Isolation of the Aspergillus nidulans sudD gene and its human homologue

Gene 211 (1998) 323–329

Isolation of the Aspergillus nidulans sudD gene and its human homologue Paul Anaya a, Susan C. Evans b, Cuiping Dai a, Guillermina Lozano b, Gregory S. May a,* a Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA b Department of Molecular Genetics, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA Received 8 January 1998; accepted 13 February 1998; Received by J.A. Engler

Abstract We have been studying the heat-sensitive bimD6 mutation of Aspergillus nidulans. At a restrictive temperature, the chromosomes of bimD6 mutant strains fail to attach properly to the spindle microtubules, and the mutant also displays a high rate of chromosome loss. We previously cloned the sudA gene, an extragenic suppressor of the heat-sensitive bimD6 mutation and showed that it coded for a DA-box or SMC protein. SMC proteins have been demonstrated to function in chromosome condensation, segregation and global gene regulation. We have now cloned the sudD gene, another of the extragenic suppressor genes of the bimD6 mutation. The predicted SUDD protein is the founding member of a widely expressed protein family. Similar proteins are found in sequence databases for Saccharomyces cerevisiae, Caenorhabditis elegans, mammals and four species of archaebacteria. We have also cloned and sequenced a human cDNA that encodes the human homologue of SUDD and mapped the gene to 18q11.2. The predicted SUDD proteins from A. nidulans, Homo sapiens and S. cerevisiae all share a variety of features. The predicted proteins are approximately 60 000 Da in mass and have a serine-plus-threonine content of about 11%. The evolutionary conservation of the proteins suggests an ancient origin and conserved function for these proteins. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Chromosome; Evolution; Fungi; Suppressor

1. Introduction Eukaryotic cells face a difficult problem of maintaining an intact genome during cell division. The large size of eukaryotic genomes makes them susceptible to breakage during mitosis. For this reason, eukaryotes have evolved a system to package their DNA into mitotic chromosomes in preparation for cell division. Condensation of the chromatin at mitosis prevents the DNA from being broken by the mechanical sheer placed on it during chromosome segregation. The process of chromosome condensation represents a topological challenge for the cell. In recent years, significant progress has been made in our understanding of how the mitotic chromosome is assembled and has led to the identification of several new polypeptides that function in chromosome condensation (Strunnikov et al., 1993; Chuang * Corresponding author. Tel: +1 713 798 4756; Fax: +1 713 798 7799; e-mail: [email protected] Abbreviations: bp, base pair(s); kb, kilobase(s) or 1000 bp; cDNA, complementary DNA; Cs-, cold-sensitive; EST, expressed sequence tag; polyA+, polyadenylated. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 11 5 - 2

et al., 1994; Hirano and Mitchison, 1994; Saitoh et al., 1994; Saka et al., 1994; Strunnikov et al., 1995, Holt and May, 1996). Among these polypeptides are topoisomerase II, the SMC proteins (stability of minichromosomes) and the recently described condensins (Anderson and Roberge, 1992; Hirano et al., 1997; Kimura and Hirano, 1997). The SMC proteins have been identified in a variety of systems and function in chromosome condensation and segregation (Strunnikov et al., 1993; Chuang et al., 1994; Hirano and Mitchison, 1994; Saitoh et al., 1994; Saka et al., 1994; Strunnikov et al., 1995, Holt and May, 1996). This progress has been possible because of the combined biochemical and genetic approaches used in a variety of systems, including budding yeast, fission yeast, A. nidulans, animal cells in culture and Xenopus oocytes. We have been working with the filamentous fungus Aspergillus nidulans to study chromosome segregation. Our work began with a characterization of the heatsensitive bimD6 mutation and cloning of the bimD gene (Denison et al., 1993). The bimD6 mutation confers a heat-sensitive mitotic defect characterized by a failure of chromosomes to correctly attach to the spindle

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Table 1 Strains used in this study Strain name

