Molecular Nature of Spontaneous Mutations in Mouse Lactate Dehydrogenase-A Processed Pseudogenes

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Molecular Nature

of

Spontaneous Mutations in Mouse Lactate

Dehydrogenase-A Processed Pseudogenes

Kayoko

M. **Fukasawa,* Masako Tanimura; Ikuya Sakai," Farida**

S. Sharief," Fu-Zon Chung*

**and Steven S.-L. Li*"**

"Laboratory of Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, and ?Center for Demographic and Population Genetics, University of Texas, Houston, Texas 77030

Manuscript received June 1 1 , 1986

Revised copy accepted September 15, 1986

ABSTRACT

The presence of at least ten mouse LDH-A pseudogenes was demonstrated in the genomic blot analysis, and four different processed pseudogenes have thus far been isolated and characterized. In this report, the nucleotide sequences of two different mouse lactate dehydrogenase-A processed pseudogenes, M 1 1 and M14, were determined and compared with the protein-coding sequences of the mouse and rat LDH-A functional genes. In the pseudogene M1 1 , the sequence of 64 nucleotides from codon no. 257 to 278 was tandemly duplicated. In the pseudogene M14, the sequence of 22

nucleotides from codon no. 68 to 75 was replaced by an inserted repetitive sequence of 242 nucleotides homologous to a mouse truncated R element. The pattern of nucleotide substitutions accumulated in mouse LDH-A pseudogenes M 1 1 and M 14, as well as that of pseudogene M 10 identified previously, was analyzed, and the substitution frequencies of the C or G at the CG dinucleotide were found to be high.

PROCESSED pseudogene is a DNA sequence

A

that is derived from the processed mRNA inter- mediate of a functional gene through retrovirus-like mechanism and that is either transcriptionally inactive because of the absence of promoter or incapable of

producing a functional protein because of the presence of frameshifts or nonsense mutations (LITTLE

1982; LUEDERS et al. 1982; NISHIOKA, LEDER a n d LEDER 1980; PROUDFOOT 1980; SHARP 1983). Thus, spontaneous mutations occurring in such pseudogenes a r e apparently not subject to functional (selective) constraints and may become randomly fixed in pop-

ulations. Processed pseudogenes have been found in

many mammalian gene families (LI 1983; WILDE

1986), and they provide a repertoire for investigating

the molecular nature of spontaneous mutations in mammals.

Lactate dehydrogenase (LDH; EC 1.1.27) catalyzes the interconversion of lactate and pyruvate with nic- otinamide adenine dinucleotide as coenzyme (EVERSE and KAPLAN 1973; HOLBROOK et al. 1975). In mammals, LDH isozymes A4 (muscle), B4 (heart) and C4

(testis) a r e encoded by three structural genes origi- nated from an ancestral gene during the course of evolution (MARKERT, SHAKELEE and WHITT 1975; LI

et al. 1983). T h e expression of these three LDH genes is developmentally regulated and tissue-specific. In order to understand the structural and evolutionary relationships of the LDH-A, B and C genes and to

' To whom correspondence should be addressed.

study the mechanism of gene regulation, we have undertaken the investigation of protein structure a n d

gene organization of mammalian LDH isozymes. We

have reported the primary structure of LDH-A4 isozymes from mouse and human as well as LDH-C4 isozymes from mouse a n d rat (LI et al. 1985; PAN et al. 1983; TSUJIBO, TIANO and LI 1985). We have also described the nucleotide sequences of LDH-A cDNA and processed pseudogenes, and the exon-intron or-

ganization of LDH-A functional genes from mouse and human (AKAI et al. 1985; LI et al. 1985; TSUJIBO,

TIANO a n d LI 1985). In this paper, we present the

nucleotide sequences of two newly-identified mouse LDH-A processed pseudogenes, the molecular nature

of spontaneous mutations in three mouse LDH-A pseudogenes, and the complexity of LDH-A gene- related sequences in the mouse genome.

MATERIALS AND METHODS

Molecular characterization of mouse LDH-A pseudo-

genes: The mouse (Mus musculus C57/B10) genomic library

was kindly provided by M. EDGELL (WEAVER et al. 1981),

and the genomic clones containing LDH-A gene-related sequences were isolated as previously described (BENTON

and DAVIS 1977; Rigby et al. 1977). The DNAs purified from clones XM 1 1 and XM 14 were analyzed by restriction mapping and Southern-blotting (SOUTHERN 1975; MANIA- TIS, FRITSCH and SAMBROOK 1982). The isolated DNA fragments containing LDH-A pseudogenes were further cleaved and subcloned into M13 mplO/mpll phages, and their nucleotide sequences were determined by the dideoxy chain termination method (MESSING, CREA and SEEBERG

198 1 ; SANGER, NICKLEN and COULSON 1977).

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178 K. M. Fukasawa et al.

