Citation
Expression of the human Delta-aminolevulinic dehydratase gene in yeast

Material Information

Title:
Expression of the human Delta-aminolevulinic dehydratase gene in yeast
Creator:
Schauer, Wren
Publication Date:
Language:
English
Physical Description:
vii, 34 leaves : illustrations ; 28 cm

Subjects

Subjects / Keywords:
Yeast ( lcsh )
Yeast ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 31-33).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Department of Integrative Biology.
Statement of Responsibility:
by Wren Schauer.

Record Information

Source Institution:
University of Colorado Denver
Holding Location:
Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
21114849 ( OCLC )
ocm21114849
Classification:
LD1190.L45 1989m .S32 ( lcc )

Full Text
EXPRESSION OF THE HUMAN DELTA-AMINOLEVULINIC DEHYDRATASE
GENE IN YEAST
by
Wren Schauer
B.S., Iowa State University, 1984
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Arts
Department of Biology
1989


This thesis for the Master of Arts degree by
Wren Schauer
has been approved for the
Department of Biology
by
Date


Ill
Schauer, Wren (M.A., Biology)
Expression of the Human Delta-aminolevulinic Dehydratase
Gene in Yeast
Thesis directed by Professor James R. Mattoon and Professor
Linda K. Dixon
ABSTRACT
A cDNA coding for human delta-aminolevulinate dehydratase
was placed in a yeast expression vector under the control
of the GAL10 promoter. This multicopy plasmid was then used
to transform a hem2 yeast strain which contains a defective
gene coding for delta-aminolevulinate dehydratase.
Expression of the human cDNA was shown in four ways: 1)
restoration of normal growth on glycerol/galactose as
primary carbon source, 2) decrease in intracellular delta-
aminolevulinic acid concentration, 3) restoration of
cytochrome biosynthesis and 4) direct, in situ assay of
delta-aminolevulinic acid dehydratase. Curing transformed
cells of plasmid restored the hem2 mutant phenotype. This
heterologous system could be used to produce large
quantities of human delta-aminolevulinic acid dehydratase
for physical and biochemical studies.
The form and content of this abstract are approved,
recommend its publication.,
lty member in charge of thesis


IV
CONTENTS
CHAPTER
I. INTRODUCTION ................................... 1
OBJECTIVE ...................................... 8
II. MATERIALS AND METHODS .......................... 8
PLASMIDS ....................................... 8
STRAINS...................................12
MEDIA AND GROWTH CONDITIONS ................... L2
TRANSFORMATION OF E. COLI AND YEAST......... 13
CONSTRUCTION OF PLASMID pSDl CONTAINING hALA-D 14
RESTRICTION MAPPING OF pSDl................. 15
CONSTRUCTION OF PLASMID pSD2 CONTAINING hALA-D 14
RESTRICTION MAPPING OF pSD2................. 17
CYTOCHROME DETERMINATIONS....................' 17
DETERMINATION OF INTRACELLULAR ALA AND ALA
DEHYDRATASE .............................. 18
III. RESULTS..................................... 18
CO-TRANSFORMATION OF hALA-D AND YEp24 .... 18
TRANSFORMATION OF C41/U1 WITH PLASMID pSDl . 19
TRANSFORMATION OF C41/U1 WITH pSD2 .......... 2 2
INTRACELLULAR ALA AND ALA DEHYDRATASE OF
TRANSFORMED AND CURED STRAINS ............ 25
IV. DISCUSSION .................................... 28


V
REFERENCES........................................... 31
APPENDIX............................................. 34


vi
TABLES
Table
1. I-ALA content of transformant and cured
strains...................................... 2 6
2. Specific activities of ALA dehydratase in
transformant and cured strains
27


Vll
FIGURES
Figure
1. Heme Biosynthetic Pathway ..................... 2
2. Outline of mechanisms of porphobolilinogen
synthesis from two molecules of delta-
aminolevulinic acid ...................... 4
3. Construction of pSDl........................... 9
4. Restriction Map of pBR3 22 11
5. Construction of pSD2.......................... 16
6. Spectra of C41/Ul-pSDl transformant .... 21
7. Effects of pSD2 transformation and curing
on cytochrome synthesis by hem2-15
strain C41/U1
24


CHAPTER 1
INTRODUCTION
The biosynthesis of heme and/or porphyrins (Fig.l)
commences with the condensation of glycine and succinyl CoA
to form delta-aminolevulinic acid (ALA). This condensation
is catalyzed by the enzyme ALA synthase. Delta-
aminolevulinate dehydratase (ALA-D) is the second enzyme
involved in this biosynthetic pathway. It is responsible
for catalyzing the condensation of two molecules of ALA to
form porphobilinogen (PBG), the monopyrrolic precursor of
the tetrapyrrolic porphyrins. Four molecules of PBG are
condensed in a reaction catalyzed by PBG deaminase to form
hydroxymethylbilane (HMB). HMB appears to be an unstable
intermediate in the conversion of PBG to uroporphyrinogen
III (Jordan et al. 1980; Battersby et al. 1979; Evans et
al. 1986a; 1986b). Uroporphyrinogen III is subsequently
decarboxylated to produce coproporphyrinogen, which is then
converted to protoporphyrinogen. Protoporphyrinogen is
then oxidized to the tetrapyrrole protoporphyrin IX.
Metalloporphyrins serve as the prosthetic groups for many
important enzymes and other proteins. The most important


