Centromeric Protein B Null Mice Are Viable with No Apparent Abnormalities

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DEVELOPMENTAL BIOLOGY 201, 135–143 (1998) ARTICLE NO. DB989005 Centromeric Protein B Null Mice Are Viable with No Apparent Abnormalities

Ana V. Perez-Castro,* Fay L. Shamanski,† Juanito J. Meneses,† TyAnna L. Lovato,* Kathryn G. Vogel,* Robert K. Moyzis,‡ and Roger Pedersen† *Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131; †Reproductive Genetics Unit, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California at San Francisco, San Francisco, California 94143; and ‡Department of Biological Chemistry, University of California at Irvine, Irvine, California 92697

The centromere protein B (CENP-B) is a centromeric DNA/binding protein. It recognizes a 17-bp sequence motif called the CENP-B box, which is found in the centromeric region of most chromosomes. It binds DNA through its amino terminus and dimerizes through its carboxy terminus. CENP-B protein has been proposed to perform a vital role in organizing chromatin structures at centromeres. However, other evidence does not agree with this view. For example, CENP-B is found at inactive centromeres on stable dicentric chromosomes, and also mitotically stable chromosomes lacking ␣-satellite DNA have been reported. To address the biological function of CENP-B, we generated mouse null mutants of CENP-B by homologous recombination. Mice lacking CENP-B were viable and fertile, indicating that mice without CENP-B undergo normal somatic and germline development. Thus, both mitosis and meiosis are able to proceed normally in the absence of CENP-B. © 1998 Academic Press Key Words: centromere; CENP-B; gene targeting; mitosis; meiosis.

INTRODUCTION amino terminus to a 17-bp motif (CENP-B box) in the human centromeric ␣-satellite DNA (Pluta et al., 1992; The centromere plays an important role in chromosome Muro et al., 1992). Immunoelectron microscopy studies segregation. This is the region of the chromosome where show that CENP-B is distributed throughout the hetero- the kinetochore is formed, allowing the binding of the chromatin beneath the kinetochore (Cooke et al., 1990). spindle microtubules, which are responsible for the separa- The mouse CENP-B gene is an intronless single-copy tion of the sister chromatids. In mammals, this centromeric gene (Sullivan and Glass, 1991) highly conserved in its region is composed of different amounts of nontranscribed DNA and protein sequences among species such as human, repetitive sequences, classically called satellite DNA (Par- African green monkey, mouse, and hamster (Sullivan and due and Gall, 1970; Grady et al., 1992; Pluta et al., 1995). Glass, 1991; Yoda et al., 1996; Bejarano and Valdivia, 1996). Centromeric proteins (CENP) were initially discovered The CENP-B box is the only apparent sequence conserved with the aid of autoantibodies from patients with calcino- in the satellite DNA of mammals such as human, Mus sis, Raynaud’s phenomenon, esophageal dysmotility, musculus, Mus caroli, tree shrews, giant panda, and diverse sclerodactyly, telangiectasia (CREST) or with scleroderma primates (Masumoto et al., 1989a; Muro et al., 1992; Pietras (Earnshaw and Rothﬁeld, 1985; Valdivia and Brinkley, et al., 1983; Kipling et al., 1995; Haaf and Ward, 1995; Wu 1985). Proteins recognized by the autoantibodies CENP-A et al., 1990; Haaf et al., 1995). This conservation suggests through -C have been localized to the centromere by that CENP-B plays an important role in centromere func- immunoelectron microscopy (Cooke et al., 1990). CENP-B tion. Recently, a CENP-B-related centromere DNA-binding is considered the major centromere antigen, since antibod- protein was reported in Schizosaccharomyces pombe, and ies to it are present in the highest titers in all patients. overexpression of this protein affects mitotic chromosome CENP-B is an 80-kDa protein which binds through its stability. In addition, deletion of this yeast homolog results

