Cloning, expression and sequence homologies of cDNA for human gamma enolase
6
Click here to load reader
Transcript of Cloning, expression and sequence homologies of cDNA for human gamma enolase
Gene, 79 (1989) 355-360 Elsevier
GEN 03037
355
Short Communications
Cloning, expression and sequence homologies of cDNA for human gamma enolase
(Recombinant DNA; glycolytic enzyme; isotypic protein; evolutionary conservation; brain; muscle; liver)
Daniele Ohs b, Giovanna Barba *, Giovanna Barbieri b, Agata Giallongo* and Salvatore Feoa
a Zstituto di Biologia deli0 Svikppo (Comiglio Nazimale defle Ricerche), Palermo (Ztaly), and b Dipartimento di Biologia Celfulare e delfo ~v~luppo, 90123 Pale~o (Italy) Tel. (091)~162632
Received by H. van Ormondt: 7 November 1988
Revised: 9 February 1989 Accepted: 13 February 1989
SUMMARY
The nucleotide sequence of the human y-enolase mRNA was determined from recombin~t cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5’-noncoding region of 74 bp and a 897-bp-long 3’-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat y-enolase. Both the 5’- and 3’-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat y-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of y-enolase mRNAs. The size of the y-enolase mRNA expressed in human brain is 2.4 kb. A crosshyb~dizing 1.5-kb message is detected in human skeletal muscle which may be derived from the fl-enolase-coding gene.
INTRODUCTION
Enolase (EC 4.2.1.11) catalyses the intercon- version of 2-phosphoglycerate and phosphoenolpy-
Correspondence to: Dr. S. Feo at his present address: Department of Eukaryotic Genome Organisation and Expression, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WCZA 3PX (U.K.) Tel. (01)2420200, ext. 2406; Fax (01)405 1556.
Abbre~ations: aa, amino acid(s); bp, base pan(s); cDNA, DNA complementary to mRNA; kb, kilobase or 1000 bp; Myr, one million years; NNE, non-neuronal enolase; NSE, neuron-specific enolase; nt, nucleotide(s); SSC, standard saline citrate (0.15 M NaCl/O.OlS M Na, . citrate pH 7.6).
ruvate in the glycolytic pathway. Three different iso- forms of enolase have been identified in both avian and mammalian tissues (Marangos and Schmechel, 1987). Each of these three isoforms is a homodimer of identical ct or fi, or y subunits. The aa homodimer, also called NNE, is the only isoenzyme expressed in liver and is the major enolase found in other tissues and in glial cells within the nervous tissue. The yy homodimer or NSE is present mainly in neuronal and neuroendocrine cells and the 198 homodimer is located in muscle tissue. During early development a switch from the acl to the B/3 isoform takes place in developing muscle (Rider and Taylor, 1974) and a switch from the aa to the yy isoform occurs in the
0378-I 119/89j~O3.50 0 1989 Ekevier Science Publishers B.V. (Biom~ic~ Division)
356
developing nervous system (Schmechel et al., 1980). While the existence of three different loci encoding CI, p and y subunits has been proposed since 1976 (Pearce et al., 1976; Chen and Giblett, 1976), cDNAs for rat a- and y-enolase mRNAs (Sakimura et al., 1985a,b; Forss-Petter et al., 1986) and the rat y-enolase-coding gene (Sakimura et al., 1987) have been cloned only recently. The molecular structure of the human enolase genes is not as well characterized as that of the rat. By somatic-cell hybridization the genes encoding the human a and y subunits have been localized on chromosome 1 and 12, respectively (Law and Kao, 1982). A cDNA for the human a-enolase mRNA has been isolated and character- ized (Giallongo et al., 1986) and a partial sequence of the 3’ end of the human y mRNA has been reported (Day et al., 1987). Here we report the iso- lation and sequence analysis of cDNA clones which define the structure of human y-enolase mRNA. A comparison of human y cDNA with that of the rat shows a high sequence conservation not only in the y-enolase-coding region but also in the 5’- and 3’-untranslated regions of the mRNAs.