2
COjH
COjH
HOjC
"I
SCoA
NH,
GLYCINE-*-
SUCCINYL CoA
nh2
A
> ALA
*
B C D
----> PBS------> HYDROXYMETHYLBILANE-
UROPORPHYRINOGEN III------> COPROPORPHYRINOGEN III------>
PROTOPORPHYRINOGEN IX-----> PROTOPORPHYRIN IX----> HEME
ENZYME YEAST GFNE SYMBOL
ah = CH^CO^H
A = ALA SYNTHASE (ALA-S) HEM1 P* = CH^CHiCO^H
V = CH=CHj,
B = ALA DEHYDRATASE (ALA-D) HEMS Me = -CH,
C = PBG DEAMINASE HEM3
D = UROPORPHYRINOGEN III SYNTHASE
E = URODECARBOXYLASE HEM6(HEM 12)
F = CDPROPORPHYRINOSEN OXIDASE HEM4
G = PROTOPORPHY RINOGEN *
H = FERROCHELATASE HEM5 '
* = YEAST MUTANTS NOT YET CHARACTERIZED
FIGURE 1
HEME BIOSYNTHETIC PATHWAY


3
iron porphyrin is heme, an iron chelate of protoporphyrin
IX. Hemoproteins have a variety of functions in energy
metabolism, as oxygen carriers (hemoglobin and myoglobin),
electron carriers (cytochromes) and respiratory enzymes.
Another important group of porphyrin derivatives are the
chorophylls, which are chelates of Mg2+. The chorophylls
serve as energy adapters in plants and photosynthetic
bacteria (Lascelles 1964).
The enzyme ALA-D has been identified in bacteria, plants
and animals (Borralho et al. 1989). ALA-D catalyzes the
condensation of two molecules of ALA to form the pyrrole
PBG with the release of two water molecules. The mechanism
of action of this enzyme (Fig.2) appears to involve a
Schiff's base formation between an episilon-amino group of
a lysine residue in the enzyme active site and one molecule
of the substrate, ALA. This enables carbon 3 of the bound
ALA molecule to act as a nucleophile towards the carbonyl
of a second ALA molecule (Battersby and McDonald 1975;
Jaffe and Hanes 1986; Jaffe and Markham 1987). Water is
then eliminated, allowing double bond formation, and a
subsequent tautomeric shift yields the final pyrrole
structure of PBG.
The ALA-D structural gene from yeast (HEM2.) and a human
liver cDNA coding for the human ALA-D have both been


S-AMINOLEVTJUNIC ACID DEHYDRATASE
ENH, +
COOH
cn,
fH,
-H*
-H,0
COOH
V
CH +
II
COOH
cn,
c
+ H
I II
COOH
COOH CH.
I I *
CH, CH,
HC------C-OH
O^C (pH, NH, 1 H /CH, NH, EN=C CH, NH, /CH, NH,
-H,0 + H*
II 1 II 1 II
ft
NH,
I
^OOH CH,
CH, CH,
Izt-%m****
(jlOOH CH,
(jTH, CH,
1*
H
?
OOH
NH, H
(pOOH
Lh,
I*
f
CH
Fiu. Z Outline of incclmniams of iwrpliobilinogen synthesis from two molecules
of 4-nminolcvulinic acid.
Reference
Shemin, D. (1972) Enzymes 3rd Ed. &, 323-337


5
isolated and sequenced (Myers et al. 1987; Wetmur et al.
1986). A comparision of the DNA-derived sequence of the
two proteins, human ALA-D (hALA^D) and yeast ALA-D (HEM2),
show that the two enzymes share a 52% homology over the
entire length of the proteins. The most highly conserved
regions, found in the middle of the proteins, have a
homology of 62% (Myers et al. 1987). This region also
contains the putative zinc binding site of the enzyme.
Physically the yeast enzyme and the human enzyme resemble
each other quite closely. Both are octamers; hALA-D is
composed of eight identical subunits of Mr 35,000 (Jaffe et
al. 1984; Wetmur et al.1986) whereas the yeast enzyme is
composed of subunits of 37,000 (Borralho et al. 1989). Zn2+
is required by both enzymes for maximum activity. In one
respect the two enzymes differ markedly: the pH optimum for
hALA-D is in the range of 6.3 7.1,(Anderson et al. 1979;
Gibbs et al. 1985) whereas the yeast ALA-D has an extremely
alkaline pH optimum ranging from 9.6 9.8 (De Barreiro
1967; Borralho et al. 1989).
A defect in the hALA-D gene, which is assigned to the
chromosome region 9ql3-qter (Wetmur et al. 1986), can lead
to an inherited disease called a porphyria. A number of
inherited porphyrias are associated with defects in the
enzymes catalyzing different biosynthetic steps. Enzyme
defects occurring late in the pathway lead to porphyrin


6
accumulation, while those defects occurring early lead to
accumulation of the porphyrin precursors (ALA and PBG).
Porphyria diseases are characterized by the excretion of
large amounts of porphyrins or their precursors in urine
(Brandt and Doss 1981; Doss et al. 1983). There are three
inherited acute porphyrias which are autosomal dominant.
Acute intermittent porphyria, variegate porphyria and
hereditary coproporphyria involve overproduction of ALA
synthase. The fourth acute porphyria involves a deficiency
in ALA-D and is an autosomal recessive. This type of
porphyria is very rare, and only two cases have been
described so far (Doss 1986). Symptoms associated with the
acute hepatic porphyrias include abdominal colics, loss of
neurological function, tachycardia and hypertension.
Exposure to lead and alcohol intake should be avoided by
individuals with porphyric syndromes as they inhibit ALA-D
(Doss et al. 1983). Lead poisoning symptoms are very
similiar to the symptoms of inherited acute porphyrias. In
some porphyrias non-acute symptoms also occur such as
photosensitivity, and arthritis due to the accumulation of
porphyrin in skin and teeth (Doss 1986).
Because the HEM2 gene in yeast is required for heme
formation, it is essential for cell respiration. Heme is
utilized as the prosthetic group of the respiratory
cytochromes aa3. b, c and cl. If a deleterious mutation


