382 Europeon Journol of Cell Biology 78, 382-390 (1999, June) . © Urbon & Fischer Verlag· Jena http://www.urbanfischer.de/journals/ejcb

Ilel

Characterization of a novel nucleolar protein that transiently associates with the condensed chromosomes in mitotic cells

Magnus Larssona, Eva Brundellb

, Pia-Marie J6rgensenb, Stefan Stahla

, Christer H66g1)b,c

a Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), Stockholm/Sweden b Department of Cell and Molecular Biology, The Medical Nobel Institute, Karolinska Institutet, Stockholm/Sweden C Center for Genomics Research, Karolinska Institutet, Stockholm/Sweden

Received October 21, 1998 Received in revised version February 22, 1999 Accepted March 22, 1999

Cell cycle - meiosis - nucleolus - spermatogenesis -chromosomes

We report the isolation and characterization of a murine gene encoding a conserved mammalian nucleolar protein. The pro­tein, called TsgllS, has a predicted molecular mass of 59.4 kDa and a high content of basic amino acids. A homologous human gene was localized to chromosome 16p12.3. The TsgllS pro­tein is predominantly expressed in proliferating somatic cells and in male germ cells. Indirect immunofluorescence micros­copy analysis using an affinity-purified anti-TsgllS serum shows colocalization ofTsgllS and a known nucleolar protein, fibrillarin, to the dense fibrillar component of the nucleolus. The nucleolar localization of the TsgllS protein appears to be temporally restricted to the interphase stages of the somatic cell cycle and to the meiotic phase of spermatogenesis. We find that the TsgllS protein localizes to the nucleolus in both prolif­erating and serum-starved cells. Interestingly, as the nucleolar signal disappears in mitotic cells, the 1Sg11S protein instead becomes associated with the surface of the condensed chro­mosomes.

Introduction

In each cell cycle, the onset of mitosis acts as a starting signal for a dramatic reorganization of the cell's internal structure. These events include the disintegration of the nuclear mem­brane, formation of polar centro somes and the condensation and distribution of chromosomes between daughter cells.

1) Prof. Dr. Christer Hoog, Center for Genomics Research, Karo­linska Institutet, S-17177 Stockholm/Sweden, e-mail: christer.hoog@cmb.ki.se, Fax: ++8313529

One chromosome-associated structure affected by the onset of mitosis is the nucleolus, the site at which ribosomal gene transcription and subunit assembly take place in eukaryotic cells. During mitosis the nucleolar territories become disorga­nized and while some nucleolar proteins either become evenly distributed in the cytoplasm, or remain associated with the nucleolar organizing region (corresponding to the fibrillar cen­ters (FCs) in interphase cells), others associate with the outer surface of the condensed mitotic chromosomes [22]. The latter is the case for the nucleolar proteins N038, N055, Ki-67 and three proteins having molecular masses of 53,66 and 103 kDa [4, 5, 20, 24-26, 29]. The function of these proteins is not known, but it has been suggested that they take part in the condensation/decondensation process that mitotic chromo­somes undergo, or that they protect the outer surface of mitotic chromosomes.

The nucleolus has been divided into three domains based on morphological criteria; the FC, the dense fibrillar component (DFC) and the granular component (GC) [23]. The biological functions of these three domains are not entirely clear, but it is believed that the FC corresponds to the site of the ribosomal genes and that ribosomal gene transcription occurs at the FCs and the DFCs. The majority of the ribosomal RNA processing and pre ribosome assembly would then take place at the GCs. An interesting question is whether the nucleolar proteins that are known to associate with the condensed chromosomes dur­ing mitosis colocalize within the nucleolus, as such a colocal­ization could indicate a common function. This does however, not turn out to be the case. Whereas the N038 and the N055 proteins preferentially localize to the GC of the nucleolus, the Ki-67 protein is mainly localized at the DFC [20, 25]. In sum­mary, the biological role of the nucleolar proteins which asso­ciate with mitotic chromosomes remains an open question.

We have identified a novel murine protein, Tsg118, and shown that while it is localized in the nucleoli of interphase cells, it becomes associated with the surface of the condensed chromosomes in mitotic cells. Furthermore, analysis of germ

0171-9335/99/78/06-382 $12.00/0

382 Europeon Journol of Cell Biology 78, 382-390 (1999, June) . © Urbon & Fischer Verlag· Jena http://www.urbanfischer.de/journals/ejcb

Ilel

Characterization of a novel nucleolar protein that transiently associates with the condensed chromosomes in mitotic cells

Magnus Larssona, Eva Brundellb

, Pia-Marie J6rgensenb, Stefan Stahla

, Christer H66g1)b,c

a Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), Stockholm/Sweden b Department of Cell and Molecular Biology, The Medical Nobel Institute, Karolinska Institutet, Stockholm/Sweden C Center for Genomics Research, Karolinska Institutet, Stockholm/Sweden

Received October 21, 1998 Received in revised version February 22, 1999 Accepted March 22, 1999

Cell cycle - meiosis - nucleolus - spermatogenesis -chromosomes

We report the isolation and characterization of a murine gene encoding a conserved mammalian nucleolar protein. The pro­tein, called TsgllS, has a predicted molecular mass of 59.4 kDa and a high content of basic amino acids. A homologous human gene was localized to chromosome 16p12.3. The TsgllS pro­tein is predominantly expressed in proliferating somatic cells and in male germ cells. Indirect immunofluorescence micros­copy analysis using an affinity-purified anti-TsgllS serum shows colocalization ofTsgllS and a known nucleolar protein, fibrillarin, to the dense fibrillar component of the nucleolus. The nucleolar localization of the TsgllS protein appears to be temporally restricted to the interphase stages of the somatic cell cycle and to the meiotic phase of spermatogenesis. We find that the TsgllS protein localizes to the nucleolus in both prolif­erating and serum-starved cells. Interestingly, as the nucleolar signal disappears in mitotic cells, the 1Sg11S protein instead becomes associated with the surface of the condensed chro­mosomes.

Introduction

In each cell cycle, the onset of mitosis acts as a starting signal for a dramatic reorganization of the cell's internal structure. These events include the disintegration of the nuclear mem­brane, formation of polar centro somes and the condensation and distribution of chromosomes between daughter cells.

1) Prof. Dr. Christer Hoog, Center for Genomics Research, Karo­linska Institutet, S-17177 Stockholm/Sweden, e-mail: christer.hoog@cmb.ki.se, Fax: ++8313529

One chromosome-associated structure affected by the onset of mitosis is the nucleolus, the site at which ribosomal gene transcription and subunit assembly take place in eukaryotic cells. During mitosis the nucleolar territories become disorga­nized and while some nucleolar proteins either become evenly distributed in the cytoplasm, or remain associated with the nucleolar organizing region (corresponding to the fibrillar cen­ters (FCs) in interphase cells), others associate with the outer surface of the condensed mitotic chromosomes [22]. The latter is the case for the nucleolar proteins N038, N055, Ki-67 and three proteins having molecular masses of 53,66 and 103 kDa [4, 5, 20, 24-26, 29]. The function of these proteins is not known, but it has been suggested that they take part in the condensation/decondensation process that mitotic chromo­somes undergo, or that they protect the outer surface of mitotic chromosomes.

The nucleolus has been divided into three domains based on morphological criteria; the FC, the dense fibrillar component (DFC) and the granular component (GC) [23]. The biological functions of these three domains are not entirely clear, but it is believed that the FC corresponds to the site of the ribosomal genes and that ribosomal gene transcription occurs at the FCs and the DFCs. The majority of the ribosomal RNA processing and pre ribosome assembly would then take place at the GCs. An interesting question is whether the nucleolar proteins that are known to associate with the condensed chromosomes dur­ing mitosis colocalize within the nucleolus, as such a colocal­ization could indicate a common function. This does however, not turn out to be the case. Whereas the N038 and the N055 proteins preferentially localize to the GC of the nucleolus, the Ki-67 protein is mainly localized at the DFC [20, 25]. In sum­mary, the biological role of the nucleolar proteins which asso­ciate with mitotic chromosomes remains an open question.

We have identified a novel murine protein, Tsg118, and shown that while it is localized in the nucleoli of interphase cells, it becomes associated with the surface of the condensed chromosomes in mitotic cells. Furthermore, analysis of germ

0171-9335/99/78/06-382 $12.00/0

EJCB

cells at different stages of spermatogenesis shows that Tsg118 associates with the nucleoli in meiotic cells. The relevance of these findings for the function of the Tsg118 protein is dis­cussed.

Materials and methods

Isolation and assembly of a composite TSG118.1 clone A cDNA clone, TSG118, was isolated from an oligo(dT)-primed testis cDNA library [11, 28]. The original TSG118 clone contained a 650bp insert representing the 3'-end of the TSG118 mRNA. An extended clone (approximately 1500bp) was initially isolated from the original cDNA library by standard screening methods (data not shown). This clone was further extended to contain the upstream part of the TSG118.1 gene by employing a novel PCR-based biotin-capture method (Graslund, Larsson, Hoog and Stahl, unpublished) using puri­fied mRNA as template.

Sequence analysis The cDNA inserts from the isolated clones were sequenced from the 5' end using semi-automated solid-phase DNA sequencing [10], to estab­lish the open reading frame (ORF). Primers RIT30, 5'-Biotin­AAAGGGGGATGTGCTGCAAGGCG-3' and RIT27 5'-GCTTC­CGGCTCGTATGTTGTGTG-3', complementary to regions down­stream and upstream of the multi linker region of pBluescript® SK­(Stratagene), were used for the PCR amplification performed directly on bacterial colonies. A two-step PCR program with denaturation at 96 DC for 15 s and annealing and extension at 72 DC for 2 min, was per­formed on a Perkin Elmer Gene Amp system 9600. The biotinylated, double stranded PCR products were immobilized on paramagnetic beads with covalently coupled streptavidin, Dynabeads M280-Streptavidin (Dynal AS, Oslo, Norway). A neodymium-iron-boron permanent magnet (Dynal AS) was used to sediment the beads. The eluted non-biotinylated strand was used as template for a fluorescein isothiocyanate (FITC)-Iabeled sequencing primer, 5'-FITC-TTC­ACACAGGAAACAGCTATGACC-3'. The sequencing reactions were performed on a robotic workstation (Biomek 1000, Beckman Instruments, Fullerton, CA) and an Automated Laser Fluorescent (A.L.F.™) DNA Sequencer (Pharmacia Biotech, Uppsala, Sweden) was used for detection of the Sanger fragments.

Subcloning, expression and affinity purification of fusion proteins The TSG 118 cDNA fragment was cleaved out from the corresponding pBluescript SK vector with EcoRI and XhoI and inserted into the pTI-TZZc and pT7-ABPc expression vectors [14]. E. coli BL21 (DE3)pLysS cells (Novagen Inc., Madison, WI) were transformed with the resulting vectors encoding the fusion protein ZZ-Tsg118 and ABP­Tsg118, and the two fusion proteins were expressed and affinity puri­fied as described previously [14].

