©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Riboflavin Biosynthesis in Saccharomyces cerevisiae
CLONING, CHARACTERIZATION, AND EXPRESSION OF THE RIB5 GENE ENCODING RIBOFLAVIN SYNTHASE (*)

(Received for publication, July 19, 1994; and in revised form, October 24, 1994)

Maria A. Santos José J. García-Ramírez (§) José L. Revuelta (¶)

From the Departamento de Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.


INTRODUCTION

Riboflavin, vitamin B(2), is commercially important as an additive in food industries. The yeast Saccharomyces cerevisiae has been widely used to analyze flavinogenesis biochemically and genetically(1) , and some yeast species have been developed for the biotechnological production of riboflavin(2) . Six complementation groups of S. cerevisiae mutants (rib1, rib2, rib3, rib4, rib5, and rib7) with different defects in riboflavin biosynthesis have been identified, and their normal genetic functions have been correlated with specific metabolic steps (3, 4) .

The last step in riboflavin biosynthesis is catalyzed by riboflavin synthase (EC 2.5.1.9). The enzyme catalyzes the dismutation of two molecules of the substrate 6,7-dimethyl-8-ribityllumazine to yield one molecule each of riboflavin and 5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine. Riboflavin synthase activity has been observed in many microorganisms and in spinach (for a review see (1) ). The protein from bakers' yeast has been partially purified, and a molecular weight in the range of 80,000 has been estimated; but its structure has not been further investigated(5, 6) .

Bacillus subtilis contains two forms of riboflavin synthase characterized by the subunit structures alpha(3) (light enzyme) and alpha(3)beta (heavy enzyme)(7) . Riboflavin synthase activity resides in the alpha subunit, whereas the beta subunit catalyzes the synthesis of the substrate 6,7-dimethyl-8-ribityllumazine(8, 9) . The complete amino acid sequence of the alpha subunit was obtained by direct sequencing of overlapping peptides. The sequence shows marked internal homology between the N-terminal and C-terminal halves, thus suggesting that the monomer forms two structurally similar, substrate-binding, domains(8) . Sequences have subsequently been reported for the genes encoding riboflavin synthase from the bacteria Photobacterium leiognathi(10) and Escherichia coli, on the basis of homology to B. subtilis sequence data.

Although early genetic studies provide useful information suggesting the role of RIB5, no further evidence has elucidated the function of the RIB5 gene and its gene product. In this paper we report the isolation and characterization of the RIB5 gene and the evidence supporting that RIB5 is the structural gene for riboflavin synthase in yeast. The gene appears to be expressed constitutively at very low levels. We also describe the purification of the enzyme to apparent homogeneity. The M(r) of the enzyme is about 75,000 with three subunits of M(r) 25,000 each. Our results support the model suggesting that the monomer would contain two structurally similar, although functionally different, domains.


EXPERIMENTAL PROCEDURES

Materials

All reagent chemicals were of the highest commercial grade. 6,7-dimethyl-8-ribityllumazine was synthesized as described(11) . Restriction enzymes and DNA modification enzymes were from Pharmacia Biotech Inc., New England Biolabs, or Boehringer Mannheim. Thermus aquaticus DNA polymerase was from Perkin-Elmer. Oligonucleotides RIB5E (5`-ATA GCA CCA TGG CTA CTG GT-3`), RIB52 (5`-CAA CGT GAC AAT CGG TGA GAA TAC TCC CCG-3`), RIB55 (5`-GTG GAT CCG AAA CCT ATT TAT GAC G-3`), RIB56 (5`-GAA TGC ATG GGG ACT GTT TTG G-3`), and RIB57 (5`-CAT TGT GCT ATG GTT ACG ATC TCA CCA A-3`) were synthesized on an Applied Biosystems synthesizer.

Strains and Media

The following S. cerevisiae strains were used in this study. X2180-1A (MATa) and TD28 (MATaura3-52 ino1) were obtained from F. del Rey (Universidad de Salamanca). JC2a (MATalpha leu2, 3-112 his3-Delta1 ura3-52) was from M. Jayaram (University of Texas). Riboflavin auxotrophs AJ17 (MATarib5-10 leu2, 3-112 his3-Delta1 ura3-52) and AJ18 (MATarib5-12 leu2, 3-112 his3-Delta1 ura3-52) were derived by ethyl methanesulfonate mutagenesis from the parental strain SI502B (MATaleu2, 3-112 his3-Delta1 ura3-52)(12) . The rib5 deletion strain AJ53 (MATalpha rib5Delta11::URA3 leu2, 3-112 his3-Delta1 ura3-52 ) was constructed in strain JC2a by the one-step gene disruption method described by Rothstein(13) . General methods for the genetic manipulations of yeast cells were carried out as described previously (14) . Yeast cells were cultured at 30 °C in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or in synthetic defined (SD) medium containing a 0.67% yeast-nitrogen base (Difco) supplemented with essential amino acids or Wickerham's synthetic minimum medium (15) supplemented with essential amino acids with or without riboflavin. Yeast strains auxotrophic for riboflavin, rib5 mutants, were cultured in media with riboflavin at a concentration of 20 mg/l. E. coli strain DH5alpha (16) was used for cloning and propagation of all plasmids. Bl21(DE3) (17) was used as a host strain for expression of the RIB5 gene. Bacterial cultures harboring plasmids were grown in LB (^1)containing 100 µg of ampicillin/ml.

Plasmids and Gene Deletion

The URA3 CEN4 plasmid YCp50 and the genomic library made in this vector have been described earlier(18) . A rib5 deletion/substitution allele, designated rib5Delta11::URA3, was constructed by inserting the 3.0-kb KpnI fragment from pJR217 into a pUC19 derivative lacking the PstI and HindIII sites. The resulting plasmid, pJR297, was digested with NcoI and PstI and treated with the Klenow fragment of DNA polymerase I to generate blunt ends, and HindIII linkers were attached. The 1.1-kb HindIII fragment containing yeast URA3 of YEp24 (19) was inserted into the new HindIII site yielding pJR298. The linear 1.9-kb KpnI fragment of this plasmid was used for the transformation of haploid strains JC2a and TD28. Ura colonies were selected, and gene deletion was confirmed by Southern blot analysis.