EXPERIMENTAL AND DISCUSSION
(a) Isolation of human y-enolase cDNA clones
Since human brain is known to contain y-enolase as well as a-enolase (Marangos and Schmechel, 1987), a human brain cDNA library in Igtll (Clontech, Palo Alto, CA) was screened (Benton and Davies, 1977), first at low stringency (60” C; hybridi- zation in 6 x SSC, final wash in 2 x SSC) with a human a-enolase cDNA probe (Giallongo et al., 1986) derived from a coding sequence, and subse- quently at high stringency (65 “C; hybridization in 4 x SSC, final wash in 0.2 x SSC) with a 3’-non- coding a-enolase cDNA probe. One of the plaques that was positive in the first screening and negative in the second was characterized further. The nucleo- tide sequence of this cDNA (B6 in Fig. 1) differs from that of human cc-enolase cDNA (Giallongo et al., 1986) and shows greater similarity to the rat y-enolase cDNA sequence (Sakimura et al., 1985b) than to that of the human a-enolase (Giallongo et al., 1986). Clone B6, however, was incomplete and lon- ger y-enolase cDNAs were not found in the library.
To obtain a full-length cDNA clone, we screened a cDNA library in JgtlO (Clontech, Palo Alto, CA) derived from a human neuroblastoma which is known to express varying amounts of y-enolase
a BarnHI
PVUII BglII
PYUII ( TaqI PvuII I HillCII XhoII XhoII
I I I I
ATG (EcoRI) TGA ATTAAA
0 0.4 0.8
I I
1.2 1.6 2.0 kb
L40
Fig. 1. Map of human y-enolase cDNA and sequencing strategy. (a) Partial restriction map derived from the cDNA clones B6 and LAO,
(EcoRI) indicates the site 3’ of B6 insert in 1gt 11 generated by addition of linkers. The protein-coding region is indicated by the blackened
box. The open box indicates the BamHI-EcoRI fragment from clone B6 used as probe to screen the human neuroblastoma cDNA library
(see section a). (b) Thick lines represent two overlapping cDNA clones. The arrows indicate the direction and the extent of sequence
determination for each fragment analyzed. The 5’ fragment of the B6 clone, which is lacking the EcoRI cloning site, was isolated from
the phage DNA taking advantage of a PvuII site present in the 1ucZ gene, 5’ to the missing EcoRI site (Galas et al., 1980).
351
(Zeltser et al., 1985). Among the clones hybridizing to a probe from clone B6 (see above) we selected the longest one (L40; 2-kb insert) for further characteri- zation; it was found to partially overlap clone B6 (Fig. lb).
(b) Nucleotide sequence analysis
B6 and L40 cDNAs were subcloned into Bluescribe or Bluescript vectors (Stratagene, La Jolla, CA) and the sequence of overlapping frag- ments on both strands was determined by the chain- termination method (Sanger et al., 1977) according to the strategy shown in Fig. lb. The nucleotide se- quence of the human y-enolase cDNA is shown in Fig. 2. The cDNA contains an open reading frame of 1299 bp encoding a protein of 433 aa with a calcu- lated h4, of 47 288. The 5’-noncoding region is 74 bp, is extremely G + C rich (65% G + C) and contains a sequence, CCACCG, repeated four times. This motif is also repeated three times in the 5’-untrans- lated region of the rat y-enolase mRNA (Sakimura et al., 1985b; Fig. 3) but its functional significance is unknown at present. The 3’-noncoding region is 897 bp and contains a cluster of six ATTT repeats (Fig. 2). This motif has been described as a feature of unstable mRNAs (Shaw and Kamen, 1986). A putative polyadenylation signal (ATTAAA) is locat- ed 13 nt upstream from a poly(A) tail indicating that the 3’-noncoding region is complete.
While this work was in progress Day et al. (1987) reported a partial nucleotide sequence for the 3’- untranslated region of human y-enolase. Our se- quence is in agreement with their partial sequence in all but seven positions, namely G, T, and C insertions at nt positions 2047, 2053 and 2086, respectively, and T to C, C to T, A to T, C to A changes at nt 1538, 1594,2099 and 2122, respectively. These differences could be due to sequence polymorphisms in the human genome or to errors generated during cloning and/or sequencing.
The sequence derived from the two overlapping clones spans a total of 2273 bp. This length is consis- tent with the 2.4-kb mRNA (assuming a poly(A) tail of almost 150 bp) detected by Northern-blot analysis in human brain (Fig. 4), suggesting that the cDNA is close to a full-length representation of the corresponding mRNA.
(c) Sequence homology with rat y-enolase cDNA
Human and rat y-enolases are highly homologous, having 91% similarity at the nt level in their coding regions. A striking similarity is observed also in their short 5’noncoding regions (82%) as well as in their 3’-noncoding ends, where an overall similarity of 68 y0 is found. The length of the untranslated regions is also conserved between the two species. Align- ment of the 3’ sequences (Fig. 3B) reveals the exis- tence of many long stretches with 75-80% similarity in the regions immediately following the stop codon and preceding the poly(A) addition site, including the polyadenylation signal. The rate of accumulation of neutral point mutations in the nucleotide sequence of DNA during evolution has been estimated to be 1% per Myr (Miyata and Yasunaga, 198 1). The expected homology of related sequences in species which diverged 75 Myr ago, like rat and human, is about 52 %, taking into account the correction for multiple changes (Miyata et al., 1980). The degree of conser- vation observed in the 5’ (82%)- and 3’ (68%)-non- coding regions of the rat and human y-enolase is therefore much higher than that expected by random point mutations.