Genotype

Source

GB20

pyrG89 pabaA1; fwA2 benA22 uaY9 pyrG89; pyroA4 sudD7 riboA1; pyroA4 sudB2; sC12

May et al. (1985)

8–16 (3–6) C25

Holt and May (1996) Holt and May (1996)

mitcrotubules, resulting in an increase in chromosome loss. We subsequently identified seven extragenic suppressors for the heat-sensitive phenotype of the bimD6 mutation that defined four genes sudA–D (Holt and May, 1996). We isolated four mutations in the sudA gene and determined that sudA codes for a member of the SMC family of proteins. This observation implicates BIMD in the process of chromosome condensation. Mutations in the sudA gene, like mutations in the bimD gene, displayed a variety of phenotypes consistent with their role in chromosome condensation and segregation. At restrictive temperatures, these mutants display elevated chromosome and spindle mitotic indexes. Mutations in both genes also display increased rates of chromosome loss in diploids homozygous for the mutations. During our studies of the sudA gene, we found that extra copies of sudA could suppress the cold sensitivity of the sudD7 mutation, suggesting that the products of these genes may interact. This suppression of the cold-sensitive sudD7 mutation thus led us to characterize the sudD7 mutant phenotype and clone the sudD gene. We report here that the product of the sudD gene is a highly conserved protein that is produced in eukaryotes

from fungi to vertebrates and that related coding sequences are found in archaebacteria, suggesting an ancient origin and conserved function for these proteins.

2. Materials and methods 2.1. Media, strains and genetics Media for A. nidulans growth and mating were as previously described (Cove, 1977; Ka¨fer, 1977). Strains carrying the pyrG89 mutation were grown on 2% malt extract, 0.2% peptone, 1% dextrose, trace elements, 5 mM uridine and 10 mM uracil and 2% agar. For microscopic studies, all strains were grown in 0.5% yeast extract, 20 mM dextrose, trace elements, 5 mM uridine, 10 mM uracil and 8.8 mg/ml riboflavin liquid medium. The A. nidulans strains used in this study are listed in Table 1 and carried markers previously described (Clutterbuck, 1974). Bacterial media and techniques were essentially as described by Sambrook et al. (1989). Sexual crosses were performed using standard genetic techniques (Pontecorvo et al., 1953). 2.2. Aspergillus nidulans transformation and molecular methods Transformation of A. nidulans was performed as previously described (Denison et al., 1993; May et al., 1992). The wild-type sudD gene was cloned from a chromosome IV-specific A. nidulans cosmid library (Brody et al., 1991) by complementation of the Cs−

Fig. 1. (A) sudD genomic clone analysis and cDNA clones. A partial restriction map for the region around the sudD gene is shown. Below the map is the minimal rescuing subclone for the sudD7 mutation and an arrow that defines the limits of the transcription unit and the direction of transcription. (B) Northern hybridization to polyA+ RNA probed with the PstI/XbaI complementing subclone showing a single band of hybridization.

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human EST (AM024h17, Research Genetics, Inc.) identified in the database was used as a probe to isolate additional cDNA clones from a human aorta library in lgt10 (Clontech Laboratories, Inc). A clone of approximately 3 kb in length was identified and fully sequenced. 2.3. Mapping of the human sudD gene

Fig. 2. Nucleotide sequence of the sudD cDNA with the deduced amino acid sequence shown below the nucleotide sequence (GenBank Accession No. AF013590).

sudD7 mutation. Pools of cosmids were cotransformed with pAB4-ARp1 ( Verdoes et al., 1994) into strain 8–16(3–6) and transformants incubated at 20°C for 5–7 days. A single complementing cosmid, L26D03, was identified from the positive pool using smaller pools and then single cosmids to complement the mutation. An ~2-kb PstI/XbaI fragment from this cosmid retained the ability to rescue the Cs− phenotype and recognized an approximately 2-kb message on Northern blots of A. nidulans poly-A+ RNA. It was later determined that this PstI/XbaI fragment did not contain the entire gene, and a larger 4.4-kb XhoI/XbaI fragment was isolated that contained the entire gene. DNA gel electrophoresis and genomic Southern analysis were performed as described by May et al. (1987). A. nidulans cDNA clones were isolated from a lgt10 library as described previously (May et al., 1992). A

