Proc. Natl. Acad. Sci. USA Vol. 94, pp. 3128–3133, April 1997 Genetics

Molecular characterization of TUB, TULP1, and TULP2, members of the novel tubby family and their possible relation to ocular diseases

MICHAEL A. NORTH*, JUERGEN K. NAGGERT†,YINGZHUO YAN*, KONRAD NOBEN-TRAUTH†, AND PATSY M. NISHINA†‡

*Sequana Therapeutics, Inc., La Jolla, CA 92037; and †The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609

Communicated by Elizabeth S. Russell, The Jackson Laboratory, Bar Harbor, ME, January 16, 1997 (received for review September 7, 1996)

ABSTRACT Tubby, an autosomal recessive mutation, sion no. U52433) and ET-3636.p01.d01 (nucleotides 1323– mapping to mouse 7, was recently found to be the 1421, 99 bp) by random hexamer priming, as described (6). result of a splicing defect in a novel gene with unknown These probes were used to screen Ϸ1.2 ϫ 106 plaque forming function. Database searches revealed that sequences corre- units of a human adult brain cDNA library in ␭gt11, plated sponding to the C terminus of the tub were highly according to the manufacturer’s instructions (CLONTECH). conserved across a number of species including humans, mice, Duplicate membrane filters were hybridized with labeled Caenorhabditis elegans, Arabidopsis, rice, and maize, and that probe in 10% dextran sulfate, 1% SDS, 1 M NaCl, 100 ␮g͞ml tub was a member of a gene family. We describe here, TUB, the of salmon testes DNA at 65ЊC for 18 hr. After hybridization, human homolog of mouse tub, and two newly characterized filters were washed at 65ЊCin2ϫSSC (1ϫ SSC ϭ 0.15 M family members, TULP1 for tubby like protein 1 and TULP2. sodium chloride͞0.015 M sodium citrate, pH 7)/0.1% SDS for These three family members, which differ in the N-terminal 45 min and twice in 0.2ϫ SSC/0.1% SDS for 45 min each and half of the protein, share 60–90% identity across positive plaques identified by autoradiography. Following their conserved C-terminal region and have distinct tissue plaque purification, cDNA inserts were PCR amplified using expression patterns. Alternatively spliced transcripts with 5؅ ␭gt11 primers (BRL) and directly cloned into pCR2.1 for variable sequences, three of which have been identified for the sequencing, according to the manufacturer’s instructions (In- tubby gene, may mediate tissue specific expression. We also vitrogen). Automated fluorescence sequencing was used to report that TUB, TULP1, and TULP2 map to human chromo- characterize the cDNA clones (Prism, Applied Biosystems). somes 11p15.4, 6p21.3, and 19q13.1, respectively. TULP1 and Cloning of TULP1 cDNA. Approximately 1 ϫ 106 plaque- TULP2 map within the minimal intervals identified for reti- forming units of human retinal cDNA ␭gt11 library (CLON- nitis pigmentosa 14 on chromosome 6p21.3 and cone-rod TECH) were hybridized with a 32P-labeled EcoRI͞SacII frag- dystrophy on chromosome 19q13.1. TULP1 and TULP2, which ment (1–962 bp) of I.M.A.G.E. EST clone 221670 (Research are expressed in the retina, make excellent candidates for Genetics, Huntsville, AL) at 65ЊC overnight. The membranes these ocular diseases as a mutation within the