Analysis of genomic complexity: T h e high-hl, DNAs were isolated f r o m t h e liver of mouse (C57BLj6J and DBA/ 25) a n d were cleaved with restriction endonucleases EcoRI,

BamHI and HindIII. After electrophoresis on 0.75% aga-

rose gel, t h e D N A fragments were transferred t o a nitrocellulose filter a n d hybridized with t h e ”P-labeled mouse

LDH-A cDNA probe (AKAI et al. 1985).

RESULTS

Nucleotide sequences of mouse LDH-A pseudo- genes M l l and M14: Six mouse genomic clones containing LDH-A gene-related sequences were isolated from Charon 4A genomic library (WEAVER et al.

1981). Clone

AM15

possesses mouse LDH-A functional gene, and its exon-intron organization was described (FUKASAWA and LI 1986; LI et al. 1985). Clone AM 10 contains an LDH-A processed pseudogene and clone

AM

16 possesses a truncated LDH-A pseudogene; their nucleotide sequences were reported (AKAI

et al. 1985; FUKASAWA et al. 1986). In this investigation, clones

AM1

1 and

AM12

exhibited identical restriction patterns, presumably containing the same LDH-A gene-related sequence; clone AM14 has a quite different restriction map. Figure 1 indicates the restriction endonuclease maps of the clones

AM

1 1

(AM1

2) and

AMI

4, along with their DNA sequencing strategies. T h e nucleotide sequences of approximately

1.8 and 2.5 kb from clones

AM1

1 and AM14, respectively, are given in Figure

2.

Sequence comparison of clones

AM

1 1 and

AM

14 with the mouse LDH-A functional gene (clone AM 15) indicated that clones

AM

1 1

and AM 14 contained different mouse LDH-A processed pseudogenes lacking all seven introns present in the LDH-A functional gene. Both pseudogenes

M11

and

M

14 contain two direct repeats of 12 base pairs (bp) and 8-bp, respectively; one located just upstream from the

5’

end and the other just downstream from the poly(A) tail. The nucleotide sequences upstream to their 5‘ direct repeat are very different from that of the promoter region of mouse LDH-A functional gene (FUKASAWA and LI 1986). Therefore, the LDH- A processed pseudogenes

M

1 1 and

M

14 are transcriptionally inactive because of the absence of a promoter. Furthermore, the “coding” sequences of both pseudogenes are incapable of producing LDH-A functional polypeptide chains as described below.

Evolutionary relationships: In order to infer the evolutionary relationships among the mouse processed pseudogenes and the mouse and rat functional genes, and also to infer the pattern of spontaneous mutations, we obtained an alignment of these sequences as shown in Figure 3. From this alignment, the number of nucleotide differences between each sequence pair can be easily inferred, and the results are shown in Table 1. It is clear that all of the three mouse pseudogenes are more closely related to the mouse functional gene (M15) than to the rat func-

I k b H

R X X _I x x R

A M I I AS- .I,

I.

AI

R 200bp

- H

l k b

R R R R R - R

AM14 AS] I A L

---’ I I

I

W b k

- - - l ~ 4 + +

* c ( A

FIGURE 1 .-Restriction endonuclease maps of the mouse LDH- A processed pseudogenes MI 1 and M14 and their nucleotide sequencing strategies. The overall maps of the mouse genomic clones AM11 and AM14 for restriction endonucleases R (EcoRI) and X ( X b a I ) were derived from single/double digestion as well as partial cleavage and Southern blot analyses with LDH-A cDNA probes from mouse and human. The “coding” sequences are indicated by solid blocks. The 5’ and 3’ untranslated regions are dotted. AS and AL indicate the short (10.5 kb) and long (20 kb) arm of Charon 4A

phage, respectively. The isolated EcoRI-XbaI fragment of 1.8 kb from clone AM1 1 was further cleaved by P (PstI) and S (SauSAI), and subcloned into M13 mplO or mpl1 phages. The isolatedEcoR1 fragment of 4.2 kb from clone AM14 was further cleaved by G

(BglII), P (PstI), S (SauSA), T (TagI), and/or A (AluI), and subcloned into M13 mplO or mpl1 phages. The ssDNA from M13 phages exhibiting positive hybridization with mouse and human LDH-A cDNA probes were purified and the nucleotide sequences of the inserted DNAs were determined by dideoxy chain termination methods.

tional gene (MATRISIAN et al. 1985). Therefore, all of these pseudogenes appear to have arisen after the mouse-rat split. Further, the number of nucleotide differences between

M

15 and

M

1 1,

M

10 or M 14 are

56, 79 and 84, respectively. For this reason, we pro- pose the branching order shown in Figure 4. Using the maximal parsimony principle, we have also inferred the number of nucleotide substitutions in each branch of the phylogenetic tree.

