The human TYROS gene and pseudogene are located in chromosome 15q14-q25

Gene, 134 (1993) 2899293 0 1993 Elsevier Science Publishers

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GENE 0741 I

The human TYR03 gene and pseudogene are located in chromosome 15q14-q25 (Tyrosine

kinase; receptor;

processed

pseudogene)

Anne Polvi”, Elina Armstronga, Cary Laib, Greg Lemkeb, Kay Huebnerc, Leticia C. Guida”, Robert D. Nicholls” and Kari Alitalo”

Richard

A. Spritzd,

“Molecular/Cancer Biology Laboratory, Department of Pathology, P.O. Box 21, Uniuersity of Helsinki, SF-00014 Helsinki, Finland; bMolecular Neurobiology Laboratory, The Salk Institute, P.O. Box 85 800, San Diego, CA 92138, USA. Tel. (619) 453-4100. Fax (619 ) 923-4498; cJefjerson Cancer Institute, Department of Microbiology, Thomas Jefferson University, Bluemle Life Science Building, 233 S. 10th Street, Philadelphia, PA 19107-5541, USA. Tel. ( 215 ) 955-4656. Fax (215 ) 923-4498; dDepartment.~ of Medical Genetics and Pediatrics, 445 Henry Mall, University of Wisconsin, Madison, WI 53706, USA. Tel. (608) 262-2832, Fax (608) 262-2976; and ‘Department of Genetics, Case Western Reserve Uniwrsity, 10900 Euclid Ave., Cleveland. OH 44106, USA. Tel. (216) 3683331. Fax (216) 368-1257 Received by T. Sekiya: 12 February

1993; Revised/Accepted:

6 May/8

May 1993; Received at publishers:

16 July 1993

SUMMARY

Partial cDNAs of the human TYR03 gene, encoding a putative receptor (TYR03P) were cloned from human teratocarcinoma cell, bone marrow kinase homologous domains of TYR03 and TYR03P were sequenced mouse TYR03 gene. Abundant levels of the 4.2-kb TYR03 mRNA were other human tissues. TYR03 and TYR03P were both assigned to human from somatic

tyrosine kinase, and its processed pseudogene and melanocyte cDNA libraries. The tyrosine and compared with each other and with the detected in human brain, and lower levels in chromosome 15q14-q25 by analysis of DNAs

cell hybrids.

INTRODUCTION

Tyrosine kinase (TK) genes comprise a large gene family important in the control of cell growth and differ-

Correspondence to: Dr. K. Alitalo, Molecular/Cancer Biology Laboratory, Department of Pathology, University of Helsinki, P.O. Box 21, SF-00014 Helsinki, Finland. Tel. (358-O) 434 6434; Fax (358-O) 434 6448; e-mail: [email protected] Abbreviations: BM, bone marrow; bp, base pair(s); cDNA, DNA complementary to mRNA; CHO, Chinese hamster ovary; CSK, c-src tyrosine kinase; FCS, fetal calf serum; FS, frame shift; IGFlR, insulin-like growth factor-l receptor; Jurkat, T-cell leukemia cell line; kb, kilobase or 1000 bp; LTK, leukocyte TK; nt, nucleotide(s); NTERA-2D1, human teratocarcinoma cell line; ORF, open reading frame; PAE, porcine aortic endothelial cell line; PCR, polymerase chain reaction; RA, retinoic acid; Tera-2, teratocarcinoma; TK, tyrosine kinase; TYR03, gene encoding TK; TPA, 12-0-tetradecanoyl-phorbol-13-acetate; TYR03P, TYR03 pseudogene.