0012-1606/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. 135 136 Perez-Castro et al. in meiotic dysfunction and chromosome loss (Halverson et viability and fertility. Genotyping was done by PCR of genomic al., 1997). Because of its association to the ␣-satellite DNA isolated from tails with primers as shown in Fig. 1A: a, heterochromatin and its dimerization domain at the car- 5Ј-CCACTTGTGTAGCGCCAAGTGCCA-3Ј;b,5Ј-CCTTCGAG- boxy terminal, CENP-B has been proposed to be important TTGGCAGTGTAGTCGC-3Ј; and c, 5Ј-CGACTTTCT- in organizing specific centromeric structures (Pluta et al., GTCGGACCAGGCCTC-3Ј. PCR amplification was carried out for 30 cycles with denaturation at 94°C for 30 s, annealing at 60°C 1992; Kitagawa et al., 1995). Also, certain IgG preparations for 30 s, and elongation at 72°C for 30 s. Southern blots were done of CREST serum that affect kinetochore function and from genomic tail DNA digested with SpeI and fractionated on a morphogenesis appeared to recognize only CENP-B in im- 0.8% agarose gel. The gel was transferred onto a Hybondϩ (Amer- munoblots of chromosomal proteins (Bernat et al., 1991). sham) membrane and probed. However, other evidence is not consistent with this view. For example, the Y chromosome in humans does not bind Tissue Culture CENP-B (Pluta et al., 1995) and mitotically stable chromosomes lacking ␣-satellite DNA have been reported (Maio et JM-1 ES cells were cultured and electroporated as previously al., 1981; Heller et al., 1996). To determine if CENP-B is described (Qui et al., 1995). CENP-B mutant and normal primary essential in mammalian centromere function, we have cell fibroblasts were freshly isolated from tissue that was minced in 2.5 mg/ml collagenase in CO -independent medium (Gibco, Cat. generated mutant mice carrying a null mutation of the 2 No. 18045-088) supplemented with 2% BSA. Minced tissue was CENP-B gene. incubated in a shaker at 37°C for 30 min, then aspirated through an 18-gauge needle and incubated for another 30 min. Cells were aspirated again through an 18-gauge needle and then diluted and MATERIALS AND METHODS washed two times. Cells were resuspended in DMEM complete medium and transferred to a 25- or 75-cm2 flask, depending on the Isolation and Characterization of the CENP-B size (age) of the specimen. Cells were grown for 5 to 8 days before Mouse Genomic DNA their first passage.

To generate a probe for the CENP-B gene, an 11.5-day pc embryonic mouse cDNA library (Clontech ML1027a) was screened Tubulin Staining by PCR, with primers CENPB7 5 -GCTGCTGGACAT- Ј Cells growing on glass coverslips were washed with 1ϫ PBS and AGCTGTACC-3Ј and CENPB8 5Ј-CAGATTGGAGGTG- fixed for 30 min in 600 mM Pipes, pH 6.9, 25 mM Hepes, 10 mM CCTGCTGT designed to produce a 626-bp fragment. PCR ampli- EGTA, and 2 mM MgCl containing 10% formaldehyde and 1% fication was carried out for 40 cycles, with denaturation at 94°C for 2 glutaraldehyde. The cells were washed 3 ϫ 5 min with PBS, and 1 min, annealing at 58°C for 1 min, and elongation at 72°C for 3 free aldehydes were blocked with 100 mM sodium borohydride in min. The amplified fragment was directly sequenced by cycle PBS for 20 min. Cells were then washed 3 ϫ 5 min and blocked for sequencing in an ABI 373A automatic DNA sequencer (Applied 30 min with 3% BSA in TST (Tris–HCl 10 mM, NaCl 0.15 M, and Biosystems, Inc., Foster City, CA) according to the protocols Tween 20 0.2%). After being washed with TST the cells were provided by the manufacturer, to verify that it was derived from the incubated with a 1/200 dilution of the anti-␤-tubulin antibody CENP-B gene. The PCR-generated probe was used to screen an (clone TUB 2.1; Sigma, T-4026) for 1 h, rinsed with TST, and then sCos-1 genomic library derived from a 129 strain embryonic stem incubated with 1/128 dilution of an anti-mouse IgG FITC- cell line (kindly provided by Dr. John Mudgett). conjugated antibody (Sigma F5262) for 1 h. Cells were washed again and mounted with Vectashield. Targeted Deletion of the CENP-B Gene in ES Cells To construct the targeting vector a 7.3-kb NotI–SalI genomic Testis Histology fragment was first subcloned into the O18S1 vector, which is a Testes samples from F3 homozygous mutants (Ϫ/Ϫ), heterozy- modification of psP73 vector (Promega). O18S1 has a NotI linker at gotes (ϩ/Ϫ), and wild-type (ϩ/ϩ) 17-week-old mice were fixed in the EcoRV site and an SfiI linker at the PvuII site. The resulting Bouins overnight at room temperature (RT) with gentle rotation. subclone was called V3. An AscI–AscI 660-bp fragment was deleted The tissue was washed several times in PBS and stored at 4°C in ϩ and a PGK (phosphoglycerate kinase)–neo poly(A) cassette was 70% ethanol. The tissue was dehydrated through a series of inserted. The resulting clone containing the neo cassette was called alcohols and then embedded in JB-4 ethyl methacrylate embedding A183. A PGK–TK cassette was inserted at the 5Ј end of A183 and medium (Polysciences). Sections (5 ␮m) were cut on a Sorvall JB4A the resulting vector, CENPB1KO, was electroporated into JM-1 ES microtome. Sections were baked for1hat75°C, stained with cells as described (Qui et al., 1995). hematoxylin Gill No. 3 and 0.6% eosin Y in distilled water, and mounted with Permount. Animals, Breeding, and Genotyping Sperm Counts C57BL/6J blastocysts (The Jackson Laboratory) were used as recipients for ES cell injection. Male chimeras generated from Male mice were sacrificed by cervical dislocation and the testes blastocyst injection were mated to Swiss Black females (Tac:N: and epididymus removed. Surrounding fat was dissected away and NIHS-BCfBR; Taconic). Null homozygous mice were generated the caudal epididymus was carefully removed. The tissue was then from heterozygous intercrosses. Crosses between F1,F2,F3, and F4 finely minced in 1 ml of PBS and allowed to incubate at RT for 15 generations of different genotypes were carefully monitored for min. The sample was then filtered through a 70-␮m nylon mesh to