tub gene is were washed sequentially for 1 hr each with 2ϫ SSC/0.1% SDS known to lead to early progressive retinal degeneration. at 50ЊC, 1ϫ SSC/0.1% SDS at 50ЊC, and 0.5ϫ SSC/0.1% SDS at 60ЊC. Thirty positive plaques were purified and phage Mice homozygous for a splicing defect in the C terminus of positives of insert size Ͼ1 kb were digested with EcoRI and the tub gene are characterized by progressive retinal and subcloned into pUC9. One of these clones was a full-length cochlear degeneration (1, 2), maturity-onset obesity with cDNA containing the entire ORF. Independently, to isolate insulin resistance, and impaired glucose tolerance (3). We the flanking 5Ј sequences, nested oligonucleotide primer pairs recently identified tub as a novel gene with high expression AP1 and hMan2-R3 5Ј-AGCTCCTCGGAGCCCTAGC-3Ј in the retina, brain, and testis (4). Analysis of the tub gene (nucleotides 2017–2035 of the TULP1 sequence), as well as revealed sequence similarity to the p46 mouse cDNA (5) and AP2 and hMan2-R1 5Ј-ACATGCCTCGATCCATGCC-3Ј an integrated molecular analysis of genomes and their (nucleotides 979–997), were used in subsequent rounds of expression (I.M.A.G.E.) expressed sequence tag (EST) amplification of Marathon–Ready brain cDNA (CLON- clone from a human retinal cDNA library, suggesting that tub TECH), according to manufacturer’s protocol. Amplification is a member of a family of . To further characterize this products were gel purified (Qiagen, Chatsworth, CA) and family and explore the possibility that its members may be sequenced automatically (Prism, Applied Biosystems) or man- candidate genes for diseases that exhibit similar phenotypes ually by dideoxy cycle sequencing (Sequitherm, Epicentre as tubby, we obtained and characterized human full-length Technologies, Madison, WI). Alternately, gel purified prod- cDNAs for TUB, for the gene corresponding to the human ucts were subcloned into the TA-cloning vector according to EST, now referred to as TULP1 for tubby like protein 1, as the manufacturer’s instruction (Invitrogen) and plasmids were well as for a new member, TULP2. isolated by a standard protocol (6) prior to sequencing. Cloning of TULP2 cDNA. A PCR product amplified from MATERIALS AND METHODS a mouse testis cDNA library using primers MP46.1 5Ј- Cloning of TUB cDNA. 32P-labeled hybridization probes TCTACAGAGACAAACTATGCCC-3Ј and MP46.2 5Ј- GGAAATGTGCTACACCATC CTC-3 was used as a were prepared from two tub exon trap products, ET- Ј probe to screen 1 ϫ 106 plaque-forming units of human testis 3636.p01.a04 (nucleotides 1422–1593, 171 bp, GenBank acces- cDNA library in ␭DR2 (CLONTECH). Hybridization, washes, and radiography were carried out as described for The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: EST, expressed sequence tag. Data deposition: The sequences reported in this paper have been Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA deposited in the GenBank database (accession nos. U82467–U8469). 0027-8424͞97͞943128-6$2.00͞0 ‡To whom reprint requests should be addressed. e-mail: pmn@aretha. PNAS is available online at http:͞͞www.pnas.org. jax.org.