entiation (Hunter and Cooper, 1985). TKs play a major role in these processes by transducing signals across the plasma membrane and in the cytoplasm (Ullrich and Schlessinger, 1990; Cantley et al., 1991). All TK genes encode highly conserved catalytic domains (Hanks et al., 1988), and they can be divided into receptor and cytoplasmic TK classes on basis of the presence or absence of sequences encoding transmembrane and extracellular domains. The expression patterns of TKs vary widely different tissues and developmental stages. among Because of their central roles in cell growth regulation many of the TKs also have an oncogenic capacity when mutated or abnormally expressed (Bishop, 1991). We have previously studied TKs involved in the growth and differentiation of human hematopoietic cells (Partanen et al., 1990). We have subsequently analyzed a human bone marrow (HBM) cDNA library by use of PCR amplification of TK-homologous sequences. Here

290 we report on two of the sequences the

found, which represent

TYR03 gene and its pseudogene,

TYR03P.

to be expressed

in developing

and mature

Lemke, 1991). A related sequence

brain (Lai and

has also been reported

from mouse fetal cerebellar cDNA (Stark et al., 1991). Using our PCR fragment as a probe, two additional EXPERIMENTAL

cDNA clones were found and partially sequenced: clone lH, a 2.8-kb cDNA from the NTERA-2Dl cell library,

AND DISCUSSION

(a) Isolation and sequence of TYR03-related TK-related

sequences

and from mRNA amplified,

cloned,

from the HBM

of normal

human

sequenced

and

clones cDNA

library

melanocytes compared

with

were se-

quences in the GenBank database (see the methods in Fig. 1 legend). One of the human PCR cDNA sequences

and clone 19X, a 4.5kb cDNA from the HBM library. A comparison of the TK-homologous domains of clone

TYR03 (Lai and Lemke, un-

1H, clone

19X and mouse

published predicted

data) cDNAs is shown aa sequences of the

domain

in Fig. 1. The nt and 1H TK-homologous

were 91.9% and 98.2% identical

with the corre-

obtained identical

from both HBM and melanocytes was almost with rat TYR03 cDNA, which was originally

sponding mouse TYR03 sequences, respectively. Thus clone 1H is likely to represent the human homologue of

identified

from the rat central

the mouse

nervous

system and found

TYR03 gene.

TTCTTACACTCCCACTCCATCCAAGCCAAGGTCTCCTTAGGCCTTCAGACGACCTTT 19x I” TY3

I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A...................................~~.. EDVYDLMYQCWSRDPKQRPSFTCLHMELENIL GAGGACGTGTATGATCTCATGTRCCAGTGCTGGRGTGCTGACCCCAAGCAGCGCCCGAGCTTTACTTGTCTGCGAATGGRACTGGAGAACRTCTTG E . . . ..A.............................C..C................. A. . . c.. G..........................TC..

Fig. I. Nucleotide sequence of TYR03P and mouse TYR03. TYR03 (TY3) (C.L. and G.L., are shown by dashes. The sites

172 816

the TK domain of human TYROS, the deduced aa sequence, and comparison with the corresponding domains of The TYR03 (1H) nt and deduced aa sequence are shown in the middle and those of TYR03P (19X) and mouse unpublished data) above and below it, respectively. Dots mark residues identical in clone IH. Deletions in clone 19X of insertions are displayed by vertical lines and the inserted sequences are indicated. FS mutations in clone 19X are

also shown. Stop=stop codon. Underlining shows the clone 19X sequences corresponding to primers used for genomic PCR. Methods: TK-related sequences from the HBM hgtl0 cDNA library (Clontech Lab.. Inc., Palo Alto. CA) and from cDNA of normal human melanocytes were amplified by PCR using degenerate primers from a conserved region (Wilks. 1988; Partanen et al., 1990) cloned into plasmid. sequenced after asymmetric PCR amplification (States et al., 1990). and compared with sequences in the GenBank database. A PCR fragment (containing nt 400-602 of the clone 19X sequence) was used as a probe to isolate the 19X clone from the HBM library and the 1H clone from the NTERA-2Dl cell IgtlO cDNA library (Skowronski et al.. 1988). Isolation, characterization and subcloning of the 1H and 19X clones were performed by standard methods (Maniatis et al.. 1982). The clones were sequenced by the dideoxy chain-termination method (Sanger et al., 1977) with SequenaseR Version 2.0 (US Biochemical, Cleveland, OH, USA) using a primer walking approach. The TYR03 (1H) and TYR03P (19X) sequences illustrated in this figure have been submitted to the EMBL

Data Bank with accession

numbers

X728X6 and X72887.