A high degree of sequence conservation in the 3’noncoding regions between diverse species has also been observed in several genes coding for iso- typic proteins like tubulin (Cowan et al., 1983), myosin (Saidapet et al., 1984), creatine kinase (Billadello et al., 1986) and actin (Ponte et al., 1983). In the latter, the presence of highly homologous 5’-untranslated regions has also been reported (Ponte et al., 1984). Although the function of 5’- and 3’-noncoding regions of genes is at present un- known, this high level of conservation suggests the existence of a strong evolutionary constraint to preserve these sequences. It has been proposed that 3’-untranslated regions might play an important role in the regulation of tissue-specific expression (Yaffe et al., 1985).
(d) Amino acid sequence comparison
The predicted amino acid sequences of the human and rat y-enolases and the human cc-enolase were compared. The overall homology is 97% between human and rat sequences and 83% with the c1 iso- form. It has been suggested that the Thr residue in
- 4
-14 GGGCCGCCGTCG~CCACCG~CCACTACCRCCC
1
3
1
ATG KC
ATA GAG AK
AK
TGG GCC CGG GAG RTC CTG GAC TCC CGC GGG AAC ccc ACA GTG
MET S%r 11% Glu Lys 11% Trp Ala Arg Glu Ile Leu Asp Ser Arg Gly Asn Pro 'Thr Val
19
61
GAG GTG GAT CTC TAT ACT GCC AAA GGT CTT TTC CGG CCT GCA GTG CCC AGT GGA GCc TCT
Glu Vail. Asp Len Tyr Thx hla Lys Gly Leu Ph$ Aq
Ala Ala Val Pro Snr Gly Ala Ser
39
121
ACG GGC ATC TAT GAG GCC CTG GAG CTG AGG GAT GGA GAC AAh CAG CGT TAC Tl'A GGC ARA
Thr Gly 11% Tyr Glu Ala L%u Glu Leu AIQ Asp Gly Asp Lys Gln Arg Tyr Leu Gly Lys
59
161
GGT WC
CTG A?& GCA GTG GAC CAC ATc AA= TCC AK
ATC CCG CCA CCC CTC ATC AGC TCA
Gly "al Le" Lys Ala Val Asp His Ile Asn Ser Thr 11% Ala Pro Ala Leu Sle Ser Ser
79
241
GGT CTC TCT GTC GTG GAG CAA GAG AAA CTG GAC AAC CTG ATG CTG GAG TTG GAT GGG ACT
Gly Leu Ser Val Val Glu Gin Glu Lys L%u Asp Asn Leu Met Leu Glu Leu Asp Gly Thr
99
301
GAG AAC AAA TCC AAG TTT GGG GCC AAT GCC AK
CTG GGT GTG TCT CTG GCC GTG TGT RAG
Glu Asn Lys .S%r Lys Phe Gly Ala Asn Ala II% I,%" Gly "al Ser Leu Ala Val Cys Lys 119
361
GCA GGG CCA GCT GAG CGG GAA CTG CCC CTG TAT CGC CAC ATT GCT CAG CTG GCC GGG AAC
Ala Gly Ala Ala Glu Arg Glu Leu Pro Leu Tyr Arq His ILe Ala Gin LE1U Ala Gly Asn 139
421
TCA GAC CTC ATC Cl% CCT GTG CCG GCC TTC AAC GTG ATC AAT GGT GGC TCT CAT GCT GGc
Ser Asp Leu :le Le" PIO Vdl Pm
Ala Phe Am, Val Iie As, Gly Gly Se* r.is Ala Gly 159
