Structure and expression of the gene encoding mouse t-complex polypeptide (Tcp-1)

Gene, 120 (1992) 207-215 0 1992 Elsevier Science Publishers B.V. All rights reserved. 037%1119/92/$Q5.00

207

GENE 06686

Structure and expression of the gene encoding mouse t-complex polypeptide WP-0 (Genomic DNA; nucleotide sequence; exon-in&on; CpG-rich region; regulatory element; mRNA; transcription start point)

Hiroshi Kubota8, Keith Willison b, Alan Ashworth b, Masami Nozaki a, Hiroshi Miyamoto at Hideyuki Yamamoto a, Aizo Matsushiro a and Takashi Morita a ’ Department of Microbial Genetics. Research Institute far Microbial Diseases. Osaka University, Yamadaoka 3-1, Suita, Osaka 565, Japan; and b Institute of Cancer Research, Chester Beatcy Laboratories. 237 Fulham Road, London SW3 6JB, UK. Tel. (44-71)352-8133 Receiwd by W. Sisk 26 November 1991; RevisedjAccepted:

12 ~~ch/~

May 1992; Received at publishers: 19 &me 1992

SUMMARY

The nucl~~de (nt) sequence of the structural gene (Tcp-l) ~co~g mouse t-complex pol~~~de 1 (TCP-1) has been determined. The nt sequence extending to 10043 bp shows that the ?@-I gene is divided into 12 exons, 11 introns and 5’and 3’-flanking regions. The Tcp-l gene has a tight cluster of major transcription start points (Q). Two EC boxes, one CCAAT box and some other possible regulatory elements are located in the region upstream from the tsp, but no TATA box was found. Extending from the 5’-flanking region to the first intron, a CpG dinucleotide-rich cluster is located. In addition, Tcp-1 gene transcripts in mouse organs, embryos and cultured cells were analyzed by Northern blotting. The Tcp-1 mRNA is enriched not only in testes, but also in early post-implantation embryos and some cultured cell lines, as compared with mouse organs other than the testis. The amount of Tcp-I mRNA in embryos decreases during development. These results suggest that the expression of the Tcp-I gene may be regulated sparely and temporally in embryonic and adult mice by transcriptional control or by mRNA stability.

INTRODUCTION

Mouse T&p--lis a gene located in the l-complex encoding a polypeptide which has two allelic farms distinguishable by their isoelectric points (Silver et al., 1979). The

Curws~ondence to: Drs. A. Matsushiro or T. Morita, Department of Microbiat Genetics, Research Institute for Microbial Diseases, Osaka Uuiversity, Y~adaoka 3-1, Suita, Osaka 565, Japan. Tel. (gl-6)g75-2913; Fax (81-6)876-2~7g. Abbreviations: aa, amino acid(s); Ad, adenovirue; AMV, avian myeIoblastosis virus; bp, base pair(s); cDNA, DNA complementary to RNA; DTT, dithiothreitol; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame(s); SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/ 0.015 M Nasxitrate pH 7.6; TCP-1, t-complex polypeptide 1; Tcp-i, gene (DNA, RNA) encoding TCP-1; fsp, transcription start point(s); ti, wild type.

acidic form, TCP-lA, is encoded by all complete t-haplotype c~omosomes, and the basic form> TCP-lB, is encoded by all wt inbred strains (Willison et al., 1986). These proteins are produced at a high level during spermatogenesis (Silver et al., 1987; Willison et al., 1990) and also at lower levels in almost all cells investigated. The nt sequence of Tcp-I cDNA has been previously published (Wiiison et al., 1986), and Tcp-I homologous genes or TCP-I homol~ous proteins have been identified in bumans (Kirchhoff and Willison, 1990)? ~~~~a~~~~~ ~.w&PB,?guster (Ursic and Ganetzky, 1988), peas (Ellis, 1990) and yeast (Ursic and Culbertson, 1991). Recently, it has been demonstrated that the TCP- 1 protein is associated with an intracellular transport system (Willison et al., 1989) and has siguif?cant homology to the ‘chaperonin’ group of proteins: GroEL of Escfreldchia cofi, Hsp60 of mitochond~~ Rub&co-binding protein of chloroplasts (Ellis, 1990;

208

100

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000 4100 4200 430& 4400 4500 4600 4700 4800 4900 5000

Gupta, 1990) and thermophiiic factor 55 (TF55) of the thermophilic archaebacterium, Sulfolobus shibatae (Trent et al., 1991). These proteins are involved in posttranslational folding, assembly and transport of proteins (Hemmingsen et al., 1988; Martin et al., 1991), suggesting that TCP-I plays a fund~ent~ role in cells. The Tcp-I structural gene was identified by Southern blot analysis and molecular cloning (Willison et al., 1986). However, its nt sequence has not been determined, and therefore the gene has remained uncharacterized. Here, we have sequenced the Tcp-1 gene from genomic DNA clones

and also have further analysed the distribution of the Tcp-1 gene transcripts.