7
occurs in the HEM2 gene, all these respiratory enzymes are
affected. The hem2 mutants in yeast belong to a group of
respiratory-deficient mutants which have certain
characteristics. Because hem2 mutants have no
ALA-D activity and produce no heme, they cannot make the
microsomal cytochrome b5 which is required for production
of unsaturated fatty acids. Consequently, a source of oleic
acid, usually in the form of Tween 80, is required for
growth. Methionine is also required because the mutant
cannot make siroheme, needed for sulfite reductase (Murphy
et al. 1973; Woods et al. 1975). The mutants also have
distinct absorption spectra which show deficiencies in all
cytochrome levels when compared to wild-type strains.
Mutants with partial defects, for example hem2-15 mutants,
will produce limited levels of cytochromes (Myers et al.
1987) .
Since the homology between ALA-D from yeast and human
liver is high, it was of interest to determine if the
human gene introduced into a yeast strain containing a hem2
mutation could function there. If such heterologous
expression occurs, yeast could be used as a practical
source of large amounts of human ALA-D for biochemical
studies similiar to those of Jaffe (1986; 1987). In order
to express the hALA-D gene in yeast, an appropriate yeast
expression signal is required. In the present study a


8
plasmid containing a cDNA coding for the human ALA-D under
the control of the yeast GAL10 promoter was constructed.
This plasmid was then introduced into a yeast containing a
hem2 mutation. Expression of the hALA-D gene was
accomplished as shown by 1) improved growth, 2) in vivo
depletion of accumulated ALA, 3) restoration of cytochrome
biosynthesis and 4) by direct enzyme assay.
OBJECTIVE
The purpose of this study was to determine whether human
delta-aminolevulinate dehydratase could be expressed in
yeast. To this end recombinant DNA methods were used to
insert a cDNA isolated from a human liver cDNA library into
a yeast expression vector.
MATERIALS AND METHODS
PLASMIDS
A pUC9 plasmid (Fig.3 and Appendix) containing a human
liver cDNA coding for delta-aminolevulinate dehydratase
(hALA-D) was obtained from Robert Desnick and James Wetmur,
Mount Sinai Hospital, New York (Wetmur et al. 1986). The
hALA-D gene was isolated from pUC9 by digestion with the
restriction enzyme Haell (Fig.3). This enzyme cuts the
pUC9 plasmid into three fragments. The largest fragment is


HPAI
LEU 2
ECORI
ECORI
CUT
PST 1
LAC Z
ECORI
Fig. 3. Construction of pSDl


10
2.0 Kb and contains part of the pBR322 sequence (Fig.4),
including a gene for ampicillin- resistance. A second
fragment, "1.6 Kb, contains the hALA-D gene and the EL. coli
lacZ gene which codes for beta-galactosidase. The third
fragment, "0.3 Kb, represents the rest of the plasmid DNA.
The restriction digest was run on a 1% agarose gel, the
band containing the hALA-D was cut out and eluted from the
gel with a sodium iodide solution and absorbed on glass
beads, using a kit called Geneclean, a product of Bio 101
Labs, La Jolla, CA. The purified DNA fragment was then
treated with T4 DNA polymerase and deoxynucleotides to
generate blunt ends for subsequent ligation into a yeast
expression vector.
Plasmid YEp62 (Fig.3) is a yeast expression vector which
contains a pBR322 fragment including ampicillin resistance,
lacZ. yeast LEU2. and the yeast GAL10 promoter (Broach et
al. 1983; St. John et al. 1979; 1981). YEp62 was chosen to
express the hALA-D cDNA in yeast because it contains the
GAL 10 promoter which is inducible by galactose (St.John and
Davis 1979; 1981). The presence of the EL. coli lacZ gene is
used for constructing fusion proteins, but was not employed
in the present study. Digestion of YEp62 with Smal
generates a single blunt-ended cut between the GAL10
promoter and lacZ.
Plasmid YEp24 is an EL. coli-veast shuttle vector


11
Cto 123
Acc I 2246
1. Bolivar. F., Rodriguez, R.L., Greene, P.J., Betlach, M.C.,
Heynecfcer, H.L and Boyer, H.W. (1977) Gene 2, 95-113
2. Sutcliffe, J.G. (1978) Cold Spring Harb. Symp. Quant. Biol.
43,77-90
3. Sutdiffe, J.G. (1978) Free. Nad. Acad. Set. USA 75.3737-
3741
4. Peden, K.W.C. (1983) Gene 22.277-280
5. Badanan, K. and Boyer, H.W. (1983) Gene 26,197-203
X Lathe, R KJeny, M.P., SKory, S. and Lecocoq, J.P. (1984)
DNA X 173-182
7.1 lauatarapiauta, M. and Davison, J. (1984) DNA 3,259-264
X Roberta, RJ. (1987) Nucleic Adda Raa. 15, Supplement.
r189-1217
X GenBank (1987) 50.0
Fig. U. Restriction Map of pBR322 DNA