Southern blots contained genomic DNA from a human/rodent somatic cell hybrid panel digested with either EcoRI or HindIII (a gift from Dr. Siciliano, M.D. Anderson Cancer Center). The membranes were prehybridized at 65°C for 1 h in hybridization buffer containing 1% SDS, 10% dextran sulfate, 1 M NaCl, and 100 mg/ml salmon sperm DNA. The blot was then hybridized overnight at 65°C with 1×106 cpm/ml of an ~1-kb KpnI/SacI fragment of human sudD cDNA random-labeled with [32P]dCTP. Membranes were washed and subjected to autoradiography at −80°C for 1–3 days. Fluorescence in-situ hybridization. Briefly, YAC 927h2 DNA was amplified by inter-Alu PCR, labelled by nick-translation with biotin using the Bionick kit (Gibco BRL) and purified with the G-50 minispin column ( Worthington). The probe was combined with 50-fold human Cot 1 DNA (Gibco BRL) dried completely and resuspended in 25 ml hybridization solution (5 ml dH O, 12.5 ml formamide, 2.5 ml 10× SSC, 5 ml 2 50% dextran sulfate). Hypermetaphase slides were prepared from normal lymphoblastoid cells. The slides were pretreated with 2× SSC at 37°C for 30 min, dehydrated in an ethanol series, denatured in 70% formamide with 2× SSC for 2 min at 72°C, dehydrated in cold ethanol and air-dried. The denatured probe was applied to slides under coverslips and incubated overnight at 37°C. Coverslips were removed, and then slides were incubated for 5 min in 2× SSC at 72°C and rinsed briefly in BST buffer (0.1 M sodium bicarbonate, 0.3 M sodium chloride, 0.05% Tween-20, pH 8.0). FITC-labelled antiavidin ( Vector) was incubated on slides for 30 min at 37°C then washed three times in BST buffer. Propidium iodide (Oncor) diluted with Antifade (Oncor) to 0.3 mg/ml was added and viewed under fluorescence.

3. Results 3.1. Allelism test for sudB2 and sudD7 mutations Both the sudB2 and sudD7 mutations map to linkage group IV, but complementation tests placed them in separate complementation groups. Because we previously observed non-complementation between the four separate mutations isolated in the sudA gene, we tested for genetic linkage between the sudB2 and sudD7 mutations (Holt and May, 1996). As would be expected for

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Fig. 3. Alignments derived from a BLAST search of the non-redundant nucleotide databases showing the most highly conserved sequences shaded in gray. GenBank Accession Nos for these sequences are P34649 for C. elegan, AA061258 for the mouse, Q03021 for T. acidophilum and T54144 for the human sequence.

mutations in unlinked genes, a cross between strains carrying the sudB2 (C25) and sudD7 [8–16(3–6)] mutations produced progeny that were not Cs−. Of 100 segregants analyzed, 58 were cold-sensitive, and 42 were not cold-sensitive. Based on these results, we conclude that the sudB2 and sudD7 mutations define two independent suppressor genes on linkage group IV. As independent proof that these two mutations define separate genes, the cloned sudD gene was not able to complement the sudB2 mutation. 3.2. Molecular cloning of the sudD gene The sudD gene was cloned by complementation of the conditionally lethal Cs− sudD7 mutation using a chromosome IV specific cosmid library, as we have described previously (May et al., 1992; Denison et al., 1993; Holt and May, 1996). A single cosmid clone that complemented the Cs− phenotype was identified (L26D03), and the smallest fragment that could rescue the Cs− phenotype was defined using restriction endonucleases (Fig. 1A). The smallest DNA fragment that rescued the cold-sensitive sudD7 mutation, an ~2-kb PstI/XbaI fragment, hybridized to a single RNA species of ~2-kb on a Northern blot (Fig. 1B). Two criteria were used to demonstrate that we had cloned the sudD gene. First, we showed in a genetic cross of a single copy transformant (#4) to the unrelated strain GB20, that there was a tight linkage between the transforming nutritional marker and the sudD gene. If we had cloned an unlinked suppressor of sudD7, then we would have