291 polyadenylation 15-bp poly(A)

signal AUAAA tail. Southern

65 bp upstream

blot hybridization

from a analysis

of both genomic DNA and the 19X cDNA clone digested with AccI and Pstl yielded identical size fragments on hybridization to internal 19X probes (data not shown). This indicates that the genomic locus corresponding to clone 19X lacks introns, at least in the regions corresponding

to the putative

TK and C-terminal

(c) Analysis of human TYR03 A e2.4

probe

B Tera-2 +FCS

RA kb

4.2 -+ a+ Fig. 2. Expression of TYR03 in adult human tissues and indicated cell lines. The Northern blot A contains 2 pg of polyadenylated RNA from different human tissues (Clontech, Palo Alto, CA). Northern blot B contains 5 pg poly(A)+RNA from the Tera-2 cell line and C from PAE cell line and mouse brain. The blots were hybridized with a 1.2-kb fragment of clone IH containing the first 387 bp of the TK-encoding region shown in Fig. I. The mobilities of the molecular weight standards and the estimated

sizes of the transcripts

are shown. The p-actin

(marked with a) is also shown in B and C. Tera-2 cells were induced with RA for 6 days as described (Pertovaara et al., comparison also shown are samples from Tera-2 cells either 24 h in the absence of FCS or grown in the presence of 10%

human

blot

mRNA expression

containing

poly(A)‘RNA

tissues was hybridized

(Fig. 2A). The predominant

from

with the clone 1H

4.2-kb

transcript

de-

tected with this probe was expressed in all tissues, most abundantly in brain. Another, less abundant transcript

C

-FCS

Northern

multiple

regions.

signal

grown and 1993). For starved for FCS. PAE

cells (Miyazono et al., 1985) were grown in F-12 medium (Gibco, Paisley, Scotland, UK) containing 10% fetal bovine serum. RNA extraction and analysis were performed as described (Pertovaara et al., 1993).

(b) Analysis of the 19X clone The TK-homologous nt sequence of clone 19X was 93.9% identical to that of clone 1H and 89.6% identical with mouse TYR03. However, the tentative reading frame of clone 19X contained inserts of 8 and 57 bp, two 1-bp deletions and one deletion of 15 bp, leading to three frame shifts (FS) when compared with the mouse and human TYR03 ORFs. Also there were numerous silent and replacement point mutations and three nonsense mutations. Analysis of a short melanocyte PCR cDNA yielded an identical sequence. Clone 19X had an unusually long (3.3-kb) noncoding tail containing an Alu element and other repetitive sequences, and an incomplete

of 8.3 kb gave strongest signals from the brain, lung, and kidney. TYR03 was also expressed in several cell lines, including human teratocarcinoma (Tera-2) cells (Fig. 2B), porcine aortic endothelial (PAE) cells (Fig. 2C) and Jurkat T-cell leukemia cells, human erythroleukemia (HEL) cells, and normal human melanocytes (data not shown). Also RNA from mouse brain gave a 4.2-kb signal (Fig. 2C). Thus the 4.2-kb transcript was identified in human, porcine and mouse species. The 4.2-kb transcript was also detected in variable amounts in several primary and metastatic melanoma cell lines (Herlyn et al., 1990) (data not shown). TYR03 expression was downregulated on differentiation of Tera-2 cells with retinoic acid (RA), whereas serum had no effect on expression (Fig. 2B).