12
containing yeast 2- micron DNA origin of replication, yeast
URA3 and pBR322 with tetracycline resistance and ampicillin
resistance for selection in Eh. coli. YEp24 was chosen as a
vector because of the URA3 gene and the presence of
compatible restriction sites. URA3 plasmids can be
specifically "cured" by growing yeast transformants on
minimal medium containing 5-fluoroorotic acid (5-FOA) and
uracil.
STRAINS
The mutant yeast strain used in this study was C41/U1,
which was obtained from Alan Myers, Department of
Biochemistry, Iowa State University. The genotype of C41/U1
is a ura3 hem2-15. The hem2-15 allele is a missense
mutation which permits slight synthesis of ALA-D (Myers et
al. 1987), so it is referred to as "leaky".
D28 (alpha lys) and D273-10B (aloha) were used as the
control strains. Eh. coli strains C600SF8 (thi-1 thr-1 leuB6
lacYl tonA21 supE44) and JM109 (recAl endAl gyrA96 thi
hsdR17 supE44 relAl (lac-proAB)/ F' [traD36 proA+ proB+
laclq lacZ M15] were also used (Ausubel et al. 1987).
MEDIA AND GROWTH CONDITIONS
The growth media included (a) YPD; 1% yeast extract, 2%
peptone, 2% dextrose. (b) YPG; 1% yeast extract, 2%
peptone, 3% glycerol, (c) YPAD; same as YPD with addition


13
of adenine sulfate, 100 mg/1. (d) YHAT; same as YPAD with
the following additions: Tween 80, 10ml/l; and hemin,
25mg/l. (e) minimal medium: 0.67% Yeast Nitrogen Base
without amino acids, 3% glycerol, 2% succinic acid, and 1%
D-galactose. Other nutritional supplements were added as
required: uracil 100mg/l; and Tween 80, 10ml/l. Cells were
grown at 30 C.
TRANSFORMATION OF E. COLI AND YEAST
Transformation of coli strains JM109 and C600SF8 was
carried out as described by Davis, Dibner and Battey
(1986) The E_j_ coli cells are grown to mid log phase and
treated with calcium choride to make them competent for
transformation. Plasmid DNA is then introduced to the cells
and they are incubated at 37 C for an hour. Amplification
of the plasmid DNA occurs upon the addition of fresh medium
and further incubation. At the conclusion of this
incubation the cells are plated on medium and incubated
overnight. Transformation of yeast was performed with
lithium acetate as described by Ito et al.(1983). The yeast
cells are grown to mid log phase and treated with lithium
acetate to generate competent cells. The plasmid DNA is
introduced to the cells along with salmon sperm DNA. Salmon
sperm DNA appears to aid in the induction of plasmid DNA
into the yeast cells, though the exact mechanism is not
known. After several incubations the cells are resuspended


14
in buffer, plated on medium and allowed to incubate at 30
C for three to six days. At this point transformant
colonies should appear.
CONSTRUCTION OF PLASMID nSDl CONTAINING hALA-D fFicr.3)
The hALA-D fragment was ligated into the Smal site of
YEp62. To obtain control of heterologous gene expression it
is necessary to place the gene of interest (in this case
hALA-D) under the control of a yeast promoter. The Smal
digest of YEp62 was treated with calf intestinal alkaline
phosphatase (CIAP) to dephosphorylate the ends of the
linearized plasmid. Ligation was achieved by using a 3:1
molar ratio of insert to linearized vector fhALA-D:YEp62)
and incubating with one unit of T4 ligase and
deoxynucleotides overnight at 14 C. A sample of the
ligation mixture was run on a gel the following morning to
estimate the amount of ligated DNA. This ligation mixture
was then used to transform the EL. coli strain JM109. DNA
was extracted from a culture derived from a transformant
colony and used to transform the E.coli strain C600SF8
which is easier to maintain than strain JM109. The chimeric
pSDl plasmid DNA was amplified in C600SF8, and the
extracted DNA was used both for restriction mapping and for
yeast transformation.


15
RESTRICTION MAPPING OF PSDl
Digestion of the amplified plasmid DNA by EcoRI. Hindlll
and PstI confirmed that the desired ligation had taken
place. A restriction map of the constructed plasmid was
drawn from the gel electrophoresis data obtained from the
digests (Fig.3). The restriction fragment sizes (in Kb)
were determined from the measurements of fragment migration
distances which were entered into a program given to us by
J. Croonenberghs from the Adolph Coors Co. This YEp62-hALA-
D constructed plasmid was given the name pSDl. The plasmid
is suitable for transforming yeast containing a leu2
marker.
CONSTRUCTION OF PLASMID PSD2 CONTAINING hALA-D (Fio.5^
This construction was begun by digesting pSDl with the
restriction enzyme EcoRI. As shown in Fig.5, this enzyme
generates three fragments from pSDl. The largest fragment,
."5.6 Kb, contains a section of the yeast LEU2 gene and 2-
micron yeast DNA. The second fragment, '2.6 Kb, contains a
second section of LEU2, and the yeast GAL10 promoter still
linked to the hALA-D gene. The third fragment, "2.5 kb,
contains lacZ and pBR322 sequences. The GALlO-hALA-D
fragment was excised from the gel and the DNA was eluted
and separated from _the agarose using the Geneclean
procedure. The isolated fragment was then treated with