Immunization of rabbits New Zeeland White rabbits were immunized intramuscularly with 500 ~g of the ZZ-Tsg118 fusion proteins in phosphate-buffered saline (PBS) and Freund's complete adjuvant (FCA). Booster injections were given 4, 8 and 12 weeks after the initial injection with the same amount of fusion protein but with Freund's incomplete adjuvant instead of FCA. The rabbits were bled 10 days after the second and third booster injection. Antisera from the last bleeding were used in this study. Poly­clonal antisera were prepared and affinity purified as described [26, 27], with the ABP-lSg118 fusion protein immobilized.

Immunobloning procedure Protein extracts were boiled in SDS-reducing buffer (62.5 mM Tris­HCI, pH 6.8,10% glycerol, 2.3% SDS, 10mM DTT). The proteins

Identification of a novel nucleolar protein 383

were separated by SDS-PAGE and transferred to an Immobilon-P membrane in transfer buffer (41 mM Tris, 192mM glycine, 0.02 % SDS, pH8.3) [12]. The filters were incubated with the affinity-purified anti-Tsg118 antiserum (1: 100) after which washing and detection were performed as described previously [26, 27].

Cell culture, cell synchronization and indirect immunofluorescence microscopy Mouse Swiss-3T3 fibroblasts were cultured in Dulbecco's modified Eagles medium (DMEM), from GIBCO, containing 10% fetal calf serum (Sigma). Cells were plated at low density and grown at 37 DC in a humidified atmosphere containing 5 % CO2 , To synchronize cells, 3T3 cells were cultured in DME medium, including 0.1 % fetal calf serum (Sigma) for 47 hours [26]. DME medium including 10% fetal calf serum was added to the serum-starved cells and aliquots were removed for immunofluorescence microscopy analysis. Germ cells at different stages of development were isolated as described before and centrifuged onto a glass slide using a cytospin centrifuge [15, 28]. Cells were fixed in freeze-cold methanol: acetone (50:50) for 5min and pre­incubated with 3 % BSA prior to addition of the first antibody. To seg­regate the nucleolus, 3T3 cells were treated with 1 ~g/ml actinomycin D-mannitol (Sigma) for 4 and 6 hours. The cells were rinsed with PBS and fixed for 10 min with 1 % paraformaldehydelPBS, 0.15 % Triton X-100. The affinity-purified anti-TSG118 serum was diluted 1:20, the monoclonal anti-fibrillarin antiserum 1: 500 (a gift from Dr. J. Steitz, Yale University) and the human anti-p80 coilin antiserum 1 :400 (a gift from Dr. E. Tan, Scripps Research Institute). The secondary anti­bodies were a fluorescein isothiocyanate-conjugated swine anti-rabbit IgG (diluted 1:50, Boehringer Mannheim), a rhodamine-conjugated goat anti-mouse IgG (diluted 1 :80, Boehringer Mannheim) and a rhodamine-conjugated goat anti-human IgG (diluted 1:100, Boehrin­ger Mannheim). The cells were also stained with 1 ~g/ml of Hoechst 33258 for 15 s. The slides were mounted in a 78 % glycerol mounting medium, containing 1 mg/ml para-phenylene diamine, examined in a Zeiss Axioscope microscope and photographed with a Kodak TMAX 400 film. Protein colocalization was analyzed using a Leica DMRXA microscope, digitalized using a Hamamatsu C4880-40 CCD camera and the Openlab software package from Improvision. The pictures were printed on a Kodak 8650 PS CMYK sublimation printer.

Results

The TSG 118.1 gene encodes a highly basic protein We have previously isolated a clone (EST-TSG 118) from a murine testis cDNA library and shown that it detects an mRNA band in Northern blots with an approximate size of 2.2 kb [11, 28]. The TSG 118 transcript was abundantly expressed in the round haploid cells of the testis, but also observed in other testicular cell types such as spermatocytes and spermatogonia. Since no RNA hybridization signal was apparent in tissues like heart, kidney or liver, this suggested that the transcript detected by the TSG 118 cDNA sequence could have a particular function during germ cell develop­ment.

In order to understand more about the function of the pro­tein encoded by the gene corresponding to the TSG 118 mRNA sequence, we decided to characterize the full-length TSG118 cDNA sequence. The TSG118 clone described previously con­tained a 650-bp-Iong insert with a poly(A) stretch at one end, indicating that the cloned sequence corresponds to the 3' part of the endogenous transcript. Using the TSG118 cDNA insert as a probe, additional cDNA clones were isolated from a murine cDNA library. Together the composite cDNA clone, TSG 118.1, was found to have a total length of 2018 bp (Fig. lA

EJCB

cells at different stages of spermatogenesis shows that Tsg118 associates with the nucleoli in meiotic cells. The relevance of these findings for the function of the Tsg118 protein is dis­cussed.

Materials and methods

Isolation and assembly of a composite TSG118.1 clone A cDNA clone, TSG118, was isolated from an oligo(dT)-primed testis cDNA library [11, 28]. The original TSG118 clone contained a 650bp insert representing the 3'-end of the TSG118 mRNA. An extended clone (approximately 1500bp) was initially isolated from the original cDNA library by standard screening methods (data not shown). This clone was further extended to contain the upstream part of the TSG118.1 gene by employing a novel PCR-based biotin-capture method (Graslund, Larsson, Hoog and Stahl, unpublished) using puri­fied mRNA as template.

Sequence analysis The cDNA inserts from the isolated clones were sequenced from the 5' end using semi-automated solid-phase DNA sequencing [10], to estab­lish the open reading frame (ORF). Primers RIT30, 5'-Biotin­AAAGGGGGATGTGCTGCAAGGCG-3' and RIT27 5'-GCTTC­CGGCTCGTATGTTGTGTG-3', complementary to regions down­stream and upstream of the multi linker region of pBluescript® SK­(Stratagene), were used for the PCR amplification performed directly on bacterial colonies. A two-step PCR program with denaturation at 96 DC for 15 s and annealing and extension at 72 DC for 2 min, was per­formed on a Perkin Elmer Gene Amp system 9600. The biotinylated, double stranded PCR products were immobilized on paramagnetic beads with covalently coupled streptavidin, Dynabeads M280-Streptavidin (Dynal AS, Oslo, Norway). A neodymium-iron-boron permanent magnet (Dynal AS) was used to sediment the beads. The eluted non-biotinylated strand was used as template for a fluorescein isothiocyanate (FITC)-Iabeled sequencing primer, 5'-FITC-TTC­ACACAGGAAACAGCTATGACC-3'. The sequencing reactions were performed on a robotic workstation (Biomek 1000, Beckman Instruments, Fullerton, CA) and an Automated Laser Fluorescent (A.L.F.™) DNA Sequencer (Pharmacia Biotech, Uppsala, Sweden) was used for detection of the Sanger fragments.

Subcloning, expression and affinity purification of fusion proteins The TSG 118 cDNA fragment was cleaved out from the corresponding pBluescript SK vector with EcoRI and XhoI and inserted into the pTI-TZZc and pT7-ABPc expression vectors [14]. E. coli BL21 (DE3)pLysS cells (Novagen Inc., Madison, WI) were transformed with the resulting vectors encoding the fusion protein ZZ-Tsg118 and ABP­Tsg118, and the two fusion proteins were expressed and affinity puri­fied as described previously [14].

Immunization of rabbits New Zeeland White rabbits were immunized intramuscularly with 500 ~g of the ZZ-Tsg118 fusion proteins in phosphate-buffered saline (PBS) and Freund's complete adjuvant (FCA). Booster injections were given 4, 8 and 12 weeks after the initial injection with the same amount of fusion protein but with Freund's incomplete adjuvant instead of FCA. The rabbits were bled 10 days after the second and third booster injection. Antisera from the last bleeding were used in this study. Poly­clonal antisera were prepared and affinity purified as described [26, 27], with the ABP-lSg118 fusion protein immobilized.

Immunobloning procedure Protein extracts were boiled in SDS-reducing buffer (62.5 mM Tris­HCI, pH 6.8,10% glycerol, 2.3% SDS, 10mM DTT). The proteins

Identification of a novel nucleolar protein 383

were separated by SDS-PAGE and transferred to an Immobilon-P membrane in transfer buffer (41 mM Tris, 192mM glycine, 0.02 % SDS, pH8.3) [12]. The filters were incubated with the affinity-purified anti-Tsg118 antiserum (1: 100) after which washing and detection were performed as described previously [26, 27].

Cell culture, cell synchronization and indirect immunofluorescence microscopy Mouse Swiss-3T3 fibroblasts were cultured in Dulbecco's modified Eagles medium (DMEM), from GIBCO, containing 10% fetal calf serum (Sigma). Cells were plated at low density and grown at 37 DC in a humidified atmosphere containing 5 % CO2 , To synchronize cells, 3T3 cells were cultured in DME medium, including 0.1 % fetal calf serum (Sigma) for 47 hours [26]. DME medium including 10% fetal calf serum was added to the serum-starved cells and aliquots were removed for immunofluorescence microscopy analysis. Germ cells at different stages of development were isolated as described before and centrifuged onto a glass slide using a cytospin centrifuge [15, 28]. Cells were fixed in freeze-cold methanol: acetone (50:50) for 5min and pre­incubated with 3 % BSA prior to addition of the first antibody. To seg­regate the nucleolus, 3T3 cells were treated with 1 ~g/ml actinomycin D-mannitol (Sigma) for 4 and 6 hours. The cells were rinsed with PBS and fixed for 10 min with 1 % paraformaldehydelPBS, 0.15 % Triton X-100. The affinity-purified anti-TSG118 serum was diluted 1:20, the monoclonal anti-fibrillarin antiserum 1: 500 (a gift from Dr. J. Steitz, Yale University) and the human anti-p80 coilin antiserum 1 :400 (a gift from Dr. E. Tan, Scripps Research Institute). The secondary anti­bodies were a fluorescein isothiocyanate-conjugated swine anti-rabbit IgG (diluted 1:50, Boehringer Mannheim), a rhodamine-conjugated goat anti-mouse IgG (diluted 1 :80, Boehringer Mannheim) and a rhodamine-conjugated goat anti-human IgG (diluted 1:100, Boehrin­ger Mannheim). The cells were also stained with 1 ~g/ml of Hoechst 33258 for 15 s. The slides were mounted in a 78 % glycerol mounting medium, containing 1 mg/ml para-phenylene diamine, examined in a Zeiss Axioscope microscope and photographed with a Kodak TMAX 400 film. Protein colocalization was analyzed using a Leica DMRXA microscope, digitalized using a Hamamatsu C4880-40 CCD camera and the Openlab software package from Improvision. The pictures were printed on a Kodak 8650 PS CMYK sublimation printer.

Results

The TSG 118.1 gene encodes a highly basic protein We have previously isolated a clone (EST-TSG 118) from a murine testis cDNA library and shown that it detects an mRNA band in Northern blots with an approximate size of 2.2 kb [11, 28]. The TSG 118 transcript was abundantly expressed in the round haploid cells of the testis, but also observed in other testicular cell types such as spermatocytes and spermatogonia. Since no RNA hybridization signal was apparent in tissues like heart, kidney or liver, this suggested that the transcript detected by the TSG 118 cDNA sequence could have a particular function during germ cell develop­ment.

