Cloning, nucleotide sequence and molecular evolution of a rabbit processed metallothionein MT-2 pseudogene

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1403-1410

November 15, 1988

CLONING, NUCLEOTIDE SEQUENCE AND MOLECULAR EVOLUTION OF A RABBIT PROCESSED METALLOTHIONEIN MT-2 PSEUDOGENE

You C. Tam

, Manuel Hassan*,

Arvind Chopra and Jean-Paul Thirion

Department of microbiology, Faculty of medicine Centre Hospitalier Universitaire, Sherbrooke Quebec, Canada JIH 5N4 Received

September

26, 1988

SUMMARY: A rabbit metallothionein-2 pseudogene (MT-2 p ) has been isolated from a partial rabbit genomic library. Its unusual sequence shows evidence of complex rearrangements involving recombination and deletion events. There are no intervening sequences , 3' poly A tract or 5' regulatory DNA sequences. The pseudogene is flanked by two sets of direct repeats (CT)3 GT(CT)4 and CTGG(G)CTC. They are most probably the sites of insertion of MT-2 ~ in the rabbit genome. In addition, a number of repetitive DNA sequences are observed flanking the MT-2~ gene. These are features of a processed retrogene. ©1988AcademicPress, lnc.

Metallothioneins (MTs) are small cysteine-rich proteins which heavy metals. Their synthesis is induced by a variety of stimuli

hind like

cadmium, zinc, copper, glucocorticoids, alpha interferons, iodoacetate, carbon tetrachloride, stress, etc.. (i). These proteins are thought to be involved in copper and zinc metabolism and metal detoxification (2). MTs are ubiquitous

in nature and much interest

has been focussed on the

of their synthesis and evolution. In this paper, we report sequence, evolution, probable divergence and unusual features MT-2 pseudogene ( M T - 2 ~ ) .

regulation

the cloning, of a rabbit

METHODS New Zealand rabbit DNA was purified as described (3). The DNA of 8 grams of a New Zealand female rabbit liver was extracted by homogenization, phenol-chloroform treatment and alcohol precipitation. The DNA was digested with EcoRl and the fragments were separated on a sucrose gradient as described (4), The region of the sucrose gradient which contained 4-6 Kb DNA fragments was isolated. This DNA was then inserted by ligation at the EcoRl site of lambda gtl0 (5). The DNA was then packaged (6) and the phages were grown in E.coli C600 Hfl (F-, lac YI, leu, B6, Ton A21, thi-l, thr-l, Hfl, lambda-). The phages were screened as described by Benton & Davis (7). About 2x106 phages DNA of the partial genomic library were probed with a nick-translated mouse MT-I cDNA (8) in 50% formamide at 37°C for 16-60 hours. The filters were washed 30 min at 25°C. Plaques that hybridized with the probe were picked and plaque-purified three more times. Lambda DNA from

* The order of these first coin.

two authors has been

1403

decided by the

flip of

a

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

these candidate plaques was digested with EcoRl. The fragments were separated on a i~ agarose gel, transferred to nitrocellulose and hybridized according to Southern (9). A clone MT-48, containing a 4.8 Kb DNA fragment that hybridized with the mouse MT-I cDNA was isolated. The 4.8 Kb fragment was sucbloned in pUCI3 (i0) and amplified. The restriction map of the 4.8 Kb fragment was determined and the appropriate fragments were subcloned in MI3 vectors (ii) mpl8 and mpl9 for sequence analysis by the chain termination procedure (12). Genomic blot was also carried out using the second and third exons of the rabbit MT-I gene (13). Specifically, the 1.4 Kb fragment spanning the Saml-Stul-Pstl-Sphl-Pstl-EcoRl sites of clone MT44 (13) was used as probe. Hybridization was carried out at 42°C and washing of the nitrocellulose paper at 58°C in 0.1xSSC, 0.i~ SDS. RESULTS AND DISCUSSION Preliminary experiments showed

that mouse

metallothionein cDNA

would

hybridize with rabbit DNA digested with EcoRl as determined by Southern blot analysis.

The size

of the

fragments that hybridize

therefore proceeded to clone these

was about

4-6 Kb.

fragments. When rabbit DNA was

We

digested

with EcoRI and Southern blot analysis carried out using the second and third exon of rabbit MT-I as probe,

6 bands were observed.

Kb fragment previously shown to fragment reported here to contain and 8 Kb were observed.

