volume 8 Number 231980
Nucleic Acids Research
Alcohol dehydrogenase in Drosophila: isolation and characterization of messenger RNA and
cDNA clone
Cheeptip Benyajati**, Nancy Wang**, Aijula Reddy + , Eric Weinberg^ and William Sofer**
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
Received 3 September 1980
ABSTRACT
The mRNA for alcohol dehydrogenase (ADH) 1n D. melanogaster has been
I d e n t i f i e d by translation In a c e l l - f r e e system. The In vntro synthesized
polypeptide, s p e c i f i c a l l y precipitated by anti-ADH antibody, has Identical
subunit molecular weight (25,000 daltons) and t r y p t i c peptide p r o f i l e to
the In y1yo synthesized ADH. The poly A containing ADH-mRNA has been
p u r i f i e d by specific precipitation of ADH-polysomes using ant1-ADH antibody
and S. aureus. Transformation of E. c o l l with the dA-ta1led ADH-mRNAcoraplementary DNA hybrid annealed to the dT-tailed pBR322 yielded one
plasmid which has been I d e n t i f i e d as the AOH-cDNA clone. The I d e n t i f i c a t i o n Involved hybridization selection of ADH-mRNA and In v i t r o
t r a n s l a t i o n , 1n s i t u hybridization to the Adh locus on salivary gland
polytene chromosomes and DNA sequencing. This ADH-cDNA plasmid contains
349 bases of the C-terrainal protein coding and 180 bases of the 3'
untranslated region.
INTRODUCTION
Alcohol dehydrogenase (ADH) 1n DrosophUa melanogaster Is a well
studied gene-enzyme system with several Interesting biochemical,
genetic and regulatory features. Because of I t s relative abundance (1t
comprises 1-2% of the total soluble protein of mature adults) ADH protein
has proven easy to purify (1,2). The a v a i l a b i l i t y of the pure protein has,
1n turn, allowed for the enzyme to be extensively characterized. These
studies have revealed DrosophUa ADH to be quite different from the
mammalian l i v e r and yeast ADH's; 1t has a smaller subunit molecular weight
(25,000 vs. 39,800 for the l i v e r enzyme and 35,300 for the yeast) and lacks
the bound Zn^+ (3) required for catalysis by the other ADH's. The
conpleted sequence of the protein (4,5) has shown very l i t t l e homology
between DrosophUa ADH and either l i v e r or yeast enzymes.
From a genetic point of view, Drosophila ADH Is v i r t u a l l y unique among
genes 1n higher organisms because chemical selection procedures have been
developed that allow screening for f l i e s that lack enzyme a c t i v i t y (6,7).
The large number of homozygous viable mutants recovered that lack active
© IRL Press Limited. 1 Falconberg Court, London W1V 5FG, U.K.
5649
Nucleic Acids Research
enzyme Indicate that enzyme a c t i v i t y 1s not required for survival or
f e r t i l i t y ( 8 ) . These mutants have also made 1t possible to precisely map
the location of the Adh_ structural gene both genetically (9,10) and
cytologically (10,11,12).
F i n a l l y , the expression of the Adh gene appears to be regulated.
A c t i v i t y Is high at certain stages of development (1n mature adults and
l a t e t h i r d Instar larvae) and low at others (In pupae). Moreover, certain
tissues, such as f a t body, Intestine and Malp1gh1an tubules have r e l a t i v e l y
high enzymatic a c t i v i t y ; others, l i k e salivary glands and 1mag1nal discs
have l i t t l e or none (13).
We have been Interested 1n the regulation of gene expression of the
Adh gene. Our strategy has been to use the selective procedures that we
developed to secure mutants at the Adh locus. Among these mutants we hope
t o f i n d some whose lesion i s 1n the regulatory machinery the gene. In
order to carry out this type of analysis we required a probe for the Adh
gene. In t h i s report, we f i r s t describe the Isolation and characterization
of ADH-mRNA. This nRNA was then used to produce an ADH-cDNA clone which
we 1n turn I d e n t i f i e d and characterized. The cloned cDNA w i l l allow us to
analyze wild type and mutant strains of DrosophUa for the presence and
structure of the Adh_ gene and ADH-mRNA.
MATERIALS AND METHODS
Organisms. The ADH-pos1t1ve s t r a i n s , Adi/ and Adh^ pr en which are
homozygous for the ADH^ and ADH" electrophoretic variants respectively,
were the sources of DrosophUa melanogaster RNAs. The symbols ££ (purple)
and £n_ (cinnabar) refer to eye color markers. E. c o l l K-12, strain
HB101, was obtained from Dr. H. Smith of the Microbiology Department, Johns
Hopkins University.
DNA and Enzymes. pBR322 DNA was used as the vector to transform
E. c o l l HB101. Plasmid DNA was p u r i f i e d according to Clewell (14). Avian
myeloblastosis virus reverse transcHptase was provided by Dr. J . W. Beard,
the Office of Program Resources and Logistics, NCI. Bacterial alkaline
phosphatase and terminal transferase were purchased from Bethesda Research
Labs. Restriction enzymes were from Bethesda Research Labs and New England
BioLabs. Polynucleotide kinase, nucleotides and p(dT)i2-18 w e r e from
PL Biochemicals.