RESULTS

AND DISCUSSION

(a) Structure of the mouse Tcp-1 gene The nt sequence of the Tcp-I gene extending to 10043 bp

(Fig. 1) shows that the gene is divided into twelve exons, eleven introns, and 5’- and 3’-flanking regions (shown schematically in Fig, 2). The first, third and seventh introns

209

6

5100 5200 5300 5400 5500 5600 5700 5800 5900 6000 6100 6200 6300 6400 6500 6600 6700 6800 6900 7000 7100 7200 7300 7400 7500 f600 7700 7800 7900 8000 8100 8200 8300 8400 8500 8600 8700 8800 8900 9000 9100 9200 9300 9400 9500 9600 9700 9800 9900 0000

6

Fig. 1. The nt sequence vector pUC19. subcloned

(10043 bp) of the Tcp-Z structural

From the pUC19

clones, restriction

into the Ml3 phage vector.

with Taq DNA polymerase,

Single-stranded

fluorescently

1986; Innis et al., 1988). The nt sequence

gene. Restriction

fragments

fragments

or deleted fragments

from Tcp-1 genomic DNA clones were subcloned

produced

by exonuclease

DNAs from the Ml3 clones were sequenced

labelled primers (ABI Dye Primers)

and an automated

from 1 to 782 was derived from a BALB/c

genomic

from 783 to 10043 was from a 129/Sv clone (TlB3; Kubota et al., 1991). Exons are underlined left of each line. Exon-intron junctions were determined according to the consensus sequence

by the dideoxynucleotide

sequencer

into the plasmid

III and mung bean nuclease (ABI 373A) (Sanger

digestion

chain-termination

were

method

et al., 1977; Smith et al.,

DNA clone (4.2; Willison et al., 1986) and the sequence with thin lines, and the exon number is shown on the for splicing junctions, 5’-exon/GT-intron-AG/exon-3’

(Breathnach et al., 1978). Other marks are as follows: dots, translation start (ATG) and stop (TGA) codons; arrowheads, major tsp; arrows, Bl (Krayev et al., 1980) and B2 (Krayev et al., 1982) mouse repetitive sequences; wavy underlining, polyadenylation signal; bold underlining, consensus sequence for the factor E2F binding (SivaRaman

and Thimmappaya,

boxes with solid lines, GC boxes (Kadonaga

1987); dashed underlining,

et al., 1986); box with dashed

similar sequence to Ad ElA enhancer

line, CCAATT

box (Chodosh

et al., 1988).

core (Hearing

and Shenk, 1983);

210 Ba

1

2

3

4

5

6

7

6

9

10

11

12

I

I lb

Fig. 2. Structure exon number

of the Tcp-1 gene. A restriction

is indicated

map of the Tcp-1 gene with the exons and introns

under the boxes. A, ApaI; Ba, BarnHI;

Bg, BglII: E, EcoRI,

are longer than 1 kb and longer than the other introns. Thus, exons 1, 2-3, 4-7 and 8-12, respectively, appear clustered. There is a CCAAT box and two GC boxes, but no TATA box, in the 5’-flanking region. A polyadenylation signal, ATTAAA, is located 14 bp upstream from the polyadenylation site as previously reported (Kubota et al., 1991). The mouse repetitive sequences, B2 (Krayev et al., 1982) and Bl (Krayev et al., 1980), are present in the seventh and ninth introns, respectively.

H, HindIII;

is shown.

Exons are shown as open boxes, and the

P, PvuI.