expected to recover Cs− segregants. Instead, we observed that only 9% of the progeny were of the recombinant class, a number similar to that which we previously observed for single copy integrants (May et al., 1985). Second, the ~2-kb PstI/XbaI transforming fragment, which lacks the amino terminal portion of the sudD gene, and the cDNA were both able to rescue the Cs− phenotype. If the gene had been a multicopy suppressor, then neither the cDNA nor the partial genomic clone should have been able to complement the sudD7 mutation. The ~2-kb PstI/XbaI fragment was used to screen a lgt10 cDNA library, and several overlapping clones were identified. The two longest clones identified were cloned into pUC19 and their sequences determined. The longest cDNA clone was 1980 bp long and coded for a predicted polypeptide of 558 amino acids (Fig. 2). The deduced SUDD protein sequence predicts a polypeptide with a pI of 5.3 and a predicted mass of approximately 63 443 Da. The predicted SUDD polypeptide displays no structural features or sequence motifs that would suggest a function, but it does have a high serine plus threonine content, 11.4%. 3.3. Homologues of sudD A search of the DNA sequence databases identified several potential homologues of the predicted SUDD protein, including one in H. sapiens, S. cerevisiae, C. elegans and archaebacteria. Alignment of these homologous sequences identified three highly conserved short

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(see below and Fig. 6). We sequenced the end of several clones and identified a candidate clone that potentially coded for the complete polypeptide. This clone overlapped with the 3∞ non-translated region of the EST clone used to isolate it, and it displayed some sequence similarity to the amino terminal coding regions of the A. nidulans and S. cerevisiae protein sequences. We completely sequenced this cDNA and determined that it was 2856 bp in length and encoded a predicted polypeptide of 519 amino acids with a mass of 59 125 Da ( Fig. 4). We think that this clone contains the complete protein coding region because antibodies prepared against a recombinant form of the protein identify a protein of ~60 000 Da on Western blots (data not shown). This is close to the predicted molecular weight of the protein based on the deduced amino acid sequence (data not shown and Fig. 4). Using the H. sapiens sequence we derived and the S. cerevisiae sequence available in the database, we conducted a detailed comparison of the protein sequences. The yeast homologue, Rio1p, is 483 amino acids in length (56 130 Da) and is 44.5% identical and 64% similar to the A. nidulans SUDD. The human SUDD is 519 amino acids long (59 125 Da) and 36% identical and 60% similar to the A. nidulans protein. Using the GCG program BESTFIT, we aligned the A. nidulans protein to that from yeast and humans ( Fig. 5A and B). These alignments show the extensive similarity between the proteins over most of their length. Each of the predicted polypeptides also has an unusual amino acid composition, containing ~18% glutamic and aspartic acid, and ~11% of serine and threonine content. The high serine and threonine content make these proteins good candidates as substrates for protein kinases.