Radioactive materials were purchased from New England Nuclear and
Amersham.
5650
Nucleic Acids Research
RNA. All glassware and solutions were either autoclaved, f i l t e r
s t e r i l i z e d or diethylpyrocarbonate treated (15). For the preparation
of total cytoplasmic RNA from DrosophUa, four- to six-day-old Imagoes
( l i v e or stored frozen In l i q u i d N2) were ground In l i q u i d N2 In a mortar
to a fine powder. They were then homogenized 1n a l o o s e - f i t t i n g Dounce
horaogenizer 1n 0.25 M sucrose, 0.25 NaCl, 0.25 M NH4CI, 0.05 M MgCl2,
0.025 M Tris-HCl, pH 7.5, 0.005 M EGTA, 0.005 M N-ethyl male1m1de,
100 iig/ml heparin, 0.5% (v/v) e-mercaptoethanol; 0.001 M cytosine
monophosphate and 50 yg/ml spermine (20 ral/g f l i e s ) . (This buffer
recipe was recommended to us by Dr. D. Sullivan, Syracuse University.) The
homogenate was centrifuged (10 m1n at 10,000 rpm, 4°C 1n a Sorvali HB4
rotor) and the resulting supernatant made 1% NP40. This solution was
extracted once with phenol (saturated with 0.01 M Tr1s-HCl, pH 7.5) and
three times with chloroform. RNA was precipitated from the aqueous phase
by adding 0.1 volume of 3 M sodium acetate and 2.5 volumes of cold ethanol
and c h i l l i n g In a dry 1ce/ethanol bath. The precipitate was collected
by centrifugation washed once with 70% ethanol, air-dried and resuspended
1n d i s t i l l e d H2O. The RNA was stored either In ethanol at -20°C or as
an aqueous solution at -20°C.
For the Isolation of polyadenylated RNA, poly U-sepharose (Pharmacia)
was employed using deionized formamide containing buffers.
Partial purification of RNA was accomplished using 15-30% (w/v)
sucrose gradients containing 0.01 M Tr1s-HCl, pH 7.5 and 0.5% SDS.
Sedimentation was for 19 hr at 26,000 rpm at 20°C 1n a Beckman SW27 rotor.
RNA was ethanol precipitated from the collected fractions and washed
several times with 70% ethanol 1n order to eliminate sucrose.
Isolation and 1mmunoprec1p1tat1on of polysomes. Flies were ground
1n l i q u i d N2, homogenized and the homogenate centrifuged as described
above. After adding NP40 to 1%, polysomes were purified by sedimentation
for 5 hr at 50,000 rpm at 4°C In a Beckman T1 60 rotor through a cushion
of 2 M sucrose 1n 0.3 M NH4CI, 0.05 M MgCl2, 0.05 M Tris-HCl, pH 7.5
and 0.5% e-mercaptoethanol. The polysomes were resuspended 1n 0.3 M
NaCl, 0.005 M MgCl2 and 0.05 M Tris-HCl, pH 7.5 at 100-200 A26o/ml •
Immunoprecipitation of polysomes was done according to the methods
of Gough and Adams (16) using goat ant1-ADH antibody. RNA was then purified
using the phenol/chloroform extraction procedure described above.
In v i t r o c e l l - f r e e translation. DrosophUa RNA was translated In
a cell-free protein synthesizing system prepared from nuclease-inactivated
5651
Nucleic Acids Research
rabbit ret1 culocyte lysates (17). Tr1t1ated leudne was used to follow
Incorporation. The translation products were electrophoresed 1n SDSpolyacrylamide gels (18) and visualized by fluorography (19).
To Isolate cross-reacting material from the translation products, a
procedure similar to that of HI! man et a l . (20) was used (21). The
translation mixture was diluted 30-fold with 0.03 M KC1, 0.01 M Tr1s-HCl,
pH 7.4, and 0.5% Triton X-100 and subjected to chromatography on a column
of Sepharose linked to goat ant1-ADH. The column was washed with 0.03 M
KC1 , 1 H NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.01 M Tris-HCl,
pH 7.5 and then with deionized H2O. ADH was eluted from the column with
0.2* SOS. The eluate was lyophHized and resuspended 1n SOS-gel sample
buffer and electrophoresed as above.
Tryptic peptide analysis. In order to show that the radioactive
product synthesized 1n v i t r o and Isolated by Immunochromatography was
ADH, the In v i t r o translation mixture was mixed with purified l4 C-leuc1ne
labelled ADH^ (labelled In vivo), Immunopredpitated and subjected to
SOS-polyacrylamide gel electrophoresis. The gel was fractionated and the
l^C-ADH containing band TCA precipitated. The TCA was removed by ether
extraction and the protein was digested with trypsin. The resultant
peptides were fractionated on an Aminex A-5 1on exchange column (22).