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FIG.1. (a) Comparison of nucleotide sequence and deduced amino acid sequence of the 5Ј splice variants of TUB and HSU54644. Break in ;Conserved identical amino acids ,ء .sequence denotes the intron͞exon junction. (b) Comparison of mouse tub and human TUB, TULP1, and TULP2 ~, conservative replacements. The alignments were produced with the computer program CLUSTAL W1.6 (13).

cloning of the TUB cDNAs. Thirty-four positive clones were amplified and sequenced from DNA of 10 unrelated indi- detected after overnight exposure and after tertiary screen- viduals using primers TULP2-F1 5Ј-ATCGTGGATC- ing, 28 positives were isolated and converted to plasmid CCAAACACC-3Ј and TULP2-R1 5Ј-GCTGGCAAGGG- DNA in pDR2. The C-terminal intron of TULP2 was PCR TATGGTATT-3Ј. Downloaded by guest on September 30, 2021 3130 Genetics: North et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Southern Blot Analysis. Southern blots of EcoRI-digested CAGGACACCTTTGCCTTCT-3Ј, hMan1-R2 5Ј-GC- genomic DNA from a number of animal species were obtained GATCTCCCTTCCTTCCT-3Ј), the 3Ј noncoding region for from CLONTECH. They were hybridized with a 32P-labeled TUB (hMan6.4-F1 5Ј-TGCCTGGGAATCCTGCTGC-3Ј and HindIII fragment (281–1833 bp) of TUB cDNA and a 32P- hMan6.4-R1 5Ј-TCCTAAGGGTCCTGCCACT-3Ј), and for labeled EcoRI͞BstXI TULP1 fragment (the first 365 bp of 5Ј TULP1 (hMan2-F7 5Ј-CGAAAACGGAGCAAGACAG-3Ј, end of clone 221670). Blots were washed in 2ϫ SSC/0.05% SDS hMan2-R7 5Ј-TATGAGGCTCTCCAGCGTC-3Ј), using the at room temperature two times for 10 min and at 60ЊC for 20 MACVECTOR computer program (Oxford). Primers for TULP2 min, then twice in 0.2ϫ SSC/0.1% SDS at 60ЊC for 20 min each. were constructed from an exon in the 3Ј portion of the gene Northern Blot Analysis. Human multiple tissue Northern (HP46.F1 5Ј-CCACTAAATGAACAGGAGTCGC-3Ј and blots (MTN I, II, and III; CLONTECH) were hybridized with HP46.R2 5Ј-TTGGAAGTTCTTCACCGAAGCC-3Ј). After the probes described above for Southern blot analyses, in 10ϫ confirming by sequencing that the appropriate product was Denhardt’s/2% SDS, 100 ␮g͞ml of sheared salmon sperm amplified, the retention patterns for each oligonucleotide pair DNA, and 50% formamide at 42ЊC for 18 hr, then washed in were obtained by PCR assay in the Stanford G3 Radiation Hybrid 2ϫ SSC/0.05% SDS at room temperature three times for 10 panel (9) as previously described (10). hMan1-F2͞R2 amplifies a min and in 0.1ϫ SSC/0.1% SDS at 50ЊC two times for 20 min. band of Ϸ480 bp, constant hamster specific bands were observed The same blots were hybridized with the 32P-labeled product at Ϸ150, 200, and 213 bp. hMan6.4-F1͞R1 amplifies a 221-bp of TULP2 (amplified using primers HP46.F1 5Ј-CCACTA- product with constant hamster DNA-specific bands at Ϸ290 and AATGAACAGGAGTCGC-3Ј and HP46.R1 5Ј-GAAACTG- 320 bp. The product of hMan2-F7͞R2 is 92 bp in length with a GACAAGCAGATGCTG-3Ј). The hybridization was done in constant hamster DNA amplified band at Ϸ230 bp. HP46.F1͞R2 ExpressHyb solution (CLONTECH) at 60ЊC for 2 hr, accord- amplifies a 162-bp fragment. Data entered into an online data- ing to the manufacturer’s instructions. Initially, the blots were base ([email protected]) was analyzed by RHMAP soft- washed three times in 2ϫ SSC/0.05% SDS at room tempera- ware developed by Boehnke et al. (11). Markers mapping in ture, followed by washing in 0.1ϫ SSC/0.1% SDS at 55ЊC for proximity to the TUB or TULP genes, available in the database, 2 ϫ 40 min and in 0.1ϫ SSC/0.1% SDS at 65ЊC for 40 min. are included in the ideograms (Fig. 4). Sequence Analyses. Sequence assembly and analysis were performed using the programs ASSEMBLYLIGN and MACVEC- RESULTS TOR (Oxford). Multiple alignments and phylogenetic trees Isolation and Characterization of the TUB, TULP1, and were generated with the program MACAW (7). Database TULP2 cDNAs. The human homolog of the mouse tubby gene searches were conducted using the BLAST programs (8). was identified by screening an adult brain cDNA library Radiation Hybrid Mapping. Oligonucleotide primers were (CLONTECH) with tub derived probes. The full-length cDNA constructed from the novel 5Ј end of TUB (hMan1-F2 5Ј- is Ϸ7,500 bp long and can code for a 561-amino acid protein

FIG.2. (a) Phylogenetic tree of family members. (b) Hybridization of Southern blot of EcoRI-digested genomic DNAs from nine eukaryotic species with TULP1. Both TUB and TULP1 genes are conserved in all species including yeast (hybridization pattern of TULP1 shown). Downloaded by guest on September 30, 2021 Genetics: North et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3131

FIG.3. (A) Expression of TUB. Northern blot analysis of adult human tissues shows a Ϸ7–7.5-kb transcript with strong expression in heart, brain, testis, ovary, thyroid, and spinal cord after 48-hr exposure. TUB is also detected in skeletal muscle, prostate, small intestine, trachea, and adrenal gland. High expression of a 2.4-kb transcript is observed in liver and thyroid. (B) Expression of the TULP1 gene. No bands were observed on the same Northern blots when hybridized with a TULP1 probe after seven days of exposure. (C) Expression of the TULP2 gene. A Ϸ1.8-kb transcript is observed in testis. (D) Actin control.