remove tissue debris and somatic cells. The resulting sperm sample was diluted in 5% formaldehyde in PBS and counted in a hemacy- tometer. Two counts were done for each sample and the average was reported. Counts are given as sperm/epididymus.

RNA Isolation and Northern Blot Analysis Total RNA was isolated from lung fibroblasts with TRI Reagent (Molecular Research Center, Inc.) according to the manufacturer’s specifications. Poly(A)ϩ RNA was isolated using the 5 Prime–3 Prime, Inc., minioligo(dT) kit, following the manufacturer’s specifications. One microgram of purified poly(A)ϩ mRNA was fractionated on a 1.5% formaldehyde–agarose gel, transferred onto Gene- screen, and hybridized at 42°C with the CENP-B-radiolabeled probe, following the manufacturer’s specifications. After being washed in 0.1ϫ SSC/0.1% SDS at 60°C for 30 min, the filter membrane was exposed to X-ray film, then rehybridized with a full-length cDNA G3PDH probe (Stratagene) and washed in 0.2ϫ SSPE/2% SDS at 65°C for 45 min, and then exposed to X-ray film.

Preparation of Primary Mouse Fibroblasts Nuclei and Nuclear Extracts Nuclear and cytoplasmic fractions were isolated according to Masumoto et al. (1989b). The nuclear pellet was resuspended in extraction buffer (20 mM Hepes, pH 8.0, 0.5 mM EDTA, 0.1 mM PMSF, 0.5 ␮g/ml pepstatin A, 0.5 M NaCl, and 15% glycerol) and sonicated 4ϫ for 10 s. FIG. 1. Disruption of the CENP-B gene. (A) Organization of the wild-type CENP-B mouse allele, the targeting vector, and the mutated allele. Solid black box represents the CENP-B coding SDS–Page and Immunoblotting region. Restriction sites are as follows: N, NotI; S, SacII; X, XhoI; C, Proteins were fractionated in a 12% SDS–PAGE gel and trans- ClaI. Probes 1 and 2 were used for Northern blot analysis and probe ferred to nitrocellulose. The membrane was stained with Ponceau 3 was used for Southern blot analysis. (B) Southern blot analysis of S and then blocked with 3% dry milk in Block Buffer (10 mM the progeny from a CENP-B heterozygous (ϩ/Ϫ) intercross. The Tris–HCl, pH 7.4, 0.15 M NaCl, 0.2% Tween 20) for 1 h. The blot 14-kb band represents the wild-type allele and the 8.4-kb band was incubated for 1 h with a rabbit anti-CENP-B antibody 1:1000 represents the mutated allele. (C) PCR analysis of genomic DNA of dilution (kindly provided by Dr. William Earnshaw) in Block buffer progeny of the different crosses. Primers a and b were used for the with 1% dry milk. Five washes of 2 min each were done with Block detection of the wild-type allele, yielding a product of 195 bp. buffer and then the blot was incubated with an HRP-conjugated Primers b and c were used for the detection of the mutated allele, goat anti-rabbit antibody, 1:2000 dilution (Sigma) in block buffer yielding a product of 240 bp. with 1% dry milk for 1 h. The blot was washed 5 ϫ 2 min and developed with ECL reagents (Amersham).