Construction of recombinant plasmids. cDNA was synthesized from both
sucrose gradient-enriched RNA and from antibody precipitated polysomal
RNA. Each 50 pi reaction mix contained 0.1 M Tr1s-HCl, pH 8.7, 6 M
MgCl2, 60 raH KC1 , 30 mM B-mercaptoethanol, 100 pg BSA/ml, 20 pg
p(dT)i2-l8/ m 1 . 8 0 0 n9 RNA/ml (heated at 68°C, 10 nrln and quick-cooled
before addition), 1 nM each of dGTP, dCTP, dATP and TTP and 10 uC1
each of 3H-dNTPs as tracers. Reactions were started by the addition of
32 units of reverse transcriptase. Incubation was for 1 hr at 45°C at
which point another 32 units of enzyme were added and the Incubation
continued for an additional hr. The reactions were stopped by centrifuging
the Incubation mixtures through packed Sephadex G-50 1n microfuge tubes to
eliminate unincorporated nucleotides and for desalting. The cDNA
synthesized under these conditions were generally greater than 600
nucleotides 1n length, and represented about 5% of the Input RNA.
The mRNA-cDNA hybrid was then " t a i l e d " (23) 1n 50 pi of a reaction
mixture which contained 0.14 H potassium cacodylate, 0.03 H Tr1s base,
1 nW C0CI2, 100 pM e-mercaptoethanol, 100 nW dATP, 200 yg BSA/ml,
200 units terminal transferase/ml and about 1 pg of 3H-CDNA-RNA hybrid.
5652
Nucleic Acids Research
After 100 rain of Incubation at 37°C, the reaction was stopped by addition
of EDTA to 5 mM. The Incubation time was calculated to yield 50-75 added
dA residues per 3' end. pBR322 DNA (8 ug) was linearized with Pst I and
t a i l e d similarly for 4 m1n at 37°C using 100 uM TTP. About 50-70 dT
residues were added per 3' end.
Equimolar quantities of the dA-ta1led mRNA-cDNA hybrids (average
cDNA length = 1 kb) and the dT-ta1led pBR322, both freed of low molecular
weight components by centrifugation through Sephadex G-50, were combined
In 0.1 M NaCl, 1 m EDTA and 0.01 M Tr1s-HCl, pH 7.5. The mixture was
heated at 68°C for 10 m1n and annealed at 43°C for 2 hr. The resultant
hybrid molecules were used to transform E. coll K-12, strain HB101
by the transfection procedure of Mandel and H1ga (24).
We obtained 74 tetracycline resistant, ampicHlin sensitive
transformants from 0.5 pg of cDHA synthesized from sucrose gradient
enriched RNA. The DNA Inserts range from 500 to 2500 bases with the
majority larger than 900 bases. Forty-six transformants were obtained from
0.4 pg of cDNA synthesized from antibody selected polysomal RNA. The
DNA Inserts range from 200 to 1300 bases with the majority being BOO bases.
Hybr1d1zat1on-select1on and translation. DNA (1 pg) from pBR322,
bacteriophage X and pBR322 containing DrosophUa sequences was Immobilized
on nitrocellulose f i l t e r s (Schleicher and Schull, BA85) and u t i l i z e d
In hybridization-selection and translation according to R1cc1ard1 et a l . (25).
In situ hybridization. DrosophUa salivary gland chromosomes were
prepared for 1n situ hybridization by a modification of procedure of Pardue
and Gall (26) as described by Strobel et a l . (27). N1ck-translat1on was
performed according to R1gby et a l . (28) using t r i t i a t e d dNTPs.
DNA sequencing. 5' terminal labelling of restriction fragments by
polynucleotide kinase and DNA sequencing were performed as described by
Haxam and Gilbert (29). Polyacrylamide gels of 0.4 im thick were used.
The sequences were analyzed by the program of the Stanford Molgen Project
and the NIH SIWEX-AIM f a c i l i t y .
Northern hybridization. RNA was glyoxalated and separated on agarose
gels according to HcMaster et a l . (30). The RNA was transferred to diazobenzyloxymethyl (DBH)-paper and hybridized with 32p_labelled nick-translated
DNA (31).
Results
Identification of ADH-mRNA.
Mature adult flies have been observed to
5653
Nucleic Acids Research
synthesize AOH (32) Indicating that they possess functional ADH-mRNA. To
Identify this ADH-mRNA we Isolated RNA from wild-type f i l e s and translated
1t 1n a mRNA dependent rabbit reticulocyte c e l l - f r e e system. The resultant
3
H-labelled translation products were separated by SDS-gel electrophoresis
and visualized by fluorography. Figure 1 shows the results of this
experiment. The polypeptides synthesized when total cytoplasmic, poly Acontaining and poly A-lack1ng RNA were translated 1n v i t r o are shown 1n
lanes C, B and A respectively. The total cytoplasmio and poly A-conta1n1ng
preparations (these show the bulk of translational a c t i v i t y ) code for a
great variety of proteins, with one prominent band, accounting for 1-2%
of the total translational a c t i v i t y , migrating to the same position on SOSpolyacrylamide gel as ADH monomer (corresponding to a molecular weight of
25,000).
Protein of this same size binds specifically to anti-ADH antibody
coupled to Sepharose (Figure 2, lanes C, D and G). The results of a
control experiment, showing that the antibody specifically binds ADH Is shown
1n Figure 2, lane B.