The rib5Delta14 allele, which lacks nucleotides 4-15 of the RIB5 ORF, was constructed by the counter polymerase chain reaction method as described by Hemsley et al.(20) . pUC19-RIB5, which contains a 2.9-kb KpnI fragment bearing RIB5, was used as the template, and oligonucleotides RIB56 and RIB57 were used as forward and reverse primers, respectively.

Transformants were confirmed for the correctly mutated rib5Delta14 allele by sequencing. The 2.9-kb KpnI fragment containing the rib5Delta14 allele was cloned into YEp352, yielding pJR932. Cell-free extracts of RIB5-disrupted yeast cells transformed with pJR932 showed the presence of a catalytically inactive, anti-Rib5p immunoreactive, protein of the expected size.

The pJR465 plasmid was constructed by inserting a 0.5-kb fragment, encompassing the 5`-half of RIB5, in the KpnI-EcoRI sites of the URA3 CEN4 plasmid YCplac33(21) .

Nucleic Acid Manipulations

Transformation of E. coli, Southern blot analysis, and other routine DNA manipulations were carried out as described by Sambrook et al.(22) . Yeast cells were transformed by the method of Ito et al.(23) . Plasmids were recovered from yeast cells by the rapid protocol described previously(24) .

RNA was extracted from yeast as described by Schmitt et al.(25) . When appropriate, poly(A) RNA was purified by oligo(dT)-Sepharose chromatography. In Northern blot analyses, RNA was electrophoresed on 1% agarose-formaldehyde gels and transferred to nylon membranes. DNA probes were P-labeled by the random priming method. Hybridization was carried out at 42 °C in 5 times SSC and in the presence of 50% formamide. Washing was carried out at 65 °C in 0.1 times SSC, 0.1% SDS. The levels of mRNA were estimated quantitatively by densitometry of autoradiographs on a Bio-Image image analyzer (Millipore Corp.).

DNA sequencing reactions were performed using the dideoxy chain reaction (26) using the T7 sequencing system (Pharmacia).

Primer extension analysis were done as described (22) using the synthetic oligonucleotide RIB52 that is complementary to nucleotides 104-130 of the RIB5 ORF. The reactions in formamide loading buffer were loaded adjacent to the dideoxy sequencing reactions of the RIB5 DNA using the same primer.

Expression of RIB5 in E. coli and Raising of an Anti-Rib5p Antiserum

Rib5p was expressed in E. coli using the T7 RNA polymerase-based expression vector pET-11d(17) . The entire coding region of RIB5 with an NcoI site at the initiation codon environment and a BamHI at the 3`-end was generated by polymerase chain reaction, using pUC19-RIB5 as template and oligonucleotides RIB5E and RIB55 as forward and reverse primers, respectively. By creating the NcoI site a Phe to Ala substitution is also created at the second codon. The 0.7-kb polymerase chain reaction product was gel-purified, digested with NcoI and BamHI, and ligated into pET-11d yielding pJR438. This plasmid was transformed into strain BL21 (DE3) and expressed following induction with isopropyl-1-thio-beta-D-galactopyranoside (17) . The expression of Rib5p was accompanied by an abundant formation of inclusion bodies that were purified from lysozyme-treated and sonicated transformants by repeated centrifugations (3 min, 4000 times g) and resuspending in 0.5 M NaCl, 100 mM Tris-HCl, pH 7.2, 20 mM EDTA, 0,5 mM phenylmethylsulfonyl fluoride, 1% Triton X-100 and finally washed and resuspended in phosphate-buffered saline. 100 µg of inclusion body protein were thoroughly suspended in Freund's adjuvant and injected subcutaneously into a rabbit four times at intervals of 2 weeks, and a final booster injection was given without adjuvant. Serum was prepared 10 days later.

Immunoblots and Immunological Detection

For immunoblots, yeast proteins were separated by SDS-PAGE and transferred to Immobilon protein-binding membrane (Millipore), and the blots were reacted with anti-Rib5p antiserum in the presence of 5% skim milk. Specifically bound antibodies were detected with alkaline phosphatase-second antibody conjugate contained in the Western-Light chemiluminiscence kit (Tropix) following the recommendations of the supplier.

Riboflavin Synthase Purification, Enzyme Assay, and N-terminal Amino Acid Sequencing

JC2a yeast cells transformed with pJR235 were grown to late log phase (A600 nm = 1.5-2) in SD medium lacking uracil to select plasmid-containing cells. All procedures described below were done at 4 °C unless otherwise stated. Cells were harvested by centrifugation, washed once with ice-cold water, and resuspended in 1/50 volume of extraction buffer (20 mM Tris-HCl, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride). Glass beads (0.5 mm) were added, and the cells were disrupted in a Braun homogenizer. Glass beads, unbroken cells, and cell wall components were removed by centrifugation at 4000 times g for 20 min. The crude extract was then centrifuged at 100,000 times g for 60 min to obtain the supernatant cytosolic fraction. Methyl alcohol (50% v/v final concentration) was added to the cytosolic fraction, incubated at 0 °C for 12 h, and centrifuged at 15,000 times g for 60 min. The supernatant fraction was then lyophilized, resuspended in 20 mM Tris-HCl, pH 7.5, and passed through a 0.45-µm sterile filter prior to the ion exchange chromatographic step. In consecutive runs, the filtered protein fraction (5 mg/ml) was loaded onto a Mono Q column (0.5 times 5-cm, HR 5/5, FPLC system, Pharmacia), which had been equilibrated with 20 mM Tris-HCl, pH 7.5. After sample passage, the column was washed with 5 ml of the same buffer, and the chromatogram was developed with a 25-ml linear gradient of 0.05-0.5 M NaCl in 20 mM Tris-HCl, pH 7.5. Fractions (0.5 ml) were analyzed by SDS-PAGE, immunoblotting, and enzyme activity. Riboflavin synthase eluted at about 250 mM NaCl in the gradient. Fractions from Mono Q chromatographies containing apparently pure enzyme were pooled.