In order to understand more about the function of the pro­tein encoded by the gene corresponding to the TSG 118 mRNA sequence, we decided to characterize the full-length TSG118 cDNA sequence. The TSG118 clone described previously con­tained a 650-bp-Iong insert with a poly(A) stretch at one end, indicating that the cloned sequence corresponds to the 3' part of the endogenous transcript. Using the TSG118 cDNA insert as a probe, additional cDNA clones were isolated from a murine cDNA library. Together the composite cDNA clone, TSG 118.1, was found to have a total length of 2018 bp (Fig. lA

384 M. Larsson, E. Brundell, P.-M. Jorgensen et al. EJCB

A 0 2kb

I I

--1 I I I I ~ tM r-B

T'"'~TTCGATGGAGTl'CATAACCCGGTCTGCAGAGTCCATTCATTCCCGGCT1'CCTCGGGCGATCTGCGGGCGGTCCGGGTCTCTACCCTGAAGC?l'CACACCACGCAACTCTCTGA

121 CCGGGTTTCCTl'CTGCGAGCCTGTTGCCTGAGGTCGCACCGGAATCCAAGACATACGCCAACC'I'CTTTCTCCGCGCGTGGGGCCTACCCTCGCAGATCTGATCCCTT'AGGCTCTTCCCCG

241 GCGGCTCGGCGACC'roCGGQGGTGACACGAGCACTTCGGGAGACCCGGGCGACACTGCGGGCACCGGCGTGCGCG'l'A~TCGGCGAATCCGCGGACCGCTCGCCTCCTCACGCAGCAC

M D A V 5 TAT Q G T G R P R P G R R R I S R G R ARK S K 30 361 GCTGCGGCTGGATACAAGTCTCGGCGCtGCATGGACGCGGTTTCTACAGCMCCCAGGGCACCGGGAGGCCGCGACCCGGAAGGAGMGGA'ITTCGAGGGGGcGGGCCAGAAAGTCGAAG

EI±E2 L T S G 5 Ft L R G C 5 R GOA W PAS E P M V S K T Q K A D L G P Q L P E ~ 70 CTGACTTCCGGATCGCGGCTGCGCGGCTGCTCACGCGGGGACGCTTGGCCTGCTAGCGAACCAG AATGGTCAGCM.GACCCAGAAAGCAGACCTGGGCCCACAACTCCCAGA~ 481

601 KKK KKK K R~ V A N V S E P E 'I' Q 'i S V L N S N D '{ F l D ASP P RAT S P 110 AMMGAAGAAGAAGAAAAAGA TGGTTGCAAATGTC'l'CAGAACCAGAGACTCAATACTCAGTCTI'AAACAGTMTGATTAT'ITI'ATAGACGCTl'CTCCTCCGAGGGCCACGTCCCCT

5 N N V D E V Q I P EMS L S K R KKK K Ie S C s '1' H LEE: C L G A E P T R A R 150 7 21 TC'TAACAATGTGGATGAGGTGCAGATCCCCGAGATGTCACTGAGCAAGAGAAAGAAGMAAAAAAGTCC'}'3TAGCACTCACTTGGAGGAGTGCCTGGGGGCTGAGCCTACACGGGCAAGA

Q K K S P S P R R Q ALE Q S A E G L IRE KKK K R R K S L 5 K A A S Q C S G 190 841 CAGAAAAAGTCACCTAGCCCTAGAAGGCAGGCTCTTGAGCAGTCAGCAGAAGGCCTCATCAGAGAGAAGAAAAAGMGAGGCGGAAGTCCTTATCAAAOOCTGCC'I'CCCAAGGCTCAGGG

L K T S PDP K H A K E V S K A G R K 5 K K Q R K E K K V PDT E ALP P Q D A 230 9 61 CTGAAGACCTCCCCAGACCCCAAGCATGCCAAGGAGGTAAGCAAAGCTGGTAGGAAGTCCAAMAACAAAGGAAGGAAAAAAAGGTTCCTGACACGGAAGCCCTTCCTCCCCAGGACGCT

W LYE A G D 5 L H S C LEG A E A E E Q A A L G Q K R K Q G S P R D H N M K K 270 1 081 'roGCTCTACGAGGCTGGGGATTCCCTGCACTCATGCTTAGAGGGGGCAGAGGCAGAGGAACAGQCAGCCTTGGGGCAGAAAAGGAAGCAGGGGAGCCCCAGAGATCACAACATGAAGAAA

KKK T H Q E G D ILL V N 5 R V S V ENS L K Ie G S K K S V K 5 E ALE F v P 310 120 1 AAGAAGA.b,GACCCA.CCAGGAGGGAGACATCCTeCTGGTCAACTCCAGGGTCTCCGTGGAGMCAGCCTGAAGMAGGGAGTAAGAAG'l'CAGTCAAAAGTGAAGCT'l"I'GGAG'M'CGTI'CCA

1321 IDS P K A P G KKK V K S KKK V E Q P V G E G L A ~ KKK KKK R ~ N 350 ATAGACAGCCCGAAGGCCCCTGGGAAGMGAAAGTCAAATCCAAGAAAAAGGTGGMCAGCCAGTTGGTGAAGGGCTGGC OOAAGMGAAG'AAGMGAAGAGG AGAAe

E2 ... "E3 E3 ... "E4 G v KED P W Q E EKE ~ SOT D LEV V L E K K G N MOE T C I 0 Q ~ R R Ie A 390 GGAGTAAAGGAAGACCCCTGGCAGGAGGAAAAGGAGGAGTCAGACACAGACTTAGAGGTGGTACTGGAGAAAAAAGGCAACATGGA'roAGACCTGCATAGACCAGGTGAGACGGAAGGCC 1441

1561 L Q EEl DR E S GU'k"\f~~"~~_ G L S V~QE5430 TTGCAAG.ilAGAGATTGATCGTGAGTCGGG ~~~ .. " . TGGGTTTGTCGGT... '. . '. MCCCAG

F G Q ~'1 D TAG FEN E E Q K L K F L K L M G G F K H L S P S F S R P P S M T I 470 1681 'I'T"I"'..:sGCCAGTGGGATACCGCCGG...---rTI'GAAA.~TGAAGAACAGAAGCTAAAATTCCTGAAGCTTATGGGTGGCTTTAAGCATCTGTCCCCGTCATTCAGCCGCCCTCCCAGCATGACTATC

R 5 N MAL D K K SSE M L Q Q S L Q Q D Y 0 RAM S W K Y S H GAG L G F N S 510 1 SO 1 AGGTCCA..;CATGGCCCTGGACAAAAAGTC'l'TCAGAAATGCTCCAGCAGAGTCTGCAGCAGGACTATGACCGGGCCATGAGTTGGAAGTACAGCCACGGGGCTGGCCTGGGC'ITTAACTCT

EARKVFYIQRNASKS IKLQD" 19 21 GAGGCCCGAA.~GGTC'ITCT.h.CA 'ITGACCGG.::Ll.TGCCTCCAAG'!'C':'A7CA.~GTI'GC.V.GATIAA.~TATTA'l"I'GTCT'I'GTTCT':'CAAAAA.v.AAAAAAAAA 2 0 18

c 68% 55 % 87 % 85 % 78 %

---\~_E_l_~r1,--E __ 2_t-J\ri __ E3--,t-J\ri,--E4_--,t-J\ri_E_5_-,)--

·25976 29094·30059 32692-32760 33546-33623 36911-

Fig. 1. Sequence of the murine gene TSG118.1 and its encoded gene product.The TSG118.1 gene and its putative product are presented schematically (A) and with nucleotide and amino acid sequences (B) as well as the homologous human gene represented by the 5 exons (C). The TSG118.1 sequence has a high content of basic amino acid resi­dues. Two such stretches are indicated by open boxes in (A) and (B). Two consecutive ll-amino-acid repeat sequences are represented by shaded boxes in (A) and (B). The exon borders indicated in (B) are deduced from a BAC clone containing a human gene homologous to

TSG118.1. Note that the region corresponding to amino acids 412-427 of lSg118 cannot be attributed to either of exons 4 or 5 of the human homologue. The level of homology on the nucleotide level between the exons of Tsg118 (as defined in B) and the exons of the correspond­ing human sequence is indicated above each exon in (C). The nucleo­tide positions in the BAC clone of each exon in the human gene are indicated below each exon. Note that the start of exon 1 and the end of exon 5 have not been determined. The accession number for the mouse TSG118.1 sequence in GenBank is AF034580.

and B). An in-frame methionine residue with similarities to a consensus Kozak translational initiation signal was found at position 391 in the nucleotide sequence. This methionine resi-

due es preceded by two in-frame stop codons, suggesting that it is acting as an translational initiator in vivo, and followed by an open reading frame of 529 amino acids. The putative pro-

384 M. Larsson, E. Brundell, P.-M. Jorgensen et al. EJCB

A 0 2kb

I I

--1 I I I I ~ tM r-B

T'"'~TTCGATGGAGTl'CATAACCCGGTCTGCAGAGTCCATTCATTCCCGGCT1'CCTCGGGCGATCTGCGGGCGGTCCGGGTCTCTACCCTGAAGC?l'CACACCACGCAACTCTCTGA

121 CCGGGTTTCCTl'CTGCGAGCCTGTTGCCTGAGGTCGCACCGGAATCCAAGACATACGCCAACC'I'CTTTCTCCGCGCGTGGGGCCTACCCTCGCAGATCTGATCCCTT'AGGCTCTTCCCCG

241 GCGGCTCGGCGACC'roCGGQGGTGACACGAGCACTTCGGGAGACCCGGGCGACACTGCGGGCACCGGCGTGCGCG'l'A~TCGGCGAATCCGCGGACCGCTCGCCTCCTCACGCAGCAC

M D A V 5 TAT Q G T G R P R P G R R R I S R G R ARK S K 30 361 GCTGCGGCTGGATACAAGTCTCGGCGCtGCATGGACGCGGTTTCTACAGCMCCCAGGGCACCGGGAGGCCGCGACCCGGAAGGAGMGGA'ITTCGAGGGGGcGGGCCAGAAAGTCGAAG

EI±E2 L T S G 5 Ft L R G C 5 R GOA W PAS E P M V S K T Q K A D L G P Q L P E ~ 70 CTGACTTCCGGATCGCGGCTGCGCGGCTGCTCACGCGGGGACGCTTGGCCTGCTAGCGAACCAG AATGGTCAGCM.GACCCAGAAAGCAGACCTGGGCCCACAACTCCCAGA~ 481

601 KKK KKK K R~ V A N V S E P E 'I' Q 'i S V L N S N D '{ F l D ASP P RAT S P 110 AMMGAAGAAGAAGAAAAAGA TGGTTGCAAATGTC'l'CAGAACCAGAGACTCAATACTCAGTCTI'AAACAGTMTGATTAT'ITI'ATAGACGCTl'CTCCTCCGAGGGCCACGTCCCCT

5 N N V D E V Q I P EMS L S K R KKK K Ie S C s '1' H LEE: C L G A E P T R A R 150 7 21 TC'TAACAATGTGGATGAGGTGCAGATCCCCGAGATGTCACTGAGCAAGAGAAAGAAGMAAAAAAGTCC'}'3TAGCACTCACTTGGAGGAGTGCCTGGGGGCTGAGCCTACACGGGCAAGA