(b) TCP-1 aa sequence encoded by the exons The aa sequence of TCP-1 is separately encoded by the twelve exons (Fig. 3). The N-terminal and C-terminal parts of the polypeptide can be divided into two theoretical domains, with the seventh intron (one of the three long introns) representing the dividing point between the two domains. These two parts are encoded by exons l-7 and exons 8-12, respectively, and are separated by a highly hydrophilic region. The mouse TCP-1 polypeptide has

MM DM SC

MM DM SC MM DM SC

RDC

Flida

RDS

K&AR

KET

KSAT

I

217

MM DM SC

MM DM SC

MM DM

SC

MM DM SC

MM

AIT

~@DL;@KLH~~!ZSKDDKHGSY~NAVHSGALDD

DM SC

AIT

@,DM@KLN’f$&DKSGK--SYADACAAGELDG rQTMI:TVD’f.E’PPKEDPHDH

Fig. 3. Comparison

CVA

of aa sequences

among TCP-1 homologues

and location

556

of the introns

of mouse Tcp-I. The aa sequences

of the TCP-1 homologues

from the mouse (MM; deduced from the exons in Fig. 1), Drosophila melanoguster (DM; Ursic and Ganetzky, 1988) and Succharomyces cerevisiue (SC; Ursic and Culbertson, 1991) are shown, and the aa residues conserved among them are shadowed. The location of the introns in the mouse is indicated with downward arrowheads, and the exon number is shown on the sides of each arrowhead. The central hydrophilic region is underlined, and the regions homologous to chaperonin family proteins (Gupta, 1990) are doubly underlined. Bent arrows indicate highly conserved regions I and II. The mouse TCP-1 has 72.37; and 62.4% aa identities to D. melunoguster and S. cerevisiue homologues of TCP-1, respectively (in comparable region, aa 2-541). The aa identities from the mouse TCP-1 to the above homologues in region I (aa 32-115) are 92% (DM) and 80% (SC). Those in region II (aa 374-468) are 85% (DM) and 78% (SC), respectively.

211 highly conserved regions similar to Drosophila melanogaster and ~accharom~es cerevisiae homologues found within regions I and II (Fig. 3). Fu~e~ore, the areas most homologous to the chaperonin family proteins (Gupta, 1990) are found in the middle of both regions I and II and may represent separate domains of TCP-1.

1234

6

6

78910 .-- .._

(c) The tsp in the Tcp-I gene The Tcp-1 gene has a pair of major tsp (Fig. 4) found 101-102 bp upstream from the tr~siation start codon ATG (Fig. 1); nt 1070 is the most preferred fsp, and nt 1069 is next. The pair of tsp are located 22 bp upstream from the 5’ end of the longest 7?~~-l~cDNA clone reported (pTlb 11; Kubota et al., 1991). No TATA box is observed in the immediate upstream region from the tsp. Many genes that do not contain obvious TATA boxes are known (see Sehgal et al., 1988 and Smale and Baltimore, 1989 for review), and some of them have one or a few tightly clustered tsp. The Tcp-I gene appears to be a member of this class of genes and must use an alternative accurate start mechanism other than a TATA box. For instance, beginning 4 bp downstream from the clustered tsp, there is a tandem repeat of 5’CCeCGCCGTGGT that may be important in determining the position of the preferred tsp. In addition, a dyadsymmetry element, 5’-GGCCGTTAAACGGTC, resides immediately after this tandem repeat and may also play a role. (d) Distribution of the Tcp-1 gene transcripts The enriched level of Tcp-I mRNA in the mouse testis as compared with spleen and liver was reported by Dudley et al. (1984). However, the mRNA distribution of Tcp-1 in other tissues and cell lines has not been thoroughly investigated. The amount of Tcp-1 mRNA in organs, embryos and cultured cells of the mouse has been analysed by Fig. 4. Primer extension analysis for the tsp of the Tcp-I gene. Total testis RNA from 129,&v mice (2.5 pg) was mixed with a 32P-labeUed synthetic primer

S’-AAG~AAAG~TCATCACTACGGCCGCAGACAACC

(2.5 x IO“ cpm) EDTA/lO

in 5 JLI of hybridization

mM Tris.HCl

5 mitt, at 60°C

buffer

(250 mM/KCI

pH 8.3). The mixture was incubated

for 1 h and cooled

to room

temperature.

mixture was added to 20 ~1 of reverse transcription 0.25 mM EDTA/ZO mM Tris.HCl dATP, dGTP, centrations), transcription electrophoresed

dCTP

rg/ml

and AMV reverse transcriptase was performed

at 42°C

of actinomycin

mM each

D; final con-

(6 units) was added. Reverse

for 1 h, and the products

on an 8 M urea 6% poiyacrylamide

quenced by the methods

The cooled

buffer (75 mM KCl/

pH 8.3/10 mM D’lT/0.25

and dTTPjlO0

1 mM

at 65°C for

were

gel. Products

se-

of Sanger et al. (1977) (lanes 1-4) and Mizusawa

et al. (1986) (lanes 7-10) were loaded

adjacent

to the primer

extension

products (lane 5) and labeled primer (lane 6). The products using Sanger’s method give the same mobility as those of primer extension (arrows indicate the zsp of the Tcp-I gene), but some nt were unreadable the secondary method

structure

(cont~n~ng

of these products.