Riboflavin synthase activity was determined by published methods(7) . Enzyme activity is expressed as nanomoles of riboflavin formed per hour.

The amino-terminal sequence of purified riboflavin synthase (approximately 5 µg) was determined on an Applied Biosystems 470A sequencer, with an Applied Biosystems 120A phenylthiohydantoin-derivative analyzer as an on-line detection system.

Other Protein Methods

The method of Laemmli (27) was used for SDS-PAGE, and proteins were detected by silver staining. Protein content was determined by the method of Lowry et al.(28) . The apparent native molecular mass of purified riboflavin synthase was determined by both gel filtration chromatography and nondenaturing pore limit PAGE. Gel filtration chromatography was developed on a Superose 12 HR 10/30 FPLC column (Pharmacia) calibrated with the Mol-Ranger protein molecular mass marker kit (Pierce). Nondenaturing pore limit PAGE was done as described previously (29) on a 4-20% polyacrylamide gradient gel (Bio-Rad).

Computer-aided Analysis of Sequence Data

The search for protein homologies was performed through the National Center for Biotechnology Information (NCBI) using the BLAST network service. Alignments of amino acid sequences were performed with the CLUSTAL algorithm(30) . The updated Dayhoff matrix described by Gonnet et al.(31) was used to define conservative (score of zero or greater) amino acid substitutions.


RESULTS

Isolation of RIB5

rib5 mutants of S. cerevisiae are unable to grow on riboflavin-free medium and accumulate the immediate green fluorescent precursor of riboflavin, 6,7-dimethyl-8-ribityllumazine(3) . On the basis of current knowledge of the biosynthetic pathway of riboflavin, it was assumed that these mutants should have a defect of riboflavin synthase. In order to clone the RIB5 gene by functional complementation, strain AJ17 (MATarib5-10 leu2, 3-112 his3-Delta1 ura3-52) was used as recipient in transformation, and a YCp50 S. cerevisiae genomic library was used as the transforming DNA. Transformants were screened for Ura and for both Ura and Rib prototrophy. The frequency of transformation to Ura prototrophy was about 5 times 10^3 colonies/µg of DNA. In the case of double selection (Ura, Rib), 3 transformants were recovered when 3 µg of DNA were used. Yeast DNA minipreparations were made from the Ura Rib transformants and used to transform E. coli DH5alpha to Amp^r. All three plasmids obtained from each Amp^r transformant conferred the Rib phenotype on yeast strain AJ17. In addition, AJ17 bearing these plasmids did not accumulate 6,7-dimethyl-8-ribityllumazine. The three plasmids isolated could be identical based on their restriction maps, and this plasmid, designated pJR211 (Fig. 1A), had an insertion of a 13-kb fragment in YCp50. In order to obtain a smaller DNA fragment capable of complementing riboflavin auxotrophy, pJR211 was used to provide restriction fragments for the construction of various subclones in the multicopy vector YEp352 (Fig. 1A). These subclones were separately transformed into the rib5 mutant strain AJ17 and tested for their abilities to complement the riboflavin auxotrophy of this strain. Four subclones, pJR214, pJR217, pJR126, and pJR235, sharing the presence of a 0.5-kb KpnI-EcoRI fragment were found that were able to complement the rib5-10 mutation. Other subclones, pJR213, pJR215, and pJR216, which do not uncover the KpnI-EcoRI region, were unable to confer riboflavin prototrophy to the AJ17 strain. Based on this analysis, the RIB5 gene was tentatively localized in a 0.5-kb region between the KpnI and the EcoRI sites.


Figure 1: Characterization and deletion of the RIB5 locus. A, restriction map of the isolated 13-kb fragment and complementation analysis of different subclones in the multicopy plasmid YEp352. Plus and minus signs indicate whether this fragment complements (+) or does not complement(-) the riboflavin auxotrophy of strain AJ17 (rib5-10). B, RIB5 gene deletion. A 2.3-kb NcoI/PstI fragment encompassing the RIB5 ORF was replaced by a 1.1-kb HindIII fragment URA3 gene (left). Genomic DNA was extracted from wild type (JC2a) and deleted rib5Delta11::URA3 mutant (AJ53) strains and digested with KpnI. The digestion products were separated on a 1% agarose gel, transferred to a nylon membrane, and probed with a P-labeled, 2.9-kb KpnI fragment (right).



To verify the identity of the RIB5 gene, a one-step gene disruption (13) was performed. A deletion of the putative RIB5 gene was performed by excision of a 2.3-kb NcoI-PstI fragment encompassing the RIB5-complementing sequences. These two sites were filled, a HindIII linker was added, and the HindIII cassette bearing the yeast URA3 gene (isolated from YEp24) was inserted. Two independent ura3 haploid strains, JC2a and TD28, were transformed with a linear fragment containing the 1.9-kb KpnI rib5Delta11::URA3 deletion construct, selecting for Ura prototrophy (Fig. 1B). Genomic integration and deletion of the RIB5 locus within Ura transformants was verified by Southern blot analysis using the 2.9-kb, P-labeled KpnI fragment as probe (Fig. 1B). Deletion of RIB5 in JC2a or TD28 strains led to riboflavin auxotrophy and accumulation of the intermediate compound 6,7-dimethyl-8-ribityllumazine. Genetic complementation analysis was used to verify that the rib5Delta11::URA3 deletion mutation in AJ53, a JC2a-derived strain, was allelic to rib5-10 . Strain AJ53 was crossed with strain AJ17. The resulting diploids had an Rib phenotype, suggesting allelism of the cloned gene and rib5-10. Strong support for allelism was obtained by sporulating these diploids and showing that all 12 tetrads analyzed gave four Rib spores. We conclude that the RIB5 was cloned.