(d) Chromosomal localization of TYR03-related sequences Southern blot analysis of somatic cell hybrid DNAs digested with BamHI showed three bands hybridizing to a labeled clone 19X cDNA fragment (Fig. 3A). Two of these bands were located in chromosome segment 15pterq25 and one in 15q22-q25 (Fig. 3A, C). The third band was not detected in the hybrid cell lines (data not shown). Analysis of DNA from the Al 5 and A15-1 hybrids using the clone 1H fragment as a probe also showed three hybridizing bands, two of which were located in chromosome segment 15q14-q25 (Fig. 3B, C). These data indicated that one locus maps to 15q22-q25 and another to 15q14-q22. PCR analysis of additional somatic cell hybrid DNAs (Bios Corporation, New Haven, CT, USA) using primers shown in Fig. 1 showed two human-specific products of 158 and 700 bp, both of which were assigned to chromosome 15 (data not shown). DNA sequence analysis demonstrated that the 158-bp PCR product corresponded to clone 19X and that the longer PCR product was identical with clone 1H in the sequenced region between nt 388 and 432.

292 (d) Conclusions (1)Our results show a close conservation of the human TYR03 and mouse TYR03 cDNA sequences. Furthermore, a 4.2-kb TYR03 transcript was detected in multiple species, and the human and rat TYR03 genes appeared

I,

t-m

t-h

e-m/C

+h

+h

to have similar

4=h I

I

patterns,

with low

(2) The TK-homologous domain of TYR03P (19X) contained a number of deletions and insertions, and also several point The apparent

c-h

expression

amounts of transcripts in several tissues and higher levels of expression in the brain (Lai and Lemke, 199 1).

mutations, structural

obliterating its reading frame. colinearity of 19X cDNA clone

with genomic DNA suggests that the TYR03P gene does not contain introns. Thus, TYR03P may be a processed pseudogene

that was generated

and inactivated

by retro-

position. The isolation of TYR03P from the different cDNA libraries further suggests that there is some element enabling its transcription, at least at a low level. (3) Our data localize both the TYR03 and TYROSP genes to chromosome segment 15q14-q25. One locus maps to 15q22-q25 and another to 15q14-q22. There also appears to be a third related locus on another human chromosome. At least four other TK genes have been localized to chromosome 15. These are the genes encoding LTK (Krolewski et al., 1990), IGFlR (Ullrich et al., 1986), FES (Dalla-Favera et al., 1982; Harper et al., 1983) and CSK (Armstrong et al., 1992). The CSK gene maps to 15q23-q25 and IGFlR and c--es map to 15q25-qter. No abnormalities of any of these genes have thus far been reported in any human genetic diseases.

C

23 ACKNOWLEDGEMENTS

24 25 26

15q14-q22 15q22-q25 Fig. 3. Localization

+ + of human

+

_^_

TYKu~

sequences

to cnromosome

seg-

ment 15q34-25. The Southern blots (A and B) contain DNAs from several somatic hybrid cell lines (Huebner et al., 1985; 1988; McDaniel and Schultz, 1992). Hybrids 11418, 10664, 10500, Al5 and A15-1, contain human chromosomes 15, 15pter-15q25, 15pter-15q22, 15 and 15pter-15q14, respectively. In C, the portions of chromosome 15 retained in the different hybrid cell lines are indicated to the right of the chromosome 15 ideogram. Methods: DNA isolation, digestion with BumHI (blot A) and EcoRl (blot B), electrophoresis, transfer to nylon membranes, hybridizations and washes were done using standard methods (Maniatis et al., 1982). The probe used for A corresponded to nt l-387 of the clone 19X sequence shown in Fig. 1, and the one used in blot B corresponded to nt 4155585 of clone 1H. The bands specific to human, mouse and Chinese hamster DNA are indicated as h. m and c, respectively.

We would like to thank Dr. Roger Schultz for providing somatic cell hybrids, Drs. Liisa Pertovaara and Eero Lehtonen for mRNA from the Tera-2 cells, Drs. Juha Partanen, Tomi Make12 and Olga Aprelikova for helpful advice, and Tapio Tainola, Kaarina Kronqvist, Kirsi Pylkkanen and Teresa Druck for expert technical assistance. This study was supported by the Finnish Cancer Organizations, The Finnish Academy, The Sigrid Juselius Foundation and USPHS Grants CA21 124 and AR39892.

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