ii-deaza-G)

Those products

gave an accurate

because

of Mizusawa’s

sequence.

of

AGCT

AGCT

212 Northern blotting (Fig. 5). A major Tcp-I transcript species of 2.0 kb and three minor species of 2.4, 3.7 and over 9 kb have been observed. The 2.4- and 3.7-kb transcripts might use different polyadenylation sites from the 2.0-kb transcripts or, alternatively, encode TCP-l-like proteins. (Proteins of higher molecular weight were detected by Western blot analysis using TCP-l-specific antibodies; unpublished observations.) The transcripts larger than 9 kb might be the hnRNA of Tcp-1 or, again, might encode one of these TCP-l-like proteins. Alternatively, it is possible that the three minor RNA species were partially denatured or incompletely spliced. The 2.0-kb mRNA has been quantified by densitometry tracings of autoradiographs. Among the mouse organs analysed (Fig. 5A), the amount of Tcp-1 mRNA in the testis is by far the most enriched. The mRNA level in bone marrow of thymus is ten times less than that in the testis,

A

while the heart and brain contain one hundred times less Tcp-1 mRNA than the testis. In postimplantation embryos (Fig. 5B), Tcp-1 mRNA is more enriched than in any of the mouse organs examined other than the testis. In particular, 11.5-day embryos contain Tcp-1 mRNA at 30% of the level found in the testis. After 7.5-9.5 days of gestation, the entire conceptus of mouse embryos including embryo proper and extra-embryo proper (amnion, parietal yolk sac, visceral yolk sac and maternal decidual tissue) were isolated and their mRNA levels of Tcp-1 analysed. The Tcp-1 mRNA level is rapidly reduced during development in the early stages, 7.5-9.5 days (70% decrease for two days). During further development in the middle and late stages, 11.5-15.5 days, the mRNA levels of Tcp-I continue to decrease (40-60% decrease per two days). The gap of mRNA levels between samples of 7.5-9.5 days and 11.5-15.5 days would result

C

6

-28s

-28s

18s -18s

123

Fig. 5. Northern isothiocyanate

45

6

7

blot of Tcp-Z mRNA.

9 IO

8

Total RNAs

prepared

and CsCl (Davis et al., 1986) were electrophoresed

11

12

from mouse

13 14

organs,

15

embryos

on 2.2 M formaldehyde-l

18

16 17

and cultured

y0 agarose

19

20

21 22

cells by ultracentrifugation

gels and transferred

to Magnagraph

with guanidine .nylon mem-

branes (Micron Separations Inc.). A 3ZP-labelled Tcp-2 cDNA (clone pTlbl1; Kubota et al., 1991) probe was hybridized to the membranes, which were subsequently washed in 0.1 x S SC containing 0.1 y0 SDS at 55°C for 15 min. (Panel A) Tcp-1 mRNA in mouse organs. The organs were prepared from 129/Sv adult (lanes l-4, 68) and 4-week-old

(lane 5) mice. (Panel B) Tcp-I mRNA

in mouse postimplantation

embryos.

The embryos

were collected

with (lanes 13-14) or without (lanes 15-17) extraembryonic membranes and decidua from 129/Sv mice. Noon on the day of which a vaginal plug was observed was designated as day 0.5 of gestation. (Panel C) Tcp-1 mRNA in cultured cells. Fibroblast L, teratocarcinoma F9 and differentiated teratocarcinoma

PYS-2 cells were cultured

in Dulbecco’s

modified Eagle’s medium supplemented

A was also used in panels B and C as control (lanes l-3, !J-11, N-19). Amount 2, 10, 19) and 0.1 pg (lanes 3, 11).