In addition to the

contain the MT-I gene (13) the MT-II~ gene,

fragments of

These fragments most probably

4.4

and the 4.8

Kb

2, 2.8,

represent rabbit

3 MT

genes not yet isolated. Figure I shows the restriction map of the 4.8 Kb insert of clone MT-48. Southern blot

analysis of

the

4.8

revealed that the hybridizing region Pvull-Pstl-Pvull

sites. The

Kb fragment

using

was in a small

cloning strategy

MT-I

mouse

eDNA,

fragment spanning

used to sequence

the

the 1.8

Kb

BamHl to BamHl fragment is as indicated (Fig. I). Figure 2A shows the sequence of encodes the M T - 2 ~

gene (the

numbered i). Fig. 2B shows flanking regions repeats,

with A,B,C,D

most probably

1.8 Kb fragment

repetitive

I)

sequences,

DRI

codons and the I to

the

coding

region has

which

codon of the gene

a schematic representation of the

Terl and Ter2 termination

show that

the BamHI-BamHl

A of the ATG initiation

gene and

and

DR2

direct

53 codons. We been

the

will

object

recombination events 2) Terl is the original termination codon and not and 3) the

gene is a

pseudogene which

has been integrated

genome at the DRI site. The gene will be called MT-2P The coding region from and

is its

in the

of Ter2

rabbit

thereafter.

including the initiation codon

ATG to

the

nearest in phase termination codon TAG (Ter 2), is 186 base pair (62 codons) long. The aspartate at position (14). Because the rabbit MT-2 shall compare

the MT-2 ~

ii indicates

coding

sequence

Comparison of the similarity of the MT-I sequence reveals

this gene is

coding sequence has not

3 regions

MT-2~

of

with that

an MT-2~

gene

been determined, of

the

MT-I

coding sequence with the

very high

homology separated

we

(13). rabbit by

two

deletions resulting from two recombination events. The first 21 codons have 84~ similarity. The 22 to 29 MT-2 ~ codons and the 27 to 34 MT-I codons have 88~ similarity. 86~ similarity.

The 30 to 53 M T - 2 ~ After the

termination Terl ATG codon.

codons and the 38 to 61 MTI codons

MT-2 ~ codon Codons

54 to

53,

2 C

are present

62 of MT-2p

whatsoever to any region of the MT-I coding sequence. coding region of

MT-2 ~ may

recombination deletions

have evolved from

(5 codons

and 3

a MT

show no

before

have the

similarity

This suggests that the mRNA or cDNA

by

two

insertion of

two

bases before the termination codon or some other process which put Terl

out

1404

codons) and the

V o l . 156, N o . 3, 1 9 8 8

B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

- - E c o RI - - B g l II

- P v u II

1 Kb

- B a m HI -Bgl

II

11l'It,i I It

--Pvu II -- Pst I Pvu I I --Pvull - - A v a II

- - B a m HI

--Ava II - - P v u II - - B g l II

- - E c o RI

Fig. i

of phase.

Restriction enzyme map of the cloned insert of lambda MT-48. The arrows indicate the direction and extent of sequencing runs. ( ~): sequencing with universal primer; (e =): sequencing with synthetic primer. Transcription proceeds from right to left. The coding sequence is shown with a thickened line.

This shifts

the

reading

frame such

that

the

next

potential

termination codon is at position 187 (Ter2), As illustrated by Figure 3, the rabbit MTI sequence overlapping the two deletions in MT-2 ~ reveals two direct repeat sequences TGCAAATGC (eodons 19 to 21 and 24 to 26) and CTGCTGC (codons 33 to 35 and 36 to 38) which by all probability must

have specified

mispairing mechanism

(15). As

these repeated sequences

the

sites for

expected,

deletion

elimination by

in a two step process

characteristics besides various genetic lesions: found in the functional counterpart are missing; the pseudogene

corresponding to

the

slipped

(Fig. 3A and 3B) yielded

deletions of 5 and 3 codons found in M T - 2 ~ . As reviewed b y Vanin (16), most-processed pseudogenes