ECORI
PBR 322
LEU 2
CUT GAL 10
LAC Z
ECORI
GAL 10
2fi
CUT
URA3
ECORI
ALA-D
Fig. 5. Construction of pSD2


17
T4 polymerase and deoxynucleotides to produce blunt- ends.
The yeast plasmid YEp24 was chosen as the URA3 vector. The
chimeric GALlO-hALA-D sequence was placed within the Smal
site of YEp24. The preparation of YEp24 for ligation
included digestion with Smal. followed by dephosphorylation
with CIAP and purification with Geneclean. The ligation
procedure was the same as that used in constructing pSDl.
The resulting ligation mixture was used to transform E.
coli C600SF8. After amplification, plasmid DNA was
extracted from C600SF8 using the Circle Prep DNA extraction
procedure (Bio 101).
RESTRICTION MAPPING OF PSD2
Restriction mapping with EcoRI. Hindlll and PstI
confirmed the ligation, and the new plasmid was designated
pSD2.
CYTOCHROME DETERMINATIONS
The cytochrome levels in the yeast controls and
transformants were estimated from whole-cell spectra. Yeast
cells were grown for 72 hours at 30 C and 300 rpm in 200 ml
of minimal medium containing galactose and glycerol in 500-
ml Erlenmeyer flasks, then centrifuged and washed with
deionized water. Suspensions of 2.5 mg dry weight per ml in
phosphate buffer were examined using a split-beam
spectrophotometer. A dilute milk suspension was used as


18
reference.
DETERMINATION OF INTRACELLULAR ALA AND ALA DEHYDRATASE
The intracellular ALA concentrations in yeast cells were
determined as described by Malamud et al. (1979). The ALA
dehydratase activity was determined in situ as described by
Borralho et al.(1983).
RESULTS
CO-TRANSFORMATION OF hALA-D AND YEP24
A preliminary experiment was made to determine whether
or not the pUC9 plasmid containing the hALA-D gene would
function in yeast. The plasmid containing hALA-D was
linearized by digestion with Hindlll and mixed with the
vector YEp24. The mixture was then used to transform strain
C41/U1 (a ura3 hem2-15). Transformants were selected on
minimal medium containing glycerol and galactose as a
carbon source. Near-normal cytochrome spectra were obtained
with suspensions of transformed cells, indicating that the
hALA-D gene complemented the hem2-l5 defect.


19
TRANSFORMATION OF C41/U1 WITH PLASMID PSD1
In order to establish that the hALA-D gene in plasmid
pSDl could function, the plasmid was used to transform
strain C41/U1. Transformants which could result from
complementation between the hem2 and plasmid-borne hALA-D
were selected by plating on minimal glycerol and galactose
medium supplemented with uracil. Galactose was used
because it activates the GAL10 promoter, which in turn
activates transcription of the hALA-D gene. Selection for
growth on glycerol as primary carbon source was used
because the plasmid did not contain the ura3 gene needed
for nutritional selection. In order to utilize glycerol,
yeast must have adequate cytochromes, which in turn,
require functional ALA-D. Although the galactose used as
inducer could serve as a fermentable carbon source and
should support growth of the hem2-15 mutant, it was
observed that strain C41/U1 grew extremely slowly in the
minimal medium, whereas transformants of this strain grew
quite well. Colonies were obtained, used to inoculate flask
cultures and spectra were run. The transformed strain
(C41/Ul-pSDl) and the untransformed mutant (C41/U1) were
both grown on minimal galactose medium containing
appropriate supplements, as needed, for 48 hours. Strain
C41/U1 had a spectrum which showed no cytochrome aa3 and
very low b, c and cl cytochromes (Fig. 6). In contrast,


20
C41/Ul-pSDl had a spectrum similiar to that of wild type
(see Fig.6) containing strong aa3. b, c + cl cytochrome
bands. To verify that these colonies were actual
transformants and not hem2 revertants, it was necessary to
select cells that had lost the pSDl plasmid. This was done
by subculturing the transformed cultures on YHAT medium
three times, selecting single colonies, then replica-
plating these subclones on YPG and YPD media. YHAT medium
contains all nutritional requirements for normal yeast
strains as well as Tween 80 and hemin to support growth of
hem2 mutants. When hem2/hALA-D transformants are permitted
to grow on this rich medium, the selective pressure to
maintain the plasmid is minimized and mitotic segregation
of cells lacking plasmid from transformed cells can occur.
Subcultures from YHAT plates were tested by replica-plating
on YPG and YPD media. YPG is a glycerol medium on which
hem2 mutants will grow extremely poorly, if at all, whereas
on YPD medium the glucose in the medium will permit
fermentative growth of these mutants. When transformants
which have grown on YHAT medium were plated, both large and
small colonies resulted. The small colonies on YPD plates
should have arisen from transformants which have lost the
plasmid. Loss of the pSDl plasmid was confirmed by running
cytochrome spectra and comparing the transformant and a
"cured" strain to the untransformed strain (Fig.6). The
results showed that the transformant gave a spectrum