Statistical Analysis subcloned (Fig. 1A). A replacement vector was con- structed by deleting a 660-bp fragment that contained the Probability (P) values were obtained using ␹2 analysis (Sigmastat start of translation and the basic–Helix I–(loop)–Helix II version 1.0) for breeding analysis in two-by-two contingency tables with 1 degree of freedom. For body weight, testes weight, and motif that binds the CENP-B box, and in its place a ϩ sperm counts the nonparametric t test was used, and P values were PGK-neo poly(A) cassette was inserted in the opposite obtained using the unpaired Mann–Whitney two-tailed test (Sig- orientation of transcription to the CENP-B gene. In mastat version 1.0). addition, a PGK-TK poly(A)ϩ cassette was inserted at the 5Ј end of the construct, also in the opposite orientation of transcription to the CENP-B gene. The resulting CENP- RESULTS B1KO clone (Fig. 1B) was linearized with SfiI, electroporated into 129Sv/J ES cells, and subjected to G418 and Targeting of the CENP-B Gene in ES Cells FIAU selection. Nine correctly targeted ES cell clones of Using a PCR-amplified mouse CENP-B probe, 8 300 doubly resistant clones were identified by Southern genomic CENP-B clones were obtained from an sCos-1/ blot analysis. The ES cell clones were expanded and 129 embryonic stem cell line library. Clone 7, which microinjected into C57BL/6J blastocysts to generate chi- contained a Ͼ35-kb insert, was further characterized and meric mice, three of which gave germline transmission.

TABLE 1

Genotypes of Offspring from CENP-B F1 Heterozygous Intercrosses

Line ϩ/ϩϩ/ϪϪ/Ϫ

41 12 21 10 55 13 28 16 59 11 19 8 Total 36 68 34

FIG. 3. Immunodetection of the CENP-B protein. Extracts from primary lung fibroblasts of wild-type (ϩ/ϩ) and homozygous mutant (Ϫ/Ϫ) mice were fractionated into cytoplasmic (C) and nuclear Generation of CENP-B-Deficient Mice (N) fractions and separated by SDS/PAGE. The Western blot was developed with an anti-CENP-B antibody. Arrowhead indicates the Male chimeras (41, 55, and 59) generated from three CENP-B protein in the nuclear fraction of the wild type (ϩ/ϩ) cells. independent ES cell lines carrying the mutation were mated KD, MW markers in kilodaltons. with Swiss Black females. Germline transmission was first followed by coat color and later confirmed by PCR and Southern blot analysis (Figs. 1B and 1C). Male and female heterozygous mice were obtained (typically F1). These mice gous mutant (Ϫ/Ϫ) cells appeared normal and were no appeared normal and were fertile. The heterozygous mice different from those in the heterozygous (ϩ/Ϫ) or wild-type were mated to obtain F2 homozygous mutants. The geno- (ϩ/ϩ) cells (Fig. 4). Analysis of microtubules in primary type was determined for each pup in 17 F1 matings of the cultures of fibroblasts isolated from homozygous mutant three lines. One-quarter of the offspring were homozygous (Ϫ/Ϫ) mice from F3 and F4 generations showed no spindle mutants (Table 1). CENP-B null mice did not show any abnormalities during cell division (Fig. 4). excessive lethality and displayed no obvious abnormal phenotype. To verify that no CENP-B transcript was being made, poly(A)ϩ mRNA was isolated from wild-type (ϩ/ϩ), Breeding Analysis of CENP-B-Deficient Mice heterozygous (ϩ/Ϫ), and homozygous mutant (Ϫ/Ϫ) ani- Despite the apparently normal somatic development of mals and Northern blot analysis was performed using two homozygous mutants, the CENP-B protein could play a role different probes. The first probe spanned the deletion and in meiosis that would impair fertility. To investigate this the second probe spanned 626 bp of the 3Ј end of the gene possibility several intercrosses between CENP-B heterozy-