481
&AC AAG CTG GCC AX
CAG GAG TTC ATG +TC CTC CCR CTG GGA GCT GAG AGC TTT CGG GAT
Asn Lys Leu Ala Met Gin Giu Phe Met 11% I.%" Pro Val Gly Ala Glu Ser Phe Arg Asp 179
541
KC
ATG CGA CTA GGT GCA GAG GTC TAC CAT AC,, CTC AAG GGA GTC ATC MG
GAC MA
TAC
Ala "et Arg Leu Gly Ala Glu Val Tyr His Thr LB" Lys Gly "al 11% LyS Asp Lys Tyr 299
601
GGC AAG GAT GCC AC= AAT GTG GGC CAT GAA GGT GGC TTT GCC CCC AA'I Al'C CTG GAG AK
Gly Lys ASP Ala Thr As" Val Gly Asp Glu Gly Gly Phe Ala Pro Am
110 L%u.Glu As.,, 219
661
AGT GM, GCC TTG GAG CTG Gl'G AAG =?,A GCC ATC GAC AAG GCT GGC TX
ACG GAA AAG ATC
Ser Giu Ala Lea Glu I.%" Val Lys Glu Ala 11% Asp Lys Ala Gly Tyr ThX Glu Lys 11% 239
721
GTT ATT GGC ATG GA= GTT GCT GCC TCA GAG TTT TAT CGT GAT GGC AAA TAT GAC TTG G&C
Val Ile Gly Met Asp Val Ala Ala Ser Glu Phe Tyr Arg Asp Gly Lys Tyr Asp Leu Asp 259
781
TTC AAG TCT CCC ACT GAT CCT XC
CGA TAC ATC ACT GGG GAC CAG CTG GGG GCA CTC TX
Phe Lys S%r Pro Thr Asp Pro Ser Arg Tyr Ile Thr Cly Asp Gln LB" Gly Ala Leu Tyz 279
841
CAG GAC TTT GTC AGG GRC TAT CCT GTG GTC KC
ATT GAG GAC CCA TTT GAC CAG GAT GAT
Gin Asp Phe Val Aq
Asp Tyr Pm
Val Val Ser 11% Glu Asp Pro Phe Asp Gin Asp Asp 299
901
'KG GCT CCC TGG TCC AAG TTC ACA GCC AAT GTA GGG AK
'ZAG ATT GTG GGT GAT Gnr CTG
Trp
A
la
Ala
T
ip
Se=
Ly
s P
he
Thr
A
la
Am
V
al
Gly
Ile
G
in
Ile
ml
Gly
Asp Asp Leu 319
961
ACA GTG ACC ARC CCA &$A CGT ATT GAG CGG GCA GE
GAA GAR AAG GCC 'KC AAC TGT CTG
Thr Val Thr Asn Pro Lys Arg
Ile
Glu
A
rq
Ala
V
al
Glu
G
lu
Lys
ALL
C
ys
Rsn
C
ys
Leu
339
1021
C
TG
C
TC
A
AG
G
TC
A
X
CA
G
AT
C
‘XC
T
CG
G
TC
A
CT
G
AA
G
CC
A
K
CA
A
GC
G
TG
C
AA
G
CT
G
GC
C
Le”
Leu
Lys
Val
A
sn
Gin
Ile
G
ly
Ser
V
al
Thr
G
lu
Ala
11
%
Gln
A
la
Cys
Ly
s Le
u A
la
359
1081 CAG GAG AAT GGC TGG GGG GTC ATG GTG AGT CAT CGC TCA GGA GAG ACT GAG GAC ACA TTC
Gin Glu Asn Gly Trp Gly Val Met Val Ser "is Arg Ser Gly Glu Thr Glu Asp Thr Phe 379
1141 ATT GCT GAC CTG GTG GTG GGG CTG TGC ACA GGC CAC ATC AAG ACT GGT KC
CCG TGC CGT
Ile Ala Asp LB" Val Val Gly Leu Cys Thr Gly Glr, Ile lys Thr Gly Ala Pro Cys Arg 399
1201 TCT GAA CGT CTG GCT MA
TAC AAC CAG CTC ATG AGA ATT GAG GAA GAG CTG GGG GAT GAA
Ser Glu Arg Leu Ala Lys Tyr Asn Gin Leu Met Arq Ile Glu Glu Glu Leu Gly Asp Gl,, 419
1261 GCT CGC TTT GCC GGA CAT AAC TTC CGT AAT CCC AGT GTG CTG TGA TTCCTCTGCTTGCCTGGAG
Ala Arg
Phe
A
la
Gly
H
is
Asn
Phe Arg Asn Pro Ser Val Leu
-
433
1325 ACGTGGN\CCTCTGTCTCATCCTCC~~~CCTTGCTGTCCTGA~TGTGATAGTTCACCCCTGAGATCCCCT~~CC