between

by

recombination

share 4

(I) intervening

of the

sequence sequences

(2) the sequence similarity

and its functional counterpart stops at the points

the beginning

and

the end

1405

of the

transcript

from

the

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A _GGA_TC_CAGACTGCCTGGTTCAGTTTCTGGCTCTGCTACTCATTAGCTGTGTGACTGGGAAAGGCAA BamHI TCTTTCTGTACTTCAGTTGTTTCCTTATATACAAATAGAGATA~GGACTTTATCCGAGAGTTATGAGAATTAAGT GCATTAATATTTTTATTTATTTATTTATTTATTTATTTATTTGACAGGCAGAGTGGACAGTGAGAGAGAGAGAGA CAGAAAGAAAGGTCTTCCTTTGCCGTTGGTTCAC CCTCCAATGG CCGCCGCGGC CGGCGCAC CGCGCTGATC CGG TCGCAGGAGCCAGGAGC CAGGTGCTTTTCCTGGCCTCCCATGGGGTGCAGGGCCCAAGCACCTGAGCCATCCTCC ACTGCACTCCCTGGCCACAGCAGAGAGCTGGCCTGGAAGAGGGGCAAC CGGGACAGAATCCGGCGCCC CGACCGG GACTAGAACCCGGTGTGCTGGCACCGCTAGGCGAGGATTAGCCTAGTGAGCCGCGGTGCCGGCCTAAGTGCATTA ATATTTAAAGTGTTG~CTAAAATGGG.AC_CGGCGCCGTGGCACAGTAGGCCAAGCCT AvaII CTGCCTGTGGGGCTGGCATCTCATATGGGCGCCAGTTCATGTCGTGGCTGCTCCTCTTCTCATCCAGCTCTCTGC TATGGCCTGGGAAGGCAGTAGAAGATGGCCCAAGTGCTTGGGCCCCTGCAC CTGCATGTGAGAC CTGGAAGAATT TCCTTGCTCCTGGCTTCGTATTGGCCCAGCTCC__AGC_TGTTGCGGCCATTTGGGGAGTGAACCAGTGGATGGAAGA PvuI I C CTTTCTCT CTGT CTCT CTCCTC TGTAACTTTACCT CTCAAATAAATGAATAAATCTTTTAAAAATATTTGAAGT GTTTAACACACCTTCCAACTGCCGGACTGACTCTTCACCTTTCGCTTTGTATTTCCAGCTTCACCTGGGCTCAAA ATGGAGCCCA_GCT_GCTCCTGCATCACCAGTGACTCCTGCACCTGTGCCAGCTCTTGCACATGCATGTCCTGCAG_G MetGluProSerCysSerCys IleThrSerAspSerCysThrCysAlaSer S erCysThrCysMe tSerCysArg PvuII PstI AAGAGCTGCTGCCCAGTGGGCTGTGTCCAGTGTGCCCAGGGCTGCATCTGCAAAGGGGCATCAAACAAATGCAGC Lys SerCysCysProValGlyCysValGlnCysAlaGlnGlyCys I leCysLysGlyAlaSerAsnLysCys Ser PvuII _TGCTGCGCCCCTGAGAGCCTGGCTCCCACATGCAAATA__GGAGCAACGTGTGCAAACCTGGATTTATACCCCCATAC CysCysAla Terl Ter2 AACCTGACTT CTATGCTG CATTCCTTCTTGTGTGAAATATGTGAATGAGAATACAAGTTGTTGAT _TT.T_ .A~_ _ GAA AhaIII TAAAGTGTTTAGAACATGGTCTAGGGGCCAATGTCGTGGCATAGCTGGTTTAGCCACTACCTGCGATGCTGGCAT CCCACATGGGTACTGGTTTGAGTCCTGGCTGCTCCACTTCCAAGCCAGCTCCTGCTAATGCACCCAGGCCTGGCT GTTGCAGCCATTTGGGGAGTGAACCATAGAATGCAAGAAGATGGCTCAGTGCTTGGGCACTTGCACCCAAGTGGG AGACCCAGATGCAGCTCCTGGTTCCTGGCTTTGGCTTGGCCCAGCCCTGGCCATTGCAGCCAGTTGGGGAATGAA CCAGCAGCTTGGAAGATCTCTCTGTCTCTCTCTCTCTCTCTGTAACTCTTTCAAATAAGCAAATCTGGCCCGGGC BglII CGCGGCTCAACAGGCTAATCCTCCGCCTAGCGGCGCCGGCACACCGGGTTCTAGTCCCGGTTG CC CCTCTTCCAG GCCAGCCCTCTGCTGTGGCCCGGGAGTGCAGTGGAGGATGGCCCAGGTGCTTGGGCCCTG CACCCCATGGGAGAC CAGGAAAAGCACCTGGATCC BamHI