(Fig. 1A). No CENP-B message was detected in the homozygous (ϩ/Ϫ) and homozygous mutant (Ϫ/Ϫ) mice of both F2 gous mutant (Ϫ/Ϫ) mice using either probe 1 (Fig. 2) or and F3 parental generations were carried out. The survival, probe 2 (data not shown). Primary ﬁbroblasts were cultured litter size, and genotype for each litter were monitored. from wild-type (ϩ/ϩ), heterozygous (ϩ/Ϫ), and homozygous There was no signiﬁcant difference in litter size in any of mutant (Ϫ/Ϫ) mice. Western blot analysis failed to detect the F2 or F3 crosses (Table 2). Sections of testes from a any CENP-B protein in either cytoplasmic or nuclear prepa- homozygous mutant (Ϫ/Ϫ)F3male were examined by light rations of the cultured cells from the homozygous mutant microscopy, but no differences in the stages of spermiogen- (Ϫ/Ϫ) mice (Fig. 3). When stained with an anti-␤-tubulin esis were seen compared to testis from heterozygous (ϩ/Ϫ) antibody, microtubules in the mitotic spindle of homozy- siblings (Fig. ) or wild-type (ϩ/ϩ) mouse (data not shown).

The 21-day survival of the progeny from matings of F2 homozygous mutant mice (Ϫ/Ϫ) was not signiﬁcantly dif-

ferent from F2 crosses involving heterozygous (ϩ/Ϫ) mice (Table 3, B, C, D, E, and F). Interestingly, the overall survival of offspring from mat-

ings of F3 homozygous mutant mice was lower than for similar crosses in the F2 generation (Table 3, G versus A, P Ͻ 0.01). This could indicate an increased loss of homozy-

gous mice in the F3 generation. However, a detailed comparison of the patterns of F2 and F3 losses did not appear to support such a view. Thirty-one litters were produced FIG. 2. Northern blot analysis of lung ﬁbroblasts from CENP-B crossing F3 homozygous mutant mice. The litter size was mutant mice. The blot was ﬁrst probed for CENP-B and then normal (mean 6.3 Ϯ 2.0, range 3–13, Table 2). All of the reprobed for G3PDH. The size of the G3PDH band is 1.27 kb and pups died in 5 of these litters and between 2 and 5 pups died the size of the CENP-B band is 2.9 kb. The locations of the 18S and in 6 other litters. This means that there was complete 28S ribosomal RNA are indicated. survival in 65% of these 31 litters. By way of comparison, in

FIG. 4. Immunostaining of microtubules in mouse primary ﬁbroblasts. Microtubules from primary lung ﬁbroblasts of wild type (ϩ/ϩ) and

F3 homozygous mutant (Ϫ/Ϫ) mice were stained with an anti-␤-tubulin antibody. The microtubules appear as ﬂuorescent green.

the case of F2 homozygous mutant crosses, there was compared to the cross of an F3 male homozygous mutant complete survival in only 50% of the 16 litters, but death of with an F3 female heterozygous (Table 3, K) indicates that all pups occurred in only 1 litter. There was complete F4 homozygous mice were not dying preferentially. Taking survival in 77% of 22 litters from the F2 heterozygous all the evidence into consideration, we conclude that crosses and death of all pups in 3 litters. Thus, the lower CENP-B null mice are normal in terms of both survival and overall survival in F3 compared with F2 homozygous mat- fertility. ings appeared to result from sporadic rather than systematic losses, with a prevalent outcome of normal development. Interestingly, there was also lower survival of offspring in DISCUSSION matings in which only the F3 male was homozygous mu- From the results of this study we conclude that the tant and the F3 female was heterozygous (Table 3, K). In this particular case, all of the pups died in 3 of the 5 litters, CENP-B gene is not essential for viability and that its making it very unlikely that only the homozygous mutant absence does not affect fertility in the mouse. This conclu- progeny were dying. In addition, the fact that survival was sion is based on the following observations of CENP-B null mice. Northern and Western blot analysis of homozygous higher in the progeny of an F3 homozygous mutant cross mutant mice showed that neither the mRNA for CENP-B nor the protein itself was present. The ratio of genotypes