1404 CAGGGTGCCCAGAACTTCCCTGATTG4CCTGCTCCGCTGCCC
1483 TCCTTTCrrTGCCCTACTCATTGGGGTTCCGCACTTTCCAC
1562 AATGTW\ATGACWLTTATTATRAIVLGGGGGTCCGTffi~G~TGATCA~ATCTGTGAT~AGCGTCA~GTT~TGT
1641 GCTGAGGTGTTAGAGAGGGACCATGTGTCACTTGTGCTTTT~TCTTG~CCACGTGTCTTCCACTTT~ATATG~CG
1720 TGAACTGTGCATAGTGCTGT~ffi~AGTGTT~~TGTGATCACG~T~T~T~~T~TAGTGT~
1799 -
GTTTTTCATTCATCCCATT~TCATT~CCCCAT~CTC~~~?~CT~CT~
1878 CTTGG~CGATGTGTCTGTATTTCATGTGGCTGTRGA~CC~G~~GAC~~~~~TCTT~TAG~~~
195f ARGGGTCATA~~CTTGACATCAG~CCTTTGTGTGTACTCACTG~~CTGCGTT~TCCAGA~GGA~~TG
2036 TGTGCC~AGTTTTCCTCTATACATCTCTCCCTAG
2115 CCTCTCACTCCCCATGCCACGTTCCACAG2TGCCACCACCACCTCTGT~ATT~TGA~ACCTCC~GTCTG~
2194 TCAGTG poly(A)
A
-60
-50
-40
-30
-20
-10
H",mn - CGCCACCGCCACCGCCACCGCCACCACCGTCTGAGTC=GA=~~~~CAG=C~C----G~~~~C~A~~~A~C~
:::: :::::
: ::::::::::
:::::: . . . . . . . . . . . . . . .
. . . . . . ..*......
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
Rat
- CGCCGCCGCCGTCACCACCGCCACCGCCACCGGCTGAGTCTGCAGTCC~CG~GGAGATCCCAGCCATC~
B
1369
Human - TTCCTCTGCTTGCCTU;RGACGTU;AACCTCTGTCTCATCC~CT~CCT------T~TG~CTGAT
:: :::::::::::
: ::
::::: ::
. . . . . . . . . . . . . . . . .
. . . . . . . . . .
. . . . . . .,......
. . .
. . . . . . . . . .
Rat
- -CCCCCTGCTTGCCTGAACACC-ffi~CATCA-TCTCATTC~CT~A~CTCTTTCTT~TGTCCC~C
1439
Human - CTGTGATAGTTCACCCCTGAGATCCCCTGAGCCCCCA~T~C~~~TCCCTGATT~CCTGCTCCG
::
::::::
:::
::: :: :::::::::
:
I.......
. . . . . . . .
::
:: : ::::::::
:
Rat
- CC~CATAGTTA---CCTT-GRTACCTTGAGCCCCACGTC~~A~~CA-CCTC~C~CA~~CTCTG
1504
Human - Cn;CTCCTTGGCTTACCTGACCTCTTGCTGTCT-Cn;CTC~~TCCTTTCTG~CCTACTCATT----
: :::
:::::
: :::
:::
::::::
: ::
:::
::::::
:::
: :::
: :::
: R
at
- GCTGTTCTTGGCTTCCACAACCCCCTT~TGTCTCCTACTCTTCCTCCTCTCT~CC----CATTTTTG
1572
Human - --~TTCCGCACTTTCCACTTCTTCCTTTCTTTCTCTCT
:
::: ::::
::
::::::
::: :::: ::
f...
. . . .
::
:::
: :::
:::::
Rat
- CXXGGATTCCAG~GCCCACTT--TCCCTTCTATT---CTCTCTAATCTT~TGA
1642
Hwoan - ~ATTATTAT~
~CG~~~T~~A~AT~TGA~~~A~TT~TG~
:: ::
: ::
::
:::
. . . . . . . . . . . .
:::: :: : ::
::::: ::::: ::::::::
Rat
- CW\CTAWIAWV\GCGG---TCCACCGCCAGCGTCCOTGT
1711
Human - TGA~TGT-TAGAGAGCGACCATGTGTCACTTGTGCTTTT~TCTTGTCCCACG~TCTTCCACTTT~AT
. . .
. . .
. . . .
. .
::::::
::::::
::::::
::::..
.....
:: :::
.,.
...
Fat
- T~GTTTAAAGTGGGGCCACGTGGCACGTGTGCTTCCCCT~CATCCATGGTGTGTTA----------
1781
Hunan - ATGAGCCGTGAACTGTGCATAGTTGGGATGGAGGGGAGTGTTGGGCATGTGATCACKCTGXTAATA
:::: :::::: :::: :: :::::
:
. . . . . . . . . . .