The cloned KpnI-EcoRI fragment, which complements the rib5-10 mutation, hybridized in a yeast chromosome blot to chromosome II (data not shown). This result is in agreement with the previously reported location of the rib5 locus at 3.7 centimorgans proximal to the his7 locus on the right arm of chromosome II(32) .

rib5 Mutant Strains Lack Riboflavin Synthase Activity

To determine whether riboflavin synthase activity was missing in rib5 mutant strains, crude extracts were assayed for the enzyme. The wild type strain X2180-1A contained 5.59 units of enzyme activity/mg of protein, whereas the rib5 mutant strains AJ17, AJ18, and AJ53 contained no detectable enzyme activity. The cloned RIB5 gene carried on a multicopy plasmid, pJR235, was able to restore enzyme activity to about 20 times more than the wild type level since three independent transformants of strain AJ53 gave 95.40, 86.41, and 92.99 units of enzyme activity/mg of protein.

Nucleotide Sequence

The nucleotide sequence was determined by the dideoxy method(26) .The initial sequencing of the 0.5-kb KpnI-EcoRI fragment revealed the presence of a single 371-base pair open reading frame interrupted at the EcoRI site. To obtain the entire nucleotide sequence of the gene, sequencing was extended to an adjacent AccI site. In all, both strands of a 1.6-kb KpnI-AccI segment of yeast genomic DNA were sequenced (Fig. 2). An ATG at position 160 and a TAA stop codon at position 874 define the complete open reading frame of 714 base pairs coding for a 238-amino acid protein with a predicted molecular mass of 25 kDa. Analysis of the sequence surrounding the putative starting ATG triplet shows that it is in good agreement with the translation start consensus proposed by Cigan and Donahue(33) .


Figure 2: Nucleotide sequence and predicted amino acid sequence of the RIB5 gene. Amino acids are shown in single-letter code. The numbering adopted for both nucleotides and amino acids is initiated at the first ATG of the RIB5 ORF. Restriction sites referred to in the text are boxed. In the 5`-flanking region, the consensus TATA element is underlined, and a pyrimidine-rich tract is double underlined. A downward arrowhead indicates the major transcription initiation site. In the 3`-flanking region, sequences matching the transcription termination and polyadenylation signals are indicated by open circles and solid dots, respectively.



The predicted encoded product is a moderately charged, acidic protein with a calculated pI of 4.96. Hydropathy analysis (34) predicts a moderately hydrophilic profile with no obvious membrane-spanning domains. From the distribution of the codons used, a codon bias index of 0.175 can be calculated(35) , which suggest a poorly expressed gene.

Analysis of the putative 5`-promoter region demonstrated a characteristic AT-rich (63%) yeast promoter with a 12-base pair-long poly(dT) stretch. Such sequence elements have been shown to serve as promoter elements for constitutive expression(36, 37) . One TATA element obeying the consensus (38, 39) occurs relatively close upstream between positions -66 and -59.

The 3`-untranslated region shows two copies of the classical polyadenylation signal, AATAAA, at positions 717-722 and 1139-1143, whereas sequences matching a yeast consensus termination motif, TAG . . . TATGT . . . TTT, were identified at a region starting at 787 and ending at 915(40) . The yeast intron splice signal, TACTAAC (41) , was not found.

RIB5 mRNA Analysis

Northern analysis provided evidence for the expression of the encoded ORF. A single band of hybridizing transcripts was detected on blots of total RNA isolated from both wild type X-21801A and mutant AJ17 strains using the KpnI-EcoRI fragment encompassing part of the RIB5 gene as probe (Fig. 3A). The size of the RIB5-specific transcripts, approximately 0.85 kb, is consistent with the predicted coding region. Deletion of the RIB5 gene eliminates the 0.85-kb transcript. Quantitatively similar levels of RIB5 transcripts were observed in the wild type strain grown on a wide variety of medium compositions including different carbon sources and the addition of purines and/or riboflavin (data not shown). The steady-state level of the RIB5 transcripts was found to be very low, 2 times 10 of total mRNA, by comparison with the known level of the URA3 transcripts(42) . These results indicate that RIB5 is constitutively expressed at a low level.


Figure 3: Analysis of the RIB5 transcript. A, Northern analysis. Total RNA (50 µg) from yeast was purified, electrophoresed, transferred to a nylon membrane, and probed with a P-labeled fragment containing part of the RIB5 ORF. Lane 1, strain JC2a (wild type RIB5); lane 2, strain AJ53 (rib5Delta11::URA3); lane 3, strain AJ17 (rib5-10). The positions of RNA markers (RNA ladder, Life Technologies, Inc.) are shown at the left. B, Primer extension analysis. A synthetic 30-mer oligonucleotide (RIB52), complementary to nucleotides 104-133 of the RIB5 ORF, was annealed to 25 µg of poly(A) RNA from the wild type strain X2180-1A (lane 1) or 25 µg of yeast tRNA (lane 2) and extended with avian myeloblastosis virus reverse transcriptase. The extension products were run on a denaturing polyacrylamide gel next to a sequencing reaction using the same oligonucleotide as sequencing primer. The position of the major extension product is indicated on the right.



We determined the transcriptional initiation sites by primer extension of an oligonucleotide complementary to the sense strand of the ORF between positions 132 and 104. One major extension product 142 nucleotides long (Fig. 3B) was detected, indicating that transcription predominantly starts at the cytosine residue at position -10 with respect to the ATG initiation codon. This predicts a 10-nucleotide untranslated leader sequence that is shorter than that normally found in yeast transcripts. In addition, several minor initiation points, lying downstream of the TATA sequence, were also detected. This finding is not unusual since heterogeneity in transcript 5`-ends is common for yeast mRNA(43) .