B -.. ATTT_..GA.._ AA...CT.._ A AA~_.CCTGG G C T C . . _ ~

A

B

Fig. 2

gene;

adenosyl

rich d i r e c t

the sites

similarity

sequences, and 9

most

3'

DR1

-301 -226 -151 - 76 -1 75 150 225 5O0 375 450 525 500 675 750 825

CCTGA__.CCTGGCTC___AAATAG__.CT.__

1....... 53

other

is the p r e s e n c e

which

the

ends;

repeats

of i n s e r t i o n to

between

MT-2 ~

regulatory 8

at

counterpart

immediately rabbit

D

(3) there

the p o i n t

functional mark

DR2

-826 - 751 -676 -601 -526 -451 -376

Ted

DR1

62 Ter2 DR2

A: DNA sequence of the MT-2~ gene region. The 1804 bp containing the regions hybridizing to a mouse MT-I cDNA clone is shown. Numbering is from the initiation codon. The direct repeats flanking the gene and the repetitive sequences which are discussed in the text are underlined. B: Structure of MT-2P gene region. The coding region is boxed in. Numbering refers to codon numbers. Ters are termination codons. Direct repeats are indicated by arrows. A, B, C and D designate repetitive sequences.

functional 3' to

C

-901

the

homology

(4) the of

poly

shows

and

and

are c o m m o n l y direct

into

the

immediately

its f u n c t i o n a l

the The

sequences,

pseudogene,

sequences alpha

etc..) and is f l a n k e d b y d i r e c t the sequence

1406

occur

ends.

no p r o m o t e r

DR1

by

which

counterpart

regulatory

processed

repeats, where

sequences,

(labelled

its

to

glucocorticoid CCTGG(G)CTC

and

flanked

genomes,

5'

no i n t e r v e n i n g

pair:

immediately

pseudogene

These

the p s e u d o g e n e s

A tract

T A T A b o x and CCAAT box, base

the

pseudogenes

of 7-17 base pairs.

the p s e u d o g e n e

sequences,

of a p o l y A s e q u e n c e between

in Figure

2B).

homology

between

(metal

interferon repeats

In contrast MT-2P

of to and

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

22 23 24 25 26 27 28 29 30 31 32 33 34 35 AAAGAATGCAAATGCACCTCCTGCAAGAAGAGCTGCTGCTCC MT -1 17 18 19 20 21 22 23 24 25 26 27 28 29 30

AGCTCCTGCAAATGCACCTCCTGCAAGAAGAGCTGCTGCTCC 17 18 19 20 21 27 28 29 30 31 32 33 34 35

MT-I after i deletion

30 31 32 33 34 35 36 37 38 39 40 41 AAGAAGAGCGTCTGCTCCTGCTGCCCCGCCGGCTGC MT-I after 1 deletion

ACCTCCTGCAAGAAGAGCTGCTG+CCTGCTGCTGC 27 28 29 30 31 32 33 34 35 36 37 38

B

MT-I after 2 deletions

ACCTCCTGCAAGAAGAGCTGCTGCCCCGCCGGCTGC 27 28 29 30 31 32 33 34 38 39 40 41

17 18 19 20 21 27 28 29 30 31 B2 33 34 38 39 AGCTCCTGCAAATGCACCTCCTGCAAGAAGAGCTGCTGCCCCGCC

MT-I after 2 deletions

AGCTCTTGCACATGCATGTCCTGCAGGAAGAGCTGCTGCCCAGTG 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

MT-2~

C

Fig. 3

Possible recombination events of the rabbit MT-I cDNA for the deletion of direct repeat sequences to give the pseudogene MT-2P. The two upper sequences in part A and B of this figure depict a region of the MT-I cDNA (numbers refer to codons from the initiation codon ATG); the lower of the two sequences are the upper sequence shifted 5 or 3 codons to the right. Dotes indicate the alignment of identical nucleotides; a row of these indicate a region of direct repeat in the cDNA; the 2 predicated sequences which would occur after deletions caused by slipped mispairing mechanism (15) are also given. Part C of the figure shows the substitutions that could have taken place for the evolution of the pseudogene MT-2~ from the recombination deleted cDNA for the region shown.

its f u n c t i o n a l the b e g i n n i n g

counterpart

sequence homology Moreover, repeats

the

the m e c h a n i s m

a repetitive repetitive

almost

of i n s e r t i o n

of

of the p o i n t s

corresponding

f r o m its f u n c t i o n a l

immediately

5'

sequence

adenosyl

repetitive sequence

and 3'

is a b s e n t

residues.

the m R N A

sequences of A T T T

GA f r o m p o s i t i o n

and a r e p e t i t i v e

( l a b e l l e d A,

These

gene.