with 10% fetal calf serum. The same testis RNA used in panel

of total RNAs loaded was 10 pg (lanes 1,4-9,12-17,20-22),

1 pg (lanes

213 from a diluting effect of low mRNA levels of extra-embryo proper including decidua in the former samples. At the final stage of embryonic development, at 17.5 days, the amount of Tcp-I mRNA in embryos is very low (Paldi and Jami, 1991). In some cultured cells (Fig. X), Tcp-1 mRNA is as abundant as that found in postimplantation embryos. Embryonal teratocarcinoma F9 cells, differentiated teratocarcinema PYS-2 cells (epithelioid cells) and fibroblast L cells, contain Tcp-I mRNA at 2%30% of the level found in the testis. These results indicate that Tcp-1 mRNA is abundant not only in the testis, but also in postimplantation embryos during early and mid-gestation and in cultured cells. These results also suggest that the expression of the Tcp-1 gene may be regulated spatially and temporally in mice and embryos by transcriptional control or by the stability of mRNA. The amount of the Tcp-I mRNA may be increased during embryogenesis and cell culture where cells proliferate rapidly. (e) The CpG-rich region in the Tcp-1 gene Actively transcribed animal genes often contain regions enriched in CpG dinucleotides which are undermethylated, whereas CpGs in the other regions are methylated. In the methylated regions, CpGs are rare because of the nt transition produced by the deamination of 5-methylcytosine to thymine (Coulondre et al., 1978). The CpG-rich unmethylated regions are termed CpG islands of HpaII tiny fragment (HTF) islands (reviewed by Bird, 1986). If the CpG islands in genes are methylated artificially, transcription from these genes is inhibited. The sequence of the Tcp-1 structural gene indicates that it contains a region enriched in CpG dinucleotides (nt lOO-nt 1900 in Fig. 1). The CpG-rich region extends from the 5’-flanking region to the first intron of the Tcp-1 gene and therefore includes exon 1 (Fig. 6A). Exon 1 has nine CpG dinucleotides, though a mouse Tcp-1 pseudogene (Kubota et al, 1992) has only one CpG in its corresponding region (Fig. 6B). These results suggest that the CpGrich region in Tcp-I has been evolutionarily conserved, possibly in an unmethylated state, indicating that this region might play a role in Tcp-1 gene expression.

Fig. 6. The CpG-rich

region in the Tcp-I gene. (A) The distribution

CpG and GpC dmucleotides Each vertical line indicates sequence. numbering

The numbers

is shown

a CpG or GpC dinucleotide given on the CpG

distribution

on the Tcp-2 gene refer to the nt

in Fig. 1. The open boxes in the gene structure

exons. (B) Comparison

of the nt sequence

the Tcp-2 gene and a mouse-processed 1992). CpG dinucleotides are boxed, Tcp-Z gene is underlined.

of

above the Tcp-I gene structure.

indicate

the

in the exon-1 region between

Tcp-2 pseudogene (Kubota et al., and the ATG start codon of the

214

(f) Possible regulatory elements of Tcp-1 gene expression Many regulatory factors of gene expression and the sequences to which they bind have been identified (reviewed by Jones et al., 1988) during the past ten years. The sequence of the Tcp-1 gene suggests several possible regulatory elements in the region upstream from the clustered tsp (Fig. 1). These include possible binding sequences of CTF/ (CPl,CP2) (CCAAT box; Jones et al., 1987; Chodosh et al., 1988), Spl (GC box; Briggs et al., 1986; Kadonaga et al., 1986), and E2F (5’-TTTCGCGC; SivaRaman and Thimmappaya, 1987; Mudryj et al., 1991) or DRTFI (5’TTTCGCGC; La Thangue et al., 1990; Bandara and La Thangue, 1991). In addition, a sequence similar to the Ad Ela enhancer core sequence (5’-GGAAGTGA; Hearing and Shenk, 1983) is observed in this region. All of these elements have been reported to regulate the expression of the Ad early genes. Thus, a portion of the transcription mechanism of the Tcp-1 gene might be similar to those of the Ad early genes. Of these factors, Mudryj et al (1991) indicated that EZF activity is dependent on the cell cycle and is almost inactive in the G, phase. Thus, E2F may play a role in Tcp-I gene transcription, since Tcp-1 mRNA is enriched in cultured cells, embryos and testes, samples which consist of rapidly proliferating cells. The level of Tcp-1 mRNAs is dependent on the cell cycle in cultured cells (Y. Takemoto, H.K., T.M., and A.M., unpublished results).

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(g) Conclusions (I) The mouse Tcp-1 gene encoding t-complex polypeptide 1 has been cloned and sequenced. The gene consists of 12 exons and 11 introns. (2) The major transcription of Tcp-I gene is initiated 101-102 nt upstream from the translation start codon of the gene, but it has no TATA box in the promoter region. (3) From the 5’4lanking region to the first intron, a CpG-rich cluster is located in the Tcp-1 gene, which might regulate gene expression. (4) The levels of transcripts of Tcp-1 gene are enriched in the testis, early postimplantation embryo and culture ceU lines, where cells proliferate rapidly.

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