Multimeric Structure of Riboflavin Synthase Suggested by Intragenic Complementation

As shown above, rib5-10 mutants are complemented by a multicopy plasmid (pJR126) carrying a 0.5-kb KpnI-EcoRI fragment. However, this fragment only encompasses an incomplete RIB5 ORF, which is expected to express a C-terminally truncated peptide lacking the C-terminal approximate half, 118 out of 238 amino acids, of the predicted Rib5 peptide. Two simple hypotheses could explain this result. First, the truncated peptide may still be active and may compensate for the absence of riboflavin synthase activity in rib5-10 mutants. Alternatively, the truncated peptide may be inactive, but intragenic complementation could exist, i.e. riboflavin synthase may be a homomultimeric protein so that the inactive truncated peptide and the inactive peptide encoded by the rib5-10 allele could mix to form multimeric proteins that contain both types of inactive peptides. In these mixed peptide multimers, the two types of mutant peptides could compensate, resulting in an enzymatically active protein, even though the multimers consisting solely of one type of mutant peptide are inactive. To test these hypotheses complementation experiments were performed on point (rib5-10 and rib5-12) and null allele (rib5Delta11::URA3) mutant strains. The EcoRI-KpnI fragment carried on a multicopy plasmid could complement the point mutant strain AJ17 (rib5-10) but failed to complement the null allele mutant strain AJ53 (rib5Delta11::URA3). By contrast, the entire RIB5 gene carried on a multicopy plasmid (pJR235) was able to complement both mutant strains (AJ17 and AJ53). These results are consistent with the intragenic complementation hypothesis and indicate that the C-terminal 118 residues are required for expression of active riboflavin synthase. Furthermore, the truncated RIB5 ORF carried on a centromeric plasmid (pJR465) also failed to complement the point mutation, suggesting that high levels of expression of the truncated protein are required for intragenic complementation, presumably because of low stability of the multimeric complex containing both types, point and truncated, of mutant peptides. As expected, intragenic complementation was allele-specific. No complementation was observed between a different point mutant strain, AJ18 (rib5-12), and either monocopy or multicopy plasmids carrying the truncated RIB5 ORF.

Purification and Structural Properties of Yeast Riboflavin Synthase

Since intragenic complementation indicates that RIB5 encodes a peptide that is the subunit of a multimeric riboflavin synthase, we further analyzed the structural properties of the enzyme. Because riboflavin synthase activity was low, even in RIB5-overproducing cells, purification of the enzyme was more easily monitored by using specific antiserum raised against a Rib5 peptide. This Rib5 peptide represented the entire RIB5 coding region with the exception of a Phe^2 Ala substitution. The expression of the recombinant Rib5 peptide in E. coli cells was accompanied by abundant formation of inclusion bodies, known to occur during high level expression of some cloned genes(44, 45) . The Rib5 peptide was purified from isolated inclusion bodies, solubilized, and used to raise polyclonal antiserum in rabbits (see ``Experimental Procedures''). Using this antiserum in immunoblots, it was possible to detect a single 24.5-kDa protein in cell extracts from the wild type and mutant rib-510 strains (Fig. 4). This protein was about 20-25-fold more abundant when the RIB5 gene was present on a multicopy plasmid (pJR235) and the protein could not be detected in deletion mutant rib511Delta::URA3 cell extracts (Fig. 4) or when preimmune serum was used. Based on these results, we conclude that this polyclonal antiserum specifically recognizes the RIB5-encoded peptide.


Figure 4: Immunochemical identification of the RIB5 gene product. Extracts (25 µg of protein) from wild type X2180-1A (lane 1), mutant AJ17(rib5-10) (lane 2), deletion mutant AJ53 (rib5Delta11::URA3) (lane 3) cells, or AJ53 cells harboring the plasmid pJR235 (lane 4) were electrophoresed, transferred to membranes, and analyzed by immunoblotting with antiserum against Rib5p. The migration of prestained molecular mass markers is shown on the right.



Riboflavin synthase was purified from soluble extracts of yeast cells harboring the RIB5 gene on a multicopy plasmid (pJR235) by successive methanol fractionation and chromatography on a Mono Q column (Table 1). The majority of the enzyme was recovered from the Mono Q column as a sharp peak when 250 mM NaCl was applied (Fig. 5). An increment in total enzyme activity was consistently observed after Mono Q chromatography, suggesting the removal of inhibitory compounds at this purification step (Table 1). On average, a 8500-fold increase in specific activity could be achieved with a recovery of about 167% as compared with the total cell lysate. The final preparation had a specific activity of 18.9 mmol/h/mg of protein and was used as the source of enzyme for further studies. As revealed by silver staining after SDS-PAGE, the final preparation migrated as a single protein band with an apparent molecular mass of 24,5 kDa (Fig. 6A). This value is in good agreement with the predicted molecular mass of Rib5p based on its deduced amino acid composition. The identity of the band was confirmed by immunoblotting using anti-Rib5p polyclonal antiserum (Fig. 6B). Purity of the Mono Q-purified enzyme was also assessed by analyzing riboflavin synthase-active fractions eluting from the Mono Q column by SDS-polyacrylamide gel electrophoresis followed by silver staining, which also revealed a single band, suggesting the homogeneity of the final preparation (data not shown). The authenticity of the 24.5-kDa immunoreactive band was further confirmed by N-terminal amino acid sequencing. The amino acids determined in 8 cycles matched the sequence predicted for Rib5p from its DNA sequence beginning at the initiator Met.




Figure 5: Elution profile of the riboflavin synthase activity from Mono Q column. The riboflavin synthase active fraction from the methanol precipitation step was applied and eluted from the Mono Q column as described under ``Experimental Procedures.'' Inset, Fractions containing enzyme activity were analyzed by immunoblotting with anti-Rib5p antiserum.




Figure 6: SDS-PAGE and immunoblot analysis of fractions collected during purification of riboflavin synthase. Proteins were resolved on 14% SDS-polyacrylamide gels and were visualized either by silver staining (A) or by immunoblotting using anti-Rib5p polyclonal antibodies (B). Molecular masses of standards (kDa) are indicated on the left. The position of Rib5p is marked with an arrow. T, total cell lysate; M, 50% methanol-soluble fraction; Q, Mono Q.



To determine the relative molecular mass (M(r)) of the native enzyme, purified riboflavin synthase was chromatographed by FPLC on a Superose 12 column. From the elution profile of riboflavin synthase and of a number of protein standards a molecular mass of 79 kDa was estimated (Fig. 7A). A similar value (72 kDa) was obtained when the native enzyme was analyzed by nondenaturing pore limit PAGE (Fig. 7B). The implication is that the native enzyme consists of three polypeptides of identical M(r) values.