obtained from F1 heterozygous crosses was 1:2:1 (ϩ/ϩ:ϩ/Ϫ: TABLE 2 Ϫ/Ϫ), showing that there was no preferential absence of the Mean of Litter Sizes for Crosses of the Different Genotypes homozygous mutant gametes or progeny in such crosses. In and Generations addition, we found that litter size did not change in matings of homozygous mutant F2 and F3 animals, meaning that Parental Genotypes of Number of Litter size there were no abnormal deaths of the homozygous mutants Generation crosses litters (mean Ϯ SD) during embryonic development in later generations. Al- though there was somewhat lower survival among the F2 fϪ/ϪϫmϪ/Ϫ 16 7.0 Ϯ 2.0 progeny of F homozygous mutant crosses, and even a lower F2 fϩ/Ϫϫmϩ/Ϫ 22 7.6 Ϯ 2.4 3 F2 fϪ/Ϫϫmϩ/ϩ 8 6.0 Ϯ 2.6 survival in an F3 cross between a male homozygote and a F2 fϪ/Ϫϫmϩ/Ϫ 5 7.8 Ϯ 1.3 female heterozygote, this could not be clearly attributed to F2 fϩ/ϩϫmϪ/Ϫ 5 7.0 Ϯ 2.0 the homozygous mutant genotype. To date we have ob- F2 fϩ/ϪϫmϪ/Ϫ 12 8.4 Ϯ 2.5 served 15 litters of normal size and viability from crosses F fϪ/Ϫϫmϩ/Ϫ 31 6.3 Ϯ 2.0 3 between F4 homozygous mutants, suggesting that the F5 F3 fϪ/Ϫϫmϩ/ϩ 6 7.0 Ϯ 2.1 homozygous mutant mice are still viable. These ﬁndings F f / m / 3 8.3 2.3 3 Ϫ Ϫϫ ϩϪ Ϯ indicate that mitosis and meiosis in the mouse are able to F fϩ/ϩϫmϪ/Ϫ 5 7.8 Ϯ 1.6 3 proceed through several generations in the absence of the F fϩ/ϪϫmϪ/Ϫ 5 6.8 Ϯ 1.6 3 CENP-B protein.

TABLE 3 36 Ϯ 5.29 g (P ϭ 0.62). The testes from the same animals a 21-Day Survival in Crosses of F2 and F3 Generations were dissected and weighed, showing values of 0.187 Ϯ

0.039 g for the F3 homozygous (Ϫ/Ϫ) mutants at 16 weeks Parental and 0.201 Ϯ 0.01 g for their heterozygous (ϩ/Ϫ) and wild- generation and type (ϩ/ϩ) siblings (P ϭ 0.4, t test). For the mice between 22 Comparison genotype Live/total % Survival P value and 24 weeks of age, the mean testes weight for F3 homozy- (1) gous mutants (N ϭ 4) was 0.181 Ϯ 0.039 g and for the