In

to the c o d i n g a n d the

region. direct

suggest

the f o r m a t i o n

to

fact,

flanking

differences

or c D N A in

of

-764 to

sequence

a number

the i n t e r a c t i o n s

and f u n c t i o n s

that r e p e t i t i v e

sequences

information

in

processed

interactions

are f o u n d 5' a n d 3' to the c o d i n g

from position

B, C and D in Figure

to be a s s o c i a t e d w i t h

desirable

short

that MT-2~

f r o m that of other p r o c e s s e d p s e u d o g e n e s .

Several

to -416

stops

3' p o l y A s e q u e n c e

are n o t r i c h in

may differ

ceases w e l l

and end of the t r a n s c r i p t

may

-755;

to

-783;

from p o s i t i o n

Repetitive

-Iii

sequences

(17-19).

of

sequences.

repetitive

facilitate

1407

no

increasing

functional

is k n o w n

the

the

units

about is

genetic

chances

(19).

-96

shown

One p o s s i b i l i t y of

of -435

to p o s i t i o n have been

Little

the d i s p e r s i o n

thereby

otherwise

region:

a sequence

of 22 A f r o m p o s i t i o n

of p s e u d o g e n e s

pseudogenes

between

a tract

of A A A T 2B).

-823

of

Besides

Vol. 156, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the direct repeats

(DR1) flanking

the coding sequence,

alternating CT are located from position to position 647

(labelled DR2

direct repeats

of

-146 to -121 and from position

in Fig. 2B).

This repetitive

CT

619

sequences

probably mark the sites of a second integration event which occurred in this region of the genome before or after the DR1 integration since repetitive CT sequences have been implicated as sites of DNA recombination and integration

(20) MT genes from human (21,22), rabbit

sheep (24), mouse

(13) and MT cDNAs from Chinese hamster

cloned.

(25,26),

rat (27)

(28) and monkey

and

(29) have been

The number of copies of MT-I gene, MT-2 genes and pseudogenes varies

in different species. Rodents appear to have one copy each of MT-I and MT-2, whereas primates have multiple copies.

Pseudo MT genes have been detected in

human (21,22) and rat (27) but not in mouse difference

in the expression of

MT genes

(2) (possibly because of species in germline cells).

This is

the

first report of the presence of an MT pseudogene in rabbit. From the isolation of rabbit MT genes, the MTs

can be

examined.

homologies between the

Table 1

coding sequence

mammalian MT genes

or cDNA.

homologies between

the coding

mammalian MT genes pseudogenes

(27)

calculations. homology

or cDNA.

with

sequence

be seen from

rabbit

identification of MT-2~

Table 1

are

than

derivative of rabbit

acid cloned

of

included

other

in

the

higher amino

acid

confirming

the

MT-I,

MT-2 mRNA.

For the

compared,

parent MT-2.

In addition to the two internal deletions and the insertion a total

the 61 amino acids. The differs from insertion,

the

8 and

between MT-2~

of 15 mutational changes have

only rabbit MT-2 protein isoform

other mammalian

between residues

12 substitutions

other

amino acids

the CC dinucleotide,

are

rabbit

acid cloned

and amino

of analysis not

MT-2Y has a

as a

amino other

MT-I and

the method

insertions with

and and

the nucleotide

MT-2 there

MT-2

of rabbit

Similar to and

nucleotide

of rabbit

Table 2 shows

deletions

As can

the evolutionary relationship of

shows the

I0

MTs

in having

(30). Because

MT-2 Y pseudogene must

have

its of

taken place

in

sequenced so

an alanine

MT-2Y

53

and

residue

DNA does

not

evolved before

the

far

inserted have

this

insertion

event.