Figure 7: Characterization of native riboflavin synthase. A, Superose 12 filtration of riboflavin synthase, with ferritin, catalase, aldolase, bovine serum albumin, ovalbumin, and chymotripsinogen A as inclusive standards. The calculated value for riboflavin synthase was 79. B, relative migration of native riboflavin synthase in nondenaturing polyacrylamide (4-20%) gradient gel. Standard proteins used as markers are shown in the illustration, with riboflavin synthase M(r) estimated as 72. (Inset: left lane, markers; right lane, purified riboflavin synthase).



Yeast Riboflavin Synthase Is Similar to the alpha Subunit of Riboflavin Synthase from Prokaryotes

A search of the current data bases showed that the nucleotide sequence of RIB5 was identical to the sequence of one of the ORFs (YBR1274) reported recently as part of the European Community project for sequencing chromosome II of S. cerevisiae(46) . A comparison of the deduced Rib5p amino acid sequence revealed significant similarity between Rib5p and the alpha subunits of riboflavin synthase from B. subtilis (ribB), E. coli (ribC), and P. leiognathi (ribB) as shown in Fig. 8A. The overall identities with Rib5p were 40% with B. subtilis RibB, 33% with P. leiognathi RibB, and 32% with E. coli RibC.


Figure 8: A, sequence comparison among riboflavin synthases from S. cerevisiae, B. subtilis, E. coli, and P. leiognathi. The amino acid sequence of yeast riboflavin synthase was aligned to the sequences from B. subtilis, E. coli, and P. leiognathi. Amino acids identical in at least two sequences are boxed. Regions showing a higher degree of homology are designated a-f. The 6,7-dimethyl-8-ribityllumazine-binding sites and the cysteinyl residue of the putative catalytic site are highlighted by a black background. B, internal homology of the N and C termini of yeast riboflavin synthase. Evolutionary conservative amino acid substitutions are highlighted by a gray shaded background, in contrast to the black background for identical residues.



Six regions, a-f (Fig. 8A), appeared highly conserved in both eukaryotic and prokaryotic enzymes. The four proteins shared a completely conserved 5-amino acid sequence at their amino termini, region a, suggesting a functional role for this region. Direct evidence of the functional significance of the N-terminal region of yeast riboflavin synthase was obtained by constructing a mutant allele, rib5Delta14, which expressed a protein lacking amino acids Phe^2 to Ile^5 (see ``Experimental Procedures''). Protein extracts of AJ53 (rib5Delta11::URA3) cells containing the rib5Delta14 allele on a multicopy plasmid showed no enzyme activity although a high amount of Rib5 protein was detected by immunoblotting (data not shown).

Ligand binding studies have shown that the mechanism for the riboflavin synthase reaction involves the formation of a ternary complex consisting of the binding of two molecules of the substrate 6,7-dimethyl-8-ribityllumazine to two sites in the enzyme(5, 47, 48) . Moreover, the alpha subunit of prokaryotic riboflavin synthases and other proteins involved in bacterial bioluminescence, which also bind 6,7-dimethyl-8-ribityllumazine, show a marked internal sequence homology between the N-terminal and C-terminal halves. Therefore, it was suggested that the monomer forms two structurally similar domains, each contributing the binding site for one substrate molecule. As shown in Fig. 8B, the eukaryotic enzyme also showed a considerable internal sequence homology between the two protein halves. Furthermore, a short sequence motif (Gly-X-X-Val-Asn-Leu-Glu) proposed as the ligand binding site(8, 44) , was doubly represented in the amino acid sequence, regions c and f, one within each proposed domain.


DISCUSSION

We have cloned and characterized a gene, RIB5, which encodes riboflavin synthase in the yeast S. cerevisiae. The accumulation of the substrate 6,7-dimethyl-8-ribityllumazine and the absence of detectable enzyme activity in a strain disrupted for RIB5 confirmed the identity of the gene. RIB5 is present as a single copy gene located on chromosome II.

The RIB5 ORF consists of 714 nucleotides beginning at the first in-frame methionine, predicting a protein of 238 amino acids. Primer extension analysis indicates that there is one major transcription and several minor transcription start sites within 70 nucleotides upstream of the start of translation. Yeast often has multiple sites for transcription initiation that vary depending on the nutritional condition of the cell. The 5`-untranslated sequence shows the presence of a poly(dA-dT) region suggested to be an upstream promoter element for constitutive expression(36) . Analysis of RIB5 transcript levels on different culture conditions indicates that RIB5 behaves as a housekeeping gene, which is constitutively expressed at low level. This pattern of gene expression is in accordance with the scarce (0.06-0.72 µmol of riboflavin/g of protein)(2) , though indispensable, necessity of riboflavin for the cell.

The complementation between the original rib5-10 mutant allele used to clone the gene and the deleted allele expressing only the amino-terminal half of the encoded polypeptide is consistent with the classical interpretations of intragenic complementation, suggesting not only that Rib5p assembles into a multimeric complex but also that there is a discrete functional domain located at the amino terminus.

The hypothesis that Rib5p forms multimers has been demonstrated by purifying the enzyme. Published methods of purification of yeast riboflavin synthase are rather cumbersome and inefficient because of the low riboflavin synthase level in cell extracts and the absence of sensitive enzymatic assays(47) . We have now developed a simple method of purification that relies on RIB5 overexpression by using a multicopy plasmid and the use of Rib5p-specific antibodies. With this approach we were able to purify the enzyme to apparent homogeneity using a three-step purification protocol, which achieved an overall purification of 8500-fold with a recovery of 167%. This fairly high yield made it possible to analyze some of its properties. From comparison of either the elution volume or the R of pure native riboflavin synthase with those of standard proteins by gel filtration analysis or nondenaturing pore limit PAGE, respectively, a molecular mass of approximately 75,000 daltons was estimated for the native enzyme. This value is in reasonable agreement with the molecular mass reported by Harvey and Plaut (5) for a partially purified riboflavin synthase preparation on the basis of its sedimentation coefficient. However, an apparent subunit molecular mass of 24,500 daltons was obtained for the pure protein by SDS-PAGE analysis, in good agreement with the predicted molecular mass of Rib5p based on its deduced amino acid composition. These findings indicate that the native riboflavin synthase of S. cerevisiae exists in the trimer form of identical subunits. Riboflavin synthase appears in B. subtilis as two different species characterized by its subunit composition. The so-called light enzyme is a trimer of alpha, riboflavin synthase, identical subunits. The heavy enzyme consists of an alpha subunit trimer enclosed within an icosahedral structure composed of 60 beta, 6,7-dimethyl-8-ribityllumazine synthase, subunits(7, 49, 50) . Unlike Bacillus, we found no evidence of a similar heavy multienzyme complex in Saccharomyces.