Further confirmation that ADH can be synthesized 1n araRNAdependent
protein synthesizing system 1s shown In Figure 3. Here ^H-leudne labelled
protein synthesized 1n v i t r o was subjected to antibody precipitation and
SDS-polyacrylamide gel electrophoresis. The material migrating at a
position corresponding to a molecular weight of 25,000 was then subjected
to tryptic digestion and the resultant peptides separated by 1on exchange
chromatography. Authentic l^C-leudne labelled ADH was present during
these manipulations to serve as a marker. As shown 1n Figure 3 the peptide
p r o f i l e s of authentic ADH matches almost exactly that of the 1n v i t r o
synthesized product.
From these experiments we conclude that DrosophUa adults contain a
polyadenylated mRNA that can direct the synthesis of ADH. ADH was
Identified by antibody precipitation, by SDS-gel electrophoresis and by
virtue of I t s t r y p t i c peptide pattern.
ADH-mRNA p u r i f i c a t i o n . We used two approaches to enrich for ADHmRNA. F i r s t , total cytoplasmic RNAs were fractionated by sedimentation
through sucrose gradients containing SDS. ADH-mRNA was assayed by
translation In a reticulocyte 1n v i t r o system and SDSgel electrophoresis
The results of a single round of enrichment yielded one fraction containing
RNAs of 11-13S that coded for polypeptides of about 23,000 to 35,000
daltons (data not shown). A second round of purification by sucrose
5654
Nucleic Acids Research
A
B
C
D
CONALBUMIN
•BSA { 6 8 K)
— OVALBUMIN
( 8 6 K)
(43K)
PURIFIED
— ADH ( 2 5 K)
(IN VIVO LABELED)
• — LYSOZYME
(15 K)
Figure 1. In v i t r o translation products of DrosophUa RNAs. Drosophila
RNAs were translated In the Inactivated rabbit reticulocyte lysate cell
free system. The ^H-labelled translation products were electrophoresed
on a 12-1/2% SDS-polyacrylamide gel and displayed by fluorography.
A: poly A-lack1ng RNA translation, 40 yg translated , B: poly
A-conta1n1ng RNA translation, 20 yg translated, C: total cytoplasm 1c
RNA translation, 20 yg translated, D: mixture of 3 H-labelled purified
protein molecular weight markers. All markers except 1n vivo 3 H-labelled
ADH were labelled by the alkylation reduction procedure (42).
5655
Nucleic Acids Research
A
B
C
D
E
F
G
43K
-ADH (25K)
Spe
pna
antibody sepharose
column.
The d i l u t e d • J H-TabeTTedTransTatTon products
were chromatographed on ant1-ADH antibody sepharose columns as described
1n the Method Section. S p e c i f i c a l l y bound m a t e r i a l s were e l u t e d and
electrophoresed on a 12-1/2% SDS-poiyacrylamide gel and displayed by
f l u o r o g r a p h y . A, E and F: 3 H - l a b e l l e d p r o t e i n molecular weight markers,
B: p u r i f i e d I n v i v o ^ H - l a b e l l e d ADH retained by the column, C: t o t a l
cytoplasmic RNA from the Adh^ s t r a i n t r a n s l a t i o n , D: poly A-conta1n1ng
RNA from the Adhf s t r a i n t r a n s l a t i o n , G: t o t a l cytoplasmic RNA from
the Adh" strain translation.
Figure 2.
gradient sedimentation yielded a preparation 4-5 fold enriched for ADH-mRNA
(Figure 4, lane D).
Second, AOH-raRNA was purified from ADH containing polysomes specifically
precipitated by ant1-ADH antibody and S. aureus (16). Purification of at
least f i f t y - f o l d was achieved; ADH polypeptide was the only 1n v i t r o
translation product detected from such RNA preparations (Figure 4, lanes B
and C).
cDNA cloning. The protocol we used to clone cDHA 1s described 1n the
Materials and Methods section. A similar approach was reported by Wood and
5656
Nucleic Acids Research
800
5
10
15
20
FRACTION NUMBER
25
400
2000
300
i
to
200 *
4
1000
100
20
40
60
FRACTION
80
100
120
NUMBER
Figure 3. Analysis of 1n v i t r o synthesized ADH. a) In v i t r o ( 3 H-labelled)
synthesized protein was Immunoprecipitated with ant1-ADH antibody 1n the
presence of In vivo ( 14 C-labelled) synthesized ADH. The 1mmunoprec1p1tate
was subjected to SDS-gel electrophoresis (10% gels) and the gels fractionated
and counted. Electrophoresis was from right to l e f t , b) Tryptic peptide
p r o f i l e of In vivo (^C-labelled) and 1n v i t r o ( 3 H-labelled) synthesized
ADH fractionated on the arainex A-5 1on exchange column.
Lee (33) and Za1n et a l . (34). The advantage of this procedure over the
conventional double-stranded cDNA method Is that fewer steps are required,
thus reducing loss of material. However, as also noted by Zain et a l .
(34), the transformation efficiency 1n E. c o l l of such molecules Is quite
5657
Nucleic Acids Research
B
C
D
86 K
68 K
43 K —
AOH
(25K)
«
Globin —
Figure 4. SDS-polyacrylamide gel electrophoresis of in v i t r o translation
products from antibody precipitated polysomal RMA. 5A260 units of
polysomes per ml were Immunoprecipitated as described by Gough and Adams
(16) using 135 and 270 yg of anti-ADH antibody and 375 and 750 yl
respectively of packed Staphylococcusaureus c e l l s . The RNAs from
phenol extracted polysomes were translated in a c e l l - f r e e system, the
translation products electrophoresed on a 12-1/2% SOS-polyacrylamide gel
and fluorographed. A: ^H-labelled protein molecular weight markers, B;
translation of polysome precipitated by 135 yg of anti-ADH antibody and
375 yl of packed Staphylococci, C: translation of polysome precipitated
by 270 yg of anti-ADH antibody and 750 yl of packed Staphylococci and
D: translation of sucrose gradient enriched RNA.
low possibly due to the nuclease sensitive nature of the hybrid molecules.