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A

M11 gaattctacatcagggacccaaagc

ccaggagtttcctaaattaaactcaaacactaaagagtcataagttcaaaccaaggtaagccagactgggagttcataggactggcct a a a a a a t

Eco R I

*

G T G T C T G G A G C C A C T G T C G E C G A G C C C A A G C C A T G C T G C ~ C C C A T C G T G E A C T A G C G G T E T C A A A A G A T T C A A A G T C C A A

GATGGCAACCCTCAAGGACCAGCTGATTGTGA4TCTTCTTAAGGAAGAGCAGGCTCCCCAGGACAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATG

GCTTGTGCCATCAGTATCTTA4TGAAGGGCTTGGCGGATGAGCTTGCCCTTGTTGACGTCATGG~GACAAACTCAAGGGCGAGATGATGGATCTCCAGC

ATGGCAGCCTCTTCCTTAAAACACC4AAAATTGTCTTCAGCAAAGACTACTGTGTAACTGCGAACGCCAAGCTGGTCATCATCACAGGGGTGTCTGTCAG

C AAGAGGGGG4GAGCCAACTCAACCTGGTCCAACGAAACATGAACATCTTCAAGTTCATCACTGCCAACATTGTGAAGCACAGTCCACACTTCGAGCTGC

TGATTGTCTCAAATCCAGTGGATATCTTGACCTACGTGGCTTGG~AATCAGTGGCTTTCCCAA4AGCGGAGTTATTGGAAGTGGTTGCAGTCTGGATTC

4GCTCGGTTCTGTTACCTfATGGAAG4AAGGCTGGGGGTTCACCTGAGCTGTCATGGCTGGGTCCTGGGAGAACATGGCAACTCCAGTGTGCCTGTGTGG

49TGGTGTGA4CGTTGCCGGTGTCTCCCTGAAGTCTCTGAACCCAGAACTGGGCACTGATGCAGAC~GGAACAGTGGAAGGAGGTTCAAAAGCAGGTGT

GGACAGTGCCTCCAAGGTGATCA4GCTGAGAGGTTACACATCCTGGGCCATTGGCCTCTCTGTGGCAGACTTGGCCGAGAGCATAATGAAGAACCTTAGG

CGGGTGC4TCCCATTTCCACC4TGATT4AGGGACTTGGTCGAGAGCATAATGAACCTTAGGTGGGTGC4TCCCATTTCCACCATGATTAAGGGTCTCTAT

GGA4TCAGTGATGACGTCTTCCTCAGTGTCCCATGTATCCTGGGACAAAATAGAATCTCAGATGTTGTGAAGGTGACTCTGACTCCCGAGGACGAGGCCC

GCCTGAAGA4GAGTGCAGACACCCTCTGGGGAATCGAGAAGGAGCTGCAGTTCTAAAGTCTTCTGAGTGTCCTAGCACTTCACTGTCCAGGCTGCAGCAG

GGTTTCTATGGAGACC4CACCCTTCTCTTCTGAACTGTGGTTAGTACAGTGGTTTTGAGGTGGTGTGGGGAAACATCTCACTCCCCACAGCTCTGTTGCC

AAGTGGTACTTGTGTAGTGGTAACCTGGTTAGTGTGAC4GTCCCGCTGTCTCCGAGACACACTGCCAACTGTGCAGGCTTCAATTACCCTGTGAGCCTGC

TGCATTGCTGCCCTGCGCACCCTCACCAAACATGCCTAGGCAACGAGTTCCCAGTTAGTCATAACCTGGCTCCAGTGTGTACGTCCATGATGCATATCTT

GTGCATGA4TGTTGTACAGGATATTTTATGTATTATATGTGTCTGTAGTGTGCATTGCAATGTTATGTGAGATGTAAAATCTGCATATGGAT~TGGAAC

t l

#257

X277U257 W277

#331

tA4GGACCCAAGTGTCATGCCAATGAAAACACCAAATAAACCTTG~CAGTGAAAAAAAAAAAAAGAGA a a a a a t t t c t a g a s i g n a l Poly (A) Xba I

B

H I a Eco R I

...

ctattcatgtctctatgaaaccaaaggaattgagatatcaagttcttcaaatttttt tgtttatgtttaaattaaatttaaaagcatactctcaactgagggggagggggaggaggagagggagagggagaggaggagacttttacccagcactccc

tcatqgtcaagaacagagacagatgagattctctqcatcttgggtcttgcaactgaggaaagccatcacgttccagcctcagagaaagtgaaaacaggc~

C G T C c A G C C i C 4 A A A G A T T E A A A G T C C A 4 ~ C C G C A A C C C ~ C A A G G A C C A ~ C T G A T T G T G ~ A T C T T C T T A ~ G G 4 A G A A C A ~ G T C C C C C A A ~ A C A A G A T T A ~ 4TTGTTGGGGTTGGTGCTGTTGTCATGGCTTGTGC TATCAGT4TCTTAATGAAGGACTT4GTGGATGAGCTTGCCCTTGTTGATGTTCTTGGAAAACAAG

CTAA4GGGAGAGATGATGTATCTCGAGCATGGA~tgggaggagattccctttgtcctatgaaggctctatggtccagtgtgggggaatggcaggg

caggagtcaggagtgggtgggttggtgagcaggggagggtggaggaaatggagggggtgtttttcggagggcaaatgaggaaagaggataacatttgaga

tgqaaataaagaaaotatccaataaaagcaaaaaacaaaacaaaacaaaacaaacaaacaaacaaaaaaccatc AAATTGTCTCCAGC4AAGACTATTG