Significant internal homology was found between the N-terminal and C-terminal halves of the Saccharomyces Rib5p, as described previously for the B. subtilis alpha subunit(8) . Since the enzyme binds two molecules of the substrate 6,7-dimethyl-8-ribityllumazine, it was suggested that the protomer forms two structurally similar, substrate-binding domains. In this context, the intragenic complementation effect observed reinforces this hypothesis by showing that a discrete functional domain exists in the N-terminal half of Rib5p. The existence of a second functional domain in the C-terminal half of Rib5p can also be predicted by homology to the N-terminal domain. Research is currently under way at our laboratory to determine whether the separate N-terminal and C-terminal domains of Rib5p are able to bind the substrate and whether the interaction of both types of separate domains can restore the enzyme activity.

Although structurally similar as a consequence of the probable common origin, by gene duplication, and endowed with the same substrate binding function, both domains should not be strictly identical because of their different catalytic properties. In the enzymatic formation of riboflavin one molecule of substrate acts as the donor and other as the acceptor of the four-carbon moiety that transforms the xylene ring of 6,7-dimethyl-8-ribityllumazine into the isoalloxazine ring of riboflavin. The respective donor and acceptor sites can be distinguished by their affinities for different ligands(5, 48) , i.e. only the acceptor site binds the product riboflavin, whereas the donor site has high affinity for analogs of the second product of the reaction, 5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine. The temporary binding of 5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine to the donor domain in the course of the reaction prompted us to compare the sequences of both N-terminal and C-terminal domains of Rib5p with that of the 6,7-dimethyl-8-ribityllumazine synthase from S. cerevisiae. This enzyme also binds 5-amino-2,6-dihydroxy-4-ribitylamino-pyrimidine as substrate, and the gene encoding the enzyme, RIB4, has recently been cloned and its nucleotide sequence determined. (^2)A substantially higher degree of similarity of the deduced amino acid sequence of RIB4 with the C-terminal domain (38.8%) than with the N-terminal domain (27.6%) of Rib5p was found. Therefore, it is tempting to speculate about the existence of an N-terminal domain binding the acceptor substrate molecule and a C-terminal binding domain binding the donor substrate molecule. Binding assays with the products of the reaction or structural analogs and the split N-terminal and C-terminal domains would be necessary to answer this question.

In the course of our analysis of the RIB5 gene, we compared the deduced amino acid sequence of the yeast enzyme with the riboflavin synthases from B. subtilis, E. coli, and P. leiognathi. Although the four proteins shared significant similarity along their entire lengths, a higher degree of similarity in the amino acid sequences from these four proteins was identified in six regions, a-f. Because of the common function of these proteins, it seems likely that these regions are of functional importance. Experimental evidence for the functional relevance of the strictly conserved amino end, region a, was obtained for the yeast enzyme by deleting part of the sequence. In this respect, it is also significant that the N terminus of Rib5p is not modified post-translationally (i.e. signal sequence processing or N-terminal amino acid modification), as deduced from the N-terminal amino acid sequencing data. Since a homologous region is symmetrically located on the C-terminal domain, region d, a role in the formation of the substrate binding site rather than in catalysis may be predicted for both conserved regions. Similarly, substrate binding function may be also attributed to the homologous symmetrical regions c and f. Moreover, both regions contain the sequence motif (Gly-X-X-Val-Asn-Leu-Glu) proposed by O'Kane et al.(44) to be involved in the binding of 6,7-dimethyl-8-ribityllumazine. Early studies by Plaut et al.(6) with sulfhydryl group-binding reagents have showed that a thiol group is involved in the enzyme action. The presence of only one cysteinyl residue conserved in all four known amino acid sequences, Cys in Rib5p, makes this residue a good candidate for proton abstraction from the 7-methyl group of the acceptor substrate molecule, which initiates the proposed reaction mechanism(6) . In agreement with this assumption, it has recently been found that mutants of Rib5p with amino acid substitutions at Cys are inactive. (^3)Since Cys is located in region b, it thus seems likely that this conserved region would participate in the formation of the catalytic center. At present, no role can be directly extrapolated for region e, although other functions such as trimerization of the enzyme or the presence of other site-active residues should be considered.


FOOTNOTES

*
This research was supported by BASF Aktiengesellschaft and by Grants FAR89-0262 and BI092-0036 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z21621[GenBank].

§
Supported by a fellowship granted by the Ministerio de Educación y Ciencia, Spain.

To whom correspondence should be addressed. Tel.: 34-23-294671; Fax: 34-23-267970.

(^1)
The abbreviations used are: LB, Luria broth; kb, kilobases(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; Rib5p, RIB5-encoded protein; FPLC, fast performance liquid chromatography.

(^2)
J. J. García-Ramírez, unpublished data.

(^3)
M. A. Santos, unpublished data.


ACKNOWLEDGEMENTS

We thank Fernando Díez for technical help and N. Skinner for correcting the manuscript.