Both sucrose gradient enriched RNA and antibody selected polysotnal RNA
yielded the same efficiency—about 1 X 102 transformants per yg of cDNA.
Similar transformation efficiencies were reported (33,34).
5658
Nucleic Acids Research
I
2 3 4 5 6 7 8 9 10 II 12 13 14
,_86 K
1-68 K
— 43 K
— ADH
(25K)
Figure 5. SDS-polyacrylI amide gel electrophoresis of In v i t r o translation
products of RNAs hybridized by various DNAs.
1 pg of each DNA was
Immobilized on nitrocellulose and hybridized to Drosophila RNA as
described by Ricdardi et a l . (25). The specifically bound RNA was
eluted and translated 1n a c e l l - f r e e system, the translation product
electrophoresed on a 12-1/2% SOS-polyacrylamide gel and fluorographed.
Lanes 1, 14: 3 H-labelled protein molecular weight markers, 2-7:
translation of RNAs hybridized to six different plasroids containing
Drosophila DMA Inserts, 8: pBR322 DNA, 9: XDNA, 10: translation of RNAs
which do not bind to a blank f i l t e r In a mock hybridization, 11:
translation of RNAs which bind to a blank f i l t e r In a mock hybridization,
12: sucrose gradient enriched RNA translation, 13: no exogenous RNA added.
5659
Nucleic Acids Research
a
a' .
i
V
0
•
\
y
|V
'i
'mm
Nucleic Acids Research
From a transformation experiment using antibody selected poiysomal
RNA-cDNA hybrid molecules, one plasmid carrying ADH-cDNA (pAOH-cDNA) has
been I d e n t i f i e d by the three following c r i t e r i a .
F i r s t , using the
technique of hybridization selection and translation ( 2 5 ) , we demonstrated
t h a t DNA from this clone was capable of hybridizing to an RNA species that
was translatable Into ADH (Figure 5, lane 6 ) .
Control DNA from bacterio-
phage x, pBR322 and other plasmids f a i l e d to do t h i s .
Second,
3
H-labelled nick-translated pADH-cDNA hybridized s p e c i f i c a l l y
to only the region 35 Bl-5 on salivary gland chromosomes from D. melanogaster
(Figure 6a, b and c ) .
This region had previously been I d e n t i f i e d as the
s i t e of the Adh structural gene by genetic and cytological analyses ( 9 , 10,
1 1 , 12).
Third, the AOH.cONA Insert has been sequenced (Figure 7a and b ) .
Translation of this sequence yields a protein sequence that corresponds to
the C-terminal amino a d d portion of ADH ( 4 ) .
The sequencing studies also
show that pADH-cDNA contains an Insert of about 710 bases; 348 code for
116 C-term1nal araino acids (45% of the ADH p r o t e i n ) , 180 represent
nucleotides located a t the 3' untranslated region of the ADH-mRNA, and the
remainders are about 150 and 30 bases of dA residues added at the 5' and 31
ends of ADH-raRNA respectively.
Characterization of ADH-mRNA.
ADH-mRNA was characterized further by
"Northern" analysis using pADH-cDNA as a probe.
RNA was denatured by
glyoxalation and subjected to electrophoresis on agarose gels.
transferring to DBM-paper, ADH-mRNA was probed with
translated cDNA clone.
32
After
P - l a b e l l e d nick-
Two RNA preparations, one prepared by sucrose gradient
centMfugation, the other was antibody precipitated polysomes, each
showed one hybridizable band of 1120 +^20 bases (Figure 8 ) .
Since about
750 bases of the mRNA are Involved with protein coding (ADH contains about
250 aroino a d d s ) , and the 3 1 untranslated region 1s 180 bases long, and
assuming the length of the 31 poly A segment 1s about 100 bases, the 51
untranslated region would consist of about 100 bases.
Figure 6. In situ hybridization of 3 H-1abelled pADH-cDNA to salivary gland
poly tene chromosomes.
Three d i f f e r e n t chromosome sets are shown 1n a ,
b" and c1 at low magnification and t h e i r 2L ( l e f t arm of second chromosome)
portions are shown at higher magnification 1n a, b and c respectively.
Arrows point to the s i l v e r grains over the 35B1-3 region.