starting at the first methionine in the ORF. Comparison of the sequence and found that it maps to the same location as TUB, tubby mouse (U52433; refs. 4 and 12) and human brain cDNA 8.1 cR from the 3Ј untranslated region (Lod score, 13.17). The sequences (HSU54644; ref. 12) with the TUB sequences ob- novel 5Ј sequence extends the ORF by 55 amino acids beyond tained here, indicates that they differ in the first 444 bases (Fig. the sequence reported for TUB (12). The first 32 amino acids 1a). The position at which the human brain TUB sequences of this ORF are predominantly hydrophobic, a distinct contrast diverge coincides with an intron͞exon splice junction, suggest- to the overall highly hydrophilic nature of the predicted ing that these cDNAs derive from alternatively spliced mR- protein. A third 5Ј splice variant of the tubby gene had been NAs. To support this hypothesis, we determined the chromo- previously observed in mouse testis (U52824; ref. 4). The somal location of the novel 5Ј end of the TUB human brain remaining TUB coding sequence, nucleotides 445-1924, is Downloaded by guest on September 30, 2021 3132 Genetics: North et al. Proc. Natl. Acad. Sci. USA 94 (1997)

highly conserved between mouse and human with 89% nucle- otide identity and 94% amino acid identity (Fig. 1b). The EST clone 221670 was used to screen a human retinal cDNA library for TULP1. Multiple partial cDNA clones encom- passing the 3Ј portion of TULP1 and one full-length clone were obtained. 5Ј rapid amplification of cDNA ends of human brain cDNA was performed to confirm the 5Ј end of the cDNA sequence. The full-length cDNA is comprised of 2116 bp that potentially encodes a 542-amino acid protein (Fig. 1b). A probe derived from amplification of mouse testis cDNA using oligonucleotide primers designed according to the pub- lished p46 mouse cDNA sequence (5) was used to screen a human testis cDNA library. A clone was isolated that con- tained a 1733-bp long cDNA insert ending in a Poly(A) tail. The cDNA can code for a 520-amino acid protein beginning at the first methionine in the ORF (Fig. 1b). The corresponding gene was named TULP2. Phylogenetic analysis over the conserved 217-amino acid C terminus, indicates that TUB is more closely related to TULP1 than to the TULP2 protein (Fig. 2a). Ninety percent amino acid identity is observed between TUB and TULP1 while 66% amino acid identity is observed between TUB and TULP2 over the conserved region. TULP1 and TULP2 have 63% amino acid identity over the same region. The conservation of this gene family apparently extends across multiple animal species, as strongly hybridizing bands were detected in a high stringency genomic Southern blot (Fig. 2b). In addition, database searches reveal tubby-like in Drosophila, Caenorhabditis elegans, Arabidopsis, rice, and maize. Such conservation across species suggests that tubby and tubby-like proteins may be fundamental to normal function of these organisms. Expression and Tissue Distribution of TUB, TULP1, and TULP2. TUB gives rise to a major 7–7.5 kb transcript by Northern analysis (Fig. 3A). Like the mouse homolog, TUB is transcribed in brain and testis but in contrast to tub it is also highly expressed in liver, heart, small intestine, and ovary. Expression was also detected in skeletal muscle, thyroid, spinal cord, trachea, and adrenal gland—tissues not tested in the mouse. In addition to the major transcript, Northern analysis also revealed the presence of a faster migrating mRNA species, Ϸ2.4 kb, with highest expres- sion in thyroid and liver. In contrast to the high expression of TUB, multiple tissue Northern blots probed with TULP1 showed either faintly hybridizing or no bands (Fig. 3B). Probing the same blots with TULP2 showed a 1.8-kb transcript in testis (Fig. 3C). Retinal mRNA was not included in the Northern tissue blots above. However, because mouse tub is also highly expressed in the eye, a human retinal cDNA library was screened with human derived TUB, TULP1, and TULP2 cDNA clones. TUB transcripts, confirmed by sequencing, comprised Ϸ0.005% of retinal cDNA clones. TULP1 had a 0.01% incidence in the retinal cDNA library, suggesting high expression of this gene in the retina. Murine multiple tissue Northern blots probed with human TULP1 confirmed the observation as only retinal mRNA showed a strongly hybridizing band of the correct transcript size (data not shown). TULP2 appears to be a very low abundance transcript in the retina as it comprised only 0.0001% of the total clones screened in the retinal cDNA library. Mapping of TUB, TULP1, and TULP2 Genes. Heckenlively et al. (1) and Ohlemiller et al. (2) have reported that the