AF2fϪ/ϪϫmϪ/Ϫ 103/114 90 heterozygous and wild -type siblings (N ϭ 3) was 0.22 Ϯ BF2fϩ/Ϫϫmϩ/Ϫ 153/169 91 n.s. 0.049 g (P ϭ 0.22). Sperm counts done on these same mice CF2fϪ/Ϫϫmϩ/ϩ 41/51 80 n.s. did not reveal any significant difference between null mice DFf/m / 38/39 97 n.s. 2ϪϪϫ ϩϪ and their control littermates (P ϭ 0.22 for 16 weeks and P ϭ EFf/m / 27/27 100 n.s. 2ϩϩϫ ϪϪ 0.4 for 22- to 24-week-old mice). Epidydimal sperm counts FF2fϩ/ϪϫmϪ/Ϫ 91/101 90 n.s. 6 (2) of 150 Ϯ 4.7 ϫ 10 for the 16-week-old F3 homozygous mutants (N ϭ 4) and of 108 Ϯ 2.6 ϫ 106 for the sibling GF3fϪ/ϪϫmϪ/Ϫ 142/190 75 controls (N 3) were obtained. Epidydimal sperm counts of HF3fϪ/Ϫϫmϩ/ϩ 41/42 97 Ͻ0.01 ϭ 6 IF3fϪ/Ϫϫmϩ/Ϫ 22/25 88 n.s. 8.85 Ϯ 1.1 ϫ 10 for 22- to 24-week-old homozygous 6 JF3fϩ/ϩϫmϪ/Ϫ 38/39 97 Ͻ0.01 mutant mice and of 12.5 Ϯ 4.5 ϫ 10 for the sibling controls KF3fϩ/ϪϫmϪ/Ϫ 10/34 30 Ͻ0.001 were obtained. Therefore, in our case the CENP-B homozygous mutants until at least the F generation also did not a 21-day survival of offspring from homozygous mutant (Ϫ/Ϫ) 3 2 show any significant difference from the controls in testis crosses of F2 (1) and F3 (2) generations were compared (by ␹ analysis) to homozygous mutant crosses, heterozygous crosses, or weight or sperm count. From the results of Hudson et al. crosses in which only the male or the female was a homozygous (1998) this discrepancy could be explained by differences in mutant. In (1) B through F are compared to A, and in (2) H to K are the strain of the mice used, as in some other null mutants compared to G. n.s., not statistical significant, P Ͼ 0.05. (e.g., EGF receptor, Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995). Among all of the centromeric proteins recognized by the CREST autoantibodies, CENP-B is the best characterized at Very recently, Hudson et al. (1998) also reported mice the nucleotide and protein levels (Earnshaw et al., 1987; lacking CENP-B, with essentially similar conclusions, Yoda et al., 1996). CENP-B dimerizes through its carboxy namely that mitosis and meiosis proceeded normally with- terminus domain, and it is associated through its amino out CENP-B. However, they in addition report lower body terminus to the CENP-B box, which is a degenerate motif weight, lower testis weight, and sperm content reduction in found in centromeric ␣-satellite sequences (Masumoto et their null mutants. In light of this finding we weighed F3 al., 1989a; Bernat et al., 1991; Pluta et al., 1992). These facts homozygous (Ϫ/Ϫ) mutant mice and compared their weight taken together led to the expectation of an important role to F3 heterozygous (ϩ/Ϫ) and wild-type (ϩ/ϩ) siblings. The for CENP-B in establishing the chromatin structure of the male mice analyzed were either 16 or 22–24 weeks old. In centromere (Masumoto et al., 1989b; Bernat et al., 1991; contrast to the findings of Hudson et al. (1998) we did not Pluta et al., 1992; Sugimoto et al., 1994). find any significant reduction in body weight in 16- or 22- to However, other evidence conflicts with a functional role for 24-week-old null mice. The body weight of our male CENP-B in mitosis. Most vertebrate species do not have a homozygous mutant mice (Ϫ/Ϫ) at 16 weeks (N ϭ 3) was CENP-B box in their centromeric DNA, and the human Y 29.3 Ϯ 5.79 g (mean Ϯ SD), not significantly different from chromosome ␣-satellite DNA array also lacks a CENP-B box that of the heterozygous (ϩ/Ϫ) and wild-type siblings (ϩ/ϩ) (Maio et al., 1981; Broccoli et al., 1990; Moens and Pearlman, (N ϭ 4) 34.5 Ϯ 2.279 g (P ϭ 0.4, nonparametric t test). At 1990; Grady et al., 1992; Goldberg et al., 1996). Moreover, 22–24 weeks male homozygous mutant (Ϫ/Ϫ) mice (N ϭ 4) functional, mitotically stable human chromosomes devoid of had a mean body weight of 32.5 Ϯ 5.09 g, which was not ␣-satellite DNA have been observed, and these centromeres significantly different from that of heterozygous (ϩ/Ϫ) and do not bind CENP-B (Heller et al., 1996; Du Sart et al., 1997). wild-type (ϩ/ϩ) siblings (N ϭ 3) with a mean body weight of Finally, in dicentric chromosomes CENP-B is found at both