3.4. Mapping of the human sudD gene and Northern analysis

Fig. 4. Nucleotide sequence of the human sudD cDNA with the deduced amino acid sequence shown below the nucleotide sequence (GenBank Accession No. AF013591).

peptide sequences. Except for the S. cerevisiae homologue, a thorough comparison of these predicted proteins was not possible because only partial sequences were available (Fig. 3). So that we could perform a detailed comparison of the predicted SUDD polypeptide to that of the human homologue, we isolated human cDNA clones using the commercially available EST as a probe. None of the cDNA clones that we isolated was capable of coding for the entire messenger RNA of ~4000 nucleotides

Southern blotting of a human/rodent cell hybrid panel showed 100% concordancy to chromosome 18. Human genomic clones isolated from the CEPH-Mega YAC library by PCR (Baylor College of Medicine, Houston, TX ) identified eight positive clones (six with known addresses). The YACs are in the WC1198 contig, which maps to chromosome 18q11.2 and was consistent with the results obtained by Southern of the human/rodent cell hybrid panels. Finally, the chromosomal localization was confirmed by fluorescence in-situ hybridization. We also examined the expression of the human sudD messenger RNA. The human sudD messenger RNA was expressed as a single transcript of ~4000 nucleotides in the spleen, thymus, prostate, testis, ovary, intestine, colon and peripheral blood lymphocytes ( Fig. 6). Two additional transcripts of ~2400 and ~3000 nucleotides were also detected in RNA from testis. Based on this

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Fig. 5. Alignment of the deduced amino acid sequences of A. nidulans SUDD with the S. cerevisiae Rio1p (A) and the human SUDD homologue (B). The two alignments were generated with the GCG program BESTFIT. In (A), the top line sequence is that of the A. nidulans SUDD and the bottom line is that of Rio1p. In (B), the top line sequence is that of the human SUDD homologue, and the bottom is that of the A. nidulans SUDD. The regions shaded gray are those identified as being highly conserved in the BLAST searches.

Northern analysis, the human sudD homologue is widely and constitutively expressed in human tissues.

4. Discussion The sudD gene was identified as one of four genes that were cold-sensitive, extragenic suppressors of the bimD6 mutation (Holt and May, 1996). The bimD6 mutation confers a heat-sensitive lethal mitotic arrest phenotype (Denison et al., 1993). We previously showed that one of the other extragenic suppressor genes, sudA, was a member of the SMC family of proteins. The SMC proteins function in chromosome condensation and mutations in these genes in budding yeast, and A. nidulans result in increased rates of chromosome loss through non-disjunction (Strunnikov et al., 1993; Holt and May, 1996). Since extra copies of the sudA gene could suppress the cold-sensitive phenotype of the sudD7 mutation, we were interested in identifying the product of sudD gene

as it was likely to have an important function in chromosome condensation. The product of sudD is a highly conserved protein that is found in a variety of eukaryotes from fungi to man, as well as in four species of archaebacteria. Proteins that show this degree of conservation and distribution among species are usually functionally conserved. Thus, the role that SUDD plays in chromosome condensation and segregation must be one that is ancient in origin. Like SUDD, the SMC protein family also includes distantly related proteins in lower organisms. Since the SMC and SUDD proteins are both widely distributed and appear to function in chromosome condensation and segregation, it is likely that the function of these proteins is also conserved. Unfortunately, the predicted polypeptides for SUDD and its homologues do not suggest a possible function. An interesting feature of the proteins is their high serine plus threonine content (~11%), which suggests that they are potential substrates for protein kinases. Since chromosome condensa-

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Fig. 6. Northern blot of human RNA probed with the human sudD cDNA homologue EST AM024h17. A human multiple tissue Northern blot (Clontech) was prehybridized for 1 h at 65°C in 6× SSC, 5× Denhardt’s solution, 0.5% SDS, and 100 mg/ml salmon sperm DNA. The membrane was then hybridized overnight at 65°C in 6× SSC, 1× Denhardt’s solution, and 100 mg/ml salmon sperm DNA containing 1×106 cpm/ml of the 1-kb fragment of human sudD cDNA randomlabeled with [32P]dCTP. The membrane was washed and submitted to autoradiography at −80°C for 3 days.

tion is a cell-cycle-regulated event, the function of SUDD may be regulated through phosphorylation. We are currently investigating this possibility.

Acknowledgement This work was supported by a grant from the National Science Foundation, MCB-9513382 to G.S.M. and NIH CA34936 to G.L.

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