5661
Nucleic Acids Research
Transcription
5'
Prottin
Coding
3 Untramlottd
349
'
x
o
I
I
180
I 3O-2OO 1 * 1
O.I kb
I J O - I O O
( I
' ^
6 T
C » J T 6 ( A T J C A » T 6 C C A T C T A C C A G 6 T 6 C C ' C 6 T C I A C ' T C C 6 G C A C C
rml l{r 6lt ri* Im tla IH TIT tin rat Pr* ral r»r lac « | rtr
AA6aCC6CC6T'6 6 T C A A C T T C A C C A 6 C T C c ' c T G G C 6 A A A C T S 6 C C C C C A T T A CCI6C6T C
Ifa
i l a l l a Fal Fal iaa P»* rkr far Xar La» i U
l«a L*a i l a Pra II* far Big
rat
A C S S C T T A C A T T 6 T 6 A A C C C C 6 6 C A T C A C c'c*6 C A C C A C C C T t C T 6 C A C A c T r T C A A C T C C
rhi*
i l a !>>• Tnr ral i«a fn
Big f l a far i i f
I>r rkp Lav ral •<• rtr Pha i*a ffi*
T 6 6 T T 6 6 A T 6 T ' T ( A G C C T C A . 6 T I ( C C 6 A 6'A'A ( C T C C T 6 S C T C A t C C C A t C C A 6 C C C T C 6
Trp
t*» laf
ral ft* ft-* 01* ral Aim Urn If a
Ltu ton l l a li«
1*F«
n r Ola fr* Itr
T T 6 6 C C T 6 C e'c'c G A 6 A A C T J C 6 T C A A G . C T V T C G A 6 C T 6 A A C C A S A A C G 6 A ' G C C A T C T G 6
£n
ila
Cf
ila
A A A C T G ( ACcTt
If
I
tak
1
iaa
•!•
i«i
Ma ' ral Ifi
8 6 C A C C C T G G AttC
taa
...
«T f
Tar
Lai.
CU
ila
/la
«la
C ATC'C'AGTGG
ila
71*
.
tin
IH
£*• i n
fla
A C C A A6CAC
Tr,
Tnr
Lff
.
ifa
«1r ila
T G 6 G ATT
fl<*
fr*
i*p
H* rra
CCI6 CAT C
br
fil|
/la
•••
[TAAlGAA.TSATACTCCCAAAAAAAAAAAAAAACATAACATIAGITCATAGGGTTCIGCtA
•«
1^
•
t«.J
A C C A 6 A A G A i T l I C A C G C A A G G C A A T A A G
G'C'T
6 A I T C 6 A T t C A C A C T C A c T t
I C T I C I C C
J*
T AA T A C . ATIA'A'T A A " A ] A C T T T C C A l tAA A A A ' T A T t 6 A A A A } A T A T 6 A A A A j T 6 A 6 A A A T C C ( A >
M
Figure 7 . Nucieotide sequence of pADH-cDNA I n s e r t , a. Diagram of
r e s t r i c t i o n map and sequencing strategy employed. The regions covered by
sequencing are Indicated by arrows. The solid dots correspond to the
32p-iabelled 5' ends. The orientation of pADH-cONA Inserted Into the
PstI s i t e (between nucleotides 3612 and 3613) of pBR322 I s as shown,
b. 529 bases of the coding strand (mRNA l i k e ) of pADH-cDNA Insert and
corresponding amino acid sequence (AdhJ strain) are shown. The termination
codon, TAA I s I n a dashed box. The AATAAA sequence, present near the 31
end 1n a l l polyadenylated eucaryotic mRNAs, 1s boxed. The 3' untranslated
symmetric region 1s shown by arrows pointing away from the axis of
symmetry. The 3' untranslated homologous region 1s underlined. The wavy
l i n e Indicates the single amino a d d difference between the Adh*7 and
Adhs f l y s t r a i n s .
5662
Nucleic Acids Research
A
B
1776
I I 75
107 I
548
4 44
Figure 8. Autoradiogram of Drosophiia RNA hybridized to pADH-cDNA.
8 ug of RNA was glyoxalated and electrophoresed on 1.2% agarose g e l ,
transferred to a DBM-paper and probed with " p - l a b e l l e d nick-translated
pADH-cDNA. A: sucrose gradient enriched RNA and B: Immunopredpitated poiysomal RNA. The size standards ( I n bases) are 32p_ un tformly labelled
SV40 DNA digested with Hind I I I .
DISCUSSION
We have I d e n t i f i e d , Isolated and characterized ADH-mRNA from Drosophila
melanogaster.
The message 1s about 1120 nucleotides long, 1s polyadenylated,
has untranslated regions at the 51 and 3 1 ends, and accounts for about 1-2$
5663
Nucleic Acids Research
of the translational a c t i v i t y of mRHA from adult f l i e s . Using the Imrrainoprecipitation procedure of Gough and Adams (16), we have obtained at least
50-fold enrichment for this mRNA as assessed by In v i t r o translation.
From this mRNA, using dA-ta1led mRNA-cDNA hybrid molecules annealed to
dT-ta1led pBR322, we have constructed a cDNA clone. The transformation
efficiency of such hybrid structures was lower than that expected for doublestranded DNA, but the resultant pADH-cDNA clone proved to be a f a i t h f u l
copy of a portion of the ADH-raRNA; the translated amino a d d sequence matched
the known C-terminal amino add sequence of ADH and the nucleotide sequence
corresponded to the sequence of a genomic clone (5) Isolated from the
Man1at1s library (35).