21
C+C1
Pig. 6. Spectra of C4l/Ul-pSDl transformant


22
similiar to that of wild type cells, whereas the "cured"
strain gave a spectrum just like that produced by the
untransformed strain. The results suggested that since the
hALA-D gene did function on a plasmid in the leaky hem2-15
strain, C41/U1, it would be worthwhile constructing a
plasmid containing URA3 which would allow for direct
nutritional selection of transformants of strain C41/U1.
Moreover, this type of selection would minimize the
selective pressure for revertants of the hem2 mutation in
the recipient strain. In addition, demonstration that
selected strains were transformants instead of revertants
could be facilitated by "curing" transformants of plasmid
using 5-fluoroorotic acid. A URA3 GALlO-hALA-D plasmid was
therefore constructed and used to transform strain C41/U1.
TRANSFORMATION OF C41/U1 WITH PSD2
The hem2-15 ura3 strain C41/U1 was transformed with pSD2,
and transformants were selected on minimal medium
containing glycerol and galactose. Successful ligation was
confirmed by the fact that pSD2 complemented both the
uracil auxotrophy and the defective growth on
glycerol/galactose medium exhibited by strain C41/U1. One
of the transformant colonies was subcultured on minimal
medium containing glycerol and galactose. The cytochrome
spectrum of the cells was compared to that of untransformed


23
C41/U1 cells grown on the same medium supplemented with
Tween 80, as shown in Fig. 7. The pSD2 transformant
exhibited a spectrum similiar to that of wild type cells,,
except that the c + cl band was relatively low. Growth of
the transformant was more efficient than that of the hem2-
15 mutant; after 48 hours incubation yields of cells (mg
dry wt/ml) were 11.6 and 6.03, respectively. To verify
that the transformants were indeed "true" transformants,
rather than revertants, the cultures were "cured" of the
plasmid by culturing on minimal medium containing 5-FOA and
uracil. The ura3 mutant strains are resistant to 5-FOA
because the mutation prevents decarboxylation of 5-FOA to
the toxic analog 5-fluorouracil, whereas URA3 transformants
carry out this conversion and die (Boeke et al. 1984). In
order for a yeast cell to survive on this medium, the URA3-
containing plasmid must be lost during the replication
process, so that the defective, chromosomal ura3 allele
can be expressed. The "cured" strain can grow on the 5-FOA
medium because the medium also contains uracil. The
"cured" transformants were then plated on uracil-deficient
medium lacking 5-FOA to confirm loss of the Ura+ plasmid.
The "cured" transformants were found to express both the
ura3 and hem2 markers of the untransformed strain C41/U1
(Fig.7). Therefore, the coincidential loss of both URA3 and
hALA-D had occurred, eliminating the possibility that the
hem2 gene could have reverted.


24
C+C1
Fig. 7. Effects of pSD2 transformation and curing on cytochrome
synthesis hy hem2-15 strain CUl/Ul


25
INTRACELLULAR ALA AND ALA DEHYDRATASE OF TRANSFORMED AND
CURED STRAINS
Growth curves were obtained for the hem2-15 mutant and
the corresponding pSD2 transformant in preparation for
intracellular delta-aminolevulinic acid (I-ALA)
determination. This assay measures the concentrations of
ALA accumulated in the yeast cells at the end of
exponential growth phase. In these assays minimal medium
with galactose and glycerol was used because expression of
the human cDNA is under control of the galactose-inducible
promoter GAL10. The presence of the uncomplemented hem2-15
mutation causes an abnormal accumulation of I-ALA, with
values ranging from 1.5-2.5 nmoles/mg. The I-ALA values for
mutant C41/U1, transformant C41/Ul-pSD2, wild type D28,
wild type D273-10B, and cured transformant C41/U1-C are
shown in Table 1.


26
TABLE 1. I-ALA content of transformant and cured strains
I-ALA
GROWTH
(nmoles/mg D.W.)
C41/U1 (mutant control) 1.93
C41/Ul-pSD2 0.43
C41/U1-C 1.8
D28 (wild type) 0.12
D273-10B (wild type) 0.11
(mg(D.W.)/ml)
6.03
11.6
7.04
12.2
12.0
Strains were grown in minimal galactose medium with
appropriate supplements for 72 hours in 500-ml Erlenmeyer
flasks, at 30 C and 300rpm.
As expected, the culture of the transformant has only about
25% as much I-ALA as the mutant, indicating that the human
hALA-D gene is being expressed and that the resulting
enzyme is metabolizing ALA. However, the I-ALA values of
the cells transformed with pSD2 are significantly higher
than wild type values. The "cured transformant exhibits an
I-ALA level similiar to that of strain C41/U1.
Further confirmation that the human gene can be expressed
in yeast was obtained by direct assay of ALA-D activity in
situ (Borralho et al. 1983), as shown in Table 2.


27
TABLE 2. Specific activities of ALA dehydratase in
transformant and cured strains
Strain
ALA-D specific activity (nmoles/mcr D.W.)
pH 9.6 pH 6.8
C41/U1 (mutant) 0.10 0.095
C41/U1pSD2 0.14 0.27
C41/U1-C 0.09 0.09
D28 (wild type) 0.47 0.47
Strains were grown in minimal galactose media at 30 C and
300rpm to the end of exponential phase. These measurements
were obtained from a assay reaction time of ten minutes.
The ALA-D activity was determined at two pH values, near
the optima for the yeast (pH 9.6) and for the human liver
(pH 6.8) enzymes, respectively. Although the activity of
the yeast enzyme in wild type cells (D28) did not appear to
change with pH, the enzyme activity in transformant cells
was twice as high at the lower pH, indicating that the