TGT4ACTGCA4ACTCCA4GCT6GTCATTATCACACTGTTGGCCTGTC4GCATAGG~G4AAGCCGACTCAGTTTGGTCCACAGA4ACGCGAACACTTTAAG

TTCATCATTGCTAACATTGTGAAGTACCTACCACACTGCA4GCTGCTTATTGTCTAATTCCAGTGGATATCTTGACCTATATGGCTTGGAAA4TCAGCGG

CTTTCCCAAAATCCGAGTTATTGGAAGTGGTTGCAGTCTGGATTCAGCTCAGTTCTGTTACCTGATGGGAGAAAGGCTGGGGGTTCAGGCGATG4GCTGT

CACGGCT65GTCCTGGGAGAACATGGCGACTCCAGTGTGCCTGTGTGGAGTGGTGTGAACGTTGCCGGCATGTCCCTGAAGTCTCTGAACCCAGAATTGG

GCACTGATGCAGAC4AGGAGCAGTGGAAGGAGGTTCACAAGCAGGAGGTGGACAGTTCCTACG4GGTGATCAAGCTGAAACTGGGCCATTGGCCTTTCTG

T~GCAGACTTGTTCGAGAGC4TAATGAAGAACCTTAGGCGGGT~CATCCCATTTCCACC4TGATT~GGGTCTCTATGGAATCAATGATGATGTCTTCCT

C4GTGTCTCATGTATCCTGGGACAAAATGGAATCTCAGATGTTGTGAAGGTGACACTGACTCCTGAGGAAGAGGGCCGCTTGAAGAAGAGTGCAGATACA

CTCTGGGGAATCCA~AA4GAGTAGTTCTAGTCTTCCCAGTGTCCTAACACTTCACCGTCCAGGCTGCAGCAGGGTTTCTATGGAGACCACAATACTTTTC

ATCTGAGTGGTGGTTAGT4CAATTGTATTGAGATGGTCTGGGGGA4AAAAAAAGCCCTCCATTCCCACAGCTCTGCCCTGCTGCCA4GTGGTACTTGTGT

AGT6GTAACCTGGT4TTGTGAT4GTCCCACTGTCTCCAAGAC4CATTGCCAACTGTGTTGCAGGCCTTGCTGCACAACATCACCAAACTTGCCTAGGCCA

ATGAGTTCCCAGTTAGTCATAACCTGGCTCCAGTGTGTAAGTCCATTATGCATATCTTGTTCTACAGGAT4TTTTATGTATTCATATGTGTCTCTAGTAT

11

Y67

- - 9 1 5

Y331

AC4TTGTACTATGTGAAATGTAAGATCTGCATACGGATGACGGAACCAATCCACTCAAGTGTCATGCCAAGGAAAACACCAAATATACCTTGAACAGTGA

s i g n a l

4A44AAAATaa a qc ttacacttgtacataacagaaacaagcaagtcaagtaaagaaaaatattttggagatgagctttattatggaacagaaacta gccttaattttaccacttatttatttatttatttatttattttttctatgacaatttcatgcaggtatataatgtattagggtcagtgtaagcctctatt

actc~ttcttggctcctatcattgtgaaggaataactttttgttccaaaatgtatctctcctttatcgtctttttgttttgttatcttcacctttgcttt

taaatgactttctgagttacttagggttgtttgtgtqatcacggatgggatcttatttactggacaacaggcagctcactgtgcctatgctactgaaga~

a a t g a c t c t c c a t c t c c a g c a a c c g g t a a c t a t c a a t a g c t c t t c a g g g a g t a g t g g t g c c t t a t a a c a t c t t c t t c a t t c a t g t t g g g a c t t t g a t g g a

t c c a a t c t t a t g c a g t c c a a a t g c a g a c a a

...

E c o ~ I P o l y ( A )

+

-

101

- 1

100

200 300 400

500

600

700

800

900 1000 1100 1200

1300

1400

1500

1600

-

201

-

in1

- 1 100

200

300 400

500

600

700

800

900 1000 1100

1200 1300

1400 1500 1600

1700

1600 1900 2000

2100

2200

FIGURE 2.-Nucleotide sequences of the mouse LDH-A pseudogenes M I 1 and M I 4 and their flanking regions. The nucleotide sequences flanked by direct repeats of the mouse LDH-A pseudogenes M 1 1 and M 1 4 are numbered from the 5 ’ to 3’ end, and the number of the last nucleotide in each row is given at the right-hand side. The flanking sequences of LDH-A pseudogenes M 1 1 and M 1 4 , as well as the insertion sequence of 242 nucleotides in pseudogene M 1 4 , are given in lower case, and the direct repeats are denoted by arrows. The numbers (#) of some codons, the presumptive polyadenylation signal and poly(A) tail are also indicated.