However, this cDNA clone was not of f u l l length. I t 1s possible that
the incomplete length was due to some sequence and/or secondary structure
peculiar to ADH-mRNA that might have affected the reverse transcription
reaction. Others have observed that reverse transcriptase makes nonrandom
stops along mRNA (36,37). He also cannot exclude the p o s s i b i l i t y that the
t a i l i n g reaction and/or transformation of E. coll using the hybrid molecules
might have adversely affected the final size of the cDNA Insert.
Several observations can be made about the nucleotide sequence of
the protein coding and the 3' untranslated regions of ADH-mRNA. Like
other eucaryotic messages, the codon usage (Table 1) of this mRNA 1s
nonrandom. There 1s underutH1zat1on of codons ending 1n A and preference
f o r codons ending 1n C and G. This observation 1s true for the complete
genoaic ADH gene as well (5). Other eucaryotic mRNAs (for example,
3-maJor globin mRNA (38)) exhibit different preferred codons. Thus, codon
usage seems to be message specific.
The difference 1n electrophoretic mobilities of ADHF and ADHS from
two naturally occurring Adhfast and Adh^low f | y strains has been
attributed to at least one amino add change (threonine 1n ADH^ for lysine
1n ADHS, (39)). This amino a d d , underlined 1n Figure 9 by a wavy l i n e ,
1s threonine 1n pADH-cDNA which comes from the AdhJ s t r a i n . A single
base change (ACG for threonine to AAG for lysine) has occurred at this
position 1n the Adh$ s t r a i n .
The 3' untranslated region, which Is AT r i c h , has several Interesting
features. The symmetric region (nucleotides 366-373; 375-382) contains the
longest stretch of A residues (15 bases). The homologous region (nucleotides 499-509; 510-519) 1s 10 nucleotides from the putative poly A-add1t1on
s i t e (the dinucleotide CC). The hexanucleotide AATAAA, which Is usually
5664
Nucleic Acids Research
Table 1 .
Codon usage of the C-terminai portion of ADH.
o/m/PHE
0/TCT/SER
0/TAT/TYR
4/TTC/PHE
4/TCC/SER
3/TAC/TYR
1/TGC/CYS
O/TTA/LEU
0/TCA/SER
0/TGA/OP
2/nG/LEU
1/TCG/SER
1/TAA/OC
0/TAG/AM
o/cn/LEu
1/CCT/PRO
0/CGT/ARG
0/TGT/CYS
1/TGG/TRP
1/CTC/LEU
5/CCC/PRO
1/CAT/His
2/CAC/His
0/CTA/LEU
0/CCA/PRO
0/CAA/GLN
0/CGA/ARG
8/CTG/LEU
0/CCG/PRO
5/CAG/GLN
0/CGG/AR6
VAn/iLE
2/ACT/THR
1/AAT/ASN
0/AGT/SER
6/ATC/ILE
9/ACC/THR
6/AAC/ASN
1/AGC/SER
0/ATA/ILE
0/ACA/THR
2/AAA/LYS
0/AGA/AflG
0/ATG/MET
2/ACG/THR
1/AAG/LYS
0/AGG/ARG
2/GTT/VAL
3/GCT/ALA
I/GAT/ASP
0/GGT/GLY
1/GTC/VAL
9/GCC/ALA
2/GAC/ASP
5/GGC/GLY
0/GTA/VAL
0/GCA/ALA
0/GAA/GLU
2/GGA/GLY
5/GTG/VAL
1/GCG/ALA
5/GAG/GLU
0/GGG/GLY
1/CGC/AR6
found In other eucaryotic mRNAs about 10-20 nucleotides p r i o r to the poly
A-add1t1on s i t e and thought t o be signal f o r t r a n s c r i p t i o n termination or
poly A addition ( 4 0 ) , 1s located 45 nucleotides from the putative poly
A-add1t1on s i t e of t h i s message. I t 1s not known, whether any of these
sequences play a role 1n gene expression and r e g u l a t i o n .
We have used pADH-cDNA as a probe f o r I d e n t i f y i n g genoraic clones of
ADH. We have now completely sequenced one such clone t h a t contains
the e n t i r e coding sequence f o r the ADH polypeptide ( 4 1 ) . There are two
Intervening sequences 1n the protein coding region. Both contain the 5'
GT and 3'AG dinucleotides c h a r a c t e r i s t i c of those a t the Intervening sequence
boundaries of eucaryotic genes.
ACKNOWLEDGEMENTS
We would l i k e t o thank David Goldberg f o r coranunieating h i s procedure
5665
Nucleic Acids Research
f o r raRNA-cDNA hybrid cloning.
This work was supported by grants GM21322-01
to E. W., GM-18257, NIEHS (ES-01527) and a contract from Department of
Energy (EY-76-S-O2-2965) to W.S.C.B. was a postdoctoral fellow of the Damon
Runyon-Walter Winchell Cancer Fund, DRG-175-F.
Present Address:
*Cancer Biology Program, Frederick Cancer Research Center, P.O. Box B,
Frederick, MD 21701
**Waksman I n s t i t u t e of Microbiology, Rutgers University, P.O. Box 759,
Piscataway, NJ 08854
+
School of L i f e Sciences, Central University of Hyderabad, Hyderabad, India
^Department of Biology, Biology Bu1ld1ng/G5, University of Pennsylvania,
Philadelphia, PA 19104
#To whom correspondence should be addressed.
REFERENCES
T.