28
"recombinant" enzyme behaves like enzyme isolated from
human liver.
DISCUSSION
In this study a human gene (cDNA) coding for ALA-D was
found to function in yeast. This was initially demonstrated
by the co-transformation of a linearized pUC9 plasmid
containing the hALA-D coding seguence with the URA3 vector
YEp24, into the yeast strain C41/U1. This was an important
experiment which demonstrated that'complementation of the
yeast hem2 by the heterologous human gene is possible. It
seems likely that site-specific recombination occurred
between the hem2-15 gene resident in the yeast chromosome
and the hALA-D DNA such that at least a segment of the
yeast gene bearing the mutation was replaced by normal
human cDNA sequence. It is also reasonable to expect that
the recombination produced a human/yeast "hybrid" gene
which remained under control of the HEM2 promoter. The
construction of plasmid pSDl and its ability to complement
hem2-15 showed that the entire hALA-D gene could be
expressed and function in yeast. The crucial experiment was
the construction of plasmid pSD2 which permitted not only
direct transformation of hALA-D into strain C41/U1, but
also specific curing to eliminate the possibility of hem2


29
reversion. The pSD2 transformant was used to examine gene
expression in several ways. First, the introduction of
hALA-D restored the ability of hem2-15 cells to grow on
nonfermentable carbon sources, reflecting the restoration
of the cytochromes and cell respiration. Second,
cytochrome restoration was observed directly, using whole-
cell spectra. Third, the presence of hALA-D largely
reversed the accumulation of. intracellular ALA resulting
from the hem2-15 block in porphobilinogen formation.
Finally, increased ALA-D activity was measured directly by
the in situ assay of Borralho et al. (1983).
The apparently incomplete restoration of ALA-D activity
to normal may reflect a limited activity of the hALA-D
protein or its expression in the yeast. Alternatively,
expression may be adequate, but a fraction of the cell
population may have lost the plasmid.
A major advantage of using the URA3 hALA-D plasmid pSD2
is that transformants can be specifically freed of this
plasmid by plating cultures on medium containing 5-FOA and
uracil. Coincident loss of Ura+ phenotype and reemergence
of the
hem2-15 phenotype clearly established that transformation,
rather than hem2 reversion, had been accomplished.
That the enzyme produced in the pSD2 transformant of strain
C41/U1 is in fact "human-type" ALA-D is indicated by the
behavior of the enzyme when it is assayed at different pH


30
values; greater activity was observed at pH 6.8, the
optimum for the human enzyme (Anderson et al. 1979; Gibbs
et al. 1985), than at pH 9.6, the optimum observed with
purified yeast ALA-D (Borralho et al. 1989). It may be
noted, however, that ALA-D activity assayed in., situ in wild
type yeast did not exhibit the reverse effect, but was the
same at either pH. The cause of this behavior is not
known. The successful heterologous expression of the human
liver ALA-D in yeast provides an opportunity to produce
large quantities of the purified human enzyme for
biochemical and physical investigations. Borralho et al.
(1989) have used yeast transformed with the HEM2 gene on
multicopy vector YEp24 as a basis for purifying yeast ALA-
D to homogeneity. A similiar approach should be applical
to purifying the human ALA-D from yeast.


31
REFERENCES
Anderson, P.M., and Desnick, R.J. (1979). Purification and
properties of delta-aminolevulinate dehydrase from human
erythrocytes. J Biol. Chem. 254, 6924-6930.
Ausubel, F.M., Brent, R. Kingston, R.E., Moore, D.D.,
Smith, J.A., Siedman, J.G. (1987). Current Protocols in
Molecular Biology in Struhl, K. (ed.), Selected topics from
classical bacterial genetics. Green Publishing Associates
and Wiley-Interscience, New York, pp 1.4-1.5.
Battersby, A.R., McDonald, E. (1975). Porphyrins and
Metalloporphyrins in Smith, K.M. (ed), Biosynthesis of
porphyrins, chlorins and corrins. Elsevier Scientific
Publishing Co, New York, pp 61-122.
Battersby, A.R., Fookes, G.J.R., Matcham, G.W.J., McDonald,
E, and (in part) Gustafson-Potter KE (1979). Chemical and
enzymatic studies on biosynthesis of the natural porphyrin
macro.cycle, formation and role of unrearranged
hydroxymethybilane and order of assembly of the pyrrole
rings. J. Chem. Soc., Chem Comm 316-319
Boeke, J.F., LaCroute, F., Fink, G.R. (1984). A positive
selection for mutants lacking orotidine-51-phosphate and
carboxylase activity in yeast: 5-fluroro-orotic acid
resistance. Mol. Gen. Genet. 197, 345-346.
Borralho, L.M., Panek, A.D., Malamud, D.R., Sanders, H.K.,
and Mattoon, J.R. (1983) In situ assay for 5-
aminolevulinate dehydratse and application to the study of
a catabolite repression-resistant Saccharomvces cerevisiae
mutant. J. Bacteriol. 156, 141-147.
Borralho, L.M., Ortiz, C.H.D., Panek, A.D., and Mattoon,
J.R. (1989) ALA dehydratse from genetically engineered
yeast, (submitted).
Brandt, A., and Doss, M. (1981). Hereditary porphobilinogen
synthese deficiency in human asociated with acute hepatic
porphyria. Human. Genet. 158, 194-197.
Broach, J., Li, Y.Y., and Wu, L.C.C. (1983). Experimental
Manipulation of Gene Expression in Inouye, M. (ed), Vectors
for high-level, inducible expression of cloned genes in
yeast, Academic Press, New York, pp 83-117.