. . . . . . . . .,.
::::
Rat
- ---AGCCTTGB9CTAnT~TGTTT_GGGGAGTGCGT----------------
1851
Human - A~CTTTAGn;TATTTATTTATTTATTTATTTTATTTGTTTTTCA~TCATCCCATT~TCAT~TCCCCAT
::::
. . . . . . . . .
. .
. . . . . . . *a. . . .
::
:: ::::
::: : ..... :
. . . . .
Rat
- --------GTGTTCACATTTGTTTGTTTGTTTATTTATTTATTCAC--------TTATTTATTTCTCA~
1921
Human - RACTCRATGGCCTAAAACTGCTGACTT~~~C~~TGTCTGTATTTCATGTG~TGTAGATCCC
:::::::
::
::::
:::
::
:::
::::::::::
::
:
Rat
-
CT
GT
CA
GT
CG
TC
TG
CC
AT
TA
CT
CT
TA
CA
GT
CT
GA
AA
GC
AT
1990
Hum
an
- ~ATGA-C~T~~TC~T~~~~T~~TCAT
~~~~GACA~C~TT~~
::::::: :: :: ::::::::
:::
. . . . . . .
::: :::::::
::::: . . . . . . .
::: :
Rat
- RAGATGACCTAGGATGGGAGGTTTTGTTAGCRTGGGAAAUA~
Human - TTGTGTGTACTCACTGAAGCCTGCGTTGGTCCRGAGCGGRT
. . . . . . . .
::: ::: ::: . . . . . . . .
:
: ::::: :: ::::::::
:: ::: :: . . . . .
. . . . .
. . . .
. . . .
Rat
- TTG-GTGCACTAACTGAAGCTCGGTACTTTACAWLGTGG-CTTTTCTCCTAT
2127
Human - ACATCTCTCCC-CAACCCTRTTCCCTGTTCTTCTTC--CTCCA~T~AC~GA~~CCTC~C~TCCCC
.
.
. ...‘.._
. . I::::::::::
. . . . . . . . . .
::::
.f..
. . . . :::::::::::::
..I. I....
..,. . . . . .
aat
- ~C~TCTCCCCCFfiCCCTAffi~nCTCAGTCTTTTTTCTCC~CT~ACCAGA~~T~CTCAT~CCC
2199
Human - A-TGCCACGTTCCACAGTTGCACCACCrrTGTGGCATTGRTG
. . . . .
. ..I. :: . . . . . . . . . . . . .
. . . . . . . . .
. . . . . . . . . . . . .
. . . . . . .._ :::::::::
:::: : :::::::::::::
::
:
Rat
- CGTGCCATGTCCCACAGTTGCACTGTCTCTGT~TTTG~TGACCACCACTATT~GTCTG~CC~AG
Fig
. 2.
F
ig.
3.
359
4.4 -
2.4 - (I)
1.4 -
B; = 3 .> -12
Fig. 4. Total RNA from human liver, skeletal muscle and fetal
brain was isolated according to Chomczynski and Sacchi (1987).
Per lane 15 pg were separated on a 2.2 M formaldehyde-l %
agarose gel, transferred to a nylon membrane and hybridized as
previously described (Giallongo et al., 1986) with 32P probes of
(A) the X/z011 fragment of the IA0 cDNA (see Fig. 1). which
represents most of the 3’noncoding region of human y-enolase
mRNA; (B) the BstEII-XbaI fragment of the pH48 cDNA, repre-
senting the 3’noncoding region of human a-enolase mRNA;
(C)the HincII-EcoRI fragment of the L40 clone (see Fig. 1).
/&RI is the 3’ site generated by addition of linkers. This last
probe includes all the 3’noncoding region plus 200 bp of the
coding region of the human y-enolase mRNA. The same filter
was washed, re-hybridized each time and autoradiographed at
-70°C using Kodak XAR films and intensifying screens. Po-
sition and size of RNA markers (RNA ladder, BRL) are
indicated on the left margin.
position 190, present in the rat y-enolase and absent in the c1 isoform, might be important for the enhanced tolerance to chloride ions of the NSE (Forss-Petter et al., 1986). The presence of a Thr residue in po- sition 190 also in human y-enolase supports this hypothesis.