Q K K S P S P R R Q ALE Q S A E G L IRE KKK K R R K S L 5 K A A S Q C S G 190 841 CAGAAAAAGTCACCTAGCCCTAGAAGGCAGGCTCTTGAGCAGTCAGCAGAAGGCCTCATCAGAGAGAAGAAAAAGMGAGGCGGAAGTCCTTATCAAAOOCTGCC'I'CCCAAGGCTCAGGG

L K T S PDP K H A K E V S K A G R K 5 K K Q R K E K K V PDT E ALP P Q D A 230 9 61 CTGAAGACCTCCCCAGACCCCAAGCATGCCAAGGAGGTAAGCAAAGCTGGTAGGAAGTCCAAMAACAAAGGAAGGAAAAAAAGGTTCCTGACACGGAAGCCCTTCCTCCCCAGGACGCT

W LYE A G D 5 L H S C LEG A E A E E Q A A L G Q K R K Q G S P R D H N M K K 270 1 081 'roGCTCTACGAGGCTGGGGATTCCCTGCACTCATGCTTAGAGGGGGCAGAGGCAGAGGAACAGQCAGCCTTGGGGCAGAAAAGGAAGCAGGGGAGCCCCAGAGATCACAACATGAAGAAA

KKK T H Q E G D ILL V N 5 R V S V ENS L K Ie G S K K S V K 5 E ALE F v P 310 120 1 AAGAAGA.b,GACCCA.CCAGGAGGGAGACATCCTeCTGGTCAACTCCAGGGTCTCCGTGGAGMCAGCCTGAAGMAGGGAGTAAGAAG'l'CAGTCAAAAGTGAAGCT'l"I'GGAG'M'CGTI'CCA

1321 IDS P K A P G KKK V K S KKK V E Q P V G E G L A ~ KKK KKK R ~ N 350 ATAGACAGCCCGAAGGCCCCTGGGAAGMGAAAGTCAAATCCAAGAAAAAGGTGGMCAGCCAGTTGGTGAAGGGCTGGC OOAAGMGAAG'AAGMGAAGAGG AGAAe

E2 ... "E3 E3 ... "E4 G v KED P W Q E EKE ~ SOT D LEV V L E K K G N MOE T C I 0 Q ~ R R Ie A 390 GGAGTAAAGGAAGACCCCTGGCAGGAGGAAAAGGAGGAGTCAGACACAGACTTAGAGGTGGTACTGGAGAAAAAAGGCAACATGGA'roAGACCTGCATAGACCAGGTGAGACGGAAGGCC 1441

1561 L Q EEl DR E S GU'k"\f~~"~~_ G L S V~QE5430 TTGCAAG.ilAGAGATTGATCGTGAGTCGGG ~~~ .. " . TGGGTTTGTCGGT... '. . '. MCCCAG

F G Q ~'1 D TAG FEN E E Q K L K F L K L M G G F K H L S P S F S R P P S M T I 470 1681 'I'T"I"'..:sGCCAGTGGGATACCGCCGG...---rTI'GAAA.~TGAAGAACAGAAGCTAAAATTCCTGAAGCTTATGGGTGGCTTTAAGCATCTGTCCCCGTCATTCAGCCGCCCTCCCAGCATGACTATC

R 5 N MAL D K K SSE M L Q Q S L Q Q D Y 0 RAM S W K Y S H GAG L G F N S 510 1 SO 1 AGGTCCA..;CATGGCCCTGGACAAAAAGTC'l'TCAGAAATGCTCCAGCAGAGTCTGCAGCAGGACTATGACCGGGCCATGAGTTGGAAGTACAGCCACGGGGCTGGCCTGGGC'ITTAACTCT

EARKVFYIQRNASKS IKLQD" 19 21 GAGGCCCGAA.~GGTC'ITCT.h.CA 'ITGACCGG.::Ll.TGCCTCCAAG'!'C':'A7CA.~GTI'GC.V.GATIAA.~TATTA'l"I'GTCT'I'GTTCT':'CAAAAA.v.AAAAAAAAA 2 0 18

c 68% 55 % 87 % 85 % 78 %

---\~_E_l_~r1,--E __ 2_t-J\ri __ E3--,t-J\ri,--E4_--,t-J\ri_E_5_-,)--

·25976 29094·30059 32692-32760 33546-33623 36911-

Fig. 1. Sequence of the murine gene TSG118.1 and its encoded gene product.The TSG118.1 gene and its putative product are presented schematically (A) and with nucleotide and amino acid sequences (B) as well as the homologous human gene represented by the 5 exons (C). The TSG118.1 sequence has a high content of basic amino acid resi­dues. Two such stretches are indicated by open boxes in (A) and (B). Two consecutive ll-amino-acid repeat sequences are represented by shaded boxes in (A) and (B). The exon borders indicated in (B) are deduced from a BAC clone containing a human gene homologous to

TSG118.1. Note that the region corresponding to amino acids 412-427 of lSg118 cannot be attributed to either of exons 4 or 5 of the human homologue. The level of homology on the nucleotide level between the exons of Tsg118 (as defined in B) and the exons of the correspond­ing human sequence is indicated above each exon in (C). The nucleo­tide positions in the BAC clone of each exon in the human gene are indicated below each exon. Note that the start of exon 1 and the end of exon 5 have not been determined. The accession number for the mouse TSG118.1 sequence in GenBank is AF034580.

and B). An in-frame methionine residue with similarities to a consensus Kozak translational initiation signal was found at position 391 in the nucleotide sequence. This methionine resi-

due es preceded by two in-frame stop codons, suggesting that it is acting as an translational initiator in vivo, and followed by an open reading frame of 529 amino acids. The putative pro-

EJCB

tein, denoted Tsg118 , has a calculated relative molecular mass of 59.4 kDa. Interestingly, the predicted Tsg118 amino acid sequence was found to have a very high content of the basic amino acids arginine and lysine, resulting in a theoretical iso­electric point of 10. The two basic amino acids together consti­tute approximately 23 % of the total amino acid content, and visual inspection of the TSG118.1 open reading frame reveals multiple poly-lysine-rich stretches distributed throughout the protein sequence (two of which are indicated in Fig. 1A and B). Further inspection of the TSG118.1 open reading frame revealed the existence of two tandemly occurring 11 amino acid residue repeats (KTEASEPKKWT) in the C-terminal domain (indicated by shaded boxes in Fig. 1A and B). Since these repeats do not display any distinctive features or homol­ogies to known sequence motifs, it is not possible to postulate a particular function for them at this time point.

A human gene homologous to TSG118.1 is found on chromosome 16 The sequence of TSG 118.1 was compared to the sequences stored in GenBank and several types of sequence matches were identified. Most importantly, a human genomic chromo­some 16 sequence (localized at position 16p12.3), deposited as a bacterial artificial chromosome (BAe) insert (accession number AC002550) was shown to contain a gene homologous to TSG118.1. The human TSG118.1-related sequence was found to span a distance of approximately 12 kb and to be divided into 5 exons (Fig. Ie). The sequence similarities at the nucleotide level between the murine cDNA sequence and the exon parts of the human counterpart varied between 78-87 % for the three 3' exons (no. 3, 4 and 5) and between 55-68 % for the two 5' exons (no. 1 and 2), whereas the amino acid sequence similarities for the N- and C-terminal exons were 52-54 % and 92-76 % respectively (Fig. Ie). A short region of the murine sequence, including the above mentioned repeat sequence (in between exons 4 and 5) was found to be missing in the human sequence, suggesting that the mouse and the human genes are not orthologues, or alternatively that the mouse gene has a different sequence.

To distinguish between these two possibilities, all human and mouse TSG 118.1-related cDNA sequences found in the EST data base were compared to the human genomic sequence and to the mouse TSG118.1 cDNA sequence. We found only one type of human cDNA sequences, identical to the exon sequences found in the human gene. The mouse cDNA sequences taken from the EST data base were identical to the TSG118.1 sequence shown in Fig. 1, except for the inclu­sion or exclusion of the sequence in between exon 4 and 5. These data suggest that the human and the mouse genes repre­sent the same gene i.e. they represent two orthologues. The difference between the mouse cDNA clones, i.e. the sequence in between exons 4 and 5, is most likely due to alternative splicing of a murine gene having a slightly different genomic organization than the human gene.

The comparison of the Tsg118 amino acid sequence to data stored in GenBank also revealed a match to a small acidic pro­tein of unknown function expressed in chick otocysts (acces­sion number U37722) [7]. The similarity between the small acidic protein identified in chick otocysts and the Tsg118 sequence (38 % amino acid identity) was restricted to a 70 amino acids long region in the C-terminus of the TSG 118 sequence (amino acids 431-508). It is not likely, however, that the chick otocyst cDNA clone represents a homologue of the

Identification of a novel nucleolar protein 385

TSG 118 sequence for several reasons. Firstly, the mRNA tran­script detected by the chick otocyst cDNA clone is 0.8 kb and the open reading frame found in the full-length cDNA sequence contains 172 amino acids. This is much shorter than the corresponding length of the TSG118.1 transcript and the length of the open reading frame found in the TSG118.1 cDNA sequence. Second, a human cDNA encoding a protein of similar length and displaying a 76 % amino acid sequence identity to the chick otocyst protein has been identified [7], and probably represents a true orthologue. This suggests that the similarity between the TSG118.1 sequence and the chick otocyst sequence would rather represent a conserved func­tional domain.

The TSG 118.1 gene encodes a protein expressed in testis and in proliferating cells In order to analyze the expression and function of the protein encoded by the murine TSG118.1 sequence, we generated a polyclonal antiserum. A 203 amino acid region from the 3' end of the TSG118.1 sequence was produced as a fusion protein in bacteria [14], affinity purified and injected into rabbits (see Material and methods). The sera from two rabbits injected with the bacterially expressed Tsg118 fusion protein were affin­ity enriched and shown to specifically detect the Tsg118 fusion protein in total bacterial protein extracts, whereas the pre­immune sera from the two rabbits did not react with the fusion protein (data not shown). The affinity-purified sera were next used to analyze protein extracts prepared from different murine sources. A main protein band with a molecular mass of approximately 85 kDa was detected in Western blots by the affinity-purified anti-Tsg118 antiserum in protein extracts pre­pared from testis, from pachytene spermatocytes and from round haploid spermatids (Fig. 2). In addition to the 85 kDa band, a weaker 82 kDa band was detected by the anti-Tsg118 antiserum in the same extracts. The molecular mass of the pro-

rI1 rI1 rI1 rI1 - - - -- - - -~ ~ ~ ~ rI1 ~ ~ rI1 ~ ~ - "0 - "0 - ..,J - ..,J ~ .... ;..., ~ . ... ;..., ~ 0 rI1 ~ 0 rI1 ~ - -= .- ~ - -= .-Q. ~ ... Q. ~ ... E-i ~ ~ rI:J E-i ~ ~ rI:J

~ ~ ~ -= c. ... ~ -= c. ...