to chromosome 6p21.3. (C) TULP2 to chromosome 19q13.1. Centiray distances (cR8,000) and lod scores are shown to the right of the ideogram. RH data of additional markers obtained from the Stanford Center were included in the data analyses (RHMAP computer software 2.01). The branch and bound approach was used to determine the most likely order (RHMAXLIK). Two point analyses FIG. 4. Radiation hybrid mapping (A)5Јnovel splice variant and were done using RH2PT to obtain an estimated distance and the lod the 3Ј untranslated region of TUB to chromosome 11p15.4. (B) TULP1 scores between markers. Downloaded by guest on September 30, 2021 Genetics: North et al. Proc. Natl. Acad. Sci. USA 94 (1997) 3133

histopathological changes in the eyes and ears of tubby mice tissue specific expression. Alternate 5Ј splicing may addition- are similar to those seen in individuals with Usher syndrome. ally modulate expression in a tissue-specific manner. In at least Because Usher syndrome, Type1C maps to human chromo- one member, TUB, alternate splice variants predicting differ- some 11p15.1, the homologous region of tub on mouse chro- ing N termini were isolated. In mouse, one splice variant was mosome 7, they suggested that tubby may be the Usher 1C observed only in testis, while two other variants are found in homolog. The chromosomal location of TUB was determined brain (4, 12). Whether they localize to different areas in the in the Stanford G3 Radiation Hybrid panel with oligonucle- brain remains to be determined. otide primers specific to the 3Ј noncoding region. TUB was The function of this family of genes is also not known. However, localized to human chromosome 11p15.4 (Fig. 4A) in close a mutation in the highly conserved C-terminal portion of the tub proximity to D11S932 (14.7 cR). Since marker D11S932 gene leads to obesity, and cochlear and retinal degeneration. resides outside the minimal region established for Usher Therefore, tub and possibly related family members, must play a syndrome, Type 1C (14), our mapping results exclude TUB as vital role in tissues in which they are expressed. Additionally, that a candidate gene for this disease locus. their function is related to basic cellular processes is suggested by Gene specific PCR primers were also constructed for the fact that these family of genes are widely distributed among TULP1 and TULP2 to determine their chromosomal location species, ranging from humans to maize. in the Stanford G3 Radiation Hybrid panel. TULP1 localizes Ultimately, the reason for studying mouse models is to to chromosome 6p21.3 (Fig. 4B). Interestingly, two markers, understand human disease. We began with the cloning of D6S439 and D2S291, that flank TULP1 have been reported mouse tub, and have subsequently identified two human tubby not to recombine with the 14 locus in a family members. We hypothesized that genes within the tub human kindred (15). TULP1 is tightly linked to the retinitis family must share similar functions and that mutations within pigmentosa 14 locus and within the region of homozygosity of some of the genes may lead to phenotypes in humans that are this autosomal recessive disease. TULP2 maps to human similar to those of tubby. Interestingly, TULP1, which is highly chromosome 19q13.1 (Fig. 4C) within the minimal region expressed in the retina, maps to human chromosome 6p21.3 in identified for a cone-rod retinal dystrophy locus (16). the same minimal interval as retinitis pigmentosa 14, and TULP2 maps within the minimal interval for rod-cone dystro- DISCUSSION phy on chromosome 19q13. Proof that the TULP genes are In this communication, we have further characterized the causative of these diseases will come from testing families human homolog of the mouse tub gene, and described two segregating at those loci. The sequences presented here pro- novel tubby gene family members. Our data indicate that TUB vide the means to test these hypotheses. encodes a protein of 561 amino acids that is highly expressed We thank Gayle Bouchard-Collin and Doug McMinimy for excel- in a number of tissues examined, including heart, brain, ovary, lent technical assistance; Alan Buckler, Spencer Emtage, Nic Dra- thyroid, spinal chord, and retina and maps to chromosome copoli, and Tim Harris for advice; and Barbara Knowles, Susan 11p15.4. TULP1, closely related to TUB, encodes a protein of Ackerman, and Wayne Frankel for careful review of the manuscript. 