(e) RNA analysis and tissue specificity
Total RNA from human liver, skeletal muscle and fetal brain was hybridized with either a y-enolase
specific probe from the 3’-untranslated region of the y-enolase cDNA (Fig. 4A), with an a-specific probe from the 3’-untranslated region of the a-enolase cDNA (Fig. 4B) or with a probe containing a portion of y-enolase-coding region (Fig. 4C). In brain, where y and a isoenzymes are expressed, the two y probes detected a 2.4-kb RNA and the a probe a 1.8-kb RNA. In liver, where only the a-enolase is expressed, no specific RNA band was detected by either of the y probes whereas the a probe detected a band of 1.8 kb. In muscle (where presumably the fl-enolase is expressed) no specific RNA band was detected by the y-enolase 3’-untranslated probe, a very faint 1.8-kb band by the a probe and, interestingly, a novel 1 S-kb band by the y-enolase coding-region probe. A band of 1.5 kb was also detected by the same probe in poly(A)+ RNA from human muscle (not shown). This result suggests that the 15kb band may be the mRNA for j-enolase, which might be expected to crosshybridize to the y-enolase coding-region se- quence. Under the same hybridization conditions the message for a-enolase is not detected by the y-enolase probe (Fig. 4C), which can be due either to a higher degree of similarity between the messages for y- and j?-enolase compared to y and a (76%) or to the existence of a unique gene from which the two messages derive by alternative splicing. Evidence of a third gene encoding the jI isoenzyme relies on biochemical and immunological studies (Pearce et al., 1976; Chen and Giblett, 1976). However, in the only enolase genomic DNA isolated to date, the rat gene encoding the y isoform, alternative splicing has not been reported (Sakimura et al., 1987), sup- porting the existence of a separate locus for b-enolase. Cloning and characterization of genomic DNAs for the different isoenzymes will clarify this issue and provide insights on the evolution of the enolase genes.
Fig. 2. Nucleotide sequence of human y-enolase cDNA and deduced amino acid sequence. The nt are numbered from the first nt of
the start codon. The aa are numbered from the Ser after the Met start codon. The putative polyadenylation signal ATTAAA and the
tandem sequence repeats ATTT are underlined. The CCACCG sequence repeated four times in the 5’-untranslated regions is indicated
by numbers 1 to 4.
Fig. 3. Comparison of the 5’-(A) and 3’-(B) untranslated regions for human and rat y-enolase cDNAs. The sequences have been aligned
for maximal homology (dashes represent gaps) with the IFIND program of Intelligenetics Inc. The human sequence is numbered as
in Fig. 2. Start codons in (A) and polyadenylation signals in (B) are underlined. Identical nt are marked by colons.
360
ACKNOWLEDGEMENTS
We are indebted to L.C. Showe for the cDNA libraries, to J.M. Kemshead for the human brain tissue, and to Y. Edwards for the human muscle tissue. We thank S. Pellegrini and G. Lennon for critical reading of the manuscript. We are grateful to M. Fried, in whose laboratory some of the experi- ments were performed, for his constant advice and the time spent in helping us during the preparation of this manuscript. This work was partially sup- ported by Grant 113/M from Progetti Sanitari Finalizzati, Regione Sicilia and a ‘Progetto Bi- laterale’ from Consiglio Nazionale delle Ricerche to A.G.
REFERENCES
Benton, W.D. and Davies, R.W.: Screening Igt recombinant
clones by hybridization to single plaque in situ. Science 196
(1977) 180-182.
Billadello, J.J., Kelli, D.P., Roman, D.G. and Strauss, A.W.: The
complete nucleotide sequence of canine brain B creatinine
kinase mRNA: homology in the coding and 3’ noncoding
regions among species. Biochem. Biophys. Res. Commun.
138 (1986) 392-398.
Chen, S. and Giblett, E.R.: Enolase: human tissue distribution
and evidence for three different loci. Ann. Hum. Genet.,
London 39 (1976) 277-280.
Chomczynski, P. and Sacchi, N.: Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162 (1987) 156-159.
Cowan, N.J., Dobner, P.R., Fuchs, E.V. and Cleveland, D.W.:
Expression of human a-tubulin genes: interspecies conser-
vation of 3’ untranslated regions. Mol. Cell. Biol. 3 (1983)
1738-1745.
Day, I.N.M., Allsopp, M.T.E.P., Moore, D.C. McN. and
Thompson, R.J.: Sequence conservation in the 3’-untrans-
lated regions of neurone-specific enolase, lymphokine and
protooncogene mRNAs. FEBS Lett. 222 (1987) 139-143.
Forss-Petter, S., Danielson, P. and Sutcliffe, J.G.: Neuron-
specific enolase: complete structure of rat mRNA, multiple
transcriptional start sites and evidence suggesting post-tran-
scriptional control. J. Neurosci. Res. 16 (1987) 141-156.