97 -

66 -

a-Tsgl18 pre-immune Fig. 2. Expression of the TSG 118 protein in somatic cells and in germ cells. Protein extracts were prepared from proliferating Swiss-3T3 cells and from adult mouse testis, as well as from isolated mouse pachytene and round haploid cells. An equal amount of protein (as determined by Coomassie Blue staining of the filter (not shown» from each extract was separated using SDS-PAGE. After immunoblotting, the filters were analyzed using the affinity-purified anti-Tsg118 antiserum or a pre-immune serum. Molecular sizes are given in kDa.

EJCB

tein, denoted Tsg118 , has a calculated relative molecular mass of 59.4 kDa. Interestingly, the predicted Tsg118 amino acid sequence was found to have a very high content of the basic amino acids arginine and lysine, resulting in a theoretical iso­electric point of 10. The two basic amino acids together consti­tute approximately 23 % of the total amino acid content, and visual inspection of the TSG118.1 open reading frame reveals multiple poly-lysine-rich stretches distributed throughout the protein sequence (two of which are indicated in Fig. 1A and B). Further inspection of the TSG118.1 open reading frame revealed the existence of two tandemly occurring 11 amino acid residue repeats (KTEASEPKKWT) in the C-terminal domain (indicated by shaded boxes in Fig. 1A and B). Since these repeats do not display any distinctive features or homol­ogies to known sequence motifs, it is not possible to postulate a particular function for them at this time point.

A human gene homologous to TSG118.1 is found on chromosome 16 The sequence of TSG 118.1 was compared to the sequences stored in GenBank and several types of sequence matches were identified. Most importantly, a human genomic chromo­some 16 sequence (localized at position 16p12.3), deposited as a bacterial artificial chromosome (BAe) insert (accession number AC002550) was shown to contain a gene homologous to TSG118.1. The human TSG118.1-related sequence was found to span a distance of approximately 12 kb and to be divided into 5 exons (Fig. Ie). The sequence similarities at the nucleotide level between the murine cDNA sequence and the exon parts of the human counterpart varied between 78-87 % for the three 3' exons (no. 3, 4 and 5) and between 55-68 % for the two 5' exons (no. 1 and 2), whereas the amino acid sequence similarities for the N- and C-terminal exons were 52-54 % and 92-76 % respectively (Fig. Ie). A short region of the murine sequence, including the above mentioned repeat sequence (in between exons 4 and 5) was found to be missing in the human sequence, suggesting that the mouse and the human genes are not orthologues, or alternatively that the mouse gene has a different sequence.

To distinguish between these two possibilities, all human and mouse TSG 118.1-related cDNA sequences found in the EST data base were compared to the human genomic sequence and to the mouse TSG118.1 cDNA sequence. We found only one type of human cDNA sequences, identical to the exon sequences found in the human gene. The mouse cDNA sequences taken from the EST data base were identical to the TSG118.1 sequence shown in Fig. 1, except for the inclu­sion or exclusion of the sequence in between exon 4 and 5. These data suggest that the human and the mouse genes repre­sent the same gene i.e. they represent two orthologues. The difference between the mouse cDNA clones, i.e. the sequence in between exons 4 and 5, is most likely due to alternative splicing of a murine gene having a slightly different genomic organization than the human gene.

The comparison of the Tsg118 amino acid sequence to data stored in GenBank also revealed a match to a small acidic pro­tein of unknown function expressed in chick otocysts (acces­sion number U37722) [7]. The similarity between the small acidic protein identified in chick otocysts and the Tsg118 sequence (38 % amino acid identity) was restricted to a 70 amino acids long region in the C-terminus of the TSG 118 sequence (amino acids 431-508). It is not likely, however, that the chick otocyst cDNA clone represents a homologue of the

Identification of a novel nucleolar protein 385

TSG 118 sequence for several reasons. Firstly, the mRNA tran­script detected by the chick otocyst cDNA clone is 0.8 kb and the open reading frame found in the full-length cDNA sequence contains 172 amino acids. This is much shorter than the corresponding length of the TSG118.1 transcript and the length of the open reading frame found in the TSG118.1 cDNA sequence. Second, a human cDNA encoding a protein of similar length and displaying a 76 % amino acid sequence identity to the chick otocyst protein has been identified [7], and probably represents a true orthologue. This suggests that the similarity between the TSG118.1 sequence and the chick otocyst sequence would rather represent a conserved func­tional domain.

The TSG 118.1 gene encodes a protein expressed in testis and in proliferating cells In order to analyze the expression and function of the protein encoded by the murine TSG118.1 sequence, we generated a polyclonal antiserum. A 203 amino acid region from the 3' end of the TSG118.1 sequence was produced as a fusion protein in bacteria [14], affinity purified and injected into rabbits (see Material and methods). The sera from two rabbits injected with the bacterially expressed Tsg118 fusion protein were affin­ity enriched and shown to specifically detect the Tsg118 fusion protein in total bacterial protein extracts, whereas the pre­immune sera from the two rabbits did not react with the fusion protein (data not shown). The affinity-purified sera were next used to analyze protein extracts prepared from different murine sources. A main protein band with a molecular mass of approximately 85 kDa was detected in Western blots by the affinity-purified anti-Tsg118 antiserum in protein extracts pre­pared from testis, from pachytene spermatocytes and from round haploid spermatids (Fig. 2). In addition to the 85 kDa band, a weaker 82 kDa band was detected by the anti-Tsg118 antiserum in the same extracts. The molecular mass of the pro-

rI1 rI1 rI1 rI1 - - - -- - - -~ ~ ~ ~ rI1 ~ ~ rI1 ~ ~ - "0 - "0 - ..,J - ..,J ~ .... ;..., ~ . ... ;..., ~ 0 rI1 ~ 0 rI1 ~ - -= .- ~ - -= .-Q. ~ ... Q. ~ ... E-i ~ ~ rI:J E-i ~ ~ rI:J

~ ~ ~ -= c. ... ~ -= c. ...

97 -

66 -

a-Tsgl18 pre-immune Fig. 2. Expression of the TSG 118 protein in somatic cells and in germ cells. Protein extracts were prepared from proliferating Swiss-3T3 cells and from adult mouse testis, as well as from isolated mouse pachytene and round haploid cells. An equal amount of protein (as determined by Coomassie Blue staining of the filter (not shown» from each extract was separated using SDS-PAGE. After immunoblotting, the filters were analyzed using the affinity-purified anti-Tsg118 antiserum or a pre-immune serum. Molecular sizes are given in kDa.

386 M. Larsson, E. Brundell, P.-M. Jorgensen et 01.

tein detected by the anti-Tsg118 antiserum is higher than expected based on the relative molecular mass as calculated from its predicted amino acid sequence. However, the high content of basic amino acids and their local clusterings could affect the relative migration of the protein in gel electrophore­sis . Indeed, arginine-rich sequences have been shown to decrease the electrophoretic mobility of proteins in SDS­PAGE [9].

Based on the expression of the TSG118.1 RNA in juvenile testis (8-12 days after birth) [28], which to a large extent con­sists of proliferating spermatogonial cells , we also tested if Tsg118 was expressed in proliferating somatic cells. Interest­ingly, a dominating 82 kDa band was detected by the anti­Tsg118 antibody in protein extracts prepared from asynchro­nously growing murine Swiss-3T3 cells. In addition, a weaker 85 kDa band was also apparent in the same Swiss-3T3 extract. It is possible that the two bands detected by the anti-Tsg118 antibody represent two differently phosphorylated or glycosy­lated forms of the same protein. Alternatively, the two protein

interphase 3T3

mitotic 3T3

bands dectected by the antibody could represent two alterna­tively spliced protein variants. The same protein labeling pat­tern was also seen using an affinity-purified serum from a sec­ond rabbit (not shown). No bands were detected using the pre­immune serum from two rabbits injected with the Tsg118 pro­tein (Fig. 2), strongly suggesting that the 82 and 85 kDa pro­tein variants detected by the anti-serum indeed represent the Tsg118 protein. We conclude that the TSG 118.1 gene encodes a protein with a molecular maS's of 82-85 kDa that is expressed in proliferating somatic cells as well as in male germ cells at different stages of development.

The T5G 118.1 gene encodes a protein that is a component of the DFC of the nucleolus The two affinity-purified anti-Tsg118 antisera were also used to investigate the expression and subcellular localization of the Tsg118 protein in Swiss-3T3 cells using indirect immunofluo­rescence microscopy. Both antisera were found to label nuclear structures with an irregular shape (Fig. 3). We tested if

a-fibrillarin Hoechst

pre-immune a - fibrillarin Hoechst

interphase 3T3

Fig. 3. The Tsg118 protein associates with the nucleoli in interphase cells and with the surface of the condensed chromosomes in mitotic cells. Asynchronously growing Swiss-3T3 cells were fixed with methanol-acetone and triple stained with the anti-Tsg118 antiserum or pre-immune serum, an anti-fibrillarin antiserum und Hoechst 33258 in indirect immunofluorescence microscopy experiments. The secondary antibodies were a fluorescein isothiocyanate-conjugated swine anti-

rabbit IgG and a rhodamine-conjugated goat anti-mouse IgG. All non­mitotic cells had the same type of nucleolar staining (one example is shown in Fig. 3, where a labeled nucleolar region has been indicated by an arrow), suggesting that the accumulation ofTsg118 in the nucleolus during the interphase stages of the cell cycle is not temporally regu­lated . One example of a typically stained mitotic cell is also shown . Bar, 20 fl.m.

386 M. Larsson, E. Brundell, P.-M. Jorgensen et 01.

tein detected by the anti-Tsg118 antiserum is higher than expected based on the relative molecular mass as calculated from its predicted amino acid sequence. However, the high content of basic amino acids and their local clusterings could affect the relative migration of the protein in gel electrophore­sis . Indeed, arginine-rich sequences have been shown to decrease the electrophoretic mobility of proteins in SDS­PAGE [9].

Based on the expression of the TSG118.1 RNA in juvenile testis (8-12 days after birth) [28], which to a large extent con­sists of proliferating spermatogonial cells , we also tested if Tsg118 was expressed in proliferating somatic cells. Interest­ingly, a dominating 82 kDa band was detected by the anti­Tsg118 antibody in protein extracts prepared from asynchro­nously growing murine Swiss-3T3 cells. In addition, a weaker 85 kDa band was also apparent in the same Swiss-3T3 extract. It is possible that the two bands detected by the anti-Tsg118 antibody represent two differently phosphorylated or glycosy­lated forms of the same protein. Alternatively, the two protein

interphase 3T3

mitotic 3T3

bands dectected by the antibody could represent two alterna­tively spliced protein variants. The same protein labeling pat­tern was also seen using an affinity-purified serum from a sec­ond rabbit (not shown). No bands were detected using the pre­immune serum from two rabbits injected with the Tsg118 pro­tein (Fig. 2), strongly suggesting that the 82 and 85 kDa pro­tein variants detected by the anti-serum indeed represent the Tsg118 protein. We conclude that the TSG 118.1 gene encodes a protein with a molecular maS's of 82-85 kDa that is expressed in proliferating somatic cells as well as in male germ cells at different stages of development.