542 amino acids that is highly expressed in retina and maps to This work was supported by a grant from Sequana Therapeutics, Inc. chromosome 6p21.3. TULP2, more distantly related to TULP1 (P.M.N.), National Retinitis Pigmentosa Foundation, Inc. (P.M.N.), and TUB, encodes a protein of 520 amino acids that is highly and National Institutes of Health Grant R01DK46977 (J.K.N.). Insti- expressed in testis and maps to chromosome 19q13.1. tutional shared services are supported by National Cancer Institute A striking feature among all known tubby related proteins is Cancer Center Support Grant, CA-34196. K.N.T. is a recipient of a Deutsche Forschungs Gesellschaft fellowship. the high conservation observed in the Ϸ250 amino acids at the C terminus. Even the most distantly related members, TULP2 and 1. Heckenlively, J. R., Chang, B., Erway, L. C., Peng, C., Hawes, N. L., the C. elegans 48.2K protein, show a 55% amino acid sequence Hageman, G. S. & Roderick, T. H. (1995) Proc. Natl. Acad. Sci. USA 92, 11100–11104. identity in this region. This suggests that this conserved region 2. Ohlemiller, K. K., Hughes, R. M., Mosinger-Olilvie, J., Speck, J. D., may impart a common function to the gene family members. The Grosof, D. H. & Silverman, M. S. (1995) NeuroReport 6, 845–849. functional importance of this conservation may not extend, 3. Coleman, D. L. & Eicher, E. M. (1990) J. Hered. 81, 424–427. however, throughout the C terminus. It appears that a mutation 4. Noben-Trauth, K., Naggert, J. K., North, M. A. & Nishina, P. M. (1996) Nature (London) 380, 534–538. has been fixed in the TULP2 gene that introduced a premature 5. Vambutas, V. & Wolgemuth, D. J. (1994) Biochim. Biophys. Acta 1217, stop codon in position 1654 of the cDNA sequence, truncating the 203–206. protein by six amino acids. This truncated protein seems to 6. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. B., predominate in humans since 10 of 10 sequenced DNAs from Smith, J. A. & Struhl, K., eds. (1995) Current Protocols in Molecular Biology (Greene/Wiley–Interscience, New York). unrelated individuals have this form. 7. Schuler, G. D., Altschul, S. F. & Lipman, D. J. (1991) Proteins Struct. Funct. In contrast, the N-terminal portions of the tubby like proteins Genet. 9, 180–190. show little sequence similarity either among each other or with 8. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) other unrelated proteins. The sequences diverge near an exon J. Mol. Biol. 215, 403–410. 9. Cox, D. R., Burmeister, M., Price, E. R., Kenn, S. & Myer, R. M. (1990) boundary present in TUB, TULP1, and TULP2.. Science 250, 245–250. The actual number of members in this novel tubby gene family 10. Collin, G. B., Mu¨nch, A., Mu, J.-L., Naggert, J. K., Olsen, A. S. & Nishina, is not known. However, low stringency Southern blot analysis P. M. (1996) Genomics 37, 125–130. indicates that there may be between six to ten members (data not 11. Boehnke, M., Lange, K. & Cox, D. R. (1991) Am. J. Hum. Genet. 49, 1174–1188. shown). Although we screened for TULP2 in a human testis 12. Kleyn, P. W., Fan, W., Kovats, S. G., Lee, J. J., Pulido, J. C., et al. (1996) cDNA library with a probe from the mouse testis specific cDNA Cell 65, 281–290. p46, it most probably is not the human homolog of this gene. The 13. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. conserved C-terminal region of TULP2 and mouse p46 show only 22, 4673–4680. 14. Keats, B. J. B., Nouri, N., Pelias, M. Z., Deininger, P. L. & Litt, M. (1994) 68% amino acid identity, whereas the mouse and human TUB Am. J. Hum. Genet. 54, 681–686. proteins are 99% identical over the same 273-amino acid region. 15. Shugart, Y. Y., Banerjee, P., Knowles, J. A., Lewis, C. A., Jacobson, S. G., Therefore, at least four unique family members have been Matise, T. C., Penchaszadeh, G., Gilliam, T. C. & Ott, J. (1995) Am. J. Hum. identified, TUB, TULP1, TULP2, and mouse p46. Genet. 57, 499–502. 16. Evans, K., Fryer, A., Inglehearn, C., Duvall-Young, J., Whittaker, J. L., Unlike many housekeeping genes that are ubiquitously Gregory, C. Y., Butler, R., Ebenezer, N., Hunt, D. M. & Bhattacharya, S. expressed, members of the tubby gene family show distinct (1994) Nat. Genet. 6, 210–213. Downloaded by guest on September 30, 2021