FIG. 5. Hematoxylin- and eosin-stained plastic testes tissue sections from F3 homozygous mutant (Ϫ/Ϫ) and heterozygous (ϩ/Ϫ) CENP-B male mice (age 17 weeks). (A and B) Variation of the seminiferous tubule sperm content within each testis is apparent. Asterisks indicate tubules with very few mature sperm, and arrowheads indicate tubules filled with mature sperm. (C and D) Higher magnification of a tubule full of sperm. There was no apparent difference in the stages of sperm differentiation between homozygous mutant (Ϫ/Ϫ) CENP-B mice or heterozygous (ϩ/Ϫ) siblings. MS, mature sperm; PS, primary spermatocyte; RS, round spermatid. Magnification: (A,B) bar indicates 100 ␮m, (C, D) bar indicates 20 ␮m.

Copyright © 1998 by Academic Press. All rights of reproduction in any form reserved. 142 Perez-Castro et al. functional and nonfunctional centromeres (Earnshaw et al., Earnshaw, W. C., Ratrie, H., and Stetten, G. (1989). Visualization of 1989), suggesting that its presence is not sufficient for centro- centromere proteins CENP-B and CENP-C on stable dicentric meric function in mitosis. chromosome in cytological spreads. Chromosoma 98, 1–12. Alternative functions for this gene are suggested by the Goldberg, I. G., Sawhney, H., Pluta, A. F., Warburton, P. E., and recent finding, based on sequence similarity, that the Earnshaw, W. C. (1996). Surprising deficiency of CENP-B binding sites in African green monkey ␣-satellite DNA: Implications for CENP-B protein is related to the Tigger transposable ele- CENP-B function at centromeres. Mol. Cell. Biol. 16, 5156–5168. ment transposase (Smit and Riggs, 1996). It has been pro- Grady, D. L., Ratliff, R. L., Robinson, D. L., McCanlies, E. C., posed that CENP-B may be involved in the evolution of Meyne, J., and Moyzis, R. K. (1992). Highly conserved repetitive satellite DNA arrays by promoting nicking and homologous DNA sequences are present at human centromeres. Proc. Natl. recombination (Kipling and Warburton, 1997). Alterna- Acad. Sci. USA 89, 1695–1699. tively, it may be a DNA binding protein, serendipitously Haaf, T., and Ward, D. C. (1995). Rab1 orientation of CENP-B box recruited for chromosome condensation, whose function sequences in Tupaia belangeri fibroblasts. Cytogenet. Cell can be easily replaced by other proteins. Since CENP-B is Genet. 70, 258–262. not essential for mitosis or meiosis in mice, it will be Haaf, T., Mater, A. G., Wienberg, J., and Ward, D. C. (1995). interesting to mate the CENP-B null mice with null mu- Presence and abundance of CENP-B box sequences in great ape tants of other centromeric proteins to test further for subsets of primate-specific alpha satellite DNA. J. Mol. Evol. 41, function and redundancy of these proteins. 487–491. Halverson, D., Baum, M., Stryker, J., Carbon, J., and Clarke, L. (1997). A centromere DNA-binding protein from fission yeast affects chromosome segregation and has homology to human ACKNOWLEDGMENTS CENP-B. J. Cell Biol. 136, 487–500. Heller, R., Brown, K. E., Burgtorf, C., and Brown, W. R. (1996). We thank Dr. John Mudgett for kindly sharing with us his ES Mini-chromosomes derived from the human Y chromosome by genomic library, and Dr. William Earnshaw for kindly sharing his telomere directed chromosome breakage. Proc. Natl. Acad. Sci. anti-CENP-B antibody. We also thank Margaret Flannery for her USA 93, 7125–7130. technical assistance with the plastic tissue sections. This work was Hudson, D. F., Fowler, K. J., Earle, E., Saffery, R., Kalitsis, P., supported by DOE Grant DE-FG03-97ER62485 to R.K.M., NIH Trowell, H., Hill, J., Wreford, N. G., de Krester, D. 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