(4)

180

M 1 5

M I 0

Mll

M14

r a t

M 1 5

Mlll M l 1 M l J

r a t

Ml5 MlO M l l

M I 4

r a t

M15 M1O M l l M14

r a t

M15 M10 M 1 1 M14

r a t

M15

M l O

M 1 1 M14

r a t

M 1 5 M l O M l l M14

r a t

MI5

M i l l

MI 1

M14

r a t

M l 5

M l l l

Ml I

Ml J

r a t

K. M. Fukasawa et al.

1 10 20 30

Het A 1 ~ T h r L ~ u l y s A s p t i l n L e i i I 1 eVa I AsnLeiiLeuLysGluGluGInAl aProGl nAsnLys I IeThrVal Val GlyVal GlyAl aVal tilyMetAl aCysAl d I 1 eSer I 1 eLeu

4 l ~ l ; C A A C C C l C A A l ~ ~ A ~ C 4 ~ C l ~ A T l t i l t i A A T C T l C ~ l A A G G A A G A G C A G G C T C C C C A G A A C A A G A T T A C A G T T G T T t i G t i t i T T t i G T G C T t i l T G t i C A T t i G C T T t i T t i C C A T C A G T A T C I T A

...

.CA..

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A... .TC...

.

.A..

...

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...

.T...

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...

G

...

A

....

TC

...

Met LjsAsgLeuAl aAspGluLeuAlaLeuVa1 AspValMet GluAspLysLeuLysGlyGl UMetMetAspLeuGlnHi sGly SerLeuPheLeuLysThrProLys I leva1 SerSer

Al(;AAGGACTTtiGCL;GATGAGCTTGCCCTTGTTGACGTCATtiGAAGACAAACTCAAGGGCGAGATGATGGATCTCCAGCATGGCAGCCT~ T KCTTAAAACACCAAAAATTtiTCTNAtiC

...

A

...

A

...

T

...

G..A

...

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.

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...

1

...

T

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...

T..G..A

...

A

...

T

...

T

...

G

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80 90 100 110

...

T..G

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G.A...A...G

...

C

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G

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.

.GTT.

...

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...

.C..

...

.T..

...

T..TA

....

G

...

A

...

A

...

..TT

...

120 130 1411 1 qn

...

4 I1 50 60 70

LysAspTyrCysVal ThrAl aAsnSerLysLeuVal I1 e I 1 eThrAl aGlyAl aArgGl nG1 nG1 uGlyGl uSerArgLeuAsnLeuVa1 G1 nArgAsnVa1 Asn I1 ePheLysPhe I 1 e

MAGACTACTtiTGTAACTGCGAACTCCAAGCTGGTCATTATCACCGCGGGGGCCCGTCAGCAAGAGGGGGAGAGCCGGCTCAACCTGGTCCAGCGAAACGTGAACATCTTCAAGTTCATC

_-.

I 1 eProAsn I leva1 LysTyrSerProHi sCysLysLeuLeu 11 eVal SerAsnProVal Asp1 1 eLeuThrTyr Val A I aTrpLys I 1 eSeTGlyPheProLysAsnAryVa1 I1 eGly

ATTCCCAACATTGTCAAGTACAGTCCACACTGCAAGCTGCTGATCGTCTCCAATCCAGTGGATATCTTGACCTACGTGGCTTtiGAAAATCAGTGGCTTTCCCAAAAACCGAtiTAATTtiGA

...

A...TC...G

...

.tiT

...

T..-..A

...

G..A...T

...

.C.G..

...

.G.. .C..

...

.T.G..

...

.T..

..

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...

.G.G..

.

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....

..

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...

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.CTA..

...

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.

.-A.T..

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TA..

...

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...

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....

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.

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...

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...

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..

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...

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.T..

....

160 170 180 190

SerGlyCysAsnLeuAspSerAl aArgPheArgTyrLeuMetGlyGluArgLeuGlyVa1 H i sAl aLeuSerCysHi sGlyTrpVal LeuGlyGl uHi sGlyAspSerSerVa1 ProVal

AGTGGTTGCAATCTGGATTCAGCGCGGTTCCGTTACCTGATGGGAGAGAGGCTGGGGGTTCACGCGCTGAGCTGTCACGGCTGGGTCCTGGGAGAACATGGCGACTCCAGTGTGCCTGT ti

...

.C..

...

.T.CA.. .TA..

...

.A..

...

.CA..

...

G

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....

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...

.G. .T..

...

..A..

...

.A..

..

.TC.A..

...

.G..

...

.G..

...

200 210 220 230

TrpSerGly Val AsnVal A1 aGly Val SerLeuLysSerLeuAsnProGl uLeuGly ThrAspAl aAspLysGl uG1 nTrpLysG1 uVal H i sLysGl nVal Val AspSerAl aTyrGl U

TGGAGTGGTGTGAATGTTGCCGGCGTCTCCCTGAAGTC TCTTMCCCAGAACTGGGCACTGACGCAGACAAGGAGCAGTGGAAGGAGGTTCACAAtiCAGGTGGTGGACAGTtiCCTACGAG

...