32
Davis, L.G., Dibner, M.D., and Battey, J.F. (1986). E. coli
transformation. Methods in Molecular Biology Elsevier
Scientific Publishing Co, New York, pp 90-93.
De Barreiro, O.L.C., (1967). 5-Aminolevulinate hydrolyase
from yeast. Isolation and purification. Biochim. Biophys.
Acta. 139, 479-486.
Doss, M., Tiepermann, R.V. and Schneider, J. (1983).
Porphobilinogen-synthase (delta-aminolevulinic acid
dehydratase) deficiency in bone marrow cells of two
patients with porphobilinogen-synthase defect acute
porphyria. Klin Wochenschr 61, 699-702.
Doss, M., (1986). Clinical biochemistry of acute hepatic
porphyrias. Porphyrins and Porphyrias, John Libbey and Co,
London, pp 175-187
Evans, J.N.S., Davies, R.C., Boyd, A.S.F., Ichinose, I.,
Mackenzie, N.E., Scott, A.I., Baxter, R.L. (1986a).
Biosynthesis of porphyrins and corrins 1. 1H and 3 C NMR
spectra of (hydroxymethyl) bilane and uroporphyrinogens I
and III. Biochem. 25, 896-901.
Evans, J.N.S., Burton, G. Fagerness, P.E., Mackenzie,
N.E., and Scott, A.I. (1986b). Biosynthesis of porphyrins
and corrins. 2. Isolation, purification and NMR
investigations of the porphobilinogen-deaminase covalent
complex. Biochem. 25, 905-912.
Gibbs, P.N.B., Chaudhry, A. and Jordan, P.M. (1985).
Purification and properties of 5-aminolevulinate
dehydratase from human erythrocytes. Biochem. J. 230, 25-
34.
Ito, H. Fukuda, Y. Murata, K. and Kimura, A. (1983).
Transformation of intact yeast cells treated with alkali
cations of Thiol compounds. J. Bacteriol. 153, 163-168.
Jaffe, E.K. and Hanes, D. (1986). Dissection of the early
steps in the porphobilinogen synthase catalyzed reaction.
J. Biol. Chem. 261, 9348-9353.
Jaffe, E.K. and Markham, G.D. (1987). 13C NMR studies of
porphobilinogen synthase: observation of intermediaties
bound to a 280,000-Dalton protein. Biochem. J. 26, 4258-
4264.
Jordan, P.M. and Berry, A. (1980)< Pre-uroporphyrinogen, a
universal intermediate in the biosynthesis of
uroporphyrinogen III. FEBS Lett. 112, 86-88.


33
Lascelles, J. (1964). The biosynthesis of tetrapyrroles:
Pathway to hemes and hemoproteins. Tetrapyrrole
Biosynthesis and Its Regulation (1964), WA Benjamin Inc,
New York, pp 38-66.
Malamud, D.R., Borralho, L.M., Panek, A.D. and Mattoon,
J.R. (1979). Modulation of cytochrome biosynthesis in
yeast by antimetabolite action of levulinic acid. J.
Bacteriol. 138, 799-804.
Murphy, M.J., Siegel, L.M., Kamin, H.and Rosenthal, D.
(1973). Reduced nicotinamide adenine dinucleotide
phosphate-sulfite reductase of enterobacteria. II.
Identification of a new class of heme prosthetic group: An
iron tetrahydroporphyrin (isobacterio chlorin type) with
eight carboxylic acid groups. J. Biol. Chem. 248, 2801-
2814.
Myers, A.M., Crivillone, M.D., Koerner, T.J. and Tzagoloff,
A. (1987). Characterization of the yeast HEM2 gene and
transcriptional regulation os C0X5 and COR1 by heme. J.
Biol. chem. 262, 16822-16829.
St John, T.P. and Davis, R.W. (1979). Isolation of
galactose-inducible DNA sequences for Saccharomvces
cerevisiae by differential plaque filter hybridization.
Cell 16, 443-452.
St John, T.P and Davis, R.W. (1981). The organization and
transcription of the galactose gene cluster of
Saccharomvces. J. Mol. Biol. 152, 285-315.
Wetmur, J.G., Bishop, D.F., Cantelmo. C. and Desnick, R.J.
(1986). Human delta-aminolevulinate dehydratase:
Nucleotide sequence of a full length cDNA clone. Proc.
Natl. Acad. Sci. USA. 83, 7703-7707.
Woods, R.A., Sanders, H.K., Briquet, M., Foury, F.,
Drysdale, B.E. and Mattoon, J.R. (1975). Regulation of
mitochondrial biogenesis: Enzymatic changes in cytochrome-
deficient yeast mutants requiring delta-aminolevulinic
acid. J. Biol. Chem. 250, 9090-9098.


34
APPENDIX
The pUC plasmids were designed to contain many restriction
sites, thus facilitating gene cloning by giving the
investigator many options for gene insertion. The early
cloning vectors were very limited as far as restriction
sites were concerned, containing only two or three sites
for gene insertion.
pUC plasmids contain ampicillin resistance as a selectable
marker. The plasmids also contain the E_i. coli lac operon as
a "reporter" gene for determining whether ligation as
occurred. They also contain multiple cloning sites (MCS)
inserted into the 5'which codes for the amino terminus of
beta-qalactosidase lacZ. The MCS are actually a large group
of restriction sites grouped into a small area on the
plasmid. These MCS allow DNA to be introduced into the
plasmid interrupting the function of the lacZ.
pUC8 and pUC9 were specifically constructed to contain a
series of single restriction sites. The restriction sites
of pUC8 are reversed relative to' those in pUC9, thus
enabling cloning of DNA fragments in both orientations
(i.e. 5'to 3' and 3'to 5').