Galas, D.J., Calos, M.P. and Miller, J.H.: Sequence analysis of
Tn9 insertion in the IacZ gene. J. Mol. Biol. 144 (1980) 19-41.
Giallongo, A., Feo, S., Moore, R., Croce, C.M. and Showe, L.C.:
Molecular cloning and nucleotide sequence of a full-length
cDNA for human a-enolase. Proc. Natl. Acad. Sci. USA 83
(1986) 6741-6745.
Law, M.L. and Kao, F.: Regional mapping of the gene coding for
enolase-2 on human chromosome 12. J. Cell Sci. 53 (1982)
245-254.
Marangos, P.J. and Schmechel, D.E.: Neuron-specific enolase, a
clinically useful marker for neurons and neuroendocrine cells.
Annu. Rev. Biochem. 10 (1987) 269-295.
Miyata, T. and Yasunaga, T.: Estimation of evolutionary dis-
tances between homologous nucleotide sequence. Proc. Nat].
Acad. Sci. USA 78 (1981) 450-453.
Miyata, T., Yasunaga, T. and Nishida, T.: Nucleotide sequence
divergence and functional constraint in mRNA evolution.
Proc. Nat]. Acad. Sci. USA 77 (1980) 7328-7332.
Pearce, J.M., Edwards, Y.H. and Harris, H.: Human enolase
isoenzymes: electrophoretic and biochemical evidence for
three loci. Ann. Hum. Genet. 39 (1976) 263-276.
Ponte, P., Gunning, P., Blau, H. and Kedes, L.: Human actin
genes are single copy for a-skeletal and a-cardiac actin but
multicopy for /?- and y-cytoskeletal genes: 3’ untranslated
regions are isotope specific but are conserved in evolution.
Mol. Cell. Biol. 3 (1983) 1783-1791.
Ponte, P., Ng, S., Engel, J., Gunning, P. and Kedes, L.:
Evolutionary conservation in the untranslated regions of
actin mRNAs: DNA sequence of human beta-actin cDNA.
Nucleic Acids Res. 12 (1984) 1697.
Rider, CC. and Taylor, C.B.: Enolase isoenzymes in rat tissue.
Electrophoretic, chromatographic, immunological and kinet-
ic properties. Biochim. Biophys. Acta 365 (1974) 285-300.
Saidapet, C., Khandekar, P., Mendola, C. and Siddiqui, M.A.Q.:
Tissue specificity of 3’untranslated sequence of myosin light
chain gene: unexpected interspecies homology with repetitive
DNA. Arch. Biochem. Biophys. 233 (1984) 565-572.
Sakimura, K., Kushiya, E., Obinata, M. and Takahashi, Y.:
Molecular cloning and nucleotide sequence of cDNA to
mRNA for non-neuronal enolase (aaenolase) ofrat brain and
liver. Nucleic Acids Res. 13 (1985a) 4365-4378.
Sakimura, K., Kushiya, E., Obinata, M., Odani, S. and
Takahashi, Y.: Molecular cloning and nucleotide sequence of
cDNA for neuron-specific enolase messenger RNA of rat
brain. Proc. Natl. Acad. Sci. USA 82 (1985b) 7453-7457.
Sakimura, K., Kushiya, E., Takahashi, Y. and Suzuki, Y.: The
structure and expression of neuron-specific enolase gene.
Gene 60 (1987) 103-113.
Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with
chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74
(1977) 5463-5467.
Schmechel, D.E., Brightman, M.W. and Marangos, P.J.:
Neurons switch from non-neuronal (NNE) enolase to neuro-
nal (NSE) enolase during development. Brain Res. 190 (1980)
95-214.
Shaw, G. and Kamen, R.: A conserved AU sequence from the
3’ untranslated region ofGM-CSF mRNA mediates selective
mRNA degradation. Cell 46 (1986) 659-667.
Yaffe, D., Nudel, U., Mayer, Y. and Neuman, S.: Highly con-
served sequences in the 3’ untranslated region of mRNA
coding for homologous proteins in distantly related species.
Nucleic Acids Res. 13 (1985) 3723-3737.
Zeltser, P.M., Marangos, P.J., Sather, H., Evans, A., Siegel, S.,
Wong, K.Y., Dalton, A., Seeger, R. and Hammond, D.: Prog-
nostic importance of serum neuron specific enolase in local
and widespread neuroblastoma. In Evans, A.E., D’Angio,
G.J., and Seeger, R.C. (Eds.), Advances in Neuroblastastoma
Research, Progress in Clinical Biological Research. Vol. 75,
Liss, New York, 1985, pp. 319-329.