Sofer, W. and Ursprung, H. (1968) J . B1ol. Chem. 243, 3110-3115.
2 . Schwartz, M., Gerace, L., O'Donnell, J . and Sofer, W. (1975) 1n
International Conference on Isozymes, Vol. 1 , pp. 725-751, Academic
Press, New York.
3. Place, A.R., Powers, D. and Sofer, U. (1980) manuscript submitted.
4 . Thatcher, O.R. (1980) Biochem. J . 187, 875-886.
5. Benyajati, C , Place, A.R., Powers, D. and Sofer, W., manuscript
In preparation.
6. Sofer, W. and Hatkoff, M. (1972) Genetics 72, 545-549.
7 . O'Donnell, J . , Gerace, L . , Leister, F. and Sofer, W. (1975) Genetics,
79, 73-83.
8. Schwartz, M. and Sofer, W. (1976) Genetics 83, 125-136.
9 . G r e l l , E . H . , Jacobson, K.B. and Murphy, J.B. (1965) Science 149, 80-82.
10. O'Donnell, J . , Mandel, H.C., Krauss, M. and Sofer, W. (1977) Genetics
86, 553-566.
1 1 . Woodruff, R.C. and Ashburner, M. (1979) Genetics 92, 117-132.
12. Woodruff, R.C. and Ashburner, M. (1979) Genetics 92, 133-149.
13. Ursprung, H . , Sofer, W. and Burroughs, N. (1970) Wlhelm Roux1
ArcMv. 164, 201-208.
14. Clewell, D.B. (1972) J . Bacteriol. 110, 667-676.
15. Palmiter, R.D. (1974) Biochemistry 13, 3606-3615.
16. Gough, N.M. and Adams, J.M. (1978) Biochemistry 17, 5560-5566.
17. Pelham, H.R.B. and Jackson, R.S. (1976) Eur. J . Biochem. 67, 247-257.
18. LaernnH, U.K.
(1970) Nature 227, 680-685.
19. Laskey, R.A. and M i l l s , A.D. (1975) Eur. J . Biochem 56, 335-341.
20. Milroan, G., Krauss S.W. and 01 sen, A.S. (1977) Proc. N a t l . Acad.
Sc1. USA 74, 926-930.
2 1 . P e l U c d a , J . G . (1980) Ph.D. Thesis, The Johns Hopkins University,
Baltimore.
22. Reddy, A.R., P e l U c d a , J.G. and Sofer, W. (1980) Biochemical
Genetics 18, 339-351.
5666
Nucleic Acids Research
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Roychoudhury, R., Jay, E. and Wu, R. (1976) Nucleic Acids Res.
3 , 863-877.
Mandel. M. and H1ga, A. (1970) J . Moi. B1ol. 53, 159-162.
R i c d a r d i , R.P., M i l l e r , J . S . and Roberts, B.E. (1979). Proc. N a t l .
Acad. Sci. USA 76, 4927-4931.
Pardue, M.L. and G a l l , J.G. (1975). 1n Methods 1n Cell Biology,
Prescott, D.M., Ed., Vol. 10, pp. 1-16, Academic Press, New York.
Strobel, E., Dunsmuir, P. and Rubin, G.M. (1979) Cell 17, 429-439.
Rigby, P.W.J., Oleckraann, H . , Rhodes, C. and Berg, P.
(1977)
J . Mol. B1ol. 113. 237-251.
Maxam, A.M. and G i l b e r t , W. (1980) 1n Methods 1n Enzymology,
Grossman, L. and Moldave, K. Eds., Vol. 65, pp. 499-560 Academic
Press, New York.
McMaster, G.K. and Carmichael, G.G. (1977) Proc. N a t l . Acad. Sc1.
USA 74. 4835-4838.
Alwine, J . C . , Kemp, D.J. and Stark, G.R. (1977) Proc. N a t l . Acad.
S c i . USA 74, 5350-5354.
P e l U c d a , J . G . , and Sofer, W. (1980) manuscript submitted.
Wood. K.0. and Lee J.C. (1976) Nucleic Acids Res. 3, 1961-1971.
Za1n, S . , Sambrook, J . , Roberts, R . J . . K e l l e r , W., F r i e d , M.
and Dunn, A.R (1979) Cell 16, 851-861.
Maniatis, T . , Hardison, R.C., Lacy, E., Lauer, J . , 0'Connell, C. and
Quon, D. (1978) Cell 15, 687-701.
Friedman, E.Y. and Rosbash, M. (1977). Nucleic Acids Res. 4, 34553471.
Buell, G.N., Wickeris, M.P., Payvar. F. and Schimke, R.T.
(1978)
J . B1ol. Chem. 253, 2471-2482.
Konkel, D.A., Tilghraan, S.M. and Leder, P. (1978) Cell 15, 1125-1132.
Fletcher, T . S . , Ayala, F . J . , Thatcher, D.R. and Chambers, G.K.
(1978)
Proc. N a t l . Acad. Sc1. USA 75, 5609-5612.
Proudfoot, N.J. and Brownlee, G.G. (1976) Nature 263, 211-214.
Benyajati, C. and Sofer, W. (1980) manuscript In preparation.
R1ce, R.H. and Means, G.E. (1971) J . B1ol. Chem. 246, 831-832.
5667
Nucleic Acids Research
5668