A..

...

.CA..

....

.T..

...

.G..

...

.T.. .C..

...

.G..

....

A. .TAC.

....

A...C..

...

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...

T...A...

...

A

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C.A..

...

C

...

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...

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...

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...

T

...

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...

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...

.C. .C..

...

..G..

..

.GC.G..

...

.G. .T..

...

.T..G..

...

.T..

...

A..

..

.A

240 250 260 270

Val 11 eLysLeuLysGlyTyrThrSrTrpAla IleGlyLeuSerVal A1 aAspLeuA1 aG1 user 11 eMet LysAsnLeuArgArgVal H i sPro I 1 eSerThrMet I leLysGl yLeu

GTGATCAAGCTGAAAGGTTACACATCCTGGGCCATTGGCCTCTCTGTGGCAGACTTGGCTGAGAGCATAATGAAGAACCTTAGGCGGGTGCATCCCATTTCCACCATGATTAAGGGTCTC

AG

...

G

...

C...G

...

AA.T

...

C

...

A

...

T

...

.G..

...

.TC..

...

.T..

...

.T..

...

.TTC.

...

c c

280 290 300 310

TyrGiy I 1 eAsnGl uAspVal PheLeuSerVal ProCys IleLeuGlyGl nAsnGlyI 1 eSerAspVal Val LysVal ThrLeuThrProGl uG1 uG1 u A I aArgLeuLysLysSerAl a TATGGAATCAATGAGGATGTC TTCCTCAGTGTCCCATG1ATCCTGGGACAAAATGGAATCTCGGATGTTI;TGAAGGTGACACTGACTCCTGAGGAAGAtiGCCCGCCTtiAAGAAGAGCGCA

...

T

...

G

...

A..G

...

C

...

C

...

T.A.

...

G...T..C

...

A

...

A

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...

C

...

C

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...

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...

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...

A

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G

....

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...

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...

G

...

C

...

A

...

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...

T...

3 20 331

AspThrLeuTrpGly I leGlnLysGluLeuG1 nPheTer

:;AC4CCClCTtiGGGAATCCAGAAGGAGClGCAGTTCrM

....

C...

...

G...

..

T..A...

...

A...-.--

...

G

. . T

...

FIGURE 3.-Sequence comparison of the mouse LDH-A processed pseudogene M10, M11 and M 1 4 with the protein-coding sequence of the mouse (M 15) and rat LDH-A functional genes. Different nucleotides are given, while identical nucleotides are indicated by dots; deletions are denoted by hyphens (-). The plus sign (+) indicates the sequence of 22 nucleotides of codon no. 68 to 75, which was replaced by an inserted repetitive sequence of 242 nucleotides. The deduced amino acid sequence of mouse LDH-A functional gene is given above its nucleotide sequence.

a deletion of 1 1 nucleotides, 8 single-base deletions, and a replacement of 22 nucleotides from codon no. 68 to 75 by a sequence of 247 nucleotides flanked by two perfect direct repeats of 5-bp. As indicated in Figure 5 , this inserted repetitive sequence (IRS) was found to be homologous to the 3’ end of the 475

(5)

Mouse LDH-A Pseudogenes

TABLE 1

Numbers of nucleotide differences between LDH-A sequences

M 1 5 M11 M I 0 M14

Mouse M 15 -

Mouse M 11 56

-

Mouse M10 79 83

Mouse M 14 84 96 103

Rat LDH-A 65 93 96 93

-

previously identified genomic clone AM 10 (FUKASAWA

et al. 1986), a mouse LDH-A processed pseudogene and a B1 repetitive element are flanked by perfect direct repeats of IO-bp, and this B1 sequence starts at 14 nucleotides 3’ to the presumptive polyadenylation signal (PROUDFOOT and BROWNLEE 1976) of the pseudogene. T h e “coding” sequence of pseudogene M 10 possesses a deletion of four nucleotides and two single- base deletions. In the genomic clone AM16 (AKAI et

al. 1985), the LDH-A processed pseudogene contained a truncated DNA sequence from codon No. 266 to the poly(A) tail of mRNA (data not shown).

We have inferred the pattern of nucleotide substitutions in the pseudogenes, following the procedure of GOJOBORI, LI and GRAUR (1982). Using the phylogenetic tree shown in Figure 4, we first inferred the ancestral sequence at each branch node and then inferred the direction of nucleotide substitutions in each pseudogene; deletions and insertions were ex- cluded from comparison. T h e results are shown in Table 2; the relative substitution frequencies were calculated according to the procedure of GOJOBORI, LI and GRAUR (1 982).

T h e three pseudogenes show very similar patterns of substitutions. For example, the frequencies of the two transitions C + T and G -+ A are the highest and the substitutions CG + CA and CG + T G are very high. Therefore, the data from the three pseudogenes are pooled together and used to calculate the average relative substitution frequencies shown in the last matrix in Table 2. T h e substitution pattern in these three pseudogenes is quite similar to that of the study by LI, Wu and Luo (1984). First, the proportion of transitions is 56.3% in the three LDH-A pseudogenes

M15 M l l M10 M14 LDH-A

rat

FIGURE 4.-A phylogenetic tree for the mouse LDH-A gene (M 15), mouse pseudogenes M 1 1, M 10 and M 14, and the rat LDH- A gene (MATRISIAN et al. 1985). The branching order is inferred from the distance matrix in Table 1.