Table i. Nucleotide and amino acid homologies between the 53 codons of the rabbit MT-2 ~with MT genes or cDNA: rabbbit MT-I (13), rabbit MT-2 (30), Chinese hamster MT-I and MT-2 (28), rat MT-I (27), mouse MT-I (25), mouse MT-2 (26), sheep MT-Ia (24), monkey MT-I and MT-2 (29), human MT-Ia (21), human MT-2a (22), Deletions and insertions are not included in the calculations. Nucleotide homologies (%)

Amino acid homologies (%)

Rabbit MT-I Chinese hamster MT-I Rat MT-I Mouse MT-I Sheep MT-la Monkey MT-I Human MT-Ia

136/159 128/159 128/159 125/159 133/159 130/159 134/159

(86) (81) (81) (79) (84) (82) (84)

Rabbit MT-2 Chinese hamster MT-2 Mouse MT-2 Monkey MT-2 Human MT-2a

123/159 127/159 137/159 140/159

(77) (80) (86) (88)

41/53 35/53 37/53 40/53 41/53

(77) (66) (70) (75) (77)

1408

39/53 38/53 40/53

(74) (72) (75)

38/53

(72)

40/53 39/53 38/53

(75) (74) (72)

Vol. 156, No. 3, 1988

Table 2.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Nucleotide and amino acid homologies between the coding sequence of rabbit MT-I with other cloned mammalian MT genes or cDNA. Deletions and insertions are not included in the calculations. References are as in table i. Nueleotide homologies (~)

Amino acid homologies (~)

Chinese hamster MT-I Rat MT-I Mouse MT-I Sheep MT-Ia Monkey MT-I Human MT-Ia

162/183 160/183 156/183 160/183 163/183 163/183

(89) (87) (85) (87) (89) (89)

52/61 52/61 50/61 50/61 54/61 53/61

(85) (85) (82) (82) (89) (87)

Rabbit MT-2 Chinese hamster MT-2 Mouse MT-2 Monkey MT-2 Human MT-2a

156/183 157/183 164/183 168/183

(85) (86) (90) (92)

54/61 51/61 50/61 54/61 56/61

(89) (84) (82) (89) (92)

All mammals Therefore,

examined

contain

two

major

forms

of

metallothionein.

it has been suggested that these isoforms have arisen from a gene

duplication prior to the mammalian

radiation.

This suggestion implies

that

interspecies homology of one isoform should be greater than the intraspecies homology of the two

isoforms. This was

rodents and primates were compared rodent MTs

are

compared,

one

found to

separately.

finds that

divergence from primate MT-2s than from

be true when

However,

the

the MTs

of

if the primate

primate

MT-Is

and

show

rodent MT-Is. Therefore,

less

it is

not

apparent that the presence of two MT isoforms preceded the speciation event that separated rodents and primates. To maintain that the two MT isoforms arose prior to the speciation for this

apparent

event,

convergence

possibility is that

in the

in

evolution of

conversion of gene between the similar. Another

two possibilities have been the direction

possibility

of

evolution

the primates,

MT-I and MT-2 making the is

that

all the

suggested (I).

there h a d

One

been

two isoforms

functional

genes

a

more

in

the

primates evolved from one isoform with the other isoforms inactivated.

Table

2 shows that the amino acid sequence of rabbit MT-I is more similar to

that

of rabbit MT-2 than to that of other rodent MT-Is.

Table 1 shows that rabbit

MT-2 ~ is more similar in coding sequence

and amino acid sequence to

MT-I than to

similar to

other rodent

rabbit MT-I and MT-2 two isoforms radiation.

have

MT-2s. Thus,

the primate

sequences do not substantiate the hypothesis arisen

Interestingly

from

a

gene

enough, rabbit

duplication MT-I

coding sequence and amino acid sequence to rodent MT-Is. Rabbit MT-2 ~ also

prior

shows higher

to

rabbit

MTs,

the

that

the

mammalian

similarity

in

primate MT-Is and MT-2s than

to

shows higher similarity in coding

sequence

and amino acid sequence to primate MT-Is and MT-2s than to rodent MT-2s.

The

explanation to this is not apparent.

REFERENGES

i. 2. 3.

Hamer, D.H. (1986) Ann. Rev. Biochem. 55, 913-951. Beach, L.R., and Palmiter, R.D. (1981) Proc. Natl. Acad. 78, 2110-2114. MeKnight, G.S. (1978) Cell 14, 403-413.

1409

Sci.

U.S,A.

Vol. 156, No. 3, 1988

4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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