The T5G 118.1 gene encodes a protein that is a component of the DFC of the nucleolus The two affinity-purified anti-Tsg118 antisera were also used to investigate the expression and subcellular localization of the Tsg118 protein in Swiss-3T3 cells using indirect immunofluo­rescence microscopy. Both antisera were found to label nuclear structures with an irregular shape (Fig. 3). We tested if

a-fibrillarin Hoechst

pre-immune a - fibrillarin Hoechst

interphase 3T3

Fig. 3. The Tsg118 protein associates with the nucleoli in interphase cells and with the surface of the condensed chromosomes in mitotic cells. Asynchronously growing Swiss-3T3 cells were fixed with methanol-acetone and triple stained with the anti-Tsg118 antiserum or pre-immune serum, an anti-fibrillarin antiserum und Hoechst 33258 in indirect immunofluorescence microscopy experiments. The secondary antibodies were a fluorescein isothiocyanate-conjugated swine anti-

rabbit IgG and a rhodamine-conjugated goat anti-mouse IgG. All non­mitotic cells had the same type of nucleolar staining (one example is shown in Fig. 3, where a labeled nucleolar region has been indicated by an arrow), suggesting that the accumulation ofTsg118 in the nucleolus during the interphase stages of the cell cycle is not temporally regu­lated . One example of a typically stained mitotic cell is also shown . Bar, 20 fl.m.

IICI

these structures could represent nucleoli by using an anti­serum against fibrillarin, a constitutively expressed nucleolar protein [8, 19]. A comparison between the anti-Tsg118 and the anti-fibrillarin labelling patterns in interphase 3T3 cells revealed an exact overlap between these two proteins (Fig. 3). No labelling of the 3T3 cells was observed using pre-immune sera from rabbits injected with the Tsg118 protein (Fig. 3). These results strongly suggest that the Tsg118 is a nucleolar protein.

Fibrillarin has been shown to be localized in the DFC domain of the nucleolus [8, 19], resulting in a non-symmetrical distribution within the nucleolus when visualized by indirect immunofluorescence microscopy (Fig. 3). The fact that Tsg118 colocalizes with fibrillarin and also displays a non-symmetrical staining pattern within the nucleolus, indicates that also Tsg118 is a nucleolar DFC component. To further test this, we compared the distribution ofTsg118 and fibrillarin in 3T3 cells that had been treated with the transcription inhibitor actino­mycin D. In such cells the nucleoli segregate into smaller sub­compartments, which enables a more detailed analysis of the relative sublocalization of a nucleolar protein [20]. We find that the Tsg118 protein remains associated with the nucleoli and that it colocalizes with fibrillarin also in actinomycin D­treated cells (Fig. 4). This strongly suggests that the Tsg118 protein represents a novel component of the DFC, in addition to the three proteins, Nopp140, NAP57 and Ki-67 [6,17,18, 26, 29], that previously have been described to reside mainly in the DFCs.

Of the known proteins residing in the DFC of the nucleolus, three of them (fibrillarin, NAP57 an Nopp140) are known to also be components of the coiled body [17,18,21], a nucleolar accessory body [1, 13]. We therefore double-labeled 3T3 cells using the anti-Tsg118 antibody and a human anti-p80 coilin serum, specifically recognizing the coiled body [1-3]. We did not observe a labelling of the coiled body structure using the anti-Tsg118 serum, suggesting that the Tsg118 protein is not part of the coiled body (data not shown).

It has previously been shown that the nucleolar proteins Ki-67 and its murine homologue Tsg126 fail to localize to the nucleolus in serum-starved cells, suggesting that the subcellu­lar distribution of these proteins is linked to the proliferative activity of the cells [26]. To test if also Tsg118 is a proliferation­related nucleolar protein, we analyzed the distribution of the Tsg118, Tsg126 and the fibrillarin protein in serum-starved

Fig. 4. The Tsg118 protein colocalizes with fibrillarin, a dense fibrillar component, in actinomycin D-treated cells. Asynchronously growing Swiss-3T3 cells were treated with actinomycin D for 6 hours and fixed with paraformaldehyde. The cells were double stained with the anti­Tsg118 antiserum and an anti-fibrillarin antiserum in indirect immuno­fluorescence microscopy experiments. The secondary antibodies were

Identification of a novel nucleolar protein 387

cells. We found that the relative levels and locations of Tsg118 and fibrillarin in serum-starved cells remained relatively unchanged as determined by immunofluorescence microscopy (Fig. 5, compare with Fig. 3). The Tsg126 protein, in contrast, became displaced from the nucleoli in serum-starved cells, but accumulated again in the nucleoli once serum was added back to the cells (Fig. 5). This shows that in contrast to the Tsg126 protein, the accumulation of the Tsg118 protein in nucleoli is not related to the proliferative activity of the cells.

The nucleolar TS9118 protein accumulates atthe surface of condensed chromosomes in mitotic cells We have also analyzed the distribution of Tsg118 in mitotic cells using indirect immunofluorescence experiments. We find the Tsg118 protein to be associated with the surface of con­densed mitotic chromosomes and that this chromosomal asso­ciation is seen independently of the substage of the mitotic process (Fig. 3 and data not shown). The pattern displayed by the anti-Tsg118 antiserum is very similar to the labelling pat­tern produced by the anti-fibrillarin antiserum (Fig. 3). No labelling of the 3T3 cells was observed using pre-immune sera from rabbits injected with the Tsg118 protein (data not shown). We therefore conclude that the Tsg118 protein is a new mem­ber of a growing family of nucleolar proteins which appear to move from the nucleoli of interphase cells to the surface of the condensed chromosomes in mitotic cells.

The similarities between the temporal and spatial localization of fibrillarin and Tsg118 suggest that these two proteins could take part in related cellular activities. To analyze if these two proteins are part of a common protein complex in asynchro­nously growing 3T3 cells, protein extracts were prepared from these cells and analyzed using an immunoprecipitation proce­dure. We were, however, not able to co-immunoprecipitate these two proteins (data not shown), suggesting that they exist separately from each other at the same cellular locations.

The TS9 118 protein and fibrillarin are part of the nucleolus in spermatocytes Based on the abundance of the Tsg118 protein in pachytene spermatocytes of the testis, we investigated if the protein was associated with nucleoli in pachytene spermatocytes. Pachy­tene spermatocytes were purified from mouse testis, fixed and analyzed by indirect immunofluorescence microscopy using

a fluorescein isothiocyanate-conjugated swine anti-rabbit IgG and a rhodamine-conjugated goat anti-mouse IgG. The individual anti­Tsg118 and fibrillarin pictures were digitalized and merged to display the extensive overlap between the two proteins in the nucleoli. The cells were also analyzed using phase-contrast microscopy to display the segregated phase-dense nucleoli. Magnification: 800 x.

IICI

these structures could represent nucleoli by using an anti­serum against fibrillarin, a constitutively expressed nucleolar protein [8, 19]. A comparison between the anti-Tsg118 and the anti-fibrillarin labelling patterns in interphase 3T3 cells revealed an exact overlap between these two proteins (Fig. 3). No labelling of the 3T3 cells was observed using pre-immune sera from rabbits injected with the Tsg118 protein (Fig. 3). These results strongly suggest that the Tsg118 is a nucleolar protein.

Fibrillarin has been shown to be localized in the DFC domain of the nucleolus [8, 19], resulting in a non-symmetrical distribution within the nucleolus when visualized by indirect immunofluorescence microscopy (Fig. 3). The fact that Tsg118 colocalizes with fibrillarin and also displays a non-symmetrical staining pattern within the nucleolus, indicates that also Tsg118 is a nucleolar DFC component. To further test this, we compared the distribution ofTsg118 and fibrillarin in 3T3 cells that had been treated with the transcription inhibitor actino­mycin D. In such cells the nucleoli segregate into smaller sub­compartments, which enables a more detailed analysis of the relative sublocalization of a nucleolar protein [20]. We find that the Tsg118 protein remains associated with the nucleoli and that it colocalizes with fibrillarin also in actinomycin D­treated cells (Fig. 4). This strongly suggests that the Tsg118 protein represents a novel component of the DFC, in addition to the three proteins, Nopp140, NAP57 and Ki-67 [6,17,18, 26, 29], that previously have been described to reside mainly in the DFCs.

Of the known proteins residing in the DFC of the nucleolus, three of them (fibrillarin, NAP57 an Nopp140) are known to also be components of the coiled body [17,18,21], a nucleolar accessory body [1, 13]. We therefore double-labeled 3T3 cells using the anti-Tsg118 antibody and a human anti-p80 coilin serum, specifically recognizing the coiled body [1-3]. We did not observe a labelling of the coiled body structure using the anti-Tsg118 serum, suggesting that the Tsg118 protein is not part of the coiled body (data not shown).

It has previously been shown that the nucleolar proteins Ki-67 and its murine homologue Tsg126 fail to localize to the nucleolus in serum-starved cells, suggesting that the subcellu­lar distribution of these proteins is linked to the proliferative activity of the cells [26]. To test if also Tsg118 is a proliferation­related nucleolar protein, we analyzed the distribution of the Tsg118, Tsg126 and the fibrillarin protein in serum-starved

Fig. 4. The Tsg118 protein colocalizes with fibrillarin, a dense fibrillar component, in actinomycin D-treated cells. Asynchronously growing Swiss-3T3 cells were treated with actinomycin D for 6 hours and fixed with paraformaldehyde. The cells were double stained with the anti­Tsg118 antiserum and an anti-fibrillarin antiserum in indirect immuno­fluorescence microscopy experiments. The secondary antibodies were

Identification of a novel nucleolar protein 387

cells. We found that the relative levels and locations of Tsg118 and fibrillarin in serum-starved cells remained relatively unchanged as determined by immunofluorescence microscopy (Fig. 5, compare with Fig. 3). The Tsg126 protein, in contrast, became displaced from the nucleoli in serum-starved cells, but accumulated again in the nucleoli once serum was added back to the cells (Fig. 5). This shows that in contrast to the Tsg126 protein, the accumulation of the Tsg118 protein in nucleoli is not related to the proliferative activity of the cells.

The nucleolar TS9118 protein accumulates atthe surface of condensed chromosomes in mitotic cells We have also analyzed the distribution of Tsg118 in mitotic cells using indirect immunofluorescence experiments. We find the Tsg118 protein to be associated with the surface of con­densed mitotic chromosomes and that this chromosomal asso­ciation is seen independently of the substage of the mitotic process (Fig. 3 and data not shown). The pattern displayed by the anti-Tsg118 antiserum is very similar to the labelling pat­tern produced by the anti-fibrillarin antiserum (Fig. 3). No labelling of the 3T3 cells was observed using pre-immune sera from rabbits injected with the Tsg118 protein (data not shown). We therefore conclude that the Tsg118 protein is a new mem­ber of a growing family of nucleolar proteins which appear to move from the nucleoli of interphase cells to the surface of the condensed chromosomes in mitotic cells.

The similarities between the temporal and spatial localization of fibrillarin and Tsg118 suggest that these two proteins could take part in related cellular activities. To analyze if these two proteins are part of a common protein complex in asynchro­nously growing 3T3 cells, protein extracts were prepared from these cells and analyzed using an immunoprecipitation proce­dure. We were, however, not able to co-immunoprecipitate these two proteins (data not shown), suggesting that they exist separately from each other at the same cellular locations.

