R P

C H C H R P

ments hybridizing with a sequence from the highly homologous fusion protein coding region are not accounted for by the sequences specific for MubG7 and MubG10, at least one additional ubiquitin fusion protein gene is probably present in the maize genome. Use of DNA fragments from the noncoding 5' regions of these genes as gene-specific probes enabled us to map the chromosomal localization of these genes. The map assignments, derived from the individual strain distribution patterns obtained from both recombinant inbred (RI) families (Burr et al. 1988), were compared with 1247 other entries in the RI data pool, and the results are listed in Table 1. Maillet's (1993) monosomic analysis identified the chromosome 8 assignment for uwol (long arm chromosome 8, position 162) in the distal portion of the arm, while uwo2 (chromosome 1L 131) maps to a subcentromeric location. These assignments, added to those reported for the three polyubiquitin genes (Liu et al. 1995), suggest dispersed locations for the individual genes, although there is evidence on the basis of current map constructions that the genes reside either in regions near to centromeres or to telomeres. Using gene-specific probes, we assessed the levels of the mRNAs encoded from these particular genes in both plumules and radicles of control and heat-shocked 2-and 5-day-old maize seedlings. In this study, as in our previous report (Liu et al. 1995), we initially used polyribosome-associated RNAs as our sources of mRNAs for Northern-blot hybridization analyses. Our results disclose that the mRNA transcripts encoded from MubG7 and MubGlO are about 0.7 kb in size. It appears that heat shock does not affect the accumulation of these

C H C H R P

C H C H R P

C H C H R P

C H C H R P

fusion protein mRNAs in the radicles or plumules of 2-dayold maize seedlings but does markedly decrease the accumulation of these transcripts on polyribosomes in tissues of 5day-old seedlings. Since the level of polyribosome-associated mRNAs encoded from MubG7 and MubG10, as well as from specific maize polyubiquitin encoding genes (MubG1, MubG5, and MubG9; Liu et al. 1995), does not change in tissue from 2-day-old seedlings exposed to heat shock while HSP70- and HSP18-mRNA levels are elevated, we decided to assess the levels of these ubiquitin-encoding mRNAs in total cellular RNA from 2- and 5-day-old control and heat-shocked seedlings. Quantitative dot-blot hybrization analyses of total c e l l u l a ' ~(not ~ ~ shown) were similar to the results obtained with polyribosome-associated mRNAs and these results suggest that neither RNA processing nor translational control plays a role in determining the relative level of these mRNAs available for translation. These studies, coupled with our earlier report on the heat shock induced response of specific polyubiquitin encoding genes in maize (Liu et al. 1995), clearly demonstrate the necessity for using gene-specific probes in evaluating the effect that a stress, such as heat shock, has on the relative level of mRNAs emenating from multigene families. The relative level of ubiquitin fusion transcripts in 2-day-old seedlings is not down-regulated following heat shock (Fig. 6D, top), as it is in 5-day-old seedlings (Fig. 6D, bottom), suggesting that the function of ubiquitin fusion proteins is as critical to the survival of 2-day-old seedlings as are the hsps. Moreover, the studies in this report establish that the levels of mRNAs

Biochem. Cell Biol. Vol. 74, 1996 encoded from different members of this multigene family are affected by heat shock in a manner that is different from and (or) more complex than the HSP70- and HSP18-mRNAs and suggest that the expression of each of the genes is regulated in a gene-specific manner.


Acknowledgments The authors are grateful to Dr. Ben Burr, Brookhaven National Laboratory, for the provision of the original seeds of the recombinant inbreds and the analyses of the strain distribution patterns and to Carol Richardson for her expert help in the mapping study. This work was supported by a grant from the Human Frontier Sciences Program to B.G.A. and D.B.W. as well as by grants from the Natural Sciences and Engineering Research Council of Canada to B.G.A. and D.B.W.

References Atkinson, B.G., Liu, L., Goping, I.S., and Walden, D.B. 1989. Expressions of the genes encoding HSW3, HSPl8 and ubiquitin in radicles of heat-shocked maize seedlings. Genome, 31: 689704. Atkinson, B.G., Raizada, M., Bouchard, R.A., Frappier, J.R.H., and Walden, D.B. 1993. The independent stage-specific expression of the 18 kDa heat shock protein genes during microsporogenesis in Zea mays L. Dev. Genet. 14: 15-26. Berg, J.M. 1986. Potential metal-binding domains in nucleic acid binding proteins. Science (Washington, D.C.), 232: 485-487. Binet, M.N., Weil, J.H., and Tessier, L.H. 1991. Structure and expression of sunflower ubiquitin genes. Plant Mol. Biol. 17: 395-407. Bouchard, R.A., Frappier, J.R.H., Liu, L., Raizada, M., Atkinson, B.G., and Walden, D.B. 1993. Developmentally-modulated expression of transcripts from stress-inducible gene families during microsporogenesis and gametophyte development in Zea mays L. Maydica 38: 135-144. Burr, B., and Burr, F.A. 1991. Recombinant inbreds for molecular mapping in maize: theoretical and practical considerations. Trends Genet. 7: 55-60. Bun; B., Burr, F.A., and Thompson, K.H. 1988. Gene mapping with recombinant inbreds in maize. Genetics, 118: 5 19-526. Callis, J., and Vierstra, R.D. 1989. Ubiquitin and ubiquitin genes in higher plants. Ox. SUN.Plant Mol. Cell Biol. 6: 1-30. Callis, J., Raasch, J.A., and Vierstra, R.D. 1990. Ubiquitin extension proteins of Arabidopsis thaliana. J. Biol. Chem. 265: 12 486 12 493. Chen, K., and Rubenstein, I. 1991. Characterization of the structure and transcription of a ubiquitin fusion gene from maize. Gene (Amsterdam), 107: 205-212. Dellaporta, S.L., Wood, I., and Hicks, J.B. 1983. Maize DNA minipreps. Maize Genet. Coop. Newsl. 57: 26-29. Devereux, J., Haeberli, P., and Smithies, 0 . 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-416. Dingwall, C., and Laskey, R.A. 1986. Protein import into the cell nucleus. Annu. Rev. Cell Biol. 2: 366-390. Dretzen, G., Bellard, M., Sassone-Corsi, P., and Chambon, P. 1981. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal. Biochem. 112: 295-298. Finley, D., Bartel, B., and Varshavsky, A. 1989. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature (London), 338: 394-401. Gausing, K., and Jensen, C.B. 1990. l b o ubiquitin-long-tail fusion genes arranged as closely spaced direct repeats in barley. Gene (Amsterdam), 4: 165-171.