M14 14s R consensus

M14 IRS R consensus

M14 I R S

----,

CATCAGTGGGAGGAGATTCCCTTTGTCCTATGAAGGCTCTATGGTCCAGT

***** ********** ***** * * ** * **** *****

CATCACTGGGAAGAGAGGCCCTTGGACTTGTAAACTTTATATGCCCCAGT

GTGGGGGAATGGCAGGG-CAGGAGTCAGGAGTGGGTGGGTTGGTGAGCA~~ ACAGGGGAACGCCAGGGCCAAAAAGTGGGAGTGGGTGGGTAGGGGAGTNGG

**a*** * ***** ** * ************* ** ***

GGAGGGTGGAGGAAATGGAGGGGGTGTTTTTCGGAGGGCAAATGAGGAAA GCAGGG-GGAGGGTAT---GGGGGACTTTT---

**** ***** ** ***** ****

GAGGATAACATTTGAGATGGAAATAAAGAAAATATCCAATAAAAGCAAAA

-GGGATAGCATTTGAAATGTAAATGAGGAAAATATCTAATAAAAA

***** ******* *** **** * ********* *******

-

AACAAAACAAAACAAAACAAACAAACAAACAAAAAACCATCA

50

100

150

200

242

FIGURE 5.-Comparison of mouse inserted repetitive sequence present in pseudogene M14 with the consensus sequence of mouse R repetitive elements. The nucleotide sequence of the inserted repetitive sequence (IRS) present in mouse LDH-A pseudogene M14 is compared with the 3’ end of consensus sequence of mouse R repeat family described previously (GEBHARD et al. 1982). The identical nucleotides are indicated by the asterisk (*) and deletions are denoted by dashes. N means A, G, C or T. The perfect 5-bp direct repeats flanking the IRS are indicated by arrows.

and 59.2% in the study of LI, WU and LUO (1984). T h e proportion of transitions excluding CpG dinucle- otides is 52.4% in the LDH-A pseudogenes and 54.4% in LI, Wu and LUO (1984). Second, the relative substitution frequencies at C or G is high; it is 66.3% in the LDH-A pseudogenes and 64.3% in LI, Wu and LUO (1984). Third, for each type of nucleotide the rate of transition is the highest while the rate of transversion between complementary bases is the low- est. For example, for the G nucleotide, the rate of the transition G + A is 16.8% in the LDH-A pseudogenes and 16.0% in LI, Wu and LUO (1984); the rate of the non-complementary transversion G + T is 7.5% in the LDH-A pseudogenes and 7.0% in LI, Wu and LUO (1 984); the rate of the complementary transversion G + C is 3.4% in the LDH-A pseudogenes and 5.5% in LI, Wu and Luo (1984).

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182 K. M. Fukasawa et al. TABLE 2

Frequencies and relative frequencies of nucleotide substitutions in mouse LDH-A processed pseudogenes

M11 M 1 0 M 1 4

Substitutions Substitutions Substitutions

A T C G Bases A T C G Bases A T C G

Bases

A 266

-

1 2 7 242

-

1 4 5 219 - 4 2 3

2 1

T 242 0 - 5 1 225 3 - 5 6 219 1

-C* 219 1 3 - 2 204 4 5 - 4 193 4 9 - 4

28 1 7 - 1 28 2 4 - 0 28 3 5 - 1

6 9 2

-*G 272 6 3 0 - 258 7 3 1 - 250

cG 28 5 0 1 - 28 4 0 2 - 28 3 0 1

-Total 1055 993 957

c g

Pool

Substitutions

A

T

C*

c g

*C

CG Total

Bases A T C G

755

-

6 8 15

686 4 - 12 8

616 9 17 - 10

84 6 16 - 2

780 19 15 3 -

84 12 0 4

-3005

Average relative frequencies (%)

Total

[56.3]

A T C G (52.4)

T 2.3

(2.7)

(7.5)

C 9.3

G 16.8

(14.2)

Total 28.4

(24.4)

3.5 (4.3)

-

21.5 (14.8)

7.5 (10.4)

32.5 (29.5)

4.8 (6.0)

(10.9) 8.4

-

3.4 (1.8) 16.7 (18.7)

9.6 17.9

(12.5) (22.9)

5.0 15.8

(6.1) (1 9.7)

7.7 38.5

(8.8) (31.0)

- 27.8

(26.4)

22.3 100.0

(27.5) (100.0)

~~

The substitutions are from the nucleotides in the left column to the nucleotides in the row. The C* indicates the C not followed by G and the *G indicates the G not preceded by C. [

1:

proportion of transitions; ( ): excluding CpG.

kb fragment of clone M 16 probably corresponds to a weak hybridization band located between those of mouse LDH-A pseudogene M11 (14 kb) and functional gene (1 3 kb). T h e other strong hybridization bands of EcoRI 16, 15, 8.2, 6.4, 6.0 and 4.3 kb are probably derived from other LDH-A pseudogenes whose genomic clones have not yet been isolated. In short, at least ten different LDH-A pseudogenes are present in mouse genome.