The TS9 118 protein and fibrillarin are part of the nucleolus in spermatocytes Based on the abundance of the Tsg118 protein in pachytene spermatocytes of the testis, we investigated if the protein was associated with nucleoli in pachytene spermatocytes. Pachy­tene spermatocytes were purified from mouse testis, fixed and analyzed by indirect immunofluorescence microscopy using

a fluorescein isothiocyanate-conjugated swine anti-rabbit IgG and a rhodamine-conjugated goat anti-mouse IgG. The individual anti­Tsg118 and fibrillarin pictures were digitalized and merged to display the extensive overlap between the two proteins in the nucleoli. The cells were also analyzed using phase-contrast microscopy to display the segregated phase-dense nucleoli. Magnification: 800 x.

388 M. Larsson, E. Brundell, P.-M. Jorgensen et al. EJCB

a - fibrillarin Hoechst

Oh

a-Tsg126 a-fibrillarin Hoechst

Oh

a-fibrillarin Hoechst

24 h

Fig. S. The accumulation of the Tsg1l8 protein in nucleoli is not related to the proliferative activity of the cells. Cells were serum starved for 48 hours and aliquots removed for immunofluorescence microscopy analysis (Oh). Serum was then added and cells grown for 24h before a second aliquot was removed for analysis (24h). Cells were fixed with methanol-acetone and triple stained with the anti-

the anti-Tsg118 and the anti-fibrillarin antisera. In both cases, the sera labeled the nucleoli in the spermatocyte (Fig. 6). No labelling of the 3T3 cells was observed using pre-immune sera from rabbits injected with the Tsg118 protein (data not shown). This suggests that Tsg118 has a nucleolar-associated function in both somatic and germ cells.

Discussion

We have shown by indirect immunofluorescence microsopy methods that Tsg118 is a new member of a growing family of nucleolar proteins. The intranucleolar distribution ofTsg118 is very similar to fibrillarin, a component of the DFC of the nucleolus, suggesting that the Tsg118 protein represents a new DFC component. We find that Tsg118 and fibrillarin localize to the same nucleolar domains in both normal cells, in serum­starved cells and in cells treated with actinomycin D. The simi­larity between the distribution patterns of fibrillarin and Tsg118 , suggests that these two proteins could take part in related cellular activities. These two proteins, however, do not seem to be part of a common protein complex, as we have not been able to co-immunoprecipitate them.

Tsg1l8 antiserum (or alternatively the anti-Tsg126 antiserum), an anti­fibrillarin antiserum and Hoechst 33258 in indirect immunofluores­cence microscopy experiments. The secondary antibodies were a fluo­rescein isothiocyanate-conjugated swine anti-rabbit JgG and a rhodamine-conjugated goat anti-mouse JgG. Bar, 20 flm.

The nucleotide sequence of the murine and human TSG118.1 genes do not display any features that help us to assign a particular function to the encoded protein. One nota­ble thing, however, is the relatively high pI of the protein, caused by a high percentage of basic amino acids. We specu­late that the clusters of basic amino acids found in the Tsg118 protein could be used to establish interactions with nucleic acid or negatively charged proteins.

One of the most interesting aspects of the Tsg118 protein is its relocation from the nucleoli in interphase cells to the con­densed chromosomes in mitotic cells. Our indirect immuno­fluorescence data suggest that the protein becomes attached to the surface of mitotic chromosomes, in resemblance with the observations that have been made for other nucleolar pro­teins, i.e., N055, N038, Ki-67 and three proteins having molecular masses of 53,66 and 103kDa [4, 5, 16,20,25,26, 30]. It has been suggested that the association of nucleolar proteins to mitotic chromosomes would ensure their symmet­rical distribution between daughter cells. This cannot be the entire explanation, however, as for example the Ki-67 protein is rapidly lost in cells early in the Gl stage of the cell cyle [26, 30]. Instead, it is possible that the nucleolar proteins that asso­ciate with the condensed chromosomes in mitotic cells have a supportive function. For example, these proteins could help in

388 M. Larsson, E. Brundell, P.-M. Jorgensen et al. EJCB

a - fibrillarin Hoechst

Oh

a-Tsg126 a-fibrillarin Hoechst

Oh

a-fibrillarin Hoechst

24 h

Fig. S. The accumulation of the Tsg1l8 protein in nucleoli is not related to the proliferative activity of the cells. Cells were serum starved for 48 hours and aliquots removed for immunofluorescence microscopy analysis (Oh). Serum was then added and cells grown for 24h before a second aliquot was removed for analysis (24h). Cells were fixed with methanol-acetone and triple stained with the anti-

the anti-Tsg118 and the anti-fibrillarin antisera. In both cases, the sera labeled the nucleoli in the spermatocyte (Fig. 6). No labelling of the 3T3 cells was observed using pre-immune sera from rabbits injected with the Tsg118 protein (data not shown). This suggests that Tsg118 has a nucleolar-associated function in both somatic and germ cells.

Discussion

We have shown by indirect immunofluorescence microsopy methods that Tsg118 is a new member of a growing family of nucleolar proteins. The intranucleolar distribution ofTsg118 is very similar to fibrillarin, a component of the DFC of the nucleolus, suggesting that the Tsg118 protein represents a new DFC component. We find that Tsg118 and fibrillarin localize to the same nucleolar domains in both normal cells, in serum­starved cells and in cells treated with actinomycin D. The simi­larity between the distribution patterns of fibrillarin and Tsg118 , suggests that these two proteins could take part in related cellular activities. These two proteins, however, do not seem to be part of a common protein complex, as we have not been able to co-immunoprecipitate them.

Tsg1l8 antiserum (or alternatively the anti-Tsg126 antiserum), an anti­fibrillarin antiserum and Hoechst 33258 in indirect immunofluores­cence microscopy experiments. The secondary antibodies were a fluo­rescein isothiocyanate-conjugated swine anti-rabbit JgG and a rhodamine-conjugated goat anti-mouse JgG. Bar, 20 flm.

The nucleotide sequence of the murine and human TSG118.1 genes do not display any features that help us to assign a particular function to the encoded protein. One nota­ble thing, however, is the relatively high pI of the protein, caused by a high percentage of basic amino acids. We specu­late that the clusters of basic amino acids found in the Tsg118 protein could be used to establish interactions with nucleic acid or negatively charged proteins.

One of the most interesting aspects of the Tsg118 protein is its relocation from the nucleoli in interphase cells to the con­densed chromosomes in mitotic cells. Our indirect immuno­fluorescence data suggest that the protein becomes attached to the surface of mitotic chromosomes, in resemblance with the observations that have been made for other nucleolar pro­teins, i.e., N055, N038, Ki-67 and three proteins having molecular masses of 53,66 and 103kDa [4, 5, 16,20,25,26, 30]. It has been suggested that the association of nucleolar proteins to mitotic chromosomes would ensure their symmet­rical distribution between daughter cells. This cannot be the entire explanation, however, as for example the Ki-67 protein is rapidly lost in cells early in the Gl stage of the cell cyle [26, 30]. Instead, it is possible that the nucleolar proteins that asso­ciate with the condensed chromosomes in mitotic cells have a supportive function. For example, these proteins could help in

EJCB Identification of a novel nucleolar protein 389

spermatocyte

Fig. 6. The Tsg1l8 protein associates with the nucleoli in meiotic germ cells . Pachytene spermatocytes were isolated from mouse testis, centrifuged onto a glass slide using a cytospin centrifuge and fixed in methanol-acetone. The cells were triple stained with the anti-Tsg118 antiserum, an anti-fibrillarin antiserum and Hoechst 33258 in indirect

the condensation/decondensation process that mitotic chro­mosomes, undergo, or they could protect the outer surface of mitotic chromosomes. The excess of positively charged amino acids in the Tsg118 protein could be important in mediating physical interactions with the mitotic chromosomes.

Acknowledgements. We thank Katarina Gell and Li Yuan for technical assistance and Mathias Uhlen for valuable discussions. This work was supported by the Swedish Natural Science Research Council , the Swedish Research Council for Engineering Sciences, Erik-Philip Sorensen Foundation , Nilsson-Ehle-Funds , the European Community (The BIOTECH Programme, BI04 CT60183) and the Karolinska Institutet.

References

[1] Andrade , L. E. c., E. K. L. Chan, 1. Raska, C. L. Peebles, G. Roos, E . M. Tan: Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J. Exp. Med . 173, 1407-1419 (1991) .

[2] Andrade , K. E. C. , E. M. Tan, E . K. L. Chan : Immunocytochem­ical analysis of the coiled body in the cell cycle and during cell proliferation. Proc. Natl. Acad. Sci. USA 90,1947-1951 (1993).

[3] Chan , E. K. L., S. Takano, L. E. Andrade , J. C. Hamel, A. G. Matera : Structure, expression and chromosomal localization of human p80-coilin gene . Nucleic Acids Res. 22,4462-4469 (1994).

[4) Gautier, T. , C. Dauphin-Villemant , C. Andre, C. Masson, J . Arnoult , D. Hernandez-Verdun: Identification and characteriza­tion of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis. Exp. Cell Res. 200,5-15 (1992) .

[5] Gautier, T., M. Robert-Nicoud, M.-N. Guilly, D. Hernandez­Verdun : Relocation of nucleolar proteins around chromosomes at mitosis: A study by confocal laser scanning microscopy. I . Cell Sci . 102,729-737 (1992) .

[6] Gerdes, J., L. Li , C. Schlueter, M. Duchrow, C. Wohlenberg, C. Gerlach, I. Stahmer, S. Kloth, E. Brandt, H.-G. Flad: Immu­nobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by mono­clonal antibody Ki-67 . Am. I. Pathol. 138, 867-873 (1991) .

[7] Gong, T. W, A. D. Hegeman, J. J . Shin , K. H. Lindberg , K. F. Barald , M.l. Lomax : Novel genes expressed in the chick otocyst during development : identification using differential display of RNA. Int. J. Neurosci. 15,585-594 (1997) .

[8] Hernandez-Verdun, D. : The nucleolus today. J. Cell Sci . 99, 465-471 (1991) .

a-fibrillarin Hoechst

immunofluorescence microscopy experiments . The secondary anti­bodies were a fluorescein isothiocyanate-conjugated swine anti-rabbit IgG and a rhodamine-conjugated goat anti-mouse IgG. All meiotic cells displayed similar staining patterns and one typical example is shown (the arrow points to a labelled nucleolar region) . Bar, 30 !lm.

[9] Hu , c. C., S. A. Ghabrial: The conserved hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. J. Virol. Methods 55, 367-379 (1995) .

[10) Hultman, T., S. Bergh, T. Moks, M. Uhlen: Bidirectional solid­phase sequencing of in vitro-amplified plasmid DNA. BioTech­niques 10,84-93 (1991).

[11] Hbbg, C.: Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy. Nucleic Acids Res. 19,6123-6127 (1991) .

[12) Laemrnli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970).

[13) Lamond, A.I., M. Carmo-Fonseca: The coiled body. Trends Cell BioI. 3, 198-204 (1993).

(14) Larsson , M. , E. Brundell , L. Nordfors , C. Hoog, M. Uhlen , S. Stahl : A general bacterial expression system for functional analy­sis of cDNA-encoded proteins. Protein Expr. Purif. 7, 447-457 (1996).