Genschik, P., Parmentier, Y., Durr, A., Marbach, J., Criqui, M.C., Jamet, E., and Fleck, J. 1992. Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses. Plant Mol. Biol. 20: 897-910. Goping, S.I., Frappier, J.R.H., Walden, D.B., and Atkinson, B.G. 1991. Sequence, identification and characterization of cDNAs encoding two different members of the 18 kDa heat shock family of Zea mays L. Plant Mol. Biol. 16: 699-71 1. Hoffmann, N.E., KO, K., Milkowski, D., and Pichersky, E. 1991. Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3. Plant Mol. Biol. 17: 1189-1201. Klug, A., and Rhodes, D. 1987. "Zinc fingers," a novel protein motif for nucleic acid recognition. Trends Biol. Sci. 12: 464-469. Liu, L. 1992. Characterization and expression of polyubiquitin and ubiquitin fusion protein genes in tissues from control and heatshocked maize seedlings. Ph.D. Thesis, The University of Western Ontario, London, Ont., Canada. Liu, L., Maillet, D., Frappier, J.R.H., Walden, D.B., and Atkinson, B.G. 1995. Characterization, chromosomal mapping, and expression of different polyubiquitin genes in tissues from control and heat-shocked maize seedlings. Biochem. Cell Biol. 73: 19-30. Lund, P.K., Moats-Staats, B.M., Simmons, J.G., Hoyt, E., D'Ercole, A.J., Martin, F., and Van Wyk, J.J. 1985. Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J. Biol. Chem. 260: 7609-7613. Maillet, D.S. 1993. Recombinant inbred mapping of ubiquitin genes in Zea mays L. M.Sc. Thesis, The University of Western Ontario, London, Ont., Canada. Maniatis, T., Fritsch, E.G., and Sambrook, J. 1982. Molecular cloning - a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp.191-193,320-325, and 371-373. McMullen, M.D., Hunter, B., Phillips, R.Z., and Rubenstein, I. 1986. The structure of the maize ribosomal DNA spacer region. Nucleic Acids Res. 14: 4953-4968. Miiller-Tanbenberger, A., Graack, H.R., Grohmann, L., Schleicher, M., and Gerisch, G. 1989. An extended ubiquitin of Dictyostelium is located in the small ribosomal subunit. J. Biol. Chem. 264: 53 19-5322. Ozkaynak, E., Finley, D., Solomon, M.J., and Varshavsky, A. 1987. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 6: 1429-1439. Pollmann, L., von Kampen, J., and Wettern, M. 1991. Ubiquitin in a lower plant: characterization of ubiquitin encoding DNA and RNA from Chlamydomonas reinhardtii. Eur. J. Biochem. 202: 197-204. Redman, K.L., and Rechsteiner, M. 1989. Identification of the long ubiquitin extension as ribosomal protein S27a. Nature (London), 338: 438-440. Rhoades, M.M. 1950. Meiosis in maize. J. Hered. 41: 58-67. Salvetsen, G., Lloyd, L., and Farley, D. 1987. cDNA encoding a human homology of yeast ubiquitin 1. Nucleic Acids Res. 15: 4585. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467. Schlesinger, M.J., and Bond, U. 1987. Ubiquitin genes. Oxf. Surv. Eukaryotic Genes, 4: 77-89. Shah, D.M., Rochester, D.E., Krivi, G.G., Hironaka, C.M., Mozer, T.J., Fraley, R.T., and Tiemeier, D.C. 1985. Structure and expression of maize HSP 70 gene. In Cellular and molecular biology of plant stress. Edited by J.L. Key and T. Kosuge. Alan R. Liss Inc., New York. Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-5 17.

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Swindle, J., Ajioka, J., Eisen, H., Sanwei, B., Jacquemot, C., Browder, Z., and Buck, G. 1988. The genornic organization and transcription of the ubiquitin genes of Trypanosoma cruzi. EMBO J . 7: 1121-1127.

19

Westphal, M., Miiller-Taubenberger, A., Noegel, A., and Gerisch, G. 1986. Transcript regulation and carboxy terminal extension of ubiquitin in Dictyostelium discoideum. FEBS Lett. 209: 92-96.