REFERENCES

  1. Bacher, A. (1991) in Chemistry and Biochemistry of Flavoproteins (Muller, F., ed) Vol. 1, pp. 215-259, Chemical Rubber Co., Boca Raton, FL
  2. Demain, A. L. (1972) Annu. Rev. Microbiol. 26, 369-388 [Medline] [Order article via Infotrieve]
  3. Oltmanns, O., Bacher, A., Lingens, F., and Zimmermann, F. K. (1969) Mol. & Gen. Genet. 105, 306-313
  4. Oltmanns, O., and Bacher, A. (1972) J. Bacteriol. 110, 818-822 [Medline] [Order article via Infotrieve]
  5. Harvey, R. A., and Plaut, G. W. E. (1966) J. Biol. Chem. 241, 2120-2136 [Abstract/Free Full Text]
  6. Plaut, G. W. E., Beach, R. L., and Aogaichi, T. (1970) Biochemistry 9, 771-785 [Medline] [Order article via Infotrieve]
  7. Bacher, A., Baur, R., Eggers, U., Harders, H.-D., Otto, M. K., and Schnepple, H. (1980) J. Biol. Chem. 255, 632-637 [Abstract/Free Full Text]
  8. Schott, K., Kellermann, J., Lottspeich, F., and Bacher, A. (1990) J. Biol. Chem. 265, 4204-4209 [Abstract/Free Full Text]
  9. Volk, R., and Bacher, A. (1988) J. Am. Chem. Soc. 110, 3651-3653
  10. Lee, C. Y., and Meighen, E. A. (1992) Biochem. Biophys. Res. Commun. 186, 690-697 [Medline] [Order article via Infotrieve]
  11. Plaut, G. W. E., and Harvey, R. A. (1971) Methods Enzymol. 18B, 515-538
  12. Lawrence, C. W. (1991) Methods Enzymol. 194, 273-281 [Medline] [Order article via Infotrieve]
  13. Rothstein, R. (1983) Methods Enzymol. 101, 202-211 [Medline] [Order article via Infotrieve]
  14. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  15. Wickerham, L. J. (1951) U. S. Dep. Agric. Tech. Bull. 1029, 1-56
  16. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580 [Medline] [Order article via Infotrieve]
  17. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  18. Rose, M. D., Novick, P., Thomas, J. H., Botstein, D., and Fink, G. R. (1987) Gene (Amst.) 60, 237-243 [CrossRef][Medline] [Order article via Infotrieve]
  19. Botstein, D., Falco, S. C., Stewart, S. E., Brennan, M., Scherer, S., Stinchcomb, D. T., Struhl, K., and Davis, R. W. (1979) Gene (Amst.) 8, 17-24 [CrossRef][Medline] [Order article via Infotrieve]
  20. Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J. (1989) Nucleic Acids Res. 17, 6545-6551 [Abstract]
  21. Gietz, R. D., and Sugino, A. (1988) Gene (Amst.) 74, 527-534 [CrossRef][Medline] [Order article via Infotrieve]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  23. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168 [Medline] [Order article via Infotrieve]
  24. Hoffman, C. S., and Winston, F. (1987) Gene (Amst.) 57, 267-272 [CrossRef][Medline] [Order article via Infotrieve]
  25. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18, 3091-3092 [Medline] [Order article via Infotrieve]
  26. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  27. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  28. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  29. Anderson, L. O., Borg, H., and Mikaelsson, M. (1972) FEBS Lett. 20, 199-201 [CrossRef][Medline] [Order article via Infotrieve]
  30. Higgins, D. G., and Sharp, P. M. (1988) Gene (Amst.) 73, 237-244 [CrossRef][Medline] [Order article via Infotrieve]
  31. Gonnet, G. H., Cohen, M. A., and Benner, S. A. (1992) Science 256, 1443-1445 [Medline] [Order article via Infotrieve]
  32. Santos, M. A., Iturriaga, E. A., and Eslava, A. P. (1988) Curr. Genet. 14, 419-423 [Medline] [Order article via Infotrieve]
  33. Cigan, A. M., and Donahue, T. F. (1987) Gene (Amst.) 59, 1-18 [CrossRef][Medline] [Order article via Infotrieve]
  34. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  35. Sharp, P. M., and Cowe, E. (1991) Yeast 7, 657-678 [Medline] [Order article via Infotrieve]
  36. Struhl, K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8419-8422 [Abstract]
  37. Struhl, K. (1989) Annu. Rev. Biochem. 58, 1051-1077 [CrossRef][Medline] [Order article via Infotrieve]
  38. Chen, W., and Struhl, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2691-2695 [Abstract]
  39. Harbury, P. A. B., and Struhl, K. (1989) Mol. Cell. Biol. 9, 5298-5304 [Medline] [Order article via Infotrieve]
  40. Zaret, K. S., and Sherman, F. (1982) Cell 28, 563-573 [Medline] [Order article via Infotrieve]
  41. Langford, C. J., Klinz, F. J., Donath, C., and Gallwith, D. (1984) Cell 36, 645-653 [Medline] [Order article via Infotrieve]
  42. Silverman, S. J., Rose, M., Botstein, D., and Fink, G. R. (1982) Mol. Cell. Biol. 2, 1212-1219 [Medline] [Order article via Infotrieve]
  43. McNeil, J. B., and Smith, M. (1986) J. Mol. Biol. 187, 363-378 [Medline] [Order article via Infotrieve]
  44. O'Kane, D. J., Woodward, B., Lee, J., and Prasher, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1100-1104 [Abstract]
  45. Marston, F. A. O. (1986) Biochem. J. 240, 1-12 [Medline] [Order article via Infotrieve]
  46. Doignon, F., Biteau, N., Crouzet, M., and Aigle, M. (1993) Yeast 9, 189-199 [Medline] [Order article via Infotrieve]
  47. Plaut, G. W. E. (1971) in Comprehensive Biochemistry (Florkin, M., and Stotz, E. H., eds) Vol. 21, pp. 11-45, Elsevier, Amsterdam
  48. Otto, M. K., and Bacher, A. (1981) Eur. J. Biochem. 115, 511-517 [Abstract]
  49. Bacher, A., Ludwig, H. C., Schnepple, H., and Ben-Saul, Y. (1986) J. Mol. Biol. 187, 75-86 [Medline] [Order article via Infotrieve]
  50. Ladenstein, R., Meyer, B., Huber, R., Labischinski, H., Bartels, K., Bartunik, H. D., Bachmann, L., Ludwig, H. C., and Bacher, A. (1986) J. Mol. Biol. 187, 87-100 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.