[15] Liu, J. G., L. Yuan , E. Brundell, B. Bjorkroth, B. Daneholt, C. Hoog: Localization of the N-terminus of SCPl to the central element of the synaptonemal complex and evidence for direct interactions between the N-termini of SCPl molecules organized head-to-head. Exp . Cell Res. 226, 11-19 (1996).

[16] Luji, S., N. Zumei , Z . Shi, W. Ge , Y. Yang: Involvement of a nucleolar component, perichromonucleolin, in the condensation and decondensation of chromosomes. Proc. Natl. Acad . Sci. USA 84, 7953-7956 (1987).

[17) Meier, U. T. , G . Blobel : Noppl40 shuttles on tracks between nucleolus and cytoplasm. Cell 70, 127-138 (1992) .

[18] Meier, U. T., G. Blobel: NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J . Cell BioI. 6, 1505-1514 (1994) .

[19] Ochs , R . L. , M. A. Lischwe , W H. Spohn, H. Busch: Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. BioI. Cell 54,123-134 (1985).

[20] Ochs, R. L., T. W Stein, E. K. L. Chan, M. Ruutu, E. M. Tan: cDNA cloning and characterization of a novel nucleolar protein. Mol. BioI. Cell 7,1015-1024 (1996) .

[21) Raska, I., L. E . C. Andrade, R . L. Ochs, E. K. L. Chan , c.­M. Chang, E. M. Tan : Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies . Exp. Cell Res. 195,27-37 (1991).

[22] Rattner, J. B.: Integrating chromosome structure with function. Chromosoma 101, 259-264 (1992) .

[23] Scheer, U., D. Weisenberger: The nucleolus. Curro Opin . Cell BioI. 6,354-359 (1994).

EJCB Identification of a novel nucleolar protein 389

spermatocyte

Fig. 6. The Tsg1l8 protein associates with the nucleoli in meiotic germ cells . Pachytene spermatocytes were isolated from mouse testis, centrifuged onto a glass slide using a cytospin centrifuge and fixed in methanol-acetone. The cells were triple stained with the anti-Tsg118 antiserum, an anti-fibrillarin antiserum and Hoechst 33258 in indirect

the condensation/decondensation process that mitotic chro­mosomes, undergo, or they could protect the outer surface of mitotic chromosomes. The excess of positively charged amino acids in the Tsg118 protein could be important in mediating physical interactions with the mitotic chromosomes.

Acknowledgements. We thank Katarina Gell and Li Yuan for technical assistance and Mathias Uhlen for valuable discussions. This work was supported by the Swedish Natural Science Research Council , the Swedish Research Council for Engineering Sciences, Erik-Philip Sorensen Foundation , Nilsson-Ehle-Funds , the European Community (The BIOTECH Programme, BI04 CT60183) and the Karolinska Institutet.

References

[1] Andrade , L. E. c., E. K. L. Chan, 1. Raska, C. L. Peebles, G. Roos, E . M. Tan: Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J. Exp. Med . 173, 1407-1419 (1991) .

[2] Andrade , K. E. C. , E. M. Tan, E . K. L. Chan : Immunocytochem­ical analysis of the coiled body in the cell cycle and during cell proliferation. Proc. Natl. Acad. Sci. USA 90,1947-1951 (1993).

[3] Chan , E. K. L., S. Takano, L. E. Andrade , J. C. Hamel, A. G. Matera : Structure, expression and chromosomal localization of human p80-coilin gene . Nucleic Acids Res. 22,4462-4469 (1994).

[4) Gautier, T. , C. Dauphin-Villemant , C. Andre, C. Masson, J . Arnoult , D. Hernandez-Verdun: Identification and characteriza­tion of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis. Exp. Cell Res. 200,5-15 (1992) .

[5] Gautier, T., M. Robert-Nicoud, M.-N. Guilly, D. Hernandez­Verdun : Relocation of nucleolar proteins around chromosomes at mitosis: A study by confocal laser scanning microscopy. I . Cell Sci . 102,729-737 (1992) .

[6] Gerdes, J., L. Li , C. Schlueter, M. Duchrow, C. Wohlenberg, C. Gerlach, I. Stahmer, S. Kloth, E. Brandt, H.-G. Flad: Immu­nobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by mono­clonal antibody Ki-67 . Am. I. Pathol. 138, 867-873 (1991) .

[7] Gong, T. W, A. D. Hegeman, J. J . Shin , K. H. Lindberg , K. F. Barald , M.l. Lomax : Novel genes expressed in the chick otocyst during development : identification using differential display of RNA. Int. J. Neurosci. 15,585-594 (1997) .

[8] Hernandez-Verdun, D. : The nucleolus today. J. Cell Sci . 99, 465-471 (1991) .

a-fibrillarin Hoechst

immunofluorescence microscopy experiments . The secondary anti­bodies were a fluorescein isothiocyanate-conjugated swine anti-rabbit IgG and a rhodamine-conjugated goat anti-mouse IgG. All meiotic cells displayed similar staining patterns and one typical example is shown (the arrow points to a labelled nucleolar region) . Bar, 30 !lm.

[9] Hu , c. C., S. A. Ghabrial: The conserved hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. J. Virol. Methods 55, 367-379 (1995) .

[10) Hultman, T., S. Bergh, T. Moks, M. Uhlen: Bidirectional solid­phase sequencing of in vitro-amplified plasmid DNA. BioTech­niques 10,84-93 (1991).

[11] Hbbg, C.: Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy. Nucleic Acids Res. 19,6123-6127 (1991) .

[12) Laemrnli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970).

[13) Lamond, A.I., M. Carmo-Fonseca: The coiled body. Trends Cell BioI. 3, 198-204 (1993).

(14) Larsson , M. , E. Brundell , L. Nordfors , C. Hoog, M. Uhlen , S. Stahl : A general bacterial expression system for functional analy­sis of cDNA-encoded proteins. Protein Expr. Purif. 7, 447-457 (1996).

[15] Liu, J. G., L. Yuan , E. Brundell, B. Bjorkroth, B. Daneholt, C. Hoog: Localization of the N-terminus of SCPl to the central element of the synaptonemal complex and evidence for direct interactions between the N-termini of SCPl molecules organized head-to-head. Exp . Cell Res. 226, 11-19 (1996).

[16] Luji, S., N. Zumei , Z . Shi, W. Ge , Y. Yang: Involvement of a nucleolar component, perichromonucleolin, in the condensation and decondensation of chromosomes. Proc. Natl. Acad . Sci. USA 84, 7953-7956 (1987).

[17) Meier, U. T. , G . Blobel : Noppl40 shuttles on tracks between nucleolus and cytoplasm. Cell 70, 127-138 (1992) .

[18] Meier, U. T., G. Blobel: NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J . Cell BioI. 6, 1505-1514 (1994) .

[19] Ochs , R . L. , M. A. Lischwe , W H. Spohn, H. Busch: Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. BioI. Cell 54,123-134 (1985).

[20] Ochs, R. L., T. W Stein, E. K. L. Chan, M. Ruutu, E. M. Tan: cDNA cloning and characterization of a novel nucleolar protein. Mol. BioI. Cell 7,1015-1024 (1996) .

[21) Raska, I., L. E . C. Andrade, R . L. Ochs, E. K. L. Chan , c.­M. Chang, E. M. Tan : Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies . Exp. Cell Res. 195,27-37 (1991).

[22] Rattner, J. B.: Integrating chromosome structure with function. Chromosoma 101, 259-264 (1992) .

[23] Scheer, U., D. Weisenberger: The nucleolus. Curro Opin . Cell BioI. 6,354-359 (1994).

390 M. Larsson, E. Brundell, P.-M. Jorgensen et 01.

[24] Schliiter, c., M. Duchrow, C. Wohlenberg, M. H. G. Becker, G. Key, H.-D. Flad, J. Gerdes: The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J. Cell BioI. 123,513-522 (1993).

[25] Schmidt-Zachmann, M. S., B. Hiigle-Dorr, W. W. Franke: A con­stitutive nucleolar protein identified as a member of the nucleo­plasmin family. EMBO J. 7, 1881-1890 (1987).

[26] Starborg, M., E. Brundell, K. Gell, C. Hoog: The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and hetero­chromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progres­sion. J. Cell Sci. 109, 143-153 (1996).

[27] Starborg, M., E. Brundell, K. Gell, C. Larsson, 1. White, B. Daneholt, C. Hoog: A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initi­ation of DNA replication. J. Cell Sci. 108, 927-934.

",. uc.

[28] Starborg, M., E. Brundell, C. Hoog: Analysis of the expression of a large number of novel genes isolated from mouse prepubertal testis. Mol. Reprod. Dev. 33, 243-251 (1992).

[29] Verheijen, R., H. J. H. Kuijpers, R. O. Schlingemann, A. L. M. Boehmer, R. vanDriel, G. J. Brakenhoff, F. C. S. Ramaekers: Ki-67 detects a nuclear matrix-associated proliferation-related anti­gen. 1. Intracellular localization during interphase. J. Cell Sci. 92, 123-130 (1989).

[30] Verheijen, R, H. J. H. Kuijpers, R. vanDriel, J. L. M. Beck, J. H. van Dierendonck, G. J. Brakenhoff, F. C. S. Ramaekers: Ki-67 detects a nuclear matrix-associated proliferation-related anti­gen II. Localization in mitotic cells and association with chromo­somes. J. Cell Sci. 92, 531-540 (1989).

390 M. Larsson, E. Brundell, P.-M. Jorgensen et 01.

[24] Schliiter, c., M. Duchrow, C. Wohlenberg, M. H. G. Becker, G. Key, H.-D. Flad, J. Gerdes: The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J. Cell BioI. 123,513-522 (1993).

[25] Schmidt-Zachmann, M. S., B. Hiigle-Dorr, W. W. Franke: A con­stitutive nucleolar protein identified as a member of the nucleo­plasmin family. EMBO J. 7, 1881-1890 (1987).

[26] Starborg, M., E. Brundell, K. Gell, C. Hoog: The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and hetero­chromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progres­sion. J. Cell Sci. 109, 143-153 (1996).

[27] Starborg, M., E. Brundell, K. Gell, C. Larsson, 1. White, B. Daneholt, C. Hoog: A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initi­ation of DNA replication. J. Cell Sci. 108, 927-934.

",. uc.

[28] Starborg, M., E. Brundell, C. Hoog: Analysis of the expression of a large number of novel genes isolated from mouse prepubertal testis. Mol. Reprod. Dev. 33, 243-251 (1992).

[29] Verheijen, R., H. J. H. Kuijpers, R. O. Schlingemann, A. L. M. Boehmer, R. vanDriel, G. J. Brakenhoff, F. C. S. Ramaekers: Ki-67 detects a nuclear matrix-associated proliferation-related anti­gen. 1. Intracellular localization during interphase. J. Cell Sci. 92, 123-130 (1989).

[30] Verheijen, R, H. J. H. Kuijpers, R. vanDriel, J. L. M. Beck, J. H. van Dierendonck, G. J. Brakenhoff, F. C. S. Ramaekers: Ki-67 detects a nuclear matrix-associated proliferation-related anti­gen II. Localization in mitotic cells and association with chromo­somes. J. Cell Sci. 92, 531-540 (1989).