DISCUSSION

The analysis of nucleotide sequences of mouse LDH-A processed pseudogenes has revealed various types of DNA alterations including base substitutions, deletions, tandem duplication and insertion of repet-

itive elements. T h e rate and the pattern of nucleotide substitutions in mammalian processed pseudogenes have been extensively analyzed and were elegantly used to illustrate the molecular evolution of mammalian genes and pseudogenes (GOJOBORI, LI and GRAUR 1982; Li 1983; Li, Gojobori and NEI 1981; LI, WU and LUO 1984; Wu et aE. 1986). The relatively higher rate of base substitutions at CG dinucleotide was

found in mouse LDH-A pseudogenes as well as other mammalian pseudogenes (GOJOBORI, LI and GRAUR 1982). The high frequency of polymorphism at MspI and Tag1 restriction sites containing the CG dinucleotide in human genomic DNA (BARKER, SCHAFER and WHITE 1984) further supports the proposal that the methylated cytosine residue is a mutational “hotspot” in mammalian DNA. The “coding” sequence of pseudogene M 1 1 contains a tandem duplication, while the pseudogene M 16 possesses only a truncated “coding” sequence and 3 untranslated region. T h e pseudogene M 10 was followed by a B 1 repetitive element (KRAYEV

et al. 1980, 1982) at its poly(A) addition site, and the “coding” sequence of the pseudogene M14 was inserted by a truncated R repetitive element (GEBHARD

(7)

Mouse Pseudogenes

least 10 mouse LDH-A pseudogenes in addition to the LDH-A functional gene. T h e chromosomal locations of these mouse LDH-A pseudogenes remain to be determined, while the mouse LDH-A functional gene has been mapped genetically to chromosome No.

7.

It should be pointed out that the weak hybridization bands of mouse genomic blot probed with mouse LDH-A cDNA may represent LDH-B/LDH-C gene- related sequences whose genomic clones remain to be isolated and characterized. As described previously (CHUNG et al. 1985; TSUJIBO, TIANO and LI 1985),

the human genome contains multiple LDH-A gene- related sequences, and several processed pseudogenes have been isolated and characterized. T h e presence of multiple pseudogenes has also been reported for a number of other mammalian gene families such as cy- tubulins (LEMISCHKA and SHARP 1982), cytochrome t

(Wu et al. 1986) and glyceraldehyde 3-phosphate dehydrogenase (BENHAM, HODGKINSON and DAVIES

1984; HANAUER and MANDEL 1984; PIECHACZK e t al.

1984;

Tso

et al. 1985).

EcoRI

BomHI

HlndPf

C D C D C D

t

FIGURE 6.-Genomic blot analysis of mouse LDH-A gene-re-

lated sequences. T h e genomic DNAs were cleaved by restriction

endonucleases EcoRl, BamHl or Hind111 and were electrophoresed

on 0.75% agarose gel. T h e DNA fragments of C57BL/6J (C) and DBA/2J (D) mice were Southern yansferred to nitrocellulose filter and hybridized with mouse LDH-A cDNA probe in 6 X SSC at 63" overnight and washed twice with 1 X SSC a t 42" for 30 min. T h e estimated sizes (kb) of the EcoRI fragments exhibiting strong hybridization are indicated.

(KLECKNER 198 I). Similarly, the interspersed repetitive elements in mammals might constitute a signifi- cant mutation liability because of the possible trans- position and rearrangement of the functional genes flanked by repetitive sequences.

I t has been proposed that the intronless pseudogenes were formed by the integration into germline DNA of reverse-transcribed processed mRNA (SHARP

1983) and that formation of processed pseudogenes may share common pathways with retroviruses and transposable elements. In Drosophila, processed pseudogenes have not yet been reported, but several types of transposable elements have been isolated and characterized (SPRADLING and RUBIN 1981). T h e abundance of mammalian processed pseudogenes might be directly associated with an existing mechanism similar to the replication of retroviruses in mammalian germ- lines. Thus, the "housekeeping" genes actively expressed in germline cells may be found to have more copies of processed pseudogenes than those expressed mainly in differentiated somatic cells. There are at

We thank M. H. EDCELL for the mouse genomic library, B. H. JUDD for his interest and support of this investigation, W. QUAT-

TLEBAUM for computer analysis of nucleotide sequences, F. JOHN-

SON, R. VOELKER, T. KUNKEL, J. DRAKE, C. LANGLEY, B. H. JUDD

and W.-H. LI for helpful discussion and reading the manuscript. This study was partly supported by National Institutes of Health grant GM30